Dissertations / Theses: '3D culture model'

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Zhao, Huizhi. "3D Cell Culture Model Synthesized By Polycaprolactone Nanofiber Electrospinning." Ohio University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1531319675295094.

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Peddagangannagari, Sreekanth Reddy. "An in vitro human 3D co-culture model to study endothelial-astrocyte interactions." Thesis, Open University, 2012. http://oro.open.ac.uk/54831/.

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At the gliovascular interface, reciprocal inductive influences between brain microvascular endothelial cells (BMVEC) and astrocytes occur. Most of the knowledge in this area of research is derived from in vitro eo-culture models in which astrocytes are cultured on a stiff, two-dimensional (2D) surface. Three-dimensional (3D) culture models closely mimic the in lJZVO cellular architecture and they bridge the gap between 2D culture models and animal models. Hence, an in vitro 3D eo-culture model was developed and characterised, to study the interactions between BMVEC and astrocytes. In this model, human astrocytes (HA) were seeded inside a collagen type--I gel while human immortalised cerebral microvascular endothelial cells (hCMEC/D3) were cultured on the gel surface. Both cell types were of human origin to improve the translatability of findings to humans in vivo. Additional important features of the model are the culture of endothelial cells on a soft matrix, and the simulation of the geometric relationship that exists in vivo i.e., the interaction of astrocytes with BMVEC from their ab luminal side. To determine the effect of the 3D environment on the HA, the proliferation rate and expression of four molecules namely, glial fibrillary acidic protein (GF AP), aquaporin-4 (AQP4), endothelin-l (Et-l) and endothelin receptor type-B (EDNRB), were compared between 3D and 2D cultured HA. The decreased expression of AQP4 and EDNRB and the much-decreased proliferation rate of 3D HA suggested their reduced reactivity and a similarity to their in lJiVO counterparts. However, 3D HA did not differ from the 2D HA in their ability to release soluble factors that induce barrier properties on BMVEC, as observed by similar levels of three expression markers of barrier phenotype on hCMEC/D3 cells namely, zonula occludens-l, claudin-5, and P-glycoprotein and similar paracellular permeability coefficients to fluorescent-dextrans (70 kDa).

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Smith, Jenny Thompson. "A 3D culture model to investigate cellular responses to mechanical loading in spinal cord injury." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/16199/.

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Spinal cord injury (SCI) can cause paralysis, loss of sensation, and respiratory dependency, which has a significant impact on the quality of life of patients, their life expectancy and is also a significant economic burden due to the high costs associated with primary care and loss of income. One of the difficulties in establishing a treatment method is the heterogeneity of SCI; there are many different types and severities of traumatic primary injury, across different age groups of patients and different locations within the spinal cord, whilst at a cellular level, there are multiple, interacting secondary injury cascades that amplify the primary damage inflicted during the traumatic insult. Many techniques have been developed to mimic particular injuries found in human SCI, however in vivo animal models can be extremely costly and time consuming. The modest translation of therapeutic treatments from animal models to successful clinical trials suggests that there is a need for simplified models of SCI, in which the complex secondary cascade can be broken down into specific cellular interactions under controlled injury parameters. It was hypothesised that in vivo injuries could be simulated using a 3D in vitro model of SCI within a tethered, self-aligned, type-I collagen gel. An in vitro model such as this could advance the understanding of cellular responses to injury and help inform animal studies which may facilitate the design of therapeutics. Initially, different matrices were investigated in order to determine their suitability for use as matrix components for a 3D in vitro model of SCI. The matrices were characterised in terms of their mechanical properties, and the cellular responses of astrocytes following culture within the matrices. A fully hydrated matrix was selected which had a lower elastic modulus in comparison to spinal cord tissue, and which maintained astrocytes in a non-reactive state, as determined by the expression of markers for reactive astrogliosis. Contusion models of SCI are thought to generate the most relevant animal models of SCI, therefore their suitability as an injury mechanism within a 3D cellular model was investigated. A pilot study using the Hatteras contusion device, demonstrated that there was potential for in vivo type contusion devices to be utilised with an in vitro 3D collagen gel SCI model. The remainder of the study utilised the Infinite Horizons (IH) in vivo impactor, which is a force controlled contusion device. The experimental parameters utilised with the IH impactor within an in vivo setting were investigated as to their suitability for collagen gel impactions. Following a detailed investigation, the in vivo parameters of an impact force of 200 kdyn and a dwell time of 0 ms, using a 2.5 mm diameter impaction tip were adopted; however the calibration start height of the impaction tip was altered to avoid full penetration of the impactor tip through the gel. The limitations of the contusion device affected the consistency of the impaction and resulted in a lack force output data. These limitations need to be resolved in order to directly compare in vivo and in vitro SCI using the IH impactor. The impaction of 3D aligned, collagen gels, seeded with primary rat astrocytes, using the IH impactor generated a 3D cavity bordered by reactive astrocytes, which was reminiscent of the glial scar and cystic cavity which forms at the lesion site in vivo. An increasing gradient of the astrocyte reactivity marker, glial fibrillary acidic protein, was expressed by cells closest to the impact zone. Astrocytes within the first 100 µm of the impact zone were highly ramified with cellular filaments aligned with the edge of the impact zone. An increase in the expression of astrocyte reactivity markers was observed over a ten-day period following impaction. In summary, a 3D model of SCI was developed that was highly adaptable, and suitable for further advancement to increase the complexity and experimental outputs that were presented in this study. More detailed analysis of the cellular responses, over longer time courses, and perhaps with the additional complexity of multiple cell types would complement investigations within in vivo models. 3D in vitro tethered collagen gel models such as this could provide valuable insights into the cellular mechanisms which may progress the translation of treatments into the clinic.

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Manzan, Martins Camilla. "EFFECT OF ENDOCRINE DISRUPTORS ON HUMAN ENDOMETRIAL STROMAL CELLS AND THEIR INTERACTION WITH TROPHOBLAST." Doctoral thesis, Università di Siena, 2022. http://hdl.handle.net/11365/1183943.

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Decidualization is crucial for embryo development and implantation, placenta formation and fetal growth. This process is characterized by morphological and biological changes in endometrial stromal cells that play a key role on fetal trophoblast migration and invasion. Successful placentation depends on the interaction between endometrial stromal cells and extravillous trophoblast cells. The trophoblast spheroids, a 3D culture model, is reported to appropriately mimic the in vivo situation, and reflect the cell to cell interaction. The Bisphenol A (BPA) and para-nonylphenol (p-NP) are endocrine disrupting chemicals (EDCs), present in the polycarbonate plastics used in many products such as food packaging, bottles and beverage cans and as an intermediate in the production of phenolic resins. Studies demonstrated that maternal exposure to EDCs, at environmentally relevant concentrations, are associated to aberrant early embryo development and uterine receptivity due to their estrogenic activity. Nowadays it is known that environmental contaminants can change stromal cell decidualization and trophoblast migration. In the present study, we developed a simple 3D culture model using transformed human endometrial stromal cells (tHESCs) and immortalized first trimester human extravillous trophoblast cells (HTR-8/SVneo). The aim of this work was to evaluate the effect of BPA and p-NP on endometrial stromal cells during decidualization and their interaction with trophoblast spheroids. The data showed that pre-exposition to p-NP of endometrial stromal cells impaired decidualization interfering on the cross-talk with trophoblast and altering lysosomes biogenesis and consequently leading to an impairment in trophoblast migration.

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Sieh, Shirly. "Development of a 3D culture system to study the skeletal metastasis of prostate cancer." Thesis, Queensland University of Technology, 2011. https://eprints.qut.edu.au/50870/1/Shirly_Sieh_Thesis.pdf.

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In the cancer research field, most in vitro studies still rely on two-dimensional (2D) cultures. However, the trend is rapidly shifting towards using a three-dimensional (3D) culture system. This is because 3D models better recapitulate the microenvironment of cells, and therefore, yield cellular and molecular responses that more accurately describe the pathophysiology of cancer. By adopting technology platforms established by the tissue engineering discipline, it is now possible to grow cancer cells in extracellular matrix (ECM)-like environments and dictate the biophysical and biochemical properties of the matrix. In addition, 3D models can be modified to recapitulate different stages of cancer progression for instance from the initial development of tumor to metastasis. Inevitably, to recapitulate a heterotypic condition, comprising more than one cell type, it requires a more complex 3D model. To date, 3D models that are available for studying the prostate cancer (CaP)-bone interactions are still lacking. Therefore, the aim of this study is to establish a co-culture model that allows investigation of direct and indirect CaP-bone interactions. Prior to that, 3D polyethylene glycol (PEG)-based hydrogel cultures for CaP cells were first developed and growth conditions were optimised. Characterization of the 3D hydrogel cultures show that LNCaP cells form a multicellular mass that resembles avascular tumor. In comparison to 2D cultures, besides the difference in cell morphology, the response of LNCaP cells to the androgen analogue (R1881) stimulation is different compared to the cells in 2D cultures. This discrepancy between 2D and 3D cultures is likely associated with the cell-cell contact, density and ligand-receptor interactions. Following the 3D monoculture study, a 3D direct co-culture model of CaP cells and the human tissue engineered bone (hTEBC) construct was developed. Interactions between the CaP cells and human osteoblasts (hOBs) resulted in elevation of Matrix Metalloproteinase 9 (MMP9) for PC-3 cells and Prostate Specific Antigen (PSA) for LNCaP cells. To further investigate the paracrine interaction of CaP cells and (hOBs), a 3D indirect co-culture model was developed, where LNCaP cells embedded within PEG hydrogels were co-cultured with hTEBC. It was found that the cellular changes observed reflect the early event of CaP colonizing the bone site. In the absence of androgens, interestingly, up-regulation of PSA and other kallikreins is also detected in the co-culture compared to the LNCaP monoculture. This non androgenic stimulation could be triggered by the soluble factors secreted by the hOB such as Interleukin-6. There are also decrease in alkaline phosphatase (ALP) activity and down-regulation of genes of the hOB when co-cultured with LNCaP cells that have not been previously described. These genes include transforming growth factor β1 (TGFβ1), osteocalcin and Vimentin. However, no changes to epithelial markers (e.g E-cadherin, Cytokeratin 8) were observed in both cell types from the co-culture. Some of these intriguing changes observed in the co-cultures that had not been previously described have enriched the basic knowledge of the CaP cell-bone interaction. From this study, we have shown evidence of the feasibility and versatility of our established 3D models. These models can be adapted to test various hypotheses for studies pertaining to underlying mechanisms of bone metastasis and could provide a vehicle for anticancer drug screening purposes in the future.

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Vazquez, Marisol. "Development of a novel in vitro 3D osteocyte-osteoblast co-culture model to investigate mechanically-induced signalling." Thesis, Cardiff University, 2013. http://orca.cf.ac.uk/56764/.

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Normal mechanical loading potently induces bone formation mediated by osteocyte effects on osteoblasts. Current in vitro bone models do not reflect these cellular interactions, either focusing on mechanical loading of osteoblasts in monolayers or in 3D and therefore not elucidating the osteocyte-osteoblast interactions that regulate mechanically-induced bone formation. Adenosine, calcium-sensing and glutamate signalling have been shown to influence bone biology, with both adenosine precursors and glutamate having been implicated in mechanotransduction. The aims were to develop a novel in vitro 3D co-culture model of bone to investigate mechanically-induced signalling, and to determine the expression of adenosine, calcium-sensing and glutamate signalling components within the 3D model and their contribution to the regulation of mechanically-induced bone formation markers. A 3D model was developed as a two-phase culture system where MLO-Y4 osteocytes were embedded within type I collagen gels and MC3T3-E1osteoblasts were layered on top. In this model, cells were viable over 7 days (100 % osteoblasts, 87 % osteocytes), maintained appropriate morphology and contacted neighbouring cells through CX43 labelled projections. RT-qPCR revealed Runx2, OCN and E11 mRNA expression in both osteoblasts and osteocytes. COL1A1 mRNA expression was significantly higher in the osteoblasts (P=0.0001), whereas ALP mRNA was higher in the osteocytes (P=0.001). RT-PCR revealed expression of adenosine receptors A2A and A2B and glutamate transporter GLAST1 in osteoblasts and osteocytes, as well as glutamate receptors AMPAR2 and KA1 in osteocytes. Immunostaining confirmed expression of A2A, GLAST1 and KA1, and revealed expression of CaSR, in both osteoblasts and osteocytes. A novel mechanical loading device was developed which was used to apply osteogenic loads (5 min, 10 Hz, 2.5 N) to 3D osteocyte mono-cultures and 3D osteocyte-osteoblast co-cultures. A minimum of 48 hr pre-load time was required for a reliable load response. 3D osteocyte mono-cultures cultured for 48-72 hr or 7 days pre-load, remained viable, significantly increased PGE2 0.5 hr after load (48-72 hr: P=0.0249, 7 days: P=0.041) and decreased their IL-6 synthesis. RT-qPCR revealed a load-induced decrease in E11 (P=0.018) and RANKL (P=0.0486) mRNA, in 48-72 hr cultures. In 7 day cultures, E11 mRNA (P=0.041) increased as a result of loading. Preliminary data showed that the same loading conditions increased PINP synthesis, a bone formation marker, in 3D co-cultures (P=0.022). The AMPA/KA receptors antagonist NBQX increased PINP synthesis by 2-fold over 5 days, similar levels induced by loading in untreated cultures, suggesting that NBQX has similar anabolic effects as mechanical stimuli. Similarly, the A2A receptor antagonist SCH 442416 increased osteoblast ALP mRNA expression by 3.5-fold at day 1 post-load and increased PINP synthesis by 1.9-fold, in co-cultures after 5 days. This 3D osteocyte-osteoblast co-culture model represents a useful in vitro model for the investigation of the osteocyte-osteoblast interactions that lead to mechanically-induced signalling and regulation of bone formation markers. Adenosine, calcium-sensing and glutamate signalling components are expressed within the model, facilitating future investigations of their roles in mechanically-induced signalling. Preliminary experiments indicated that adenosine and glutamate signalling may each contribute individually to the regulation of mechanically-induced bone formation markers.

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Smolina, Margarita. "Breast cancer cell lines grown in a three-dimensional culture model: a step towards tissue-like phenotypes as assessed by FTIR imaging." Doctoral thesis, Universite Libre de Bruxelles, 2018. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/267686.

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Despite the possible common histopathological features at diagnosis, cancer cells present within breast carcinomas are highly heterogeneous in their molecular signatures. This heterogeneity is responsible for disparate clinical behaviors, treatment responses and long-term outcomes in breast cancer patients. Although the few histopathological markers can partially describe the diversity of cells found in tumor tissue sections, the full molecular characterization of individual cancer cells is currently impossible in routine clinical practice. In this respect, Fourier transform infrared (FTIR) microspectroscopic imaging of histological sections allows obtaining, for each pixel of tissue images, hundreds of independent potential markers, which makes this technique a particularly powerful tool to distinguish cell types and subtypes. As a complement to the conventional clinicopathological evaluation, this spectroscopic approach has the potential to directly reveal molecular descriptors that should allow identifying different clonal lineages found within a single tumor and therefore provide knowledge relevant to diagnosis, prognosis and treatment personalization. Yet, interpretation of infrared (IR) spectra acquired on tissue sections requires a well-established calibration, which is currently missing. Conventionally, mammary epithelial cells are studied in vitro as adherent two-dimensional (2D) monolayers, which lead to the alteration of cell-microenvironmental interplay and consequently to the loss of tissue structure and function. A number of key in vivo-like interactions may be re-established with the use of three-dimensional (3D) laminin-rich extracellular matrix (lrECM)-based culture systems. The aim of this thesis is to investigate by FTIR imaging the influence of the in vitro growth environment (2D culture versus 3D lrECM culture and 3D monoculture versus 3D co-culture with fibroblasts) on a series of thirteen well-characterized human breast cancer cell lines and to determine culture conditions generating spectral phenotypes that are closer to the ones observed in malignant breast tissues. The reference cell lines cultured in a physiologically relevant basement membrane model and having undergone formalin fixation, paraffin embedding (FFPE), a routine treatment used to preserve clinical tissue specimens, could contribute to the construction of a spectral database. The latter could be ultimately employed as a valuable tool to interpret IR spectra of cells present in tumor tissue sections, particularly through the recognition of unique spectral markers.To achieve the goal, we developed and optimized, in a first step, the preparation of samples derived from traditional 2D and 3D lrECM cell cultures in order to preserve their morphological and molecular relevance for FTIR microspectroscopic analysis. We then highlighted the importance of the influence of the growth environment on the cellular phenotype by comparing spectra of 2D- and 3D-cultured breast cancer cell lines between them. A particular focus was placed to establish a correlation between FTIR spectral data and publicly available microarray-based gene expression patterns of the whole series of breast cancer cell lines grown in 2D and 3D lrECM cultures. Our results revealed that, although based on completely different principles, gene expression profiling and FTIR spectroscopy are similarly sensitive to both the cell line identity and the phenotypes induced by cell culture conditions. We also identified by FTIR imaging changes in the chemical content occurring in the microenvironment surrounding cell spheroids grown in 3D lrECM culture model. Finally, we illustrated the impact of the in vivo-like microenvironment on the IR spectra of breast cancer cell lines grown in 3D lrECM co-culture with fibroblasts and compared them with spectra of cell lines grown in 3D lrECM monoculture. Unsupervised statistical data analyses reported that cells grown in 3D co-cultures produce spectral phenotypes similar to the ones observed in FFPE tumor tissue sections from breast carcinoma patients. Altogether, our results suggest that FFPE samples prepared from 3D lrECM cultures of breast cancer cell lines and studied by FTIR microspectroscopic imaging provide reliable information that could be integrated in the setting up of a recognition model aiming to identify and interpret specific spectral signatures of cells present in breast tumor tissue sections. Le cancer du sein est une maladie très hétérogène, tant au niveau clinique que biologique. Cette hétérogénéité rend impossible la caractérisation moléculaire complète des cellules cancéreuses individuelles dans la pratique clinique courante. Dans ce contexte, l’imagerie infrarouge à transformée de Fourier (FTIR) des coupes tissulaires permet d'obtenir pour chaque pixel d'une image de tissu des centaines de marqueurs potentiels indépendants, ce qui pourrait faire de cette technique un outil particulièrement puissant pour identifier des différents types et sous-types cellulaires. L'interprétation des spectres infrarouges (IR) enregistrés à partir des coupes histologiques nécessite cependant une calibration qui fait actuellement défaut. Cette calibration pourrait être obtenue à partir de lignées cellulaires tumorales bien caractérisées. Traditionnellement, les cellules épithéliales mammaires sont étudiées in vitro sous forme de monocouches adhérentes bidimensionnelles (2D), ce qui conduit à l'altération de la communication entre les cellules et leur environnement et, par conséquent, à la perte de l’architecture et de la fonction du tissu épithélial. Un certain nombre d'interactions physiologiques clés peuvent être rétablies en utilisant des systèmes de culture tridimensionnelle (3D) dans une matrice extracellulaire riche en laminine (lrECM). L'objectif de cette thèse consiste à étudier par imagerie FTIR l'influence du microenvironnement (via une comparaison entre les cultures 2D et 3D lrECM ou les cultures 3D lrECM en présence ou en l’absence de fibroblastes) sur une série de treize lignées de cellules tumorales mammaires humaines bien caractérisées et à déterminer les conditions de culture générant des phénotypes spectraux qui se rapprochent le plus de ceux observés dans les tissus tumoraux. Au cours de ce travail, nous avons mis au point la culture des lignées cellulaires dans un modèle 3D lrECM ainsi qu’une méthodologie de préparation des échantillons offrant la possibilité de les comparer de manière pertinente avec les cellules cancéreuses présentes dans les coupes histologiques. De même, nous avons étudié par imagerie FTIR les effets du microenvironnement sur les lignées de cellules tumorales et inversement. Pour les lignées investiguées, le passage d’une culture 2D à une culture 3D lrECM s’accompagne, en effet, de modifications du spectre IR étroitement corrélées aux modifications du transcriptome. Les marqueurs spectraux indiquent également que l’environnement 3D génère un phénotype cellulaire proche de celui trouvé dans les coupes histologiques. De manière intéressante, cette proximité est d’autant plus renforcée en présence de fibroblastes dans le milieu de culture. Doctorat en Sciences agronomiques et ingénierie biologique info:eu-repo/semantics/nonPublished

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Sorrentino, Rita. "Three dimensional oral mucosa models: development and applications." Doctoral thesis, Università del Piemonte Orientale, 2020. http://hdl.handle.net/11579/114910.

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Animal experimentation has been extensively and for a long time applied in several research fields, but since 2011 it has been substantially limited by the Commission of the European Parliament to ensure people/animals safety and reduce research costs. To respond to these directives, many attempts have been focused on the development and validation of new in vitro 3D systems, bypassing the traditional 2D cell cultures. In this regard, diverse approaches to tissue-engineered bone and oral mucosa have been developed. Despite the promising premises and the cutting-edge results, the used 3D in vitro bone-oral mucosal models still lack interaction between the mucosal and the bone components. Therefore, this project aimed to create 3D models, entirely made with primary human cells (keratinocytes, fibroblasts, and osteoblasts), able to mimic the natural structure and interaction of bone and oral mucosa. In the present work, the regulatory role of the mesenchymal tissue onto epithelia was evaluated. The main results showed that that during the differentiation hMSC produce and secrete factors that induce the keratinization and the expression of the marker of differentiation CK10; in particular in the middle stage of differentiation (OB14). The proteomic analysis revealed that this effect can be ascribable to KGF secretion. This finding may impact the design of new implantable devices able to induce, alone, the epithelial growth and keratinization to improve implant graft avoiding epithelial graft linked to the morbidity of another zone. Moreover, we also showed that OM might have a pro-innervation effect, at least during the last stages of keratinocytes stratification. Finally, we obtained and characterized an innervated mucoperiosteal model that could open new in vitro frontiers for oral biomaterials validation as well as improve knowledge regarding the mesenchymal stem cells roles onto oral mucosa development.

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Cho, Hyung Joon. "Pro-oxidative and Pro-inflammatory Mechanisms of Brain Injury in Experimental Animal and 3D Cell Culture Model Systems." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/73476.

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The pro-oxidative and pro-inflammatory mechanisms have been implicated in various human diseases including neurological and psychiatric disorders. However, there is only limited information available on the etiology in the progression of neurological damage to brain. The emergence of tissue engineering with the growing interest in mechanistic studies of brain injury now raises great opportunities to study complex physiological and pathophysiological process in vitro. Therefore, the prime goals of this study include: (1) Determination of the molecular and cellular mechanisms responsible for blast- and radiation-induced brain injuries and (2) Development of a three-dimensional (3D) model system in order to mimic in vivo-like microenvironments to further broaden our knowledge in pro-oxidative and pro-inflammatory mechanisms and their cellular responses within 3D constructs. In the first study, we demonstrated that blast exposure induced specific molecular and cellular alterations in pro-oxidative and pro-inflammatory environments in the brain and neuronal loss with adverse behavioral outcome. The results provide evidence that pro-oxidative and pro-inflammatory environments in the brain could play a potential role in blast-induced neuronal loss and behavioral deficits. In the second study, we investigated that fractionated whole-brain irradiation induced specific molecular and cellular alterations in pro-oxidative and pro-inflammatory environments in the brain along with elevation of reactive oxygen species (ROS)-generating protein (NOX-2) and microglial activation. Additionally, the contribution of NOX-2 in fractionated whole-brain radiation-induced oxidative stress was observed by dramatic amelioration of ROS generation after pharmacological inhibition of NOX-2. These results support that NOX-2 may play a pivotal role in fractionated whole-brain radiation-induced pro-oxidative and pro-inflammatory pathways in mouse brain. In the third study, we developed an in vitro 3D experimental model of brain inflammation by encapsulating microglia in collagen hydrogel with computational analysis of 3D constructs. The results indicated that our newly developed in vitro 3D model system provides a more physiologically relevant environment to mimic in vivo responses. In conclusion, these data may be beneficial in defining a cellular and molecular basis of pathophysiological mechanisms of brain injuries. Furthermore, it may provide new opportunities for preventive and therapeutic interventions for patients with brain injuries and associated neurological disorders. Ph. D.

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Lee, Si Yuen. "Culture of human pluripotent stem cells and neural networks in 3D using an optogenetic approach and a hydrogel model." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:5cecda23-6208-4c0f-a800-d5ddccae24d3.

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Development of optogenetically controllable human neural network models can provide an investigative system that is relevant to the human brain. Conventional cultures of neural networks in two-dimensions (2D) have major limitations of scale. For instance, the soma of neurons in 2D is unrealistically flattened and both axon and dendrite outgrowth is restricted. Using a combination of tissue engineering techniques and the inclusion of optogenetically modified human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs), the development of a three-dimensional (3D) human neural culture model within a defined 3D microenvironment is investigated in this study. Light-sensitive neurons were successfully generated by transducing Channelrhodopsin-2 (ChR2) into human iPSC-derived NPCs and neuroblastoma cells (SH-SY5Y) using lentiviral transduction. The use of neuron specific promoters for synapsin-1 (SYN1) and calcium-calmodulin kinase II (CaMKII) in driving the expression of ChR2-Yellow Florescent Protein (YFP) within the mixed neuronal populations from hiPSC-derived neurons (Axol cells) were compared. Viability of the cells at 7 day-post-infection was 80&percnt; - 97&percnt; in all conditions tested. In line with published literature, transduction efficiency of neurons at day 14 was found to be 3&percnt; - 7&percnt; for plasmids containing the SYN1 promoter and 2&percnt; - 5&percnt; for plasmids containing the CaMKII promoter. An increase in promoter driven ChR2-YFP expression was evaluated over 28 days as the neural subpopulations matured. Stably ChR2 expression continued through-out higher passages (≥ P10) and possibly for periods up to several months. Both SYN1 and CaMKII promoters were found to drive the expression of ChR2 in Axol cells targeting inhibitory and excitatory neurons, respectively. 3D culture systems to support cell growth and optogenetic application were developed and characterised. Alginate hydrogel functionalised with short peptide sequence arginine-glycine-aspartate (RGD), and small molecules such as Rho Kinase inhibitor (ROCKi) and ZVAD were incorporated to increase the viability of human pluripotent stem cells (hPSCs). Investigation of cell response reveals that a flow rate of 3 ml/min and an alginate concentration of 1.8&percnt; (w/v) are optimal and that stem cell survival is significantly improved through incorporation of RGD and ROCKi. Interestingly, ChR2-YFP expression of Axol and SY5Y cells was detectable when transferred to the 3D culture system. The optogenetically modified neurons were found responsive to light stimulation, showing firing patterns and calcium events typical of early developing neurons (e.g. mixed and burst waves; single and multipeak spikes). Neuronal activities were assessed using calcium imaging. Higher numbers of calcium events were associated with CaMKII driven ChR2-YFP expression than with SYN1 in Axol cells. However, calcium activity in SH-SY5Y cells was most noticeable in neurons expressing ChR2-YFP driven by the SYN1 promoter. In primary rodent neuronal cultures, synchronous calcium firing with repetitive action potentials (APs) resulted from ChR2-YFP expression was driven by both SYN1 and CaMKII promoter upon light stimulation. By combining multi-approaches, we report for the first time on the generation of an in vitro hiPSC-derived neural network model in 3D using functionalised alginate hydrogel and involving optogenetic targeting. Expression of ChR2-YFP was found driven by both SYN1 and CaMKII promoter in the RGD-alginate bead system that cultured with Axol cells.

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Ståhl, Emmy. "Identification of changes in biomarkers relevant for breast cancer biology occurring in a novel 3D-Biosilk model." Thesis, KTH, Proteinvetenskap, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-294248.

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Bröstcancer är den vanligaste formen av cancer som drabbar kvinnor. Det är en heterogen och komplex sjukdom som består av flera undergrupper, var och en med distinkt morfologi och kliniska implikationer [1]. För att modellera och studera cellbiologi, vävnadsmorfologi, molekylära mekanismer och läkemedels effekter används cellkulturer [2]. Idag är tvådimensionella (2D) modeller fortfarande den mest använda metoden för att odla celler in vitro [3]. En nackdel med 2D-modeller är att mikromiljön i dessa modeller inte imiterar in vivo strukturen av tumörer och vävnader, då de saknar tre dimensionella (3D) cell-cell och cellextracellulär matrix (ECM) interaktioner [2]. På grund av nackdelarna med 2D-modeller, har 3D-modeller blivit mer intressanta som alternativ för att lösa behovet av en pålitlig preklinisk modell för läkemedelstestning och för studier av cancerbiologi. För att utveckla ett redskap som är relevant för cancerforskning etablerar professor My Hedhammars laboratorium en 3D-modell av bröstcancer. I en sådan ny modell används Biosilk som byggnadsställning för att odla odödliga cellinjer som är representativa för de tre huvudklasserna av bröstcancer (i.e. MCF-7 (luminal-lik), SKBR-3 (HER2-överuttryckt) och MDAMB- 231 (trippel-negativ)). Eftersom transkriptions signaturer kan användas för att klassificera och studera bröstcancer är det viktigt att undersöka om och hur tillväxt i 3D-Biosilk kan påverka genuttrycksprofiler. Hypotesen som testades i denna studie var om cellkulturer i 3DBiosilk kan ha signifikanta skillnader i uttryck av biomarkörer, relevanta för bröstcancerbiologi, vid jämförelse av samma cellinje kultiverad i 2D. För att testa detta utvärderades kvalitén och reproducerbarheten av 3D-Biosilk konstruktionen med hjälp av olika kvalitetstester. Strukturen granskades med brightfield mikroskopi, arean av konstruktionen mättes med ImageJ, infärgning med phalloidin bekräftade cellnärvaro och cellvidhäftning till modellen. Alamar blue utfördes för att bedöma den cellulära metaboliska aktiviteten i modellen. Förändringarna av målgenernas genuttryck undersöktes med kvantitativ omvänd transkription PCR (RT-qPCR) och detta påvisade en statistiskt signifikant skillnad i genuttrycket beroende på om cellerna odlats i 2D- eller 3D-Biosilk modeller. I cellinje MDA-MB-231 hittades tre gener, i cellinje SKBR-3 hittades två gener och i cellinje MCF-7 hittades fyra gener. Genuttrycket för en av dessa gener i cellinje MCF-7, som var kultiverad i 3D-Biosilk, var nedreglerad (i.e. ZO-1). Detta kunde valideras på proteinnivå med immunofluorescens. Sammanfattningsvis, celler odlade i 3D-Biosilk visar på en mer aggressiv fenotyp. Breast cancer is the most common cancer among women. It is a heterogenous and complex disease composed of several subtypes, each with distinct morphological and clinical implications [1]. To model and study cell biology, tissue morphology, molecular mechanisms and drug actions, cell cultures are canonically used [2]. Today two-dimensional (2D) models are still widely the preferred method for culturing cells in vitro [3]. A drawback with 2D models is that the microenvironment in these models does not mimic the in vivo structure of tumors and tissues, lacking three-dimensional (3D) cell-cell and cell-extracellular matrix (ECM) interactions [2]. Due to the disadvantages of 2D models, 3D cultures have become an increasingly interesting alternative to solve the need for a reliable preclinical model for drug testing and the study of cancer biology. To develop a relevant tool for cancer research, the laboratory of professor My Hedhammar is currently establishing a 3D model of breast cancer. In such novel model, Biosilk is used as scaffold to grow immortalized cell lines representative of the three major classes of breast cancer (i.e. MCF-7 (luminal-like), SKBR-3 (HER2-overexpression) and MDA-MB-231 (triplenegative)). Since transcriptional signatures can be used to classify and study breast cancers, it is important to investigate if and how growth in 3D-Biosilk can impact gene expression profiles. The hypothesis tested in this study was that cells cultured in 3D-Biosilk have differences in expression of biomarkers relevant to breast cancer biology, when compared to the same cell lines cultured in 2D. To examine this, 3D-Biosilk models were created and evaluated to ensure their quality and reproducibility, for instance, the scaffold structure was monitored by brightfield microscopy, the construct’s area was measured with ImageJ, staining with phalloidin confirmed the presence of cells as well as their attachment to the construct, and Alamar blue was used to assess the cellular metabolic activity. Differences in gene expression of target genes were investigated using reverse transcription quantitative PCR (RTqPCR), which revealed statistically significant changes depending on whether the cells were cultivated in 2D or a 3D-Biosilk model. For cell line MDA-MB-231 three genes were found, for SKBR-3 two genes were found and for MCF-7 four genes were found. The expression of one gene which was found downregulated in MCF-7 cultured in 3D-Biosilk (i.e. ZO-1) was validated at protein level by immunofluorescence. In conclusion, cultivating cells in 3D-Biosilk indicates a more aggressive phenotype.

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Gérémie, Lauriane. "Development of an in-vitro intestinal model featuring peristaltic motion." Thesis, Sorbonne université, 2019. http://accesdistant.sorbonne-universite.fr/login?url=http://theses-intra.upmc.fr/modules/resources/download/theses/2019SORUS118.pdf.

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Le Gut-on-chip fait partie d'un thème de recherche plus générale, appelez Organ-on-chip qui a pour objectif de développer des modèles in-vitro qui récapitulent des caractéristiques essentielles de l'organe d'intérêt. Dans le cas de l'intestin, les Gut-on-chip plateformes ont été principalement développés pour reconstituer soit l'architecture 3D de l'intestin, soit sa dynamique et plus particulièrement le péristaltisme. Durant ma thèse j'ai développé une nouvelle et polyvalente Gut-on-chip, présentant ces deux aspects du micro-environnement intestinale. Cette Peristalsis-on-chip nous a permis d'étudier l'influence du mouvement péristaltique sur le comportement cellulaire en fonction de la géométrie de la structure. Pour cette étude nous avons ensemencé des cellules Caco2 sur des substrats 2D ou 3D recouvert de laminine et les avons soumis à un étirement cyclique (à 0.2 Hz et 10\%) pendant 2, 5, 8, 16, 24 et 48 heures. Lors de ces expériences nous avons pu observer une réorientation cellulaire perpendiculaire à l'axe d'étirement que nous avons caractérisé en fonction des conditions de recouvrement, de la confluence initiale, du temps d'étirement et de la géométrie de la structure. Il est intéressant de noter que la réponse cellulaire la plus importante a été obtenue par la combinaison de la géométrie 3D et de l'étirement, ce qui illustre bien le besoin de ces deux éléments pour mieux mimer les conditions intestinales in vivo My PhD work is part of the organ-on-chip field, and more precisely part of the gut-on-chip field. It is in line with the main objective of this field, which is the development of in-vitro models recapitulating as faithfully as possible the intestinal micro-environment. Through my PhD work I first developed a versatile gut-on-chip platform recapitulating the intestinal 3D architecture as well as its dynamic micro-environment. Therefore, this platform allows us to study the influence of the intestinal dynamic, especially the peristalsis, on cellular behavior in function of the 3D architecture of the scaffold. For this study Caco2 cells have been seeded either on a 2D or a 3D scaffold coated with laminin and submitted to a cyclic stretching (at 0.2 Hz and 10%) for 2, 5, 8, 16, 24 and 48 hours. Our main observation was the cellular reorientation induced by the stretching, therefore we characterized the cell behavior in function of the coating condition, the initial confluency, the stretching time and the scaffold geometry. Interestingly, the strongest cellular response was obtained when the 3D geometry and the stretching was combined illustrating the need of these two stimuli to better mimic the intestinal in vivo conditions

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Mitterer, Chantal. "The role of inflammation induced by radiation or lipopolysaccharides in the metastatic process in a mouse model of breast cancer." Mémoire, Université de Sherbrooke, 2012. http://hdl.handle.net/11143/6343.

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Mortality from breast cancer is primarily due to metastatic disease, which often appears years after treatment of the primary tumor. Radiation as well as bacterial infection induces inflammation, which by releasing cytokines can be implicated in metastatic processes. Using in vitro and in vivo models, the ability of radiation to awaken dormant lung metastases was assessed as well as the capacity of a bacterial infection to enhance metastatic progression in already proliferating lung metastases. As models, we used the D2.0R (dormant) and D2A1 (proliferative) cell lines, which are derived from spontaneous murine mammary tumors. The ability of radiation to awaken dormant D2.0R mammary cancer cells was assessed in a 3-dimension (3D) cell culture system, which resulted in the formation of microspheres of cancer cells. The addition of prostaglandin E 2 (PGE2 ; 100ng/m1) or conditioned media from irradiated (5 Gy) CALU-3 human bronchial epithelial cells stimulated the proliferation of the dormant D2.0R cells resulting in microspheres with a larger diameter compared to the untreated cells. Regarding the proliferative D2A1 microspheres, their rate of proliferation was not further increased by adding PGE2 or the conditioned media of irradiated CALU-3 cells. In Balb/c mice bearing dormant lung D2.0R micrometastases, our data showed that a fractionated radiation dose (5x7.5 Gy) to the mammary gland resulted in a significant increase in the development of metastases, as measured 42 days post-irradiation by bioluminescent reaction. We also evaluated whether a bacterial infection could stimulate the growth of D2A1 cancer cells. Gram-negative bacteria release the lipopolysaccharide (LPS) that induces an inflammatory response. In lungs of mice treated with LPS, a higher level of interleukin-1? (IL-1?) was measured supporting the induction of an inflammation. This was accompanied by an increase of cell adhesion molecules (VCAM-1 and ICAM-1) 5 hours after treatment. The ability of LPS mediated-inflammation to stimulate the quantity and size of the proliferating D2A1 lung metastases was also demonstrated by optical imaging. In aged mice, a significant increase in total surface area covered by the lung metastases was measured as well as a tendency to have more numerous metastases. Conversely, no difference in tumor size or quantity was observed in young mice, which nevertheless had increased expression in pro-inflammatory mediators and adhesion molecules. In conclusion, our study demonstrated that inflammation increases the awakening of dormant D2.0R microspheres in a 3D in vitro model, while in mice treated with LPS, an age-dependent stimulation of the proliferation and number of D2A1 lung metastases was measured.

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Bianchi, Arianna. "Epithelial-mesenchymal interactions in the cornea : development of a novel 3D culture cornea model and progress towards environmental reprogramming of cornea epithelium." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/3033.

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The unmet need for corneal epithelial cells for treating human eye diseases makes the cornea important in the cell therapy field. Additionally cornea tissue engineering has become valuable for clinical use, research, and for creating representative models replacing animals for chemical/drug testing. This study initially used qPCR to investigate the expression levels of key markers produced by 2D corneal epithelial cell cultures after wounding in a scratch assay. Then an attempt was made to environmentally reprogram human hair follicle keratinocytes into corneal epithelial cells using limbal epithelial stem cell media. Immunohistochemical and qPCR analysis revealed no changes in signature genes but rather a similarity between HFC and LSC’s when cultured in LSC’s culture conditions. Attention then focused on developing a novel three-dimensional bilayered spheroid cornea model using hanging drop culture. It is widely accepted that cells in 3D culture more closely mimic their in vivo counterparts than 2D cultures, and qPCR and immunofluorescence analysis of 3D spheroids made from cultured rabbit corneal stromal cells revealed that they partially reverted back to a quiescent in vivo phenotype. Coating the spheroids with cultured rabbit limbal epithelial cells produced a bilayered model of the cornea. Multiple iterations were produced incorporating variations in media and cell origin, leading to a cornea model that could be maintained for 10 days, expressed appropriate cytokeratins and other corneal markers including Pax6 and that, upregulated the expression of key cornea signature proteins including Aldh1a1 as a result of epithelial-mesenchymal interactions. Preliminary versions of a human bilayered cornea model were then created from equivalent human cell types. Generally this 3D model displays advantages over other in vitro cornea equivalents and has potential, but needs further refinement. The methodology was also used to coat stromal spheroids with skin keratinocytes, highlighting the possibility of reprogramming the former into corneal epithelial cells through epithelial-mesenchymal interactions.

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Lebeko, Maribanyana Robert. "The use of in vitro 2d co-culture models to determine the optimal keratinocyte: melanocyte ratio to be used in the development of pigmented 3d skin model." Doctoral thesis, University of Cape Town, 2015. http://hdl.handle.net/11427/16564.

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Includes bibliographical references Burn injuries are among the most devastating of all injuries and a major global public health crisis, with fire related burns accounting for approximately 265 000 deaths annually. The African continent, most especially Sub-Saharan Africa, has the second highest mortality rates (15% of global mortality rates). In South Africa, 3.2 % of the total population sustains burn injuries, with 50 % of these cases as children under the age of20 years. Studies have also shown that most of these incidences are prevalent within the age groups of 0-5 years, and account for the 3rd most common cause of mortality in children under the age of 15 years. In depth knowledge and understanding of cellular facets of wound healing has allowed for a greater stance in the interventions aimed at circumventing problems associated with development of effective wound defects treatment regimen. Burn treatment options are largely dependent on the degree and extensiveness of burns. A wide body of literature exists with regards to traditional as well as current treatment options. These include, for instance the use of various forms of skin auto-grafts. Despite such great success with all kinds of innovative ideas surrounding the use of autologous skin grafting, lack of available donor sites for skin grafts still remains a problem, more so in cases where patients suffer burns spanning more than 70% TBSA. This therefore has inspired the design and use of bioengineered skin substitutes as well as cultured/non-cultured autologous epidermal cells. Unfortunately, to date, no tissue engineering technique has fully been able to recapitulate the anatomy and physiology of the skin, or has attained the biological stability as well as achieving the aesthetic outcome. Several hurdles are yet to be overcome to achieve this. Amongst many, inclusion of melanocytes, other skin appendages as well as potential progenitor cells is some of the attributes of an ideal 3D skin equivalent. Therefore pigmented 3D skin constructs are of great interest as they address not only the issues of complete wound healing, but also the aesthetic outcomes. In light of this, correct keratinocyte to melanocyte ratios are also of great importance in designing such pigmented 3D constructs. Therefore the major aim of this study was to isolate skin melanocytes and keratinocytes, and co-culture them at different ratios in order to attain optimal pigment production and/or consequent improved wound healing outcome. To determine the best keratinocyte to melanocyte ratio to use in developing pigmented3D skin constructs, the following co-culture ratios were used: 5:1, 10:1 and 20:1.Proliferation assays were employed to further elucidate the growth dynamics of both human skin melanocytes and keratinocytes in either mono- or co-culture system. Secondly, FACS was used to develop a reliable technique to be used to separate the two cell types from a co-culture system in order to perform downstream analyses. Thirdly, to establish the roles of the co-cultured cells in wound healing (with regards to proliferation and migration), scratch wound healing assays were employed. Lastly, FACS was used to infer the effect of such ratios on pigment production. Our results demonstrated that keratinocytes, compared to melanocytes mono-cultures have higher proliferation capacity. On the contrary melanocyte's proliferation is up-regulated by the presence of keratinocytes in a co-culture, whereas higher numbers of melanocytes in co-culture with keratinocytes resulted in less proliferative keratinocyte phenotype. The FACS separation technique worked excellently in identifying keratinocyte population from melanocytes, with an almost 100% accuracy. This is shown by melanocytes being sorted as 93% of MART-1 + cells in a mono-culture, followed by an approximately 5:1 separation of keratinocytes from melanocytes (77% Kc and 17% Mc). In vitro scratch assays demonstrated that none of the co-culture ratios was significantly superior with regards to wound healing capacities and pigment production, in the absence of fibroblast-conditioned medium. In conclusion, the 5:1 co-culture ratio seemed to yield a non-significant, yet best outcome with regards to wound healing capacity (only in the presence of fibroblast-derived factors), thus conferring it as a potential optimal ratio of keratinocytes to melanocytes, to be used in development of our pigmented 3D constructs.

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Valentin, Loïse. "Développement de modèles de culture en 3D pour l’étude des maladies et des infections hépatiques humaines." Electronic Thesis or Diss., Sorbonne université, 2022. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2022SORUS442.pdf.

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Les infections et maladies hépatiques sont une cause majeure de morbidité et de mortalité. Elles incluent des infections causées par des pathogènes hépatotropiques, comme le parasite Plasmodium, et des maladies d’origine non infectieuse causées notamment par des traitements médicamenteux, telle que la cholestase. L’hépatocyte humain primaire (PHH) est le type cellulaire de choix pour étudier ces infections et pathologies in vitro, mais il est nécessaire de développer de nouveaux modèles de culture plus proches de la physiologie des cellules in vivo et plus pertinents que les modèles 2D. C’est dans ce contexte que s’inscrit mon projet de thèse visant à développer des systèmes de culture en 3D de cellules hépatiques humaines. Les travaux menés ont conduit à la mise en place d’un modèle 3D sphéroïde scaffold-free de PHH viable et plus fonctionnel que le modèle 2D+. Les résultats montrent également que les sphéroïdes de PHH sont un modèle approprié pour la culture du stade hépatique de Plasmodium et suggèrent une meilleure sensibilité de ce modèle pour la prédiction d’activité de molécules antipaludiques. Dans le but d’améliorer la manipulation des sphéroïdes, la magnétisation des sphéroïdes à l’aide de nanoparticules (NS) a été testée. Les NS n’affectent ni la viabilité, ni les fonctions hépatiques des PHH, ni leur susceptibilité à l’infection par Plasmodium. Enfin, un modèle identique avec des cellules HepaRG a été utilisé pour tester une méthode de détection de la cholestase médicamenteuse à l’aide d’une nouvelle sonde fluorescente. Les résultats suggèrent que cette sonde peut être utilisée dans des modèles sphéroïdes pour l’évaluation de l’activité de molécules cholestatiques Liver infections and diseases are a major cause of morbidity and mortality. They include infections caused by hepatotropic pathogens, such as the Plasmodium parasite, and diseases of non-infectious origin caused in particular by drug treatments, such as cholestasis. The primary human hepatocyte (PHH) is the cell type of choice to study these infections and pathologies in vitro, but it is essential to develop new culture models closer to the physiology of cells in vivo and more relevant than 2D models. In this context, my thesis project aims to develop 3D culture systems for human liver cells. The work allowed to the establishment of a viable 3D spheroid scaffold-free model of PHH more functional than the 2D+ model. The results also demonstrate that PHH spheroids are an appropriate model for the culture of the liver stage of Plasmodium and would be more sensitive to antimalarial molecules. In order to improve the manipulation of spheroids, the magnetization of spheroids using nanoparticles (NS) was tested. NS do not affect the viability or hepatic functions of PHHs or their susceptibility to Plasmodium infection. Finally, an identical model with HepaRG cells was used to test a method for detecting drug induced cholestasis using a new fluorescent probe. The results proved that this probe can be used in spheroid models for the evaluation of the activity of cholestatic molecules

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Rontard, Jessica. "Evaluation expérimentale du risque prion lié aux porteurs asymptomatiques chez l'Homme et le macaque." Thesis, Paris Sciences et Lettres (ComUE), 2018. http://www.theses.fr/2018PSLET008/document.

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La détection de la protéine prion anormale dans les tissus lymphoïdes de patients britanniques suggère qu’après exposition à l’agent de la variante de la maladie de Creutzfeldt-Jakob (vMCJ) plus de 99% des contaminations pourraient demeurer cliniquement silencieuses. Ces données soulignent un risque de transmission secondaire par transfusion sanguine ce qui nous a conduit à une étude expérimentale. En parallèle des formes classiques de vMCJ, nos modèles murins et simiens de retransmission ont mis en evidence des phenotypes atypiques. Ces phénotypes échappent actuellement aux critères de diagnostic puisqu’aucune protéine prion anormale (PrPres) n’est détectée.Nos travaux ont eu pour but principal d’évaluer expérimentalement le risque sanguin au travers d’études de retransmission et de caractérisation de la replication des souches classiques et atypiques aux niveaux périphérique et central.Nous observons une très forte hétérogénéité dans la réplication de la PrP anormale dans les différents tissus lymphoïdes des macaques transfusés développant une vMCJ. Le niveau de contamination des tissus lymphoïdes apparait proportionnel à l’infectiosité sanguine de ces animaux et au risque de transmission de la maladie in vivo. Concernant les formes atypiques, la majorité des macaques transfusés n’ont pas de réplication dans les tissus lymphoïdes bien que ces phénotypes soient transmissibles expérimentalement à des modèles murins. Des transmissions à des souris immunodéficientes révèlent que les souches atypiques sont transmissibles par voie périphérique en l’absence d’un système immunitaire fonctionnel.Une alternative à l’expérimentation animale a été réalisée grâce aux « mini-brains » mimant la complexité du cerveau humain. Ces organoïdes cultivés en trois dimensions sont sensibles à au moins un isolat de prion associé aux formes sporadiques humaines. Les mini-brains pourraient ainsi constituer un nouvel outil d’étude des maladies à prions et permettre à termes la caractérisation des souches atypiques The detection of abnormal prion protein in the lymphoid tissues of UK patients suggests that after exposure to the agent of variant Creutzfeldt-Jakob disease (vCJD), more than 99% of contaminations may remain clinically silent. These data highlight a risk of secondary transmission through blood transfusion. In parallel to the classical vCJD forms, our experimental models in mice and macaques revealed another group which avoids the current diagnostic criteria, including the absence of abnormal prion protein (PrPres).The main goal of our work was to experimentally assess the risk of blood through retransmission studies and characterization of the abnormal replication of classical and atypical strains examined at peripheral and central levels.We observed a high heterogeneity of the distribution of the abnormal PrP in the lymphoid tissues of vCJD transfused macaques. The global level of contamination in lymphoid tissues seems proportional to the blood infectivity in these animals and to the risk of in vivo transmission of the disease. Regarding atypical forms, despite an absence of replication in lymphoid tissues, these phenotypes are experimentally transmissible. Transmissions to immunodeficient mice reveal that atypical strains are transmissible through peripheral routes in the absence of functional immune system.An alternative to animal testing has been achieved using to "mini-brains" mimicking the complexity of the human nervous system. These organoids cultured in three dimensions are sensitive to at least one prion isolate associated with human sporadic forms. Thus, mini-brains could constitute a new tool for studying prion diseases and improve the characterization of atypical strains

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Mosaad, Eman Mohamed Othman. "Three dimensional prostate cancer model systems." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/118287/1/Eman%20Mohamed%20Othman_Mosaad_Thesis.pdf.

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The prediction of drug efficacy is a major limitation in the cancer research field. This thesis was a step forward in developing an in vitro 3-dimensional prostate cancer model as a potential high throughput drug-screening platform. The merits of using a high throughput microwell platform to efficiently manufacture hundreds of multicellular spheroids were evaluated. The improved Microwell-mesh platform was evaluated as a drug-screening platform. A critical factor was the discovery of the cell-specific bioluminescence assay instability, which was promoter and/or cell line dependent. The first multicellular co-culture micro-tumour system as a potential drug-screening platform for bone metastatic prostate cancer was developed.

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Groult, Jessica. "Expansion ex vivo des Cellules Tumorales Circulantes comme modele de pharmacologie predictive des cancers." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS236/document.

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L'émergence des thérapies ciblées dans le traitement des cancers a rendu indispensable la mise au point de marqueurs plus spécifiques et sensibles pour la surveillance des patients. Dès le stade invasif, des cellules tumorales peuvent passer dans le sang où elles constituent les Cellules Tumorales Circulantes (CTC). Les CTC sont accessibles par une simple prise de sang, évitant les biopsies invasives. De plus, elles représentent le seul matériel tumoral résiduel après traitement. C'est la raison pour laquelle les CTC constituent un axe de recherche très actif avec plus de 400 essais cliniques incluant ces cellules comme biomarqueurs. Ces essais apportent des renseignements importants sur le risque de récidive ou de progression métastatique, et ont pour objectif de pouvoir gérer en temps réel la conduite thérapeutique. Cependant, les CTC potentiellement métastatiques ne représentent qu'une fraction très minoritaire de ces cellules circulantes. Les technologies existantes, essentiellement basées sur une simple numération, ne suffisent pas pour guider efficacement la stratégie thérapeutique. Ce projet a évalué un ensemble de critères pouvant être utile pour la prise de décisions thérapeutiques pertinentes, adaptées à chaque patient, et la mesure de l'efficacité des traitements. Ce projet sera centre sur le mélanome. Les stades d'évolution de ce cancer sont bien définis, et dans les stades avances, le risque de développer des métastases est très élevé et la détection précoce de celles-ci est un enjeu important. Par ailleurs, ce cancer bénéficie de rapides progrès thérapeutiques, les CTC constituent donc un outil intéressant pour tester l'efficacité de ces nouveaux traitements The emergence of targeted therapies in cancer treatment has made essential the development of more specific and sensitive markers for monitoring patients. At the invasive stage, tumor cells can pass to blood. These cells are called Circulating Tumor Cells (CTC). CTCs are accessible through a simple blood test, avoiding invasive biopsies. Moreover, they represent the only residual tumor after treatment. It is why CTCs are a very active center of research with more than 400 clinical trials involving these cells as biomarkers. These tests provide important information on the risk of recurrence or metastatic progression and aim to manage in real time the therapeutic conduct. But the CTC potentially metastatic represents only a fraction very minority of these circulating cells. Existing technologies, mainly based on simple enumeration, are not enough to effectively guide therapeutic strategy. This project has evaluated a set of criteria to make appropriate therapeutic decisions, adapted to each patient, and able to measure the effectiveness of treatments. This project will focus on melanoma. Evolution stages of this cancer are well defined, and in advanced stages, the risk of developing metastases is very high and the early detection is an important issue. Moreover, CTC could be is an interesting tool to test the effectiveness of these new treatments

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Prudon, Nicolas. "Integrative study, from the cell to the animal model, of the development of a cell therapy for Parkinson's disease." Electronic Thesis or Diss., Bordeaux, 2024. http://www.theses.fr/2024BORD0071.

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Un ensemble d'études précliniques soutient désormais le développement de thérapies de remplacement cellulaire dérivées de cellules souches pluripotentes pour soulager les symptômes moteurs chez les patients parkinsoniens. Le remplacement de la principale population cellulaire dysfonctionnelle au sein de la maladie, les neurones dopaminergiques A9, est l'objectif principal de ces thérapies. Pour y parvenir, la plupart des approches thérapeutiques impliquent la greffe de suspensions cellulaires de progéniteurs dopaminergiques. Cependant, une quantité considérable de cellules meurent pendant le processus de transplantation, les cellules étant confrontées à l'anoïkis. Une potentielle solution consiste à greffer des préparations solides, c'est-à-dire adopter un format cellulaire 3D. La cryopréservation de ce format reste un obstacle majeur et n'est pas exempte de causer des retards dans le délai avant l'apparition des effets, comme observé avec l'utilisation de suspensions unicellulaires de progéniteurs dopaminergiques cryopréservés. Le travail de cette thèse se concentre sur le développement de microtissus neuraux 3D en tant que thérapie cellulaire pour la maladie de Parkinson. L'utilisation d'une technologie d'encapsulation cellulaire à haut débit associée à des bioréacteurs pour fournir un environnement de culture 3D a permis la différenciation dirigée des hiPSC en microtissus neuraux. La différenciation correcte des microtissus neuraux vers une identité mésencéphalique a été confirmée à l'aide de méthodes orthogonales, en utilisant notamment la qRT-PCR, le RNAseq, la cytométrie en flux et la microscopie fluorescente. L'efficacité des microtissus neuraux a été démontrée de manière dose-dépendante dans des études précliniques, en utilisant le modèle de rat hémiparkinsonien lésé par la 6-OHDA. Les greffes ont été caractérisées par une analyse histologique post-mortem, démontrant la présence de neurones dopaminergiques humains projetant dans le striatum hôte. Le travail présenté ici est la première bioproduction d'une thérapie cellulaire pour la maladie de Parkinson dans un bioréacteur mis à échelle, conduisant à une récupération fonctionnelle complète des animaux 16 semaines après la transplantation d’un format cellulaire 3D cryopréservé A breadth of preclinical studies is now supporting the rationale of pluripotent stem cell-derived cell replacement therapies to alleviate motor symptoms in Parkinsonian patients. Replacement of the primary dysfunctional cell population in the disease, i.e. the A9 dopaminergic neurons, is the major focus of these therapies. To achieve this, most therapeutical approaches involve grafting single-cell suspensions of DA progenitors. However, a considerable number of cells die during the transplantation process, as cells face anoïkis. One potential solution to address this challenge is to graft solid preparations, i.e. adopting a 3D format. Cryopreserving such format remains a major hurdle and is not exempt from causing delays in the time to effect, as observed with the use of cryopreserved single-cell DA progenitors. The work of this thesis focus on the development of 3D neural microtissues as a cell therapy for PD. The use of a high-throughput cell-encapsulation technology coupled with bioreactors to provide a 3D culture environment enabled the directed differentiation of hiPSCs into neural microtissues. The proper patterning of these neural microtissues into a midbrain identity was confirmed using orthogonal methods including qPCR, RNAseq, flow cytometry and immunofluorescent microscopy. The efficacy of the neural microtissues was demonstrated in a dose-dependent manner in non-clinical studies, using the 6-OHDA-lesioned hemiparkinsonian rat model. The grafts were characterized by post-mortem histological analysis, demonstrating the presence of human dopaminergic neurons projecting into the host striatum. The work reported here is the first bioproduction of a cell therapy for Parkinson’s disease in a scalable bioreactor, leading to a full behavioural recovery 16 weeks in the animal model after transplantation using cryopreserved 3D cell format

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Hammoudi, Taymour Marwan. "3D micropatternable hydrogel systems to examine crosstalk effects between mesenchymal stem cells, osteoblasts, and adipocytes." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/45972.

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Poor skeletal health results from aging and metabolic diseases such as obesity and diabetes and involves impaired homeostatic balance between marrow osteogenesis and adipogenesis. Tissue engineering provides researchers with the ability to generate improved, highly controlled and tailorable in vitro model systems to better understand mechanisms of homeostasis, disease, and healing and regeneration. Model systems that allow assembly of modules of MSCs, osteoblasts, and adipocytes in a number of configurations to engage in signaling crosstalk offer the potential to study integrative physiological aspects and complex interactions in the face of changes in local and systemic microenvironments. Thus, the overall goal of this dissertation was to examine integrative physiological aspects between MSCs, osteoblasts, and adipocytes that exist within the marrow microenvironment. To investigate the effects of intercellular signaling in different microenvironmental contexts, methods were developed to photolithographically pattern and assemble cell-laden PEG-based hydrogels with high spatial fidelity and tissue-scale thickness for long-term 3D co-culture of multiple cell types. This platform was applied to study effects of crosstalk between MSCs, osteoblasts and adipocytes on markers of differentiation in each cell type. Additionally, responses of MSCs to systemic perturbations in glucose concentration were modulated by mono-, co-, and tri-culture with these cell types in a model of diabetes-induced skeletal disease. Together, these studies provided valuable insight into unique and differential effects of intercellular signaling within the niche environment of MSCs and their terminally differentiated progeny during homeostatic and pathological states, and offer opportunities further study of integrative physiological interactions between mesenchymal lineage cells.

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Bonda, Ulrich [Verfasser], Carsten [Akademischer Betreuer] Werner, and Dietmar [Gutachter] Hutmacher. "The prostatic tumour stroma : Design and validation of a 3D in vitro angiogenesis co‐culture model / Ulrich Bonda ; Gutachter: Carsten Werner, Dietmar Hutmacher ; Betreuer: Carsten Werner." Dresden : Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-208301.

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Bonda, Ulrich [Verfasser], Carsten [Akademischer Betreuer] [Gutachter] Werner, and Dietmar [Gutachter] Hutmacher. "The prostatic tumour stroma : Design and validation of a 3D in vitro angiogenesis co‐culture model / Ulrich Bonda ; Gutachter: Carsten Werner, Dietmar Hutmacher ; Betreuer: Carsten Werner." Dresden : Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2016. http://d-nb.info/1114067938/34.

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Morales, Delphine. "Modèles 3D de mélanome métastatique pour l’évaluation in vitro de l’efficacité de molécules de thérapies ciblées." Thesis, Compiègne, 2019. http://www.theses.fr/2019COMP2498.

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La sensibilité des cellules de mélanomes aux molécules de thérapies ciblées dépend du microenvironnement tumoral (interactions cellule-cellule et cellule-matrice extracellulaire). Les systèmes tridimensionnels (3D) de culture in vitro reflètent mieux l’architecture structurelle native des tissus et sont attrayants pour l’étude des interactions cellulaires. Nous avons développé et comparé plusieurs modèles de mélanome métastatique : les cellules de mélanomes (SK-MEL-28 et SK-MEL-3, mutées BRAF V600E et SK-MEL-2, BRAF sauvages) cultivées en monocouche (2D) et co-cultivées en 3D sur des équivalents de derme avec des fibroblastes, afin de mieux comprendre les facteurs modulant la sensibilité cellulaire à un inhibiteur de BRAF (BRAFi, Vémurafenib) et au Vémurafenib associé à un inhibiteur de MEK (MEKi, Cobimetinib). La sensibilité cellulaire aux traitements a été évaluée sous différents aspects : prolifération cellulaire (numération cellulaire, incorporation d'EdU, test MTS), analyse des voies de signalisation MAPK et PKB / Akt (Western-blot), apoptose (TUNEL), libération de cytokines et de facteurs de croissance (ELISA) et histologie (modèles 3D). Un effet cytostatique de BRAFi a été observé sur les cellules SK-MEL-28 et SK-MEL-3 cultivées dans les modèles 2D et 3D. La lignée cellulaire SK-MEL-2 était résistante au BRAFi lorsqu'elle a été cultivée en monocouche, mais sensible lorsqu'elle a été co-cultivée avec des fibroblastes incorporés dans une matrice de collagène de type I. Les milieux conditionnés par les fibroblastes 3D (équivalents de derme) ont sensibilisé les cellules SK-MEL-2 (2D) au BRAFi. L'analyse des surnageants de culture cellulaire a révélé que les équivalents de derme libéraient certains facteurs solubles (IL-6, IL-8, HGF, TGF-β) : ces sécrétions ont été modifiées au cours du traitement par Vémurafenib. La combinaison du traitement avec MEKi a renforcé l'action du Vémurafenib sur les cellules de mélanomes métastatiques tout en diminuant la capacité de prolifération des fibroblastes. Des populations de cellules contenant des cellules de mélanomes ou des fibroblastes associés au cancer (CAFs) ont été isolées à partir d'une biopsie de métastase cutanée provenant d'une patiente atteinte d'un mélanome métastatique. Ces cellules ont permis de réaliser des modèles de mélanome métastatique patient-spécifique afin d’étudier in vitro la sensibilité des cellules de la patiente aux traitements dans un microenvironnement tumoral (sécrétion paracrine de cellules stromales et matrice de collagène). Ces modèles prédictifs 3D patient-spécifique pourront être utilisés pour déterminer des stratégies de thérapies personnalisées, ainsi que pour comprendre les phénomènes de résistance des cellules de mélanomes aux traitements Melanoma cell sensitivity to targeted therapy molecules is dependent on the tumor microenvironment (cell-cell and cell-extracellular matrix interactions). Three dimensional (3D) in vitro cell culture systems better reflect the native structural architecture of tissues and are attractive to investigate cellular interactions. We have developed and compared several metastatic melanoma models: melanoma cells (SK-MEL-28 and SK-MEL-3, BRAF V600E mutant and SK-MEL-2 BRAF wt) cultured as a monolayer (2D) and co-cultured on 3D dermal equivalents with fibroblasts to better unravel factors modulating cell sensitivity to a BRAF inhibitor (BRAFi, Vemurafenib) and a BRAFi combined with a MEK inhibitor (MEKi, Cobimetinib). Cell sensitivity to treatments was evaluated under various aspects: cell proliferation (cell counting, EdU incorporation, MTS assay), MAPK and PKB/Akt signaling pathway analysis (Western-blotting), apoptosis (TUNEL), cytokine and growth factor release (ELISA) and histology (3D models). A cytostatic effect of BRAFi was observed on SK-MEL-28 and SK-MEL-3 cells in both models. SK-MEL-2 cell line was clearly resistant to BRAFi when cultured as a monolayer but not when co-cultured with 3D fibroblasts embedded in a type I collagen matrix. Conditioned media provided by 3D fibroblasts (dermal equivalents) underlined 2D SK-MEL-2 sensitivity to BRAFi. Cell culture supernatant analysis revealed that dermal equivalents released some soluble factors (IL-6, IL-8, HGF, TGF-β): these secretions were modified during vemurafenib treatment. The combination of treatment with MEKi enhances the action of Vemurafenib on metastatic melanoma cells while decreasing the proliferation capacity of fibroblasts. Cell populations containing melanoma cells or fibroblasts associated with cancer (CAFs) were isolated from a cutaneous metastasis biopsy of a patient with metastatic melanoma. These cells allowed the realization of patient-specific models of metastatic melanoma in order to study in vitro the sensitivity of the patient’s melanoma cells to treatments in a tumor microenvironment (paracrine secretion of stromal cells and collagen matrix). These 3D predictive patient-specific models could be used to determine personalized therapy strategies, as well as to understand the resistance phenomena of melanoma cells to treatments

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CERQUENI, GIORGIA. "In vitro strategies and development of bioengineered approaches for studying age-related osteochondral diseases." Doctoral thesis, Università Politecnica delle Marche, 2021. http://hdl.handle.net/11566/292220.

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The purpose of this PhD project is the development of in-vitro culture models to study different aspects of Osteoarthritis (OA), an entire joint degenerative disease that involved articular cartilage, synovium and subchondral bone. The global incidence of knee OA is 203 per 10,000 person-year that grows with the increase of age, reaching a peak between 70-79 years old, thus dragging the peak of prevalence in old age. The main OA disabling symptom is pain that is typically intermittent and weight-bearing (mechanical) and can lead to psychological stress. Contrary to the common description of a wear-related pathology, OA is the consequence of an active and unbalanced process of repair and destruction. The actual causes of OA are still unidentified and there is still debating on the precise order of events that trigger its onset. There is currently no predetermined in-vitro model of OA disease. This is essentially due to the defective knowledge on the onset of this pathology and to the numerous tissue modifications involved that make difficult to in-vitro reproduce OA. A clear comprehension of the various mechanisms engaged in the pathology is necessary to understand its progression, as well as the targets to restore joint function. Here, two different in-vitro culture models to study different aspects of OA were applied to investigate: (i) the involvement of adult stem/stromal cells of the synovial membrane in bone remodeling, through an indirect 2D co-culture approach and (ii) the crosstalk between chondrocytes and subchondral bone in normal and pathological condition, developing an engineered 3D scaffold and simulating an inflamed microenvironment. These two models allowed the investigation of the cell behaviours from the three tissues involved in the pathogenesis of OA, and the development of a possible in-vitro platform for future studies encompassing the three components simultaneously. Lo scopo di questo progetto di dottorato è lo sviluppo di modelli di coltura in-vitro per studiare diversi aspetti dell'osteoartrosi (OA), una malattia degenerativa che coinvolge tutti i tessuti dell’articolazione tra cui la cartilagine articolare, la membrana sinoviale e l'osso subcondrale. L'incidenza globale dell'OA del ginocchio è di 203 per 10.000 persone all’anno e cresce con l'aumentare dell'età, raggiungendo un picco tra i 70-79 anni. Il principale sintomo disabilitante dell'OA è il dolore, tipicamente intermittente e portante (meccanico) che può, in alcuni casi, a stress psicologico. Contrariamente alla comune descrizione di una patologia correlata all'usura, l'OA è la conseguenza di un processo attivo e sbilanciato di riparazione e distruzione. Le cause che portano all’insorgenza di tale patologia non sono ancora state del tutto identificate e si dibatte ancora sull'ordine preciso degli eventi coinvolti nella sua progressione. Attualmente, la scarsa conoscenza della patogenesi dell’OA e le numerose modificazioni tissutali che la caratterizzano hanno reso complicato lo sviluppo di un modello in-vitro. È quindi necessario comprendere i meccanismi coinvolti nella progressione della patologia, nonché i target di nuovi trattamenti per il ripristino della funzionalità articolare. Qui, due diversi modelli di coltura in-vitro per indagare diversi aspetti dell'OA: (i) il coinvolgimento di cellule staminali / stromali adulte della membrana sinoviale nel rimodellamento osseo, attraverso un approccio di co-coltura 2D indiretta e (ii) il crosstalk tra condrociti e osso subcondrale in condizioni normali e patologiche, sviluppando uno scaffold 3D ingegnerizzato e simulando un microambiente infiammato. Questi due modelli hanno permesso lo studio dei comportamenti cellulari di tre tessuti coinvolti nella patogenesi dell'OA e lo sviluppo di una possibile piattaforma in-vitro per futuri studi che potranno comprendere simultaneamente queste tre componenti.

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COSTABILE, FRANCESCA. "Development of an in vitro murine three-dimensional tumor model to study the micro-environment ability to tune cell’s features." Doctoral thesis, Università degli studi di Genova, 2022. http://hdl.handle.net/11567/1079876.

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Background: The tumor microenvironment (TME) is a complex system, shaped by direct interactions among different cell types and by the interactions between cells and extracellular matrix (ECM). Given the emerging importance of the TME in modulating cells’ morphology and function, more sophisticated tumor models incorporating TME features are needed to elucidate cellular, molecular, and immunologic mechanisms of tumor response or resistance. An intensive investigation of in vitro models able to study tumor biology has led to the generation of different three-dimensional (3D) culture methods that better mimic in vivo conditions compared to the usual 2D culture methods. The 3D mono- and co-cultures are able to reproduce some “in vivo features” such as 3D cell morphology, which permits cells to better execute their function and enables them to deposit significant increased amount of ECM. Aim of this study is the development of an accurate in vitro 3D tumor model to study the impact of tumor ECM on the cells of the microenvironment. The understanding of the specific contributions that ECM proteins make to the tumor microenvironment became crucial due to the emergency of find alternative cures to the many cancer harboring patients resistant to the existing therapies. Methods: Our experimental approach can be divided in two main experimental plans: • the development of tumor spheroid model, helpful to understand the beginning stage of the nascent tumor; at day 7, 14 and 21 of spheroids culture we evaluated ECM deposition through immunofluorescence (IF) analysis of collagen type VI; we also analyzed how much the 3D co-culture model we generated was similar compared to the in vivo microenvironment regarding gene expression; • the development of a scaffold obtained by murine tumor tissue decellularization, utilized for the study of the advanced tumor stage, with a more complex ECM compared to the spheroid model; some scaffolds underwent to collagen type VI IF analysis while other scaffolds were used for recellularization experiments with B16f10 or NIH/3T3 cell lines. Some scaffolds were also used for scanning electron microscopy analysis (SEM). Results and Discussion: Spheroids generated by the co-culture of B16f10 and NIH/3T3 cells were the only ones lasting for 21 days and the only ones where collagen type VI was detected, compared to spheroids made just by B16f10. These results let us deduce that ECM deposition is fibroblast-dependent. According to the 3D morphology classification of Kenny et al. 2007, B16f10 + NIH/3T3 derived spheroids we generated belong to the Mass class, characterized by cells organized in a regular manner around the center of the colony; B16f10 derived spheroids seems to belong instead to the Grape-like class, characterized by colonies with poor cell-cell contacts and distinguished by their grape-like appearance. B16f10-spheroid’s morphology clearly show a lack of robust cell-cell adhesion, result that has to be overlapped with the absence of collagen type VI. Therefore, fibroblasts are needed for the generation of an early tumor stage 3D model because of their major role as ECM producers and tumor micro-environment organizers. We even investigated spheroid gene expression signature. We focused on the genes that were expressed at the same level between the tumor cells derived from the 3D coculture and the actual in vivo tumor microenvironment. Clusters analysis highlights the similarity between the 2 groups showing 23.1% of the found pathways referring to cancer microenvironment, giving grand importance to the spheroid as in vitro model compared to the 2D systems. To define an in vitro 3D model suitable to the experimentation on advanced solid tumor phase, murine B16f10 derived tumors were processed for decellularization and studied to verify the efficacy as a natural scaffold for 3D culture system. As observed also from other research groups, cell attachment was limited to the border of the tissue, and it seems cells were not able to pass through the ECM. Control experiment with polyurethane demonstrate the capability of cells to infiltrate a synthetic matrix. To have a better understanding of the ECM structure, frozen sections of decellularized tissue were analyzed by confocal microscope for detection of collagen type VI. Interestingly, both immunofluorescence and histochemistry analysis showed an increase of ECM in decellularized tissue compared to the not decellularized one. This may be due to a slight collapse of the tumor structure since cells have been removed from it. We implemented the ECM observation with the support of SEM: after the decellularization treatment the extracellular fibres are exposed and as hypothesized, the lack of cells makes the fibres twist on themselves, generating a complex net, probably impenetrable from cells. To create a method to measure the collapse of the extracellular matrix that a decellularization treatment may provokes, we compared a fresh and a decellularized specimen of mouse pulmonary parenchyma, where structural modifications are easy to be detected. In this way we were able to quantify the collapse of the ECM through alveoli area reduction. Conclusions: The development of the spheroid made by tumor and fibroblast cells is a more realistic environment compared to the usual 2D culture and to the spheroid made simply by tumor cells. Fibroblasts are needed for the generation of a better 3D model because of their major role as ECM producers and tumor micro-environment organizers. The limit of the spheroid model is the time, since, to date, the system does not provide oxygen import, necessary for the tumor bulk growth. Tumor derived 3D scaffolds by decellularization most truthfully represents the real in vivo scenario of a tumor 3D model, but the recellularization lack is very often a big limit. Our data demonstrate that the decellularizzation process provokes the collapse of the matrix that does not allow the tissue to be utilized as scaffold for in vitro cell experimentation. Here we also provide, for the first time, a method to test the damage that the treatment provokes on the samples, avoiding the loss of precious materials.

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Latour, Simon. "Rôle des protéines Orai1 et STIM1 dans les lymphomes B non-Hodgkiniens, établissement d'un modèle d'étude en 3D." Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0038.

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Les lymphomes B non-Hodgkiniens (LNHB) représentent le type d’hémopathie maligne le plus fréquent. Ces pathologies sont traitées par l’association de chimiothérapies conventionnelles et d’immunothérapies dirigées contre le CD20. Bien qu’efficace, 40% des patients résistent ou rechutent après le traitement. Deux raisons peuvent expliquer ces échecs thérapeutiques : 1) l’absence de cibles thérapeutiques impliquées dans plusieurs processus oncogéniques et 2) l’absence de modèles pré-cliniques de LNHB pertinents pour le test de molécules thérapeutiques et la compréhension de la lymphomagenèse. Le calcium est un messager ubiquitaire qui est impliqué dans de nombreux processus cellulaires en condition physiologique et pathologique. La principale voie d’entrée de calcium dans les lymphocytes B est l’entrée capacitive de calcium médiée par Orai1 et STIM1. Ces deux protéines ont été largement décrites pour être impliquées dans les processus tumoraux de nombreux cancers, cependant leurs rôles dans la lymphomagenèse restait à élucider. Nos travaux ont révélé l'implication de la signalisation calcique dans la mort induite par le GA101, un anti CD20 de nouvelle génération actuellement en essai clinique. De plus, nous avons mis en évidence l’implication des protéines Orai1 et STIM1 dans la migration des cellules cancéreuses de LNHB. De manière intéressante, l’implication de ces deux protéines dans la migration cellulaire est calcium indépendante, suggérant donc un nouveau rôle de ces protéines. Enfin, grâce à la technologie des capsules cellulaires nous avons établi un nouveau modèle 3D de lymphome mimant la niche tumorale en incluant des cellules du microenvironnement et de la matrice extracellulaire. Ce modèle semble particulièrement pertinent pour le screening de molécules et la compréhension des mécanismes de la lymphomagenèse. Ce travail de thèse révèle ainsi le ciblage de Orai1 et STIM1 comme potentiellement intéressant dans le traitement du LNHB B-cell non-Hodgkin lymphomas (BNHL) are the most common hematological malignancies, usually treated with a combination of chemotherapy and anti CD20 immunothérapie. However, 40% of patients are resistant or relapse after treatment. These therapeutic failures could be due to 1) lack of therapeutic targets implicated in several oncogenic processes, 2) lack of relevant preclinical BNHL models for drug screening and lymphomagenesis studies. Calcium is an essential second messenger involved in various cell functions. In B cells, calcium entry is mainly due to Orai1 and STIM1 proteins, both of which have been associated with oncogenesis on solid tumors. However, their role in lymphomagenesis still remains to be elucidated. Our work shows that calcium signaling in BNHL cells participates in cell death induced by GA101, a novel anti-CD20 monoclonal antibody. We also demonstrate that Orai1 and STIM1 play a role in BNHL cell migration. Interestingly, both proteins controlled cell migration in a calcium-independent manner, suggesting a new role for these proteins. Finally, using cellular capsule technology, we established a new BNHL 3D model mimicking tumoral niche by including extracellular matrix and stromal cells. This new model could be used for drug screening and understanding lymphomagenesis. In summary, this work suggests that targeting of Orai1 and STIM1 is promising for BNHL treatment

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Ham, Stephanie Lemmo. "Engineering Tumor Models Using Aqueous Biphasic 3D Culture Microtechnology." University of Akron / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=akron150470381711759.

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Capra, J. (Janne). "Differentiation and malignant transformation of epithelial cells:3D cell culture models." Doctoral thesis, Oulun yliopisto, 2018. http://urn.fi/urn:isbn:9789526218236.

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Abstract The epithelial cells form barriers that compartmentalize the organs. Carcinomas are cancers stemming from epithelial cells and are the most common cancer type. The aim of this study was to understand the differentiation and malignant transformation of epithelial Madin-Darby canine kidney (MDCK) cells and to analyse the electrophysiological parameters which regulate their transport capacity. Emphasis was placed on comparing different culture environments, both in 2D and 3D. First, the effects of drugs or basal extracellular fluid composition on MDCK cell, cyst and lumen volumes were analysed using time-lapse microscopy. The results showed that MDCK cells were capable of both water secretion and reabsorption. The cells were able to perform these functions in a hyperpolarizing or depolarizing environment; change in osmolality of basal fluid was not required. Taken together, these results validate MDCK cells as a good basic model for studying kidney function. Next, the aim was to analyse the effect of 2D and 3D culture environments on the gene expression of untransformed MDCK and temperature sensitive ts-Src -transformed MDCK cells and the changes a single oncogene can induce. Microarray analysis revealed a decrease in the expression of survivin, an inhibitor of apoptosis protein, when switching the untransformed cells from 2D environment to 3D. This downregulation of survivin occurs in adult tissues as well, indicating that the cells grown in 3D are closer to the in vivo state than 2D cells. Src oncogene induced disintegration of cell junctions, but did not downregulate E-cadherin expression. The last part was to study further the factors controlling survivin expression and its significance to cell survival. MDCK cells grown in 3D did not suffer apoptosis if the cells remained in contact with the extracellular matrix. If MDCK cells were denied of ECM contacts they were more susceptible to apoptosis than survivin-expressing ts-Src MDCK cells. Finally, if cells were denied of cell-cell junctions, cells lacking survivin suffered apoptosis even though they had proper cell-matrix contacts. Taken together, these results highlighted the importance of cellular contacts to the cells: MDCK cells needed ECM contacts to differentiate and cell-cell contacts to avoid apoptosis Tiivistelmä Epiteelisolut ovat erikoistuneet toimimaan rajapintana elimen ja ympäristön välillä. Ihmisten yleisin syöpä on epiteelisoluista alkunsa saanut karsinooma. Tämän tutkimuksen tarkoituksena oli ymmärtää Madin-Darby-koiran munuaisen solujen (MDCK) erilaistumista ja pahanlaatuistumista sekä analysoida sähköfysiologisia tekijöitä, jotka säätelevät näiden solujen kuljetustoimintaa. Erityisenä kiinnostuksen kohteena oli erilaisten kasvuympäristöjen vertailu. Farmakologisten aineiden tai basaalisen, solunulkopuolisen nesteen koostumuksen vaikutusta MDCK-solujen, -kystan sekä luumenin kokoon tutkittiin valomikroskooppisten aikasarjojen avulla. Tulokset osoittivat MDCK-solujen olevan kykeneviä sekä veden eritykseen että absorptioon, niin hyperpolarisoivassa kuin depolarisoivassakin ympäristössä. Basaalisen nesteen osmolaliteetin muutosta ei tarvittu. Nämä tulokset osoittavat MDCK-solujen olevan hyvä munuaisen tutkimuksen perusmalli. Seuraavaksi analysoitiin kaksi- ja kolmiulotteisten (2D ja 3D) viljely-ympäristöjen vaikutusta ei-transformoitujen MDCK-solujen ja lämpötilaherkkien ts-Src-transformoitujen MDCK-solujen geenien ilmentymiseen sekä yhden onkogeenin aktivoimisen aikaansaamia muutoksia. Microarray-analyysi osoitti apoptoosin estäjän, surviviinin, ilmentymisen vähenemisen, kun kasvuympäristö vaihdettiin 2D-ympäristöstä 3D-ympäristöön. Koska surviviinin väheneminen on normaali tapahtuma aikuisissa kudoksissa, voitiin todeta, että 3D-ympäristössä kasvatetut solut ovat lähempänä luonnonmukaista olotilaa kuin 2D-ympäristössä kasvaneet. Src-onkogeeni sai aikaan soluliitosten hajoamisen, mutta ei vähentänyt E-kadheriinin ilmentymistä. Tutkimuksen viimeinen osa keskittyi surviviinin ilmentymistä säätelevien tekijöiden analysoimiseen ja surviviinin merkitykseen solujen eloonjäämiselle. 3D-ympäristössä kasvaneet MDCK-solut eivät kärsineet apoptoosista edellyttäen, että solut pysyivät kosketuksissa soluväliaineeseen. Jos solut irtautuivat soluväliaineesta, ne päätyivät herkemmin apoptoosiin kuin surviviinia ilmentävät ts-Src MDCK-solut. Mikäli solujen väliset liitokset pakotettiin avautumaan, solut joutuivat apoptoosiin, vaikka ne olivat kosketuksissa soluväliaineeseen. Yhteenvetona nämä tulokset korostavat solujen kontaktien merkitystä: MDCK-solut tarvitsevat soluväliainekontakteja erilaistumiseen ja solujen välisiä kontakteja välttyäkseen apoptoosilta

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Giudice, Fernanda Salgueiredo. "Análise da expressão das proteínas Akt, NF-kB & Ciclina D1 em linhagens celulares de carcinoma epidermóide de cabeça e pescoço em ambiente tridimensional e câmara de invasão." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/23/23141/tde-29092009-081311/.

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O carcinoma epidermóide representa mais de 90% das neoplasias malignas de cabeça e pescoço, apresentando taxas elevadas de morbi-mortalidade, porém pouco se sabe sobre as vias de sinalização que estão envolvidas na progressão tumoral. Têm sido relatado na literatura, que alguns estímulos podem ativar a holoenzima PI3K que, por sua vez, desencadeia um processo que induz a fosforilação da proteína Akt (pAkt) que leva a ativação e a translocação do NF-B do citoplasma para o núcleo, onde ocorre a transcrição de genes envolvidos na proliferação e invasão celular. Assim, esse estudo analisou, através dos métodos de Imunofluorescência e Western Blot, a expressão das proteínas pAkt, NF-B e Ciclina D1 em três linhagens de células de carcinoma epidermóide de cabeça e pescoço (HN6, HN30 e HN31) submetidas a cultivo tridimensional e ensaio de invasão (gerando clones invasivos HN6.1, HN30.1 e HN31.1), ambos realizados com Matrigel®. O pAkt apresentou marcação citoplasmática e nuclear nas linhagens celulares HN6, HN30, HN6.1 e na HN30 submetida ao cultivo tridimensional, todavia, as linhagens celulares HN31, HN30.1, HN31.1 e HN6/HN31 cultivadas tridimensionalmente, apresentaram positividade predominantemente nuclear. No caso do NF-B, a localização foi marcadamente citoplasmática em todas as linhagens celulares cultivadas bi ou tridimensionalmente, porém, com exceção da HN31.1, o padrão de marcação foi citoplasmático e nuclear nos clones invasivos. Por fim, a Ciclina D1 exibiu imunopositividade apenas nuclear. A técnica de Western Blot mostrou diminuição nos níveis de expressão das proteínas analisadas quando as células foram cultivadas em Matrigel®, sendo, na maioria das vezes, essa redução estatisticamente significante, exceto no caso da linhagem celular HN6, que apresentou um aumento significativo de Ciclina D1. Os clones invasivos HN6.1 e HN30.1 exibiram elevação significativa dos níveis de expressão de pAkt e NF-B porém, a HN31.1 apresentou aumento de pAkt e discreta diminuição de NF-B, mas ambos os valores não estatisticamente significantes. Em relação à Ciclina D1, houve um aumento dos seus níveis em todas as linhagens invasivas, sendo que apenas na HN6.1 foi estatisticamente significante. Esses resultados mostraram que, nas linhagens celulares avaliadas, quando cultivadas em ambiente tridimensional, a significativa redução dos níveis de expressão de proteínas da via de sinalização PI3K ocorreu devido a uma possível fase adaptativa dessas células ao Matrigel® que seria seguida provavelmente por uma etapa de aumento do potencial proliferativo e posterior transdiferenciação de algumas células para ganho de fenótipo mais agressivo. Além disso, este estudo mostrou a participação da via de sinalização Akt/NF-B/Ciclina D1 no processo de invasão das células de carcinoma epidermóide de cabeça e pescoço (HN6.1 e HN30.1). Squamous cell carcinoma represents more than 90% of the head and neck malignant tumors, with high mortality rates. Nevertheless, the knowledge about signaling pathways involved in tumor progression remains unclear. The relationship between pAkt and NF-B is well established in carcinogenesis. It is known that the activation of PI3K can be induced by some factors what leads to Akt phosphorilation (pAkt). This further event starts an activation cascade that induces NF-B translocation from the cytoplasm into the nucleus where the transcription of genes enrolled in cellular proliferation and invasion will be done. Therefore, this study analyzed the status of pAkt, NF-B and Cyclin D1 proteins by Immunofluorescence and Western Blot methods in three head and neck squamous cell carcinoma cell lines (HN6, HN30 and HN31) submitted to three-dimensional culture model and an in vitro invasion assay (invasive clones HN6.1, HN30.1 e HN31.1), both with Matrigel®. pAkt expression was detected in cytoplasm and nucleus in HN6, HN30, HN6.1 and HN30 cultured with Matrigel®, however, HN31, HN30.1, HN31.1 cell lines and HN6/HN31 submitted to three-dimensional culture model showed nuclear expression. NF-B localization was strongly cytoplasmatic in all cell lines cultured with or without Matrigel®, but, regarding HN31.1, NF-B protein expression was nuclear and cytoplasmatic in the invasive clones. Cyclin D1 was nuclear in all cell lines analyzed. Western blot assays showed a decrease in pAkt, NF-B and Cyclin D1 expression levels in all cells lines in three-dimensional culture model, most of them statistically significant, but It was detected a considerable increase in Cyclin D1 expression in HN6 cultured in threedimensional model. Moreover, HN6.1 and HN30.1 showed a significant enhance in pAkt and NF-B levels but, HN31.1 demonstrated a raise in pAkt and a slight decline in NF-B, both were not statistically significant. Finally, an increase in Cyclin D1 expression levels was illustrated in all cell lines but, only HN6.1 showed a statistic signal. These results suggest that, in the cell lines studied, when cultured in threedimensional model, a noteworthy reduction in expression levels of PI3K signaling pathway proteins happened for the reason that a possibly adaptation phase of these cells to Matrigel® was occurring, in the first moment, but it would be probably followed by an increase proliferative potential stage and subsequent transdifferentiation of some cells that could show, consequently, a more aggressive phenotype. At last, this study revealed the participation of Akt/NF-B/Cyclin D1 signaling pathway in invasion process of head and neck squamous cell carcinoma (HN6.1 and HN30.1).

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Teixeira, Marta Carolina Jesus. "Patient-derived explant cultures for cancer modelling." Master's thesis, Universidade Nova de Lisboa. Instituto de Tecnologia Quimica e Biológica António Xavier, 2018. http://hdl.handle.net/10362/130073.

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"Cancer remains one of the deadliest diseases in the world. The process of drug discovery to find new drugs for oncology treatment takes more than a decade of research and it is associated with high attrition rates during clinical trials. One of the most common reasons claimed for the poor efficiency of new developed drugs is the lack of physiological relevance of the in vitro models used to select the novel compounds and to evaluate their potency. The role of tumor microenvironment on tumor progression and drug sensitivity is being increasingly studied and it is expected to provide significant clues for the development of novel therapies. Therefore, the incorporation of microenvironment features on cancer cell models during pre- clinical stages of research has the potential to improve significantly their predictive value. (...)" N/A

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ES, SEBAR LEILA. "Metrology for Cultural Heritage: multispectral 3D models by photogrammetry." Doctoral thesis, Politecnico di Torino, 2022. http://hdl.handle.net/11583/2959961.

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Held, Christoph. "Creating 3D models of cultural heritage sites with terrestrial laser scanning and 3D imaging." Master's thesis, University of Cape Town, 2012. http://hdl.handle.net/11427/12076.

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Includes abstract. Includes bibliographical references. The advent of terrestrial laser-scanners made the digital preservation of cultural heritage sites an affordable technique to produce accurate and detailed 3D-computermodel representations for any kind of 3D-objects, such as buildings, infrastructure, and even entire landscapes. However, one of the key issues with this technique is the large amount of recorded points; a problem which was even more intensified by the recent advances in laser-scanning technology, which increased the data acquisition rate from 25 thousand to 1 million points per second. The following research presents a workflow for the processing of large-volume laser-scanning data, with a special focus on the needs of the Zamani initiative. The research project, based at the University of Cape Town, spatially documents African Cultural Heritage sites and Landscapes and produces meshed 3D models, of various, historically important objects, such as fortresses, mosques, churches, castles, palaces, rock art shelters, statues, stelae and even landscapes.

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Kim, Leejin. "Analysis and Construction of Engaging Facial Forms and Expressions: Interdisciplinary Approaches from Art, Anatomy, Engineering, Cultural Studies, and Psychology." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/567.

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The topic of this dissertation is the anatomical, psychological, and cultural examination of a human face in order to effectively construct an anatomy-driven 3D virtual face customization and action model. In order to gain a broad perspective of all aspects of a face, theories and methodology from the fields of art, engineering, anatomy, psychology, and cultural studies have been analyzed and implemented. The computer generated facial customization and action model were designed based on the collected data. Using this customization system, culturally-specific attractive face in Korean popular culture, “kot-mi-nam (flower-like beautiful guy),” was modeled and analyzed as a case study. The “kot-mi-nam” phenomenon is overviewed in textual, visual, and contextual aspects, which reveals the gender- and sexuality-fluidity of its masculinity. The analysis and the actual development of the model organically co-construct each other requiring an interwoven process. Chapter 1 introduces anatomical studies of a human face, psychological theories of face recognition and an attractive face, and state-of-the-art face construction projects in the various fields. Chapter 2 and 3 present the Bezier curve-based 3D facial customization (BCFC) and Multi-layered Facial Action Model (MFAF) based on the analysis of human anatomy, to achieve a cost-effective yet realistic quality of facial animation without using 3D scanned data. In the experiments, results for the facial customization for gender, race, fat, and age showed that BCFC achieved enhanced performance of 25.20% compared to existing program Facegen , and 44.12% compared to Facial Studio. The experimental results also proved the realistic quality and effectiveness of MFAM compared with blend shape technique by enhancing 2.87% and 0.03% of facial area for happiness and anger expressions per second, respectively. In Chapter 4, according to the analysis based on BCFC, the 3D face of an average kot-mi-nam is close to gender neutral (male: 50.38%, female: 49.62%), and Caucasian (66.42-66.40%). Culturally-specific images can be misinterpreted in different cultures, due to their different languages, histories, and contexts. This research demonstrates that facial images can be affected by the cultural tastes of the makers and can also be interpreted differently by viewers in different cultures.

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Schmittlein, Carina [Verfasser], and Joachim [Akademischer Betreuer] Wegener. "Sensors and Actuators for 2D and 3D Cell Culture Models based on Oxygen Sensitive Culture Substrates / Carina Schmittlein ; Betreuer: Joachim Wegener." Regensburg : Universitätsbibliothek Regensburg, 2018. http://d-nb.info/1170955738/34.

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CAMPIONI, GLORIA. "Monolayers and three-dimensional cultures to investigate metabolic reprogramming in breast and bladder cancer." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2022. http://hdl.handle.net/10281/375413.

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La riprogrammazione metabolica è stata osservata in molti tipi di cancro ed è considerata un tratto distintivi di questa malattia eterogenea multifattoriale. Comprendere i meccanismi che portano al riarrangiamento metabolico e come queste attività promuovono l’attivazione di proprietà maligne nel cancro può aiutare a sfruttare le alterazioni metaboliche a beneficio terapeutico. Nei tumori solidi le cellule tumorali interagiscono con il complesso habitat del microambiente tumorale (TME) che può modulare il metabolismo delle cellule tumorali e la loro sensibilità o resistenza al trattamento farmacologico. I modelli tridimensionali (3D), come gli sferoidi, gli organoidi e gli organ-on-chip stanno cambiando il paradigma della ricerca preclinica sul cancro poiché rappresentano più fedelmente la complessità dell’ambiente e dell’architettura tissutale che si trova nei tumori in vivo rispetto alle colture cellulari bidimensionali (2D). Perciò l’utilizzo dei modelli 3D potrebbe migliorare la robustezza e l’affidabilità dei dati della ricerca preclinica riducendo la necessità di test sugli animali e favorendone la traslazione alla pratica clinica. In questa tesi, abbiamo eseguito una caratterizzazione metabolica di linee cellulari di carcinoma mammario luminale (MCF7) e triplo negativo (SUM159PT e MDAMB231) e abbiamo confrontato la loro diversa risposta alle perturbazioni metaboliche attraverso la valutazione della proliferazione cellulare (in 2D) e della capacità di formare sferoidi. I principali risultati di questo capitolo suggeriscono che le deprivazioni nutrizionali e i trattamenti farmacologici contro il metabolismo energetico hanno un impatto maggiore sulla proliferazione delle cellule che crescono in 2D rispetto alla formazione di sferoidi (3D). Inoltre, la perturbazione del metabolismo del glucosio ha mostrato l'effetto più forte sul processo di formazione degli sferoidi, riducendo gravemente la vitalità e la morfologia degli sferoidi, specialmente sulla linea cellulare altamente glicolitica MDAMB231. Inoltre, abbiamo sviluppato un flusso di lavoro affidabile e riproducibile per l'analisi metabolica di colture tridimensionali mediante la tecnologia Seahorse XFe96, un analizzatore di flusso extracellulare che misura simultaneamente il tasso di consumo di ossigeno (OCR) e il tasso di acidificazione extracellulare (ECAR) delle cellule viventi. L'ottimizzazione del protocollo di formazione degli sferoidi ha consentito di produrre sferoidi di forma altamente regolare e di dimensioni omogenee, riducendo drasticamente la variabilità nelle misurazioni di OCR ed ECAR tra i replicati tecnici sperimentali, sia in condizioni basali che di trattamento farmacologico. La normalizzazione per cellula ci ha permesso di confrontare direttamente questi parametri metabolici tra sferoidi di diverse dimensioni e tra colture 2D e 3D, rivelando che le differenze metaboliche tra gli sferoidi studiati sono per lo più legate alla linea cellulare piuttosto che alla dimensione dello sferoide. Infine, abbiamo caratterizzato il metabolismo energetico e le proprietà cellulari associate alla diffusione e alla progressione del tumore nelle linee cellulari tumorali di Grado 2 derivanti da vescica umana, RT112 e 5637. Nonostante le due linee cellulari mostrassero proprietà metaboliche e invasive distinte, entrambe presentavano una respirazione considerevole e il trattamento con metformina ha causato una down-regolazione globale della proliferazione, della migrazione e della capacità di formare sferoidi. Complessivamente i risultati di questa tesi aprono nuove prospettive di ricerca per l'identificazione di nuovi potenziali bersagli terapeutici contro il cancro, avvicinandosi alla comprensione della plasticità metabolica del cancro che può essere sfruttata per sviluppare strategie terapeutiche efficienti per il trattamento di pazienti oncologici. Metabolic reprogramming has been observed in many types of cancer, and it is considered a hallmark of this heterogeneous multifactorial disease. Understanding the mechanisms leading to metabolic rewiring and how these activities promote the activation of cancer's malignant properties can help exploit metabolic alterations for therapeutic benefit. In solid tumors, cancer cells interact with the complex habitat of the tumor microenvironment (TME), which can modulate cancer cells' metabolism and their sensitivity or resistance to drug treatment. Three-dimensional (3D) models, such as spheroids, organoids, and organ-on-chips, are changing the paradigm of preclinical cancer research as they more closely resemble the complex tissue environment and architecture found in tumors in vivo than bidimensional (2D) cell cultures. Therefore, 3D models could potentially improve the robustness and reliability of preclinical research data, reducing the need for animal testing and favoring their transition to clinical practice. In this thesis, we performed a metabolic characterization of luminal (MCF7) and triple-negative (MDAMB231 and SUM159PT) breast cancer cell lines and compared their different response to metabolic perturbations through the evaluation of cell proliferation (in 2D) and spheroid formation ability. The main results of this chapter suggest that nutritional deprivations and pharmacological treatments targeting energetic metabolism have a more significant impact on the proliferation of cells growing in 2D than on spheroid formation (3D). Moreover, the perturbation of glucose metabolism by glucose deprivation and 2-deoxy-glucose treatment showed the most potent effect on the spheroid formation process, severely reducing spheroid vitality and morphology, especially on the highly glycolytic MDAMB231 cell line. Furthermore, we developed a reliable and reproducible workflow for the metabolic analysis of three-dimensional cultures by Seahorse XFe96 technology, an extracellular flux analyzer that simultaneously measures the Oxygen Consumption Rate (OCR) and the Extracellular Acidification Rate (ECAR) of living cells. The optimization of the spheroid formation protocol enabled the production of spheroids highly regular in shape and homogenous in size, dramatically reducing variability in the OCR and ECAR measurements among the experimental technical replicates, both under basal and drug treatment conditions. Furthermore, the normalization on a per-cell basis allowed us to directly compare these metabolic parameters between spheroids of different sizes and between 2D and 3D cultures, revealing that metabolic differences among the studied spheroids are mostly related to the cell line rather than to the size of the spheroid. Finally, we characterized energy metabolism and cellular properties associated with spreading and tumor progression of RT112 and 5637, two cell lines from Grade 2 human bladder cancer. Although the two cell lines displayed distinct metabolic and invasive properties, both exhibited sizable respiration, and the metformin treatment gave a global downregulation of the proliferation, migration, and the ability to form spheroids. Altogether, the findings of this thesis open new research perspectives for identifying novel potential therapeutic targets against cancer, getting closer toward the understanding of cancer metabolic plasticity that can be exploited for developing efficient therapeutic strategies for the treatment of oncologic patients.

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Kós, José Ripper. "Urban spaces shaped by past cultures : historical representation through electronic 3D models and databases." Thesis, University of Strathclyde, 2003. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=21518.

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Digital tools have been increasingly used, in the last decades, for the study and representation of the city history. As the available instruments develop and the researchersb ecome more familiar with them, their use turns out to be more effective and provides richer results. This study aims to explore the use of information technology, particularly 3D models, for the city history research. When this study was elaborated, few initiatives effectivelly applied those new tools to convey the history of the city. A smaller number of published scientific enterprises investigated that operation. Therefore, the study is structured mainly on the analysis of some precedents based on those tools, together with others selected for applying creatively traditional methods. These analyses also raise questionings on related issues such as historical narratives, traditional methods of historical graphic representation or other digital representation modes. The examination of those subjects constitutes the thesis' theoretical part. The conclusion is presented in the form of a digital alternative for the representation of the city history. The tool developed as a prototype is grounded on 3D models representing different periods of the city linked to a database of a great diversity of historical documents. Thus, the city history is accessed through images of the significant sites from the 3D models. The prototype development is based on the assumptions that this process of retrieving historical information related to city spaces facilitates the understanding of the past culture. Furthermore, when the readers associate the space they know in the city to the historical information, they understand better the past culture that shaped it, strengthen their identity and intensify the relationship to the place they dwell in.

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FUCARINO, Alberto Giuseppe. "APPLICATION OF NOVEL 3D CULTURE MODELS OF HUMAN MUCOSAE TO STUDY THE EFFECTS OF ENVIRONMENTAL FACTORS ON NON-COMMUNICABLE DISEASES." Doctoral thesis, Università degli Studi di Palermo, 2014. http://hdl.handle.net/10447/90906.

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BOSIO, ALESSIA GRAZIANA. "IDENTIFICATION OF NOVEL BIGUANIDE BASEDCOMPOUNDS AS ANTI TUMOR AGENTS FOR GLIOBLASTOMA: PHARMACOLOGICAL ASSESSMENT IN 3D CULTURE MODELS." Doctoral thesis, Università degli studi di Genova, 2022. http://hdl.handle.net/11567/1090330.

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Glioblastoma (GBM) is the most common malignant and aggressive adult brain tumour characterized by its clinical behaviour, with high growth rate, diffuse invasiveness, and low response to therapies. Despite of multimodal treatment, which consists in extensive surgery followed by radiotherapy and chemotherapy (temozolomide, TMZ) the mean life expectancy for patients with GBM is less than 2 years. Although remarkable research efforts have been done in the last decades, no significant improvement in patient survival have been obtained from 2005, also due to the lack of appropriate models to study the role of cellular heterogeneity and microenvironment in GBM growth, invasiveness and drug response. Among the main causes of therapeutic failure there is the ability of GBM cells to rapidly invade the brain parenchyma, greatly limiting successful surgical tumour debulking and the presence of cancer stem cells (CSC, also called tumour-initiating cells, TICs) which were identified over a decade ago also in GBM. Cancer stem cells are responsible for the malignant properties of tumours, have stem properties (self-renewal and differentiation), chemo- /radio-resistance and are able to expand to re-initiate tumours, promoting tissue infiltration, metastasis and relapse. CSC display cellular plasticity that is the ability to move between cell states to efficiently adapt to signals from the tumour microenvironment, such as terminal differentiation into a nontumorigenic state, transition into an invasive mesenchymal phenotype (epithelial–mesenchymal transition, EMT), or trans-differentiation into endothelial-like cells, leading to tumour angiogenesis. Conventional chemotherapies can eliminate bulk tumour cells while CSC evade most therapies, thus in order to GBM eradication, it will be crucial to find compounds able to effectively target CSC. Metformin, a widely used antidiabetic drug shows an antiproliferative effect on GBM CSC (GSC), via the inhibition of the CLIC-1 (Chloride Intracellular Channel 1) mediated ion current. Since other biguanides (both linear or cyclic) have demonstrated to act via CLIC-1 inhibition, this mechanism of action has been proposed to be a pharmacological class effect. Thus, we tested novel biguanide derivatives to enhance the metformin antitumour effect and pharmacological profile. Firstly, we performed a screening of the antiproliferative activity (by MTT assay and cell count) of the novel biguanide in-vitro, on patient-derived GSC and to assess the absence of off-target activity we used umbilical cord mesenchymal stem cells. We identified two compounds, Q54 (IC50 0.43 mM) and Q48 (IC50 0.083 mM) which exhibit a more potent antiproliferative effect as compared to metformin, the absence of off-target activity and the selectivity towards CLIC-1 (tested by electrophysiology recordings). Conversely, Q46 which didn’t show any significant effect was chosen as a positive control. By Boyden chambers assay and Matrigel™ invasion assay, we assessed the impairment of migration and invasion. In zebrafish, Q54 nor Q48 display aspecific toxicity, but Q54 was able to reduce the proliferation of GSCs xenotransplanted in their hindbrain. Moreover, we characterized two 3D models (GSC 3D cultures and tumoroids) by assessing cell proliferation (by EdU labelling), cell subpopulations and drug response. Q54 and Q48 were able to inhibit cell proliferation on GSC 3D cultures. By screening our GSC cultures for the CLIC-1 protein content we found that 2 cultures which spontaneously express low CLIC-1, were able to grow in vivo and to retain stem-like phenotype and functional features in vitro, but in these cultures, Q48 and Q54 displayed reduced potency and efficacy as antiproliferative agents as compared to high CLIC-1-expressing tumours. Thus, this data highlight the potential of Q48 and Q54 to target GSC with a better pharmacological profile as compared to metformin in CLIC-1 expressive culture; indeed, CLIC-1 acts as a booster for GSC proliferation but it is not required for GBM development. In addition, our compounds were tested on three different models that allow us to obtain different information that integrate each other, aiming to obtain more predictive results of what could happen in a GBM. We suggest that this approach could be useful in order to try to overcome the lack of reliable models in GBM research.

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Hörr, Christian, Elisabeth Lindinger, and Guido Brunnett. "Considerations on Technical Sketch Generation from 3D Scanned Cultural Heritage." Universitätsbibliothek Chemnitz, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:ch1-200901463.

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Drawing sketches is certainly one of the most important but at the same time elaborate parts of archaeological work. Currently, 3D scanning technology is affording a number of new applications, and only one of them is using virtual copies instead of the originals as the basis for documentation. Our major contribution are methods for automatically generating stylized images from 3D models. These are not only intuitive and easy to read but also more objective and accurate than traditional drawings. Besides some other useful tools we show several examples from our daily work proving that the system accelerates the whole documentation process considerably.

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Barnes, Devon. "In vitro bioengineering applications of melt electrowritten and hydrogel composite scaffolds." Thesis, Queensland University of Technology, 2021. https://eprints.qut.edu.au/212352/1/Devon_Barnes_Thesis.pdf.

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Two-dimensional cell cultures provide an inaccurate representation of how cells develop and are affected by disease and injury. Scaffold-based tissue engineering techniques that combine novel biomaterials and printing methods could assist in the design of more physiologically relevant, three-dimensional experimental tissue models. This thesis investigated the application of carbohydrate glass as a sacrificial material toward producing perfusable hydrogel devices using melt electrowriting, the development and optimisation of a three-dimensional bioengineered bone marrow microenvironment, and a literature review of the approaches toward the development, imaging and analysis of resulting three-dimensional models.

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DI, LISA DONATELLA. "Biopolymeric microbeads as a 3D scaffold for soft tissue engineering." Doctoral thesis, Università degli studi di Genova, 2020. http://hdl.handle.net/11567/1005298.

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The increase of different types of cell cultures, which can be used for the in vitro studies of physiological and/or pathological processes, has introduced the need to improve culture techniques through the use of materials and culture media that promote growth, recreating a cellular micro-environment that can be asserted in in vivo condition. Therefore, it is important to design and develop new biologically sustainable methods, such as to contribute to the “closer-to-in vivo” condition. In particular, the design of a 3D in vitro model of neuronal culture is an important step to better understand the mechanisms of cell-cell communication, synaptogenesis and neurophysiological circuits. In order to mimic the ECM environment, a granular, porous and soft structure is preferred in the design of an artificial neural network. The granular structure is preferred due to the fact that CNS tissue seems to be organized as a greater proportion of the microscale tissue, that can be thought of as granular. For this reason, the thesis is focused on the production and characterization of bipolymeric microbeads as a 3D scaffold for soft tissue engineering. The biopolymer Chitosan is presented as an alternative adhesion factor and support for 2D and 3D neuronal cell cultures. Chitosan is a copolymer of glucosamine and N-acetyl-glucosamine, obtained by the deacetylation of chitin; it is well known for its low-cost, biocompatibility, biodegradability, muco-adhesiveness, antibacterial activity as well as its bioaffinity. Chitosan backbone shows positive charges of primary ammines that favor the electrostatic interactions with the negatively charged cell membranes promoting cell adhesion and growth. The standard studies focoused on the development of nervous system, have been performed using traditional monolayer culture onto supports modified by extracellular matrix components or synthetic biopolymers such as poly-ornithine and poly-lysine which are expressed at stages critical for neuronal differentiation in situ and are functional in neurite outgrowth in vitro, acting as adhesion proteins. Morphological and functional characterization of 2D neuronal culture grew up onto chitosan susbtrates are carried out and compared with the gold standard reported in literature, in order to validate the ability of chitosan to support neuronal adhesion, networks development and the differentiation capacity. 3D cultured neurons on chitosan microbeads based-scaffold, showed a structural development of a functional network that are more representative of the in vivo environment. The studies reported in this thesis, successfully demonstrate the alternative use of the polysaccharide chitosan as adhesion factor and as a structural component for 2D/3D neuronal cultures. Definitely, thanks to its low cost and versatility, it could be easily functionalized for the fabrication of personalized of in vitro models. In this thesis, a new technology to converts monodisperse microbead hydrogels to fine powders, is reported. Microengineered emulsion-to-powder (MEtoP) technology generates microgels with all the molecular, colloidal, and bulk characteristics of fresh microbeas upon resuspension in aqueous media. GelMA microbeads are fabricated by microfluidic technique, that is one of the most effective techniques, and allows precise tuning of the compositions and geometrical characteristics of microbeads. Gelatin-methacryloyl (GelMA) is a semi-synthetic hydrogel which consists of gelatin derivatized with methacrylamide and methacrylate groups. These hydrogels provide cells with an optimal biological environment (e.g., RGD motifs for adhesion) and can be quickly photo-crosslinked, which provide shape fidelity and stability at physiological temperature. MEtoP technology is based on protecting the dispersed phase of an emulsion to preserve its physical and chemical cues during harsh freezing and lyophilization procedures. This technology avoids the persistent problems of colloids, including difficulty in sterilization, bacterial and viral contamination, impaired stability, high processing costs, and difficult packaging and transportation.

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Rambani, Komal. "Thick brain slice cultures and a custom-fabricated multiphoton imaging system: progress towards development of a 3D hybrot model." Thesis, Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/22702.

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Development of a three dimensional (3D) HYBROT model with targeted in vivo like intact cellular circuitry in thick brain slices for multi-site stimulation and recording will provide a useful in vitro model to study neuronal dynamics at network level. In order to make this in vitro model feasible, we need to develop several associated technologies. These technologies include development of a thick organotypic brain slice culturing method, a three dimensional (3D) micro-fluidic multielectrode Neural Interface system (ÂµNIS) and the associated electronic interfaces for stimulation and recording of/from tissue, development of targeted stimulation patterns for closed-loop interaction with a robotic body, and a deep-tissue non-invasive imaging system. To make progress towards this goal, I undertook two projects: (i) to develop a method to culture thick organotypic brain slices, and (ii) construct a multiphoton imaging system that allows long-term and deep-tissue imaging of two dimensional and three dimensional cultures. Organotypic brain slices preserve cytoarchitecture of the brain. Therefore, they make more a realistic reduced model for various network level investigations. However, current culturing methods are not successful for culturing thick brain slices due to limited supply of nutrients and oxygen to inner layers of the culture. We developed a forced-convection based perfusion method to culture viable 700Âµm thick brain slices. Multiphoton microscopy is ideal for imaging living 2D or 3D cultures at submicron resolution. We successfully fabricated a custom-designed high efficiency multiphoton microscope that has the desired flexibility to perform experiments using multiple technologies simultaneously. This microscope was used successfully for 3D and time-lapse imaging. Together these projects have contributed towards the progress of development of a 3D HYBROT. ----- 3D Hybrot: A hybrid system of a brain slice culture embodied with a robotic body.

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Picone, Giovanna <1992&gt. "Study of Bone Biomineralization in 2D and 3D Cultures of a Human Osteosarcoma Cell Line As Osteoblast-like Model." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2022. http://amsdottorato.unibo.it/10235/1/Giovanna_Picone_tesi.pdf.

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This PhD project focuses on the study of the early stages of bone biomineralization in 2D and 3D cultures of osteoblast-like SaOS-2 osteosarcoma cells, exposed to an osteogenic cocktail. The efficacy of osteogenic treatment was assessed on 2D cell cultures after 7 days. A large calcium minerals production, an overexpression of osteogenic markers and of alkaline phosphatase activity occurred in treated samples. TEM microscopy and cryo-XANES micro-spectroscopy were performed for localizing and characterizing Ca-depositions. These techniques revealed a different localization and chemical composition of Ca-minerals over time and after treatment. Nevertheless, the Mito stress test showed in treated samples a significant increase in maximal respiration levels associated to an upregulation of mitochondrial biogenesis indicative of an ongoing differentiation process. The 3D cell cultures were realized using two different hydrogels: a commercial collagen type I and a mixture of agarose and lactose-modified chitosan (CTL). Both biomaterials showed good biocompatibility with SaOS-2 cells. The gene expression analysis of SaOS-2 cells on collagen scaffolds indicated an osteogenic commitment after treatment. and Alizarin red staining highlighted the presence of Ca-spots in the differentiated samples. In addition, the intracellular magnesium quantification, and the X-ray microscopy on mineral depositions, suggested the incorporation of Mg during the early stages of bone formation process., SaOS-2 cells treated with osteogenic cocktail produced Ca mineral deposits also on CTL/agarose scaffolds, as confirmed by alizarin red staining. Further studies are underway to evaluate the differentiation also at the genetic level. Thanks to the combination of conventional laboratory methods and synchrotron-based techniques, it has been demonstrated that SaOS-2 is a suitable model for the study of biomineralization in vitro. These results have contributed to a deeper knowledge of biomineralization process in osteosarcoma cells and could provide new evidences about a therapeutic strategy acting on the reversibility of tumorigenicity by osteogenic induction.

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Dean, Zachary S. "Collective Migration Models: Dynamic Monitoring of Leader Cells in Migratory/Invasive Disease Processes." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/560817.

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Leader cells are a fundamental biological process that have only been investigated since the early 2000s. These cells have often been observed emerging at the edge of an artificial wound in 2D epithelial cell collective invasion, created with either a mechanical scrape from a pipette tip or from the removal of a plastic, physical blocker. During migration, the moving cells maintain cell-cell contacts, an important quality of collective migration; the leader cells originate from either the first or the second row, they increase in size compared to other cells, and they establish ruffled lamellipodia. Recent studies in 3D have also shown that cells emerging from an invading collective group that also exhibit leader-like properties. Exactly how leader cells influence and interact with follower cells as well as other cells types during collective migration, however, is another matter, and is a subject of intense investigation between many different labs and researchers. The majority of leader cell research to date has involved epithelial cells, but as collective migration is implicated in many different pathogenic diseases, such as cancer and wound healing, a better understanding of leader cells in many cell types and environments will allow significant improvement to therapies and treatments for a wide variety of disease processes. In fact, more recent studies on collective migration and invasion have broadened the field to include other cell types, including mesenchymal cancer cells and fibroblasts. However, the proper technology for picking out dynamic, single cells within a moving and changing cell population over time has severely limited previous investigation into leader cell formation and influence over other cells. In line with these previous studies, we not only bring new technology capable of dynamically monitoring leader cell formation, but we propose that leader cell behavior is more than just an epithelial process, and that it is a critical physiological process in multiple cell types and diseases.

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Ferreira, Luís Pedro Correia Pinto. "Development of multicelular 3D cancer testing platforms for evaluation of new anti-cancer therapies." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22713.

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Mestrado em Bioquímica Clínica O cancro do pulmão (CP) é um dos cancros mais diagnosticados a nível mundial e também um dos mais mortíferos. Atualmente, as terapias administradas a nível clínico para o tratamento do CP são ainda extremamente ineficazes e limitadas no que diz respeito ao aumento da taxa de sobrevivência dos pacientes oncológicos. Esta realidade demonstra a necessidade de investigar ativamente novas terapias para o tratamento desta neoplasia. No entanto a validação pré-clínica de terapias inovadoras para o CP tem-se revelado extremamente difícil devido à inexistência de plataformas que sejam adequadas para testes a nível laboratorial, uma vez que as culturas celulares in vitro bidimensionais (2D), recomendadas pelas agências regulatórias são incapazes de mimetizar as caraterísticas principais dos tumores humanos. Estas limitações têm originado uma fraca correlação entre a performance das terapias nos estudos in vitro e a obtida em ensaios clínicos controlados. Neste contexto, os modelos de tumores tridimensionais (3D) in vitro têm vindo a ser reconhecidos como uma solução para este problema, pois podem recapitular várias componentes do microambiente tumoral. Das várias plataformas 3D in vitro de CP investigadas atualmente muito poucas avaliaram o papel da inclusão de células estaminais mesenquimais (MSCs). Para colmatar esta lacuna, o trabalho de investigação desenvolvido no âmbito desta dissertação descreve a produção e otimização de novos modelos hétero-celulares 3D in vitro. Estas plataformas são compostas por células tumorais do CP (A549) e do seu estroma, nomeadamente fibroblastos da pele e células estaminais mesenquimais derivadas da medula óssea (BM-MSCs). Estes três tipos de células foram co-cultivadas em micropartículas poliméricas de policaprolactona revestidas por ácido hialurónico, com o objetivo de incluir este componente da matriz extracelular que se encontra presente no microambiente do CP. Esta abordagem permitiu formar a nível laboratorial microtecidos multicelulares 3D híbridos que melhor mimetizam a heterogeneidade celular das neoplasias pulmonares. Os resultados obtidos demonstraram que os microtumores formados através da técnica de sobreposição-líquida são reprodutíveis em termos de morfologia e tamanho, apresentaram núcleos necróticos, organização celular 3D e produziram proteínas do microambiente tumoral. Além destas caraterísticas, os dados obtidos através de microscopia de fluorescência revelaram que as BM-MSCs migram para o interior dos microtumores ao longo do tempo. A avaliação da citotoxicidade da Doxorubicina, um fármaco anti-tumoral rotineiramente utilizado a nível clínico, demonstrou que a inclusão de micropartículas aumenta a resistência das células tumorais em modelos homotípicos. Nos modelos tri-cultura heterotípicos a citotoxicidade foi comparável à obtida em microtumores sem micropartículas. Estes resultados evidenciam assim o papel importante dos fibroblastos e das BM-MSCs na resposta dos microtumores. Numa visão global, os modelos 3D formados recapitulam com mais exatidão o microambiente do cancro do pulmão e poderão servir no futuro como plataformas de teste para descobrir ou aperfeiçoar novas terapias, ou combinações de terapêuticas, para este tipo de neoplasia. Lung cancer (LC) is one the most commonly diagnosed cancers worldwide, being also one of the deadliest. Currently, clinically administered therapies for treatment of LC are still extremely ineffective and limited in increasing oncologic patients survival rates. This reality evidences the necessity of actively investigating novel therapies for the treatment of LC. However, preclinical validation of novel therapies as revealed itself as an extremely arduous process, due to the lack of suitable laboratory testing platforms since the recommend in vitro bi-dimensional (2D) cell cultures are unable to fully mimic the main hallmarks of human tumors. In this context, in vitro tridimensional (3D) tumor models are being increasingly recognized as a solution due to their ability to correctly recapitulate several characteristics of the tumor microenvironment (TME). Amongst currently developed 3D in vitro platforms for the study of LC, few have included or studied the role of mesenchymal stem cells (MSCs). To provide further insights into this hypothesis, the research work developed in this thesis describes the production and optimization of novel heterotypic in vitro 3D models, comprised by non-small-cell lung cancer cells (A549) and stromal cells, namely skin fibroblasts (HFs), and bone-marrow derived mesenchymal stem cells (BM-MSCs). These three diverse cell populations were co-cultured in hyaluronic acid coated polymeric polycaprolactone microparticles (LbL-MPs) as to include this key extracellular matrix component of LC TME. This approach allowed the formation of 3D multicellular heterotypic microtissues (3D-MCTS) that better recapitulate the cellular heterogeneity of LC TME in the laboratory. The obtained findings demonstrate that these models formed via the liquid-overlay technique were reproducible in terms of morphology and size, presented necrotic core formation, 3D cellular organization, and deposited matrix proteins in a similar manner as in the TME. Besides this, fluorescence microscopy data revealed that BM-MSCs migrated overtime into the microtumors core . Performed doxorubicin in vitro cytotoxicity assays revealed that the inclusion of LbL-MPs lead to an increased resistance of homotypic A549 monoculture models against this anti-cancer drug commonly used in clinical treatments. Alongside, the cytotoxicity obtained in triculture heterotypic models was comparable to that of microtumors without LbL-MPs inclusion, showcasing the role of HFs and BM-MSCs in microtumors response to therapy. Globally, the herein bioengineered 3D models were able to recapitulate with an increased precision the TME of LC, making them suitable test platforms for development or improvement of standalone or combinatorial therapies for this type of neoplasia.

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UREÑA, MARTÍN Carlos. "Study of Caveolae Mechanotransduction Under 3D Compressive Stresses : Comparative Analysis of 2 Models Mimicking Structural and Mechanical Tumor Characteristics." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS525.

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La mécanique et le stress compressif jouent un rôle important dans la progression tumorale. Récemment, plusieurs approches ont été développées pour tester le stress en compression dans des modèles 3D in vitro. Dans le présent travail, nous montrons d’abord la pertinence de la compression dans l’organisation des fibroblastes associés au cancer (CAF), en enveloppant les cellules cancéreuses lors d’une compression isotrope 3D dans des capsules d’alginate creux. Dans ce système, les CAF couvrent les cellules cancéreuses en présence de compression selon un processus impliquant vraisemblablement une réorganisation du dépôt de fibronectine et non un réarrangement passif des deux sphéroïdes. Dans la deuxième partie de ce travail, nous avons étudié la réaction des composants de la cavéole au stress en compression.Les cavéoles sont des invaginations de la membrane plasmique capables d'amortir la tension de la membrane, protégeant ainsi la cellule de son éclatement. Nous montrons ici comment les cavéoles réduisent leur présence lors de la compression3D à court terme et comment cette compression inhibe l'activation de STAT1 et STAT3 induite par l'interféron. De plus, les effets à long terme des contraintes de compression sur les sphéroïdes entraînent également la perte du composant cavéole EHD2, une ATPase centrale pour la stabilité des cavéoles sur la membrane. Enfin, nous avons trouvé différentes voies avec une transcription modifiée du gène après un stress compressif. Parmi eux, nous avons caractérisé l'effet de la perte decavéoline-1 sur la libération d'exosomes sous compression 3D Mechanics and compressive stress play an important role in tumor progression. Recently, several approaches have been developed to test compressive stress in 3D in vitro models. In the present work, we first show the relevance of compression in the organization of cancer associated fibroblasts (CAFs), enwrapping cancer cells upon 3D isotropic compression in capsules of hollow alginate. In this system, CAFs cover cancer cells in the presence of compression by a process which most likely involves fibronectin deposition reorganization, and not a passive rearrangement of the two spheroids. In the second part of this work, we investigated the response of caveolae components to compressive stress. Caveolae are plasma membrane invaginations which are able to buffer membrane tension, thus protecting the cell from bursting. Here, we show how caveolae reduce their presence under 3D short term compression, and how this compression inhibits interferon induced STAT1 and STAT3 activation. Moreover, long term effects of compressive stress in spheroids result also in loss of the caveolae component EHD2, acentral ATPase for caveolae stability on the membrane. Lastly, we found different pathways with altered gene transcription after compressive stress. Among them, we characterized the effect of caveolin-1 loss on the release of exosomes under 3Dcompression

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Bertacchi, Gianna <1990&gt. "Guidelines for the Management of Cultural Heritage using 3D models for the insertion of heterogeneous data." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2022. http://amsdottorato.unibo.it/10046/1/bertacchi_gianna_tesi.pdf.

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The Management of Cultural Heritage (MCH) is a very complex operation aimed at protecting the physical integrity of CH assets, while promoting their historical value and development of tourism industry. In recent years, the use of digital technologies has become an essential part of the MCH delicate process, but the use of 3D models is still limited to few academic research to date. Furthermore, very few supra-national standard guidelines regulating their use are available to date and the operator who decides to use a 3D model as a basis for management is faced with the scarcity and fragmentation of standards and guidelines. The aim of the PhD research is to develop guidelines to produce 3D models for MCH, with the purpose to efficiently entry, store and manage digital data. The here provided guidelines investigate every aspect of the process leading from data acquisition to cataloguing and archiving, processing and creation of a simplified information system for the management. In order to elaborate guidelines that could be suitable for as many typologies of CH as possible an international approach was chosen, developing the thesis in joint supervision under the University of Bologna and the Universitat Politècnica de València, and by applying the state-of-the-art technologies of acquisition, processing and use of 3D models to a variety of case studies. The investigation, by highlighting the problems inherent to the MCH, made it possible to identify the main open issues that need to be explored in future lines of research, such as the application of standards to a large number of cultural assets; the automatic classification of raw data; the processing of collected data for the creation of relations, strategies and methods for the classification, integration and optimisation of heterogeneous data. La Gestione del Patrimonio Culturale (GPC) è un’operazione molto complessa mirata alla conservazione dell’integrità fisica dei Beni Culturali e alla contemporanea divulgazione dei valori storici e fruizione del Patrimonio. Nel corso degli ultimi anni l’applicazione delle tecnologie digitali ai Beni Culturali è diventata una parte imprescindibile nella GPC, ma l’uso dei modelli 3D per la gestione è finora limitato ad alcune ricerche e applicazioni accademiche. Inoltre, ad oggi sono pochi gli standard con carattere sovranazionale che guidano gli Enti nel procedimento di creazione e uso dei modelli tridimensionali per la GPC. Il gestore che decide di utilizzare un modello 3D come base per la gestione si scontra con la scarsezza e la frammentarietà di standard e indicazioni in tale ambito. L’obiettivo della tesi è quello di elaborare linee guida per la produzione di modelli 3D per un’efficace gestione, inserimento e conservazione dei dati. Tali linee guida indagano ogni aspetto del processo che porta dall’acquisizione del dato, alla sua catalogazione e archiviazione, al suo processamento e alla creazione di un sistema informativo semplificato per la gestione. Per arrivare all’elaborazione di linee guida che si adattino a più tipologie possibili di Beni Culturali è stato scelto un approccio interdisciplinare e internazionale, sviluppando la tesi in regime di cotutela tra l’Università di Bologna e l’Universitat Politècnica de València, e applicando lo stato dell'arte delle tecnologie di acquisizione, elaborazione e utilizzo dei modelli 3D ad una varietà di casi studio. Il percorso di ricerca ha permesso di individuare le principali questioni aperte che devono essere approfondite in futuro, come l’applicazione di standard a un alto numero di Beni Culturali; la ricerca di sistemi per la classificazione automatica dei dati grezzi; l’elaborazione dei dati raccolti per la creazione di relazioni, strategie e metodi per la classificazione, l’integrazione e l’ottimizzazione di dati eterogenei. La Gestión del Patrimonio Cultural (GPC) es una operación muy compleja cuyo objetivo es la conservación de la integridad física del Patrimonio y la difusión de los valores históricos. En los últimos años, la aplicación de las tecnologías digitales se ha convertido en una parte indispensable de la GPC, pero el uso de modelos 3D para la gestión se limita a algunas investigaciones y aplicaciones académicas. Además, hasta la fecha existen pocas normas y directrices de carácter supranacional que guíen a las instituciones en el proceso de creación y uso de modelos 3D para la GPC. El objetivo de la tesis es desarrollar directrices para la producción de modelos 3D del Patrimonio Cultural con el fin de gestionar, introducir y preservar eficazmente los datos. Estas directrices investigan todos los aspectos del proceso que va desde la adquisición de datos, pasando por su catalogación y archivo, hasta su tratamiento y la creación de un sistema de información simplificado para su gestión. Se ha optado por un enfoque interdisciplinar e internacional con el fin de elaborar directrices que se adapten al mayor número posible de tipos de Bienes Culturales, desarrollando la tesis en el marco de un acuerdo de cotutela entre la Universidad de Bolonia y la Universitat Politècnica de València, y aplicando las tecnologías más avanzadas para la adquisición, procesamiento y uso de modelos 3D a una variedad de casos de estudio. La investigación ha permitido identificar las principales cuestiones abiertas que se deben explorar en futuras líneas de investigación, como la aplicación de estándares a un gran número de Bienes Culturales; la búsqueda de sistemas para la clasificación automática de los datos brutos; el tratamiento de los datos recogidos para la creación de relaciones, estrategias y métodos de clasificación, integración y optimización de datos heterogéneos.

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Eggert, Sebastian. "Design and development of an open-source technology platform for automated manufacturing and screening of 3D cell culture models: A systems engineering approach." Thesis, Queensland University of Technology, 2021. https://eprints.qut.edu.au/212977/1/Sebastian_Eggert_Thesis.pdf.

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This PhD thesis developed, engineered and applied a novel open-source technology platform for automated manufacturing and screening of 3D cell culture models. The modular approach not only enabled agile and inexpensive development of automated solutions, but also facilitated reproducible, scalable, and efficient workflows. The results indicated that such automated solutions are a much-needed tool for in vitro research applications as well as pharmaceutical studies to increase reproducibility and throughput, resulting in time, labour, and consequently cost reductions.

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Amaral, Jonatas Bussador do. "Células MCF-7 como modelo 3D no estudo de câncer de mama humano." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/42/42134/tde-21072011-134443/.

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O diferencial da cultura de células em 3-dimensões é permitir que as células explorem as 3-dimensões do espaço, aumentando assim as interações com o ambiente e entre as células. Em estudos relacionados à biologia do câncer de mama, vem ganhando espaço a utilização de esferóides para estudos que visam à compreensão da morfogênese do espaço luminal. Neste trabalho foi mostrado que as células MCF-7 reorganizam-se em estruturas tubulares e acinares. Em ambas as situações, a formação do lúmen veio acompanhada pelo estabelecimento de uma camada de células polarizadas, arranjo este muito semelhante ao encontrado em glândulas mamárias. Os resultados apresentados apontam para a existência de uma população de células na linhagem MCF-7 que não estão totalmente comprometidas ao fenótipo tumoral. Mantidos diferenciados, os esferóides de células MCF-7 apontam como um novo modelo para estudos relacionados à formação do lúmen, permitindo assim explorar o papel de diferentes vias como as relacionadas a apoptose, autofagia, diferenciação e sobrevivência celular. As a particularity, a 3D cell culture permits cells to explore the three dimensions of the space thereby increasing cell-cell interactions, as well as interaction with the environment. In studies related to breast cancer biology, spheroids are becoming widely used in the aim to comprehend luminal space morphogenesis. We showed that MCF-7 cells reorganize themselves in tubular and acinar structures. In both situations, lumen formation was accompanied by the establishment of a layer of polarized cells, an arrangement that is very similar to that of breast glands. The presented results suggest the existence of an MCF cell line population not completely committed to the tumor phenotype. When maintained as differentiated, MCF-7 cell spheroids can be a new model for studies regarding lumen formation, thereby exploring the role of diiferent pathways, such as those related to cell apoptosis, autophagy, differentiation and survival.

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Dissertations / Theses on the topic '3D culture model'

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