Дисертації з теми "In vitro platform"
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1
Harrison, Olivia Jane. "Integrated platform to assay melanoblast development in vitro." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31164.
Анотація:
Melanoblasts are the embryonic precursors of melanocytes, the pigment producing cells of the skin and hair. Melanoblasts are of key interest to developmental biologists for numerous reasons, including their ability to migrate throughout the body from a single origin in the neural crest (NC). Current methods for the study of the melanocyte lineage are limited by the heavy reliance on animal models. To challenge this, a platform of in vitro tools were designed to replace and complement current studies. A major obstacle is the transition from 2D cultures, which provide only limited behavioural information, to 3D models which are able to recapitulate the environmental conditions. 3D cultures are regularly created using tissue samples and synthetic matrices for attachment, but building a model from cell lines only has not been achieved. A co-culture model using immortalised keratinocyte (COCA) and melanoblast cell lines proved unsuitable for observing developmental processes, due to lack of movement at high cell densities, but may be practical in pigmentation research. Other methods were explored to examine melanoblast behaviour, including the use of cell derived matrices (CDMs) integrated with melanoblast cell lines, and aggregates formed by hanging drop (HD) culture. CDMs were successfully generated from the COCA line, as well as NIH3T3 fibroblasts which has been shown previously. These structures are denuded of cells to leave the deposited extracellular matrix (ECM) components intact, representative of the dermal (fibroblast) and epidermal (keratinocyte) layers of the skin. HDs were prepared from cultured melanoblast cell lines, and form tight aggregates which disseminate when plated, in a manner similar to the dissemination of cells from the NC in explant cultures. The receptor tyrosine kinase KIT and its ligand (KITL), are vital for melanoblast development. Previous study of this signalling complex has often focussed on the haematopoietic lineage and spermatogenesis, where they perform essential roles. KITL is expressed in a membrane localised form found on the surface of keratinocytes thought to promote melanoblast/melanocyte survival, and a soluble isoform found sequestered in the ECM which promotes cell migration. Cell lines expressing fluorescently tagged KIT and KITL were created to visualise their interactions using live-cell confocal imaging. Firstly, cell lines were generated to perform co-culture experiments with KIT and KITL, and we showed that these constructs are able to interact by uptake of KITL into KIT cells. Secondly, tandem fluorescent protein timers of KIT and KITL were generated which were used to observe protein kinetics. We showed that these protein timers can be manipulated using cycloheximide to block protein production, or by increasing ligand availability. These protein timers reveal that soluble KITL (sKITL) has a faster turnover than membrane bound KITL (mKITL), and that in all three proteins, there is distinct change in spatial localisation as the proteins age. Using a novel melanoblast reporter mouse, Pmel-CMN, primary mouse melanoblasts between E12.5 and E14.5 were isolated for RNA sequencing. This time period is the earliest reported for melanoblast isolation for use in gene expression analysis. We show that within this time course, there are significant changes in the RNA expression profiles, including decreasing expression of other NC cell markers, and huge increasing expression of pigmentation genes. To assess the biological relevance of using in vitro assays, cells of the immortalised melanoblast cell line, melb-a, were cultured under different conditions and examined via RNA sequencing. Results reveal differences in several areas between primary cells and those in culture, including loss of melanocyte specificity. The different tools described in this thesis provide a platform on which to study various aspects of cell behaviour, including migration, morphology and cell adhesion at both the individual cell and population levels.
2
Jeon, Jessie Sungyun. "In vitro study of cancer cell extravasation in microfluidic platform." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/87976.
Анотація:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2014.Cataloged from PDF version of thesis.
Includes bibliographical references.
Cancer metastases arise from the cancer cells that disseminate from the primary tumor, intravasate into the vascular system and eventually transmigrate across the endothelium into to a secondary site through a process of extravasation. Microfluidic systems have a major advantage in studying cancer extravasation since they can mimic aspects of the 3D in vivo situation in a controlled environment while simultaneously providing in situ imaging capabilities for visualization, thereby enabling quantification of cell-cell and cell-matrix interactions. Moreover, microfluidics enable parametric study of multiple factors in controlled and repeatable conditions. This thesis describes novel 3D microfluidic models to mimic the tumor microenvironment and vasculature during cancer cell extravasation in order to investigate the critical steps of extravasation. First, a general non-organ-specific cancer cell extravasation model is developed in which the endothelial cells that cover the walls of the microfluidic channel represent the vessel endothelium, and the entire extravasation process including tumor cell adhesion to the endothelium and subsequent transmigration can be observed. A second model is then introduced to mimic organ-specific extravasation and investigate the preference of certain types of cancer to target specific organs for metastass. The improved model was used to study the specificity of human breast cancer metastases to bone, by recreating a vascularized bone-mimicking microenvironment. The tri-culture system allowed us to study the transendothelial migration of highly metastatic breast cancer cells and to monitor their behavior within the bone-like matrix. Next, functional microvascular networks were generated in the microfluidic system through vasculogenesis with addition of mural cells and pro-angiogenic factors to better replicate the normal physiological vasculature of the remote site for metastasis. Lastly, the vasculogenesis approach was combined with the bone-mimicking model to develop a functional osteo-cell conditioned vasculature model to study physiologically relevant extravasation in a bone-like microenvironment. In addition to the quantification of extravasation rates and subsequent tumor cell migration into the model tissue, the vascular networks were characterized by measuring permeability, and immunostaining of proteins secreted by osteo-cell and mural cell markers confirmed the creation of microenvironments and the presence of multiple cell types within the matrix. This study provides novel 3D in vitro quantitative data on cancer cell extravasation and micrometastasis of breast cancer cells within a bone-mimicking microenvironment. The developed microfluidic system represents an advanced in vitro model to study complex biological phenomena such as extravasation involving functional microvascular networks under organ-specific conditions and demonstrates the potential value of microfluidic technologies to better understand cancer biology and screen for new therapeutics.
by Jessie Sungyun Jeon.
Ph. D.
3
Chen, Michelle B. (Michelle Berkeley). "Tumor cell extravasation in an in vitro microvascular network platform." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/93857.
Анотація:
Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2014.Cataloged from PDF version of thesis.
Includes bibliographical references at the end of each chapter.
A deeper understanding of the mechanisms of tumor cell extravasation is essential in creating therapies that target this crucial step in cancer metastasis. Extravasation assays exist, but with limitations; data from in vivo models are frequently inferred from low-resolution end-point assays while most in vitro platforms are limited in their physiological relevance of the tumor microenvironment. To address this need, we developed a microfluidic platform to study tumor cell extravasation from in vitro microvascular networks formed via vasculogenesis. Various techniques to yield optimal networks were assessed in order to achieve an appropriate balance between vascular growth, remodeling and stabilization. These include the application of various soluble biochemical factors and both paracrine and juxtacrine co-culture with stromal cells. We demonstrate that out of all methods attempted, paracrine non-contact co-culture with human lung fibroblasts yield the most interconnected and stable networks. Vasculatures developed exhibit tight endothelial cell-cell junctions, basement membrane deposition and physiological values of vessel permeability. Employing our assay, we demonstrate impaired endothelial barrier function and increased extravasation efficiency with inflammatory cytokine stimulation, as well as positive correlations between the metastatic potentials of tumor cells lines and their extravasation capabilities. High-resolution time-lapse microscopy reveals the highly dynamic nature of extravasation events, beginning with thin tumor cell protrusions across the endothelium followed by extrusion of the remainder of the cell body through the formation of sub nuclear sized openings in the endothelial barrier. No disruption to endothelial cell-cell junctions is discernible at 60X, or by changes in local barrier function after completion of transmigration. Using our platform, we also elucidate the extravasation patterns of different tumor cell subpopulations, including mechanically lodged cells, single arrested non-trapped cells, and tumor cell clusters. Our platform offers key advantages over existing in vitro extravasation models by enabling all of the following: (1) high temporal and spatial resolution of extravasation events, (2) the ability to perform parametric studies in a tightly controlled and high throughput microenvironment and (3) increased physiological relevance compared to 2D and 3D planar monolayer models. Findings from our platform result in a deeper understanding of tumor cell extravasation mechanisms and demonstrate our assay's potential to be employed for the discovery of factors that could inhibit this crucial step in metastasis.
by Michelle B. Chen.
S.M.
4
Kim, Jae Eung. "In Vitro Synthetic Biology Platform and Protein Engineering for Biorefinery." Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/86645.
Анотація:
In order to decrease our dependence on non-renewable petrochemical resources, it is urgently required to establish sustainable biomass-based biorefineries. Replacing fossil fuels with renewable biomass as a raw feedstock for the production of chemicals and biofuels is a main driving force of biorefinering. Almost all kinds of biomass can be converted to biochemicals, biomaterials and biofuels via continuing advances on conversion technologies. In vitro synthetic biology is an emergent biomanufacturing platform that circumvents cellular constraints so that it can implement some biotransformations better than whole-cell fermentation, which spends a fraction of energy and carbon sources for cellular duplication and side-product formation. In this work, the in vitro synthetic (enzymatic) biosystem is used to produce a future carbon-neutral transportation fuel, hydrogen, and two high-value chemicals, a sugar phosphate and a highly marketable sweetener, representing a new portfolio for new biorefineries.
Hydrogen gas is a promising future energy carrier as a transportation fuel, offering a high energy conversion efficiency via fuel cells, nearly zero pollutants produced to end users, and high mass-specific and volumetric energy densities compared to rechargeable batteries. Distributed production of cost-competitive green hydrogen from renewable biomass will be vital to the hydrogen economy. Substrate costs contribute to a major portion of the production cost for low-value bulk biocommodities, such as hydrogen. The reconstitution of 17 thermophilic enzymes enabled to construct an artificial enzymatic pathway converting all glucose units of starch, regardless of the branched and linear contents, to hydrogen gas at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. Using a biomimetic electron transport chain, a maximum volumetric productivity was increased by more than 200-fold to 90.2 mmol of H2/L/h at a high starch concentration from the original study in 2007.
In order to promote economics of biorefineries, the production of a sugar phosphate and a fourth-generation sweetener is under development. D-xylulose 5-phosphate (Xu5P), which cannot be prepared efficiently by regular fermentation due to the negatively charged and hydrophilic phosphate groups, was synthesized from D-xylose and polyphosphate via a minimized two-enzyme system using a promiscuous activity of xylulose kinase. Under the optimized condition, 32 mM Xu5P was produced from 50 mM xylose and polyphosphate, achieving a 64% conversion yield, after 36 h at 45 °C. L-arabinose, a FDA-approved zero-calorie sweetener, was produced from D-xylose via a novel enzymatic pathway consisting of xylose isomerase, L-arabinose isomerase and xylulose 4-epimerase (Xu4E). Promiscuous activity of Xu4E, a monosaccharide C4-epimerase, was discovered for the first time. Directed evolution of Xu4E enabled to increase the catalytic function of C4-epimerization on D-xylulose as a substrate by more than 29-fold from the wild-type enzyme. Together, these results demonstrate that the in vitro synthetic biosystem as a feasible biomanufacturing platform has great engineering, and can be used to convert renewable biomass resources to a spectrum of marketable products and renewable energy.
As future efforts are addressed to overcome remaining challenges, for example, decreasing enzyme production costs, prolonging enzyme lifetime, engineering biomimetic coenzymes to replace natural coenzymes, and so on. This in vitro synthetic biology platform would become a cornerstone technology for biorefinery industries and advanced biomanufacturing (Biomanufacturing 4.0).Ph. D.
5
MELLE, GIOVANNI. "Development of a Novel Platform for in vitro Electrophysiological Recording." Doctoral thesis, Università degli studi di Genova, 2020. http://hdl.handle.net/11567/1000590.
Анотація:
The accurate monitoring of cell electrical activity is of fundamental importance for pharmaceutical research and pre-clinical trials that impose to check the cardiotoxicity of all new drugs.
Traditional methods for preclinical evaluation of drug cardiotoxicity exploit animal models, which tend to be expensive, low throughput, and exhibit species-specific differences in cardiac physiology (Mercola, Colas and Willems, 2013). Alternative approaches use heterologous expression of cardiac ion channels in non-cardiac cells transfected with genetic material.
However, the use of these constructs and the inhibition of specific ionic currents alone is not predictive of cardiotoxicity.
Drug toxicity evaluation based on the human ether-à-go-go-related gene (hERG) channel, for example, leads to a high rate of false-positive cardiotoxic compounds, increasing drug attrition at the preclinical stage. Consequently, from 2013, the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative focused on experimental methods that identify cardiotoxic drugs and to improve upon prior models that have largely used alterations in the hERG potassium ion channel.
The most predictive models for drug cardiotoxicity must recapitulate the complex spatial distribution of the physiologically distinct myocytes of the intact adult human heart. However, intact human heart preparations are inherently too costly, difficult to maintain, and, hence, too low throughput to be implemented early in the drug development pipeline. For these reasons the optimization of methodologies to differentiate human induced Pluripotent Stem Cells (hiPSCs) into cardiomyocytes (CMs) enabled human CMs to be mass-produced in vitro for cardiovascular disease modeling and drug screening (Sharma, Wu and Wu, 2013). These hiPSC-CMs functionally express most of the ion channels and sarcomeric proteins found in adult human CMs and can spontaneously contract.
Recent results from the CiPA initiative have confirmed that, if utilized appropriately, the hiPSC-CM platform can serve as a reliable alternative to existing hERG assays for evaluating arrhythmogenic compounds and can sensitively detect the action potential repolarization effects associated with ion channel–blocking drugs (Millard et al., 2018).
Data on drug-induced toxicity in hiPSC-CMs have already been successfully collected by using several functional readouts, such as field potential traces using multi-electrode array (MEA) technology (Clements, 2016), action potentials via voltage-sensitive dyes (VSD) (Blinova et al., 2017) and cellular impedance (Scott et al., 2014). Despite still under discussion, scientists reached a consensus on the value of using electrophysiological data from hiPSC-CM for predicting cardiotoxicity and how it’s possible to further optimize hiPSC-CM-based in vitro assays for acute and chronic cardiotoxicity assessment. In line with CiPA, therefore, the use of hiPSC coupled with MEA technology has been selected as promising readout for these kind of experiments.
These platforms are used as an experimental model for studying the cardiac Action Potentials (APs) dynamics and for understanding some fundamental principles about the APs propagation and synchronization in healthy heart tissue. MEA technology utilizes recordings from an array of electrodes embedded in the culture surface of a well. When cardiomyocytes are grown on these surfaces, spontaneous action potentials from a cluster of cardiomyocytes, the so called functional syncytium, can be detected as fluctuations in the extracellular field potential (FP). MEA measures the change in FP as the action potential propagates through the cell monolayer relative to the recording electrode, neverthless FP in the MEA do not allows to recapitualte properly the action potential features.
It is clear, therefore, that a MEA technology itself is not enough to implement cardiotoxicity assays on hIPSCs-CMs. Under this issue, researchers spread in the world started to think about solutions to achieve a platform able to works both at the same time as a standard MEA and as a patch clamp, allowing the recording of extracellular signals as usual, with the opportunity to switch to intracellular-like signals from the cytosol.
This strong interest stimulated the development of methods for intracellular recording of action potentials. Currently, the most promising results are represented by multi-electrode arrays (MEA) decorated with 3D nanostructures that were introduced in pioneering papers (Robinson et al., 2012; Xie et al., 2012), culminating with the recent work from the group of H. Park (Abbott et al., 2017) and of F. De Angelis (Dipalo et al., 2017). In these articles, they show intracellular recordings on electrodes refined with 3D nanopillars after electroporation and laser optoporation from different kind of cells. However, the requirement of 3D nanostructures set strong limitations to the practical spreading of these techniques. Thus, despite pioneering results have been obtained exploiting laser optoporation, these technologies neither been applied to practical cases nor reached the commercial phase.
This PhD thesis introduces the concept of meta-electrodes coupled with laser optoporation for high quality intracellular signals from hiPSCs-CM. These signals can be recorded on high-density commercial CMOS-MEAs from 3Brain characterized by thousands of electrode covered by a thin film of porous Platinum without any rework of the devices, 3D nanostructures or circuitry for electroporation7. Subsequently, I attempted to translate these unique features of low invasiveness and reliability to other commercial MEA platforms, in order to develop a new tool for cardiac electrophysiological accurate recordings.
The whole thesis is organized in three main sections: a first single chapters that will go deeper in the scientific and technological background, including an explanation of the cell biology of hiPSCs-CM followed by a full overview of MEA technology and devices. Then, I will move on state-of-the-art approaches of intracellular recording, discussing many works from the scientific literature.
A second chapter will describe the main objectives of the whole work, and a last chapter with the main results of the activity. A final chapter will resume and recapitulate the conclusion of the work.
6
Rotolo, Jimmy A. "Ceramide-mediated platform generation regulates apoptosis in vitro and in vivo /." Access full-text from WCMC:, 2007. http://proquest.umi.com/pqdweb?did=1428842781&sid=10&Fmt=2&clientId=8424&RQT=309&VName=PQD.
7
Petrucci, Teresa. "Building a platform for flexible and scalable testing of genetic editors." Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1143160.
Анотація:
Cell-free systems allow to perform in-vitro transcription-translation reactions without requiring living organisms, revolutionising scientific research over the last decade.
This allows to easily synthesise a variety of molecular components for genetic editing applications without requiring expensive and time-consuming procedures such as cell culture, animal maintenance etc.
In this work, I aimed to develop a high-throughput platform for the rapid, flexible and scalable in-vitro testing of various genetic editors, such as those part of the CRISPR/Cas repertoire. I used the commercially available E. coli cell extract (MyTXTL) in combination with a fully customisable design to generate fluorescent reporters, that allow standardised testing of various CRISPR components against any predesigned target or protospacer adjacent motif (PAM) sequences.
In order to increase the scalability of this screening platform, I utilised automated liquid handling technologies (Echo 525) and explored the possibility to introduce a high throughput cloning method (BASIC assembly).
I believe that this approach will be highly valuable for the screening of CRISPR components prior than their final application in in-vivo systems, such as humans or animals.
These genetic editors could then be used in many biological and artificial systems, such as gene editing, metabolomics and genetic engineering.
8
Nordh, Nicki. "Development of a cell cultureplatform in PDMS : Microfluidic systems for in vitro productionof platelets." Thesis, Uppsala universitet, Mikrosystemteknik, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-261711.
Анотація:
To be able to effectively study blood platelets in different environments adevelopment of an in vitro model of a microfluidic system for plateletproduction was started. The purpose of this thesis was to fabricate systemsand then characterize them and visualize the flow. The system consists of twochannels, one in the middle and the other one enclosing it. They are connectedthrough pores where Megakaryocytes can protrude through and produce platelets.The designs were produced in PDMS. This was done by first transfer the designsas structures onto a silicon wafer through UV lithography. The wafer served asa mould for casting PDMS that later was bonded to glass. The systems were thenstudied with three different methods. Computer simulations, flow tests andultimately tests with cells. From the results new designs were made andfabricated. The new designs were then tested the same ways as the first ones.The systems can most probably produce platelets with some optimisation of thetest parameters. No definite results were gathered to prove plateletproduction. Different flow speeds were tested and the flow profile atdifferent flow rates was visualised. The full capability of the new designscould not be fully studied due to unforeseen debris of PDMS clogging thechannels. A few things need to be done to achieve better results and establishfor sure if this method of producing platelets is possible. This thesis is agood ground for future work to stand on.
9
Kim, Kihwan. "MULTICEULLULAR TUMOR HEMI-SPHEROID: A NOVEL IN VITRO 3D MODEL PLATFORM FOR ACCELERATED DRUG DEVELOPMENT." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1481900120946458.
10
FICULLE, ELENA. "DEVELOPMENT OF AN IN-VITRO HUMANIZED MICROFLUIDIC PLATFORM TO STUDY NEURONAL TAU AGGREGATION AND PROPAGATION." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/807615.
Анотація:
Introduzione: la patologia di Alzheimer (AD) è la causa più comune di demenza ed è caratterizzata da depositi extracellulari di -amiloide e da aggregati intracellulari chiamati grovigli neurofibrillari (NFTs) formati dalla proteina tau iperfosforilata. Poiché è stato dimostrato che il declino cognitivo dei pazienti di AD correla con la diffusione degli aggregati di tau più che con la presenza di -amiloide, è fondamentale capire i meccanismi molecolari che causano la propagazione di tau.
Studi hanno dimostrato come la diffusione temporale e spaziale degli NFTs sia costante nei vari pazienti e che sia dovuta alla propagazione attraverso il network assonale tra neuroni connessi a livello sinaptico. Ad oggi infatti, alcuni degli “early phase Clinical Trials” e degli studi si stanno concentrando sulla propagazione di tau al fine di poter prevenire la sua internalizzazione da parte delle cellule riceventi, ancora sane, sia con uso di molecole che con anticorpi.
Ci sono stati diversi studi per riprodurre in vitro un modello di network neuronale al fine di valutare in maniera appropriata delle terapie mirate prima che esse venissero testate in vivo, e la maggior parte di questi si basano sull’uso di microfluidica. Tuttavia, in questi modelli spesso sono stati utilizzati degli approcci poco fisiologici come l’over-espressione o la mutazione di tau o l’utilizzo di tags fluorescenti che possono compromettere l’affidabilità di questi sistemi. Per questo motivo è necessario lo sviluppo di una nuova metodologia che possa migliorare questo approccio e che potrà in fine essere utilizzata per un efficiente ed affidabile screening di nuove terapie per la malattia di Alzheimer.
Scopo del lavoro: il principale obiettivo di questa ricerca è stato quello di stabilire una nuova piattaforma neuronale umanizzata di microfluidica, che potesse modellare e che permettesse lo studio, dell’aggregazione e propagazione di tau in modo sia qualitativo che quantitativo.
I dispositivi di microfluidica rappresentano un’alternativa miniaturizzata per ricapitolare la propagazione di aggregati proteici come quelli di tau; infatti, essi permettono la coltura di popolazioni neuronali sinapticamente connesse ma fisicamente isolate che possono essere indotte allo sviluppo di aggregati e, conseguentemente, alla loro propagazione. Questo modello potrebbe essere ideale per testare gli effetti di potenziali terapie contro tau che vadano a contrastare la propagazione trans neuronale.
Materiali & Metodi: al fine di sviluppare un modello di propagazione nei dispositivi di microfluidica, è stato in primo luogo validato un protocollo per stimolare l’aggregazione di tau endogena in neuroni corticali di ratto (RCN) utilizzando come induttore del materiale derivato da cervelli umani di pazienti AD (hAD seed). Successivamente, usando le stesse condizioni, sono stati sviluppati diversi protocolli di microfluidica per RCN che hanno mostrato non solo aggregazione ma anche propagazione di tau utilizzando tecniche quali High Content Imaging (HCI) ed un software di immagine che è stato sviluppato internamente. Infine, è stato sviluppato una versione umanizzata e miniaturizzata di questo protocollo al fine di avere una piattaforma fisiologicamente rilevante per poter testare terapie contro tau umana.
Risultati: in questo progetto è stato sviluppato un modello cellulare umanizzato che permette lo studio della propagazione endogena di tau, da neurone a neurone, usando dispositivi di microfluidica.
Dopo una prima fase di ottimizzazione, è stato dimostrato che utilizzando hAD seed è possibile indurre in neuroni corticali piastrati in dispositivi di microfluidica, l’aggregazione della proteina tau endogena e la sua propagazione in maniera trans-neuronale in modo quantificabile.
Inoltre, questo modello è stato validato statisticamente ed è stato successivamente convertito in uno miniaturizzato per aumentare il “throughput” della piattaforma passando così da 6 a 16 unità di microfluidica contenute in una piastra.
Infine, questo metodo è stato umanizzato al fine di studiare l’aggregazione e la propagazione della forma umana di tau usando una cultura primaria di neuroni murini che esprimono solo la proteina tau umana.
E’ stato dimostrato che Anle 138b, una molecola conosciuta in letteratura per le sue capacità antiaggreganti, può bloccare l’aggregazione e il trasferimento trans-neuronale di tau, suggerendo che questo nuovo sistema possa essere usato per valutare i meccanismi di propagazione di tau e trovare nuove terapie.
Conclusioni: In questo lavoro è stato sviluppato con successo un metodo di microfluidica quantitativo e riproducibile che può modellare le taupatie sporadiche umane come la malattia di Alzheimer. Usando sia RCN che neuroni corticali hTau trattati ed indotti con hAD seed, è stata osservata e quantificata la formazione e la propagazione di aggregati endogeni di tau ed è stato testato l’effetto di una molecola inibitoria su questi due meccanismi. È stato inoltre dimostrato come questi modelli possano essere anche utilizzati per condurre studi esplorativi come quelli volti al monitoraggio dell’attività e della connettività neuronale e gli studi sul ruolo dei diversi tipi cellulari presenti in cultura, nell’aggregazione e propagazione di tau. Soprattutto, è stato dimostrato come questo modello possa essere utilizzato come piattaforma di screening per testare potenziali inibitori della propagazione di tau murina ed umana.Background: Alzheimer’s disease (AD) is the most common cause of dementia, characterized by the presence of extracellular -amyloid plaques and intracellular neurofibrillary tangles (NFTs) composed of aggregated and hyperphosphorylated tau. Since it has been shown that tau aggregates correlate with cognitive decline much better than -amyloid formations, it is important to understand how tau can spread in the brain. Moreover, the spatiotemporal spread of tau observed during clinical manifestation suggests that it propagates along the axonal network between synaptically connected neurons. On these bases, some early-phase Clinical Trials are aiming to target tau during transcellular spreading in order to prevent its internalization by recipient neurons, using both compounds and antibodies. For the experimental evaluation of candidates before in vivo studies, there have been many attempts to replicate this network in vitro, most of which used microfluidic approaches; however, these experiments have often utilized parameters that may reduce the physiological relevance of the assay, such as by overexpressing tau, using fluorescent tags, or by introducing MAPT mutations. New methods that can improve these assays are required to help the screening of efficient and effective treatments. Aim of the work: The main purpose of this research project has been to establish a humanized, in vitro neuronal microfluidic platform to recapitulate and study tau aggregation and propagation in a qualitative and quantitative way. Microfluidic devices represent a miniaturized alternative tool to recapitulate tau spreading conditions, by enabling the culture of synaptically connected, but environmentally isolated, neuronal populations that can be seeded, thereby inducing endogenous tau aggregation and subsequent propagation. This model system could be ideal for testing the effect of potential tau therapeutics that modulate transneuronal tau propagation. Material & Methods: Before developing the microfluidic propagation assay, a rat cortical neuron (RCN) aggregation assay that uses seeding-competent material from human AD brains (hAD seed) to induce endogenous aggregation was validated. Subsequently, the same conditions were used to develop a RCN microfluidic assay that can show endogenous tau aggregation, and consequent propagation, using High Content Imaging (HCI) and a proprietary interactive computer program for image quantification. Finally, using a transgenic mouse line that expresses human MAPT, a humanized and miniaturized version of the assay has been developed in order to have a physiologically relevant, medium-throughput platform to test tau therapies. Results: After a phase of optimization, it has been shown that hAD seed induces endogenous rodent tau aggregation and transneuronal propagation in a quantifiable manner in a microfluidic culture model. Moreover, this assay was statistically validated and further converted to a medium-throughput format allowing the user to handle 16 two-chamber devices simultaneously in the footprint of a standard 96 well plate. Furthermore, this assay was humanized in order to study hTau aggregation and propagation using primary neurons from a mouse model that expresses human tau only. It has been proved that Anle 138b, a literature small molecule that has been previously shown to impair protein aggregation, can block the transneuronal transfer of tau aggregates, suggesting that this novel system can be used to evaluate mechanisms of tau spreading and to find therapeutic interventions. Moreover, preliminary experiments have shown that the aggregation of endogenous tau induces not only an increase of neuronal excitability but also an activation of astrocytes which might also have a role in tau pathology. Conclusions: This work has been successfully developed a robust and quantitative microfluidic assay that can model an isolated mechanism of tau propagation. Using both RCNs and hTau mouse cortical neurons seeded with hAD seed, it was possible to quantify the formation and propagation of endogenous tau inclusions and demonstrate that a putative inhibitor of tau propagation is active in this assay. It was also shown that these models can be further employed for exploratory studies, such as monitoring the functional activity and connectivity of the neuronal cultures, as well as investigating the role of different cell types in tau aggregation and propagation. Most importantly, this manuscript exhibits the latent potential of microfluidic assays as screening platforms for the preclinical evaluation of tau propagation inhibitors.
11
Reichert, Verena Maria Charlotte. "Application of a human bone engineering platform to an in vitro and in vivo breast cancer metastasis model." Thesis, Queensland University of Technology, 2011. https://eprints.qut.edu.au/53212/1/Verena_Reichert_Thesis.pdf.
Анотація:
Breast cancer in its advanced stage has a high predilection to the skeleton. Currently, treatment options of breast cancer-related bone metastasis are restricted to only palliative therapeutic modalities. This is due to the fact that mechanisms regarding the breast cancer celI-bone colonisation as well as the interactions of breast cancer cells with the bone microenvironment are not fully understood, yet. This might be explained through a lack of appropriate in vitro and in vivo models that are currently addressing the above mentioned issue.
Hence the hypothesis that the translation of a bone tissue engineering platform could lead to improved and more physiological in vitro and in vivo model systems in order to investigate breast cancer related bone colonisation was embraced in this PhD thesis.
Therefore the first objective was to develop an in vitro model system that mimics human mineralised bone matrix to the highest possible extent to examine the specific biological question, how the human bone matrix influences breast cancer cell behaviour. Thus, primary human osteoblasts were isolated from human bone and cultured under osteogenic conditions. Upon ammonium hydroxide treatment, a cell-free intact mineralised human bone matrix was left behind. Analyses revealed a similar protein and mineral composition of the decellularised osteoblast matrix to human bone. Seeding of a panel of breast cancer cells onto the bone mimicking matrix as well as reference substrates like standard tissue culture plastic and collagen coated tissue culture plastic revealed substrate specific differences of cellular behaviour. Analyses of attachment, alignment, migration, proliferation, invasion, as well as downstream signalling pathways showed that these cellular properties were influenced through the osteoblast matrix.
The second objective of this PhD project was the development of a human ectopic bone model in NOD/SCID mice using medical grade polycaprolactone tricalcium phosphate (mPCL-TCP) scaffold. Human osteoblasts and mesenchymal stem cells were seeded onto an mPCL-TCP scaffold, fabricated using a fused deposition modelling technique. After subcutaneous implantation in conjunction with the bone morphogenetic protein 7, limited bone formation was observed due to the mechanical properties of the applied scaffold and restricted integration into the soft tissue of flank of NOD/SCID mice. Thus, a different scaffold fabrication technique was chosen using the same polymer. Electrospun tubular scaffolds were seeded with human osteoblasts, as they showed previously the highest amount of bone formation and implanted into the flanks of NOD/SCID mice. Ectopic bone formation with sufficient vascularisation could be observed. After implantation of breast cancer cells using a polyethylene glycol hydrogel in close proximity to the newly formed bone, macroscopic communication between the newly formed bone and the tumour could be observed.
Taken together, this PhD project showed that bone tissue engineering platforms could be used to develop an in vitro and in vivo model system to study cancer cell colonisation in the bone microenvironment.
12
Carletti, Angelo. "Development of a machine learning algorithm for the automatic analysis of microscopy images in an in-vitro diagnostic platform." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2021.
Анотація:
In this thesis we present the development of machine learning algorithms for single cell analysis in an in-vitro diagnostic platform for Cellply, a startup that operates in precision medicine.
We researched the state of the art of deep learning for biomedical image analysis, and we analyzed the impact that convolutional neural networks have had in object detection tasks.
Then we compared neural networks that are currently used for cell detection, and we chose the one (i.e. Stardist) that is able to perform a more efficient detection also in a crowded cells context.
We could train models using Stardist algorithm in the open-source platform ZeroCostDL4Mic, using code and GPU in Colab environment.
We trained different models, intended for distinct applications, and we evaluated them using metrics such as precision and recall. These are our results:
• a model for single channel brightfield images taken from samples of Covid patients, that guarantees a precision of about 0.98 and a recall of about 0.96
• a model for multi-channel images (i.e. a stack of multiple images, each one highlighting different contents) taken from experiments about natural killer cells, with precision and recall of about 0.81
• a model for multi-channel images taken from samples of AML (Acute Myeloid Leukemia) patients, with precision and recall of about 0.73
• a simpler model, trained to detect the main area (named "well") on which cells can be found, in order to discard what is out of this area. This model has a precision of about 1 and a recall of about 0.98.
Finally, we wrote Python code in order to read a text input file that contains the necessary information to run a specified trained model for cell detection, with certain parameters, on a given set of images of a certain experiment. The output of the code is a .csv file where measurements related to every detected “object of interest” (i.e. cells or other particles) are stored.
We also talk about future developments in this field.
13
Börnicke, Carl Jonathan [Verfasser]. "In vitro Reconstitution and Characterization of Soluble Complexes of the Salmonella Type III Secretion System Sorting Platform / Carl Jonathan Börnicke." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2020. http://d-nb.info/1227853734/34.
14
Canuto, Gisele André Baptista. "Avaliação metabolômica comparativa in vitro de fármaco candidato ao tratamento de leishmaniose." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46136/tde-14092016-090130/.
Анотація:
A leishmaniose é uma doença negligenciada provocada por parasitas do gênero Leishmania, que ocorre em mais de 90 países no mundo, afetando milhões de pessoas. A doença é tratável, mas ainda não tem cura, e o arsenal terapêutico comumente utilizado é limitado, e consiste do uso de fármacos de alta toxicidade. Sendo assim, a busca por novas terapias tem sido encorajada e o uso de compostos ativos isolados de produtos naturais, como plantas, tem se mostrado eficaz. Para uso futuro destes princípios ativos no tratamento, faz-se necessário o entendimento do seu mecanismo de ação. As ciências \"ômicas\", mais especificamente a metabolômica, que é a análise comparativa dos metabólitos alterados em um sistema biológico após intervenção externa, tem sido bastante aplicada com este objetivo. A abordagem metabolômica é multidisciplinar e as análises são realizadas com auxílio de plataformas analíticas modernas, como as técnicas de separação acopladas a espectrometria de massas (MS). Para tratamento dos dados são utilizadas ferramentas estatísticas avançadas, e as substâncias alteradas são correlacionadas com rotas metabólicas. Neste trabalho, o mecanismo de ação em Leishmania infantum da substância ativa (metildihidrodieugenol B) isolada da planta Nectandra leucantha, foi avaliado por abordagem metabolômica global, com multiplataforma de análise (cromatografia gasosa e líquida em fase reversa hifenadas, GC-MS e RPLC-MS, respectivamente). Otimizações de preparo de amostra, extração, e derivatização para GC-MS, para obter o maior número possível de metabólitos foram realizadas nas amostras em estudo. Promastigotas de L. infantum foram crescidos em meio de cultura e após 72 h foram tratados com metildihidrodieugenol B (na concentração de 58.18 µg x mL-1); passadas 48 h de tratamento, a ação enzimática foi interrompida, as células lavadas e congeladas para análise posterior. Metanol:água (1:1) e metanol 100% foram utilizados como solventes extratores para acessar os metabólitos intracelulares nas análises por GC-MS e RPLC-MS, respectivamente. Derivatização com O-metoxiamina em piridina (15 mg x mL-1) à temperatura ambiente por 90 min, seguida de sililação com BSTFA + 1% TMCS por 30 min à 40 ºC foi realizada para tornar voláteis e estáveis os metabólitos para análise por GC-MS. Os resultados de ambas plataformas de análise apontou grandes diferenças entre os grupos de amostras tratadas e não tratadas com a substância ativa, e os metabólitos alterados com significância estatística foram correlacionados com o metabolismo de Leishmania. Diferentes aminoácidos, ácidos graxos, carboidratos, glicerolipídeos e fosfoslipídeos foram encontrados, em sua maioria, diminuídos com o tratamento. Devido a complexidade do metabolismo do parasita e a grande diversidade dos metabólitos alterados, um mecanismo de ação multi-target pode ser atribuído ao dímero de fenilpropanoide. Mudanças no metabolismo da glicólise e gluconeogênese são destacadas, indicando alterações nas fontes de energia da célula. Modificações na composição lipídica da membrana plasmática do parasita também foram observadas, sugerindo variações em sua flexibilidade e fluidez. Os resultados obtidos são preliminares, mas muito importantes para dar o primeiro passo na elucidação da ação desta substância no tratamento da leishmaniose, pleiteando a busca por novas terapias.Leishmaniasis is a neglected disease caused by Leishmania parasites, which occurs in more than 90 countries worldwide, affecting millions of people. The disease is treatable but not curable, and the commonly used therapeutic arsenal is limited and consists in the use of highly toxic drugs. The search for new therapies has been encouraged and the use of active compounds, isolated from natural products, such as plants, has been proven effective. For future use of these active compounds in the treatment, it is necessary to understand its mechanism of action. \"Omics\" sciences, specifically metabolomics, which is the comparative analysis of altered metabolites in a biological system after external intervention, has been widely applied for this purpose. The metabolomic approach is multidisciplinary and the analyzes are performed using modern analytical platforms, such as separation techniques coupled to mass spectrometry (MS). Data treatment are performed with advanced statistical tools and altered metabolites are correlated with metabolic pathways. In this work, the mechanism of action, in Leishmania infantum, of an active compound (methyldehydrodieugenol B), isolated from Nectandra leucantha plant, was evaluated by global metabolomics approach with multiplatform analysis (gas and reversed phase liquid chromatography, GC-MS and RPLC-MS, respectively). Optimizations of sample preparation, extraction and derivatization for GC-MS to obtain the maximum number of metabolites in the samples under consideration were performed. L. infantum promastigotes were grown in culture medium and after 72 h they were treated with metildihidrodieugenol B (at a concentration of 58.18 µg x mL-1); after 48 h of treatment, enzyme activity was quenched, cells washed and frozen for further analysis. Methanol:water (1:1) and 100% methanol are used as solvent extractor to assess intracellular metabolites for analysis by GC-MS and RPLC-MS, respectively. Derivatization with O-methoxyamine in pyridine (15 mg x mL-1) at room temperature for 90 min, followed by silylation with BSTFA + 1% TMCS for 30 min at 40°C was performed to make volatile and stable metabolites for analysis by GC- MS. The results of both analytical platforms showed significant differences between treated and non-treated samples with the active substance, and altered metabolites with statistical significance were correlated with Leishmania metabolism. Different amino acids, fatty acids, carbohydrates, and glycerolipids phospholipids were found, mostly decreased with treatment. Due to the complexity of the parasite metabolism and the great diversity of altered metabolites, a multi-target mechanism can be assigned to the methyldehydrodieugenol B. Changes in the metabolism of glycolysis and gluconeogenesis are highlighted, indicating changes in the cell energy sources. Changes in the lipid composition of the parasite plasma membrane were also observed, suggesting variations in its flexibility and fluidity. The results are preliminary, but very important to take the first step into elucidation of the action of this substance in the treatment of leishmaniasis, aiming the search for new pharmaceutical therapies.
15
Lee, Bill. "Preclinical antimicrobial drug discovery : development and evaluation of a platform for high-throughput screening in vitro and an immunocompromised animal model." Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=100745.
Анотація:
The incidence of infections caused by antibiotic-resistant bacteria and fungi is rising rapidly. Once considered as little more than a nuisance, antibiotic resistance has become a serious threat. The mortality rate for some infections is approaching that of the pre-antibiotic era. New antimicrobials are needed urgently. Prior to the introduction of any new antimicrobial, comprehensive toxicity and efficacy profiles are assessed in preclinical studies. This thesis focuses on two key stages of preclinical antimicrobial drug development, specifically compound screening in vitro and animal efficacy testing in vivo. We developed a sensitive colorimetric platform with high-throughput capacity for the rapid screening of candidate antimicrobials. This platform could be adapted to assess compounds targeting a range of bacteria, fungi (such as Candida albicans), and protozoan parasites (such as Leishmania major). When this assay was modified to measure minimum inhibitory concentrations (MICs) for bacteria, 100% agreement within one dilution was achieved compared to the gold-standard method. A novel antifungal compound was taken forward to animal testing in an immunocompromised mouse model. We demonstrated herein that a histone deacetylase inhibitor in combination with an imidazole can synergise to produce a potent antifungal effect. A dose-dependent response, defined as a lower fungal burden and a higher survival rate, was achieved with increasing concentrations of the novel inhibitor.
16
Chung, Git Weng. "Development of an in vitro rat proximal tubule cell model as a platform for drug transporter and drug-drug interaction studies." Thesis, University of Newcastle upon Tyne, 2014. http://hdl.handle.net/10443/2755.
Анотація:
The kidney plays a vital role in the elimination of many endogenous metabolites and xenobiotics. Drug transporters expressed in the proximal tubule cells are key factors in the ability of the organ to successfully carry out its function. Previously, primary human proximal tubule cells have been shown to retain a remarkable degree of differentiation in culture and provide a realistic model of the proximal tubule. To address the challenge of extrapolation of drug transporter data from animal and human, this project was set out to develop a parallel rat proximal tubule cell model. This would allow direct comparison of the handling of candidate drugs in both species, and provide better understanding of the mechanisms of drug transport. A technique to isolate primary rat proximal tubule cells (PTCs) was successfully developed using a collagenase digest/Percoll gradient approach. Rat PTCs cultured for 6 days were shown to exhibit cobberstone morphology, typical of many epithelial cells. A range of transport proteins including Mdr1a/b, Bcrp, Mrp2, Oat1, Oct2, Oatp4c1, Slc2a9, Urat1, Mate1, and Mct1 were detected at the mRNA level in these cells. Functional expression of Mdr1a/b, Bcrp, Mrp2, Oct2 and Mct1 was also detected using fluorescence substrate retention assays. In addition, Mdr1a/b, Bcrp and Mrp2 transporters were found localised on the apical membrane of polarised rat PTC monolayer, and Oct2 was found on the basolateral membrane. The handling of urate by rat PTC monolayers was investigated. The monolayers showed absorptive and secretory pathways for urate, although the absorptive pathway was 3.2-fold higher in magnitude. Similarly, 3.4-times more urate was predominant across the apical than across the basolateral membrane. Oat1 and Bcrp were deduced as the transporters responsible for the secretory pathway, and Urat1 and Slc2a9 in the absorptive pathway. This was in accordance with the human PTC monolayers, and both models were representative of urate transport in vivo. Digoxin transport exhibited a net absorptive flux in rat PTC monolayers; absorptive flux was 1.7-fold higher in magnitude than the secretory flux. In contrast, in human PTC monolayers, digoxin secretory flux was 4.2-fold higher than the absorptive flux. In human PTC monolayers, digoxin secretion consisted of OATP4C1-mediated digoxin uptake by the basolateral membrane and MDR1-mediated efflux across the apical membrane. In rat PTC monolayers in addition to these pathways, a significant Oatp-mediated absorptive flux of digoxin located on the apical membrane of rat PTC monolayer was identified as the difference between rat and human digoxin handling, resulting in a dominant absorptive flux of digoxin in rat compared to net secretion in human PTC monolayers. These data alone highlight the importance of developing realistic in vitro human and rat PTC models to understand species difference in renal drug handling.
17
Allenby, Mark Colin, Athanasios Mantalaris, and Nicki Panoskaltsis. "Development of a bio-inspired in silico-in vitro platform: Towards personalised healthcare through optimisation of a bone-marrow mimicry bioreactor." Thesis, Imperial College London, 2017. https://eprints.qut.edu.au/199969/1/Allenby_MC_2017_PhD_Thesis.pdf.
Анотація:
Human red blood cell production, or erythropoiesis, occurs within bone marrow. Living animal and human cadaver models have demonstrated the marrow production of red blood cells is a spatially-complex process, where cells replicate, mature, and migrate between distinct niches defined by biochemical nutrient access, supportive neighboring cells, and environmental structure. Unfortunately, current research in understanding normal and abnormal human production of blood takes place in petri dishes and t-flasks as 2D liquid suspension cultures, neglecting the role of the marrow environment for blood production. The culture of blood on marrow-mimetic 3D biomaterials has been used as a laboratory model of physiological blood production, but lacks characterization. In this work, a 3D biomaterial platform is developed and to capture the in vivo blood production process and manufacture red blood cells from human umbilical cord blood. First ceramic hollow fibres were designed and tested to be incorporated and perfused in a 3D porous scaffold bioreactor to mimic marrow structure, provide a better expansion of cell numbers, a better diffusion of nutrients, and allow for the continuous, non-invasive harvest of small cells in comparison to static, unperfused biomaterials. Quantitative 3D image analysis tools were developed to spatially assess bioreactor distributions and associations of and between different cell types. Using these tools, the bioreactor distribution of red blood cell production were characterized within niches in collaboration with supportive, non-blood cell types and designed miniaturised, parallelised mini-bioreactors to further explore bioreactor capabilities. This thesis presents a hollow fibre bioreactor able to produce blood cells alongside supportive cells at 1,000-fold higher cell densities with 10-fold fewer supplemented factor than flask cultures, without serum, with one cell source, and continuously harvest enucleate red blood cell product to provide a physiologically-relevant model for cell expansion protocols.
18
BEZUKLADOVA, SVETLANA. "Development of an in vitro functional platform to discover new drugs for progressive multiple sclerosis and translation to preclinical animal models." Doctoral thesis, Università Vita-Salute San Raffaele, 2022. http://hdl.handle.net/20.500.11768/133063.
Анотація:
Multiple sclerosis (MS), an inflammatory autoimmune disease characterised by myelin and axonal damage, is the major cause of progressive neurological disability in young adults. Most MS patients have a continuous accumulation of disability throughout their lifespan leading to progressive MS (PMS), still an incurable disease. The urgent challenge is the development of novel effective therapies for progressive forms of MS, which could slow or prevent ongoing neurodegeneration by targeting neuroprotection and remyelination. One of the rational approaches to achieve this goal is drug repurposing. Drug repurposing focuses on identifying novel targets for generic medications with the indication for other diseases. In addition to drug repurposing, in silico drug prediction offers a complementary approach for the efficient selection of successful drug candidates. To better reproduce the human biological tissue, iPSCs can provide a valuable tool for in vitro disease modelling and almost an infinite cellular source with the preserved genetic background of the donor. The iPSC-derived oligodendrocyte and neuronal cultures represent a unique patient-specific “disease-in-a-dish” tool for evaluating drug candidates’ pro-myelinating and neuroprotective effects. The aim of the project is to design and conduct a comprehensive and well-characterized pharmacological screening to ultimately identify a handful of lead compounds with therapeutic potential for PMS. Therefore, in vitro phenotypic drug screening of drug repurposing library using primary murine and hiPSC-derived neuronal cultures was set up. The library of repurposed drugs in this project was selected by machine learning-based tool SPOKE in collaboration with S. Baranzini lab at UCSF. The pharmacological activity of two selected drugs was evaluated in vivo in the preclinical model of MS (EAE). Target identification and mechanism of action of hit compounds neuroprotective/remyelinating properties are currently ongoing. In addition, we have characterized the molecular profiles and functional phenotypes of hiPSC-derived neurons from three twin pairs discordant for MS that will provide additional knowledge on the potential impact of genetic and epigenetic changes on neuronal susceptibility to damage in MS. By better understanding the underlying causes of progressive nerve damage and impaired myelin regeneration, the development of novel treatments for people with PMS will be eventually accelerated.La sclerosi multipla (SM), una malattia infiammatoria autoimmune caratterizzata da alterazione della mielina e da danno assonale, è la principale causa di disabilità causata da neurodegenerazione progressiva nei giovani adulti. La maggior parte dei pazienti con SM ha un accumulo continuo di disabilità per tutta la durata della vita che porta alla SM progressiva (SMP), una malattia ancora incurabile. La sfida urgente è lo sviluppo di nuove terapie efficaci per le forme progressive di SM, che potrebbero rallentare o prevenire la neurodegenerazione in corso, favorendo meccanismi di neuroprotezione e di rimielinizzazione. Uno degli approcci per raggiungere questo obiettivo è il riposizionamento di farmaci già presenti sul mercato, e che prevede l'identificazione di nuovi bersagli per i suddetti medicinali allo scopo di trattare altre malattie. Oltre al riutilizzo dei farmaci, la predizione dei farmaci in silico offre un approccio complementare per la selezione di validi candidati farmacologici di successo. Per riprodurre meglio il tessuto biologico umano, le cellule staminali pluripotente indotte (CSPi) possono fornire uno strumento prezioso per la modellazione della malattia in vitro e una fonte cellulare quasi infinita, preservando il background genetico del donatore. Le colture di oligodendrociti e di cellule neuronali derivate da CSPi rappresentano uno strumento unico per modellare le patologie in modo personalizzato per il paziente e per valutare gli effetti pro-mielinizzanti e neuroprotettivi dei farmaci candidati. Lo scopo del progetto è condurre uno screening farmacologico completo e ben caratterizzato, per identificare in definitiva un set di composti con potenziale terapeutico per la SMP. Pertanto, è stato condotto uno screening in vitro su una libreria di farmaci candidati per riposizionamento, utilizzando colture primarie di cellule neuronali murine e da CSPi di derivazione umana. La libreria di farmaci da riposizionare in questo progetto è stata prodotta dallo strumento bioinformatico SPOKE, sviluppato dal laboratorio di S. Baranzini dell'UCSF. L'attività farmacologica di due farmaci selezionati è stata valutata in vivo nel modello preclinico di SM (EAS). Sono attualmente in corso l'identificazione del bersaglio e lo studio del meccanismo d'azione delle proprietà neuroprotettive/rimielinizzanti di questi composti. Inoltre, abbiamo caratterizzato i profili molecolari e i fenotipi funzionali dei neuroni derivati da CSPi da tre coppie gemelle discordanti per la SM al fine di ottenere ulteriori conoscenze sul potenziale impatto dei cambiamenti genetici ed epigenetici sulla suscettibilità del danno neuronale nella SM. Comprendendo meglio le cause alla base della progressione del danno nel sistema nervoso centrale (SNC) e, in particolare, della ridotta capacità di rigenerazione della mielina, lo sviluppo di nuovi trattamenti per i pazienti di sclerosi multipla progressiva sarà infine accelerato.
19
VISENTIN, CRISTINA. "Use of a technological platform to screening in vitro and in vivo anti-amyloidogenic drugs able to prevent early neurodegenerative process." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/158278.
Анотація:
Amyloidoses are protein misfolding diseases caused by deposition of fibrillar proteins in target organs. Nowadays, most of them are still incurable and their relevance to public health system is growing, especially as a consequence of population aging. Spinocerebellar ataxia type 3 is a member of this group of pathologies and its causative agent is ataxin-3 (ATX3). This is consists of a globular N-terminal (JD), followed by a flexible tail carrying a poly-glutamine (polyQ) tract. An expanded polyQ tract triggers the aggregation. In this work, I have investigated the capability of tetracycline (Tetra), epigallocatechin-gallate (EGCG), epigallocatechin (EGC), gallic acid (GA) and trifluoroethanol (TFE) to interfere with ATX3 amyloid deposition. Tetra is an antibiotic recently re-evaluated as anti- amyloidogenic compound. EGCG, EGC and GA, which are natural polyphenols, are already known in literature for their anti-amyloidogenic effect; finally, TFE is an osmolyte that stabilizes secondary structure, preferentially α-helix. Data obtained by aggregation assay, spectroscopic analyses (NMR, FTIR) and morphologic characterisation clearly demonstrated Tetra capability of increasing ATX3 aggregates solubility, without a substantial remodelling of the internal structure. Nevertheless, this antibiotic reduced the toxicity of the oligomeric species and ameliorated ataxic C. elegans phenotype. On the contrary, the analysed polyphenols were capable to interfere with ATX3 aggregation but, instead of preventing, they accelerated the aggregation rate redirecting the process towards the formation of soluble, not toxic, off-pathway aggregates. All compounds were also active against the JD in isolation, but only the polyphenols were capable to bind the monomeric form. In particular, they overlapped specific aggregation-prone regions directly involved in the fibrillation. This could explain their capability of redirecting the aggregation pathway and the different mode of action with respect to Tetra. These polyphenols showed a remarkable reduction of ATX3-mediated cytotoxicity and mitigation of ataxic phenotype in C. elegans and E. coli models. However, the compounds displayed a different efficacy, whereby EGCG was the most and GA the least effective. All data strongly support the idea that GA is the minimal functional unit of EGCG. TFE did not show the capability of preventing aggregation; in fact, even at very low concentration it promotes a faster amyloid-like aggregation. Biophysical characterization of its effect on JD aggregation, instead, provided evidence that ATX3 aggregation proceeds along a new identified pathway by which protein misfolding follows protein aggregation. In fact, TFE induces the formation of a native-like state almost indistinguishable from fully native protein, but more aggregation prone.Amyloidoses are protein misfolding diseases caused by deposition of fibrillar proteins in target organs. Nowadays, most of them are still incurable and their relevance to public health system is growing, especially as a consequence of population aging. Spinocerebellar ataxia type 3 is a member of this group of pathologies and its causative agent is ataxin-3 (ATX3). This is consists of a globular N-terminal (JD), followed by a flexible tail carrying a poly-glutamine (polyQ) tract. An expanded polyQ tract triggers the aggregation. In this work, I have investigated the capability of tetracycline (Tetra), epigallocatechin-gallate (EGCG), epigallocatechin (EGC), gallic acid (GA) and trifluoroethanol (TFE) to interfere with ATX3 amyloid deposition. Tetra is an antibiotic recently re-evaluated as anti- amyloidogenic compound. EGCG, EGC and GA, which are natural polyphenols, are already known in literature for their anti-amyloidogenic effect; finally, TFE is an osmolyte that stabilizes secondary structure, preferentially α-helix. Data obtained by aggregation assay, spectroscopic analyses (NMR, FTIR) and morphologic characterisation clearly demonstrated Tetra capability of increasing ATX3 aggregates solubility, without a substantial remodelling of the internal structure. Nevertheless, this antibiotic reduced the toxicity of the oligomeric species and ameliorated ataxic C. elegans phenotype. On the contrary, the analysed polyphenols were capable to interfere with ATX3 aggregation but, instead of preventing, they accelerated the aggregation rate redirecting the process towards the formation of soluble, not toxic, off-pathway aggregates. All compounds were also active against the JD in isolation, but only the polyphenols were capable to bind the monomeric form. In particular, they overlapped specific aggregation-prone regions directly involved in the fibrillation. This could explain their capability of redirecting the aggregation pathway and the different mode of action with respect to Tetra. These polyphenols showed a remarkable reduction of ATX3-mediated cytotoxicity and mitigation of ataxic phenotype in C. elegans and E. coli models. However, the compounds displayed a different efficacy, whereby EGCG was the most and GA the least effective. All data strongly support the idea that GA is the minimal functional unit of EGCG. TFE did not show the capability of preventing aggregation; in fact, even at very low concentration it promotes a faster amyloid-like aggregation. Biophysical characterization of its effect on JD aggregation, instead, provided evidence that ATX3 aggregation proceeds along a new identified pathway by which protein misfolding follows protein aggregation. In fact, TFE induces the formation of a native-like state almost indistinguishable from fully native protein, but more aggregation prone.
20
Billington, Sarah Faye. "Development of in-vitro human and rat proximal tubule cell models as a platform for drug transporter and drug-drug interaction studies." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/3031.
Анотація:
The kidney plays a key role in the systemic clearance of new molecular entities (NMEs). Approximately 32% of drugs exhibit significant renal elimination. It is estimated that nephrotoxicity accounts for 8 % of pre-clinical and 9% of clincal safety failures in drug development. Current pre-clinical models used to screen NMEs are poor predictors of human nephrotoxicity. The focus of my project is thus to develop predictive in-vitro rat and human primary proximal tubule cell models as a platform drug transporter and drug-drug interaction (DDI) studies. Primary human and rat proximal tubule cells (PTCs) were isolated from renal cortex using a combination of enzymatic digestion and density centrifugation. The isolation procedure was optimised to maximise cell yield and viability. Human and rat PTCs cultured on Transwell® inserts formed confluent monolayers with low paracellular permeability. Quantitative PCR showed mRNA expression of key renal transporters, OAT1, OAT3, OATP4C1, OCT2, BCRP, MATE1, MATE2-K, MDR1, MRP2, URAT1, NaPi-IIa, NaPi-IIc and PiT2, in human PTC monolayers. Orthologs of these transporters were also detected in rat PTC monolayers. The utility of human and rat PTC monolayers as predictive in-vitro models of proximal tubular drug handling were demonstrated using radiolabeled [3H]-tenofovir (TFV). Human and rat PTC monolayers exhibited a cell-to-media ratio greater than 1, which indicated uptake and accumulation of TFV across the basolateral membrane. We also observed a predominant absorptive pathway of TFV. The transporters mediating the transport of TFV were identified using a cocktail of transporter inhibitors. The basolateral uptake of TFV was mediated by OATP4C1 and OAT1. TFV had low affinities for the apical efflux transporters MRP2, MRP4, MDR1 and BCRP. The novel identification of OATP4C1 as a TFV transporter has led Gilead to develop assays for investigating OATP4C1-mediated DDIs, and the FDA to recognise OATP4C1 as a key renal transporter. The handling of radiolabelled inorganic [32P]-phosphate (Pi) by human and rat PTC monolayers was also investigated. Pi flux measurement revealed a net absorptive pathway of Pi across human and rat PTC monolayers. Uptake of Pi across the apical membrane was sodium dependent, saturable, and inhibited by parathyroid hormone, fibroblast growth-factor 23 and -klotho. Apical uptake of Pi was also inhibited by TFV in a saturable manner. This suggests that the mechanisim of TFVinduced hypophosphatemia is not via TFV-induced nephrotoxicity, but TFV inhibition of Pi reabsorption. The outcomes of this work have initiated a patient clinical trial. This finding could have a large translational impact, as over 14.9 million HIV-patients are pescribed TFV and TFV-related hypophosphatemia affects 30 % of patients. The data highlight the importance of developing holistic cell based models of the proximal tubule. The outcomes of this work demonstrate the power of translational science to have an impact on how the pharmaceutical industry operates.
21
Kinfu, Birhanu Mekuaninte [Verfasser], and Wolfgang [Akademischer Betreuer] Streit. "Function-based searches for selected phosphotransferases and establishing in vitro transcription platform for cell-free metagenomics / Birhanu Mekuaninte Kinfu ; Betreuer: Wolfgang Streit." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2018. http://d-nb.info/1171427328/34.
22
Körfer, Georgette [Verfasser], Ulrich [Akademischer Betreuer] Schwaneberg, and Lothar [Akademischer Betreuer] Elling. "Development of a flow cytometer-based in vitro compartmentalization screening platform for directed protein evolution / Georgette Dorothea Johanna Körfer ; Ulrich Schwaneberg, Lothar Elling." Aachen : Universitätsbibliothek der RWTH Aachen, 2016. http://d-nb.info/1159380074/34.
23
Wang, Eu Sheng. "Construction and molecular characterisation of an improved chloroplast transformation vector system as a versatile delivery and expression platform for in-vitro propagated Nicotiana benthamiana." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/30486/.
Анотація:
The objective of this study is to develop a versatile vector system for the delivery and expression of transgenes in the chloroplast genome of N. benthamiana. The successful advent of such a system would vastly streamline the construction process of chloroplast transformation vectors for the expression of recombinant proteins, such as vaccine candidates, in the chloroplasts of N. benthamiana. Transgenes targeted to the chloroplasts of higher plants are expected to be expressed at considerably higher levels as compared to nuclear expression, resulting in more significant accumulation of recombinant proteins. In this study, a 2-part chloroplast transformation vector system was developed and two new GFP vector prototypes, pEXPR-G and pEXPR-UG were generated for preliminary evaluation of functionality. The aadA and GFP expression cassettes of pEXPR-G and pEXPR-UG were evaluated in E. coli prior to actual delivery into N. benthamiana via particle bombardment. Particle bombardment parameters were optimised with particular emphasis on minimising excessive damage to the target tissue in order to facilitate the recovery of antibiotic resistant shoots and calli following transformation. To further evaluate the versatility of the developed system for the expression of vaccine antigens, recombinant vectors, pEXPR-HA and pEXPR-NA were constructed for the delivery of hemagglutinin (HA) and neuraminidase (NA) genes of avian influenza strain H5N1 into the chloroplast genome of N. benthamiana. Experimental results indicated that pEXPR-G and pEXPR-UG were fundamentally functional in E. coli and both the aadA and GFP expression cassettes were active, allowing the bacteria to withstand 500mg/l spectinomycin and express the transgene of interest at the protein level. Similar results were also observed in transplastomic N. benthamiana transformed with pEXPR-UG and pEXPR-NA. In essence, the developed 2-part chloroplast transformation vector system was found to be highly versatile and could be conveniently applied for the construction of transformation vectors for the delivery and expression of HA and NA in the chloroplast of N. benthamiana.
24
Kabulski, Jarod L. "Development of Au-immobilized P450 platform for exploring the effect of oligomer formation on P450-mediated metabolism for In vitro to In vivo drug metabolism predictions." Morgantown, W. Va. : [West Virginia University Libraries], 2010. http://hdl.handle.net/10450/10892.
Анотація:
Thesis (Ph. D.)--West Virginia University, 2010.Title from document title page. Document formatted into pages; contains xiv, 180 p. : ill. (some col.). Includes abstract. Includes bibliographical references.
25
Kirichuk, Oksana. "Avancées dans les études in vitro des interactions cellule-glycocalyx : développement d'une plateforme définie mécaniquement et biochimiquement." Electronic Thesis or Diss., Université Grenoble Alpes, 2023. http://www.theses.fr/2023GRALY084.
Анотація:
L'adhésion des cellules à la paroi des vaisseaux sanguins est un processus complexe. Les globules rouges doivent s'éloigner de la paroi du vaisseau pour éviter la formation de caillots, tandis que les cellules immunitaires peuvent migrer dans les tissus. Ce processus repose sur le glycocalyx, une couche de macromolécules couvrant la paroi des vaisseaux. Cependant, nous ne comprenons pas complètement comment les propriétés du glycocalyx (souplesse, épaisseur, composition des récepteurs) affectent cette régulation. Notre hypothèse est que l'adhésion sélective des cellules fait intervenir des facteurs mécaniques et biochimiques. Il est difficile d'étudier ce phénomène dans de vrais vaisseaux sanguins, c'est pourquoi mon étude s'est concentrée sur le développement d'une plateforme in vitro. Cette plateforme combine un modèle de glycocalyx avec des modèles synthétiques de globules blancs sous flux, permettant un contrôle précis des paramètres physiques et biochimiques du modèle de glycocalyx et des modèles de cellules.Le modèle de glycocalyx nouvellement développé comprend plusieurs ingrédients clés dont les propriétés sont étroitement contrôlées : une brosse d’acide hyaluronique (HA, un composant essentiel du glycocalyx endothélial) est combinée à la sélectine P (une molécule d'adhésion à la surface des cellules endothéliales qui joue un rôle essentiel dans l'orientation des leucocytes). En m'appuyant sur l'expérience précédente de mon groupe de recherche, j'ai utilisé une bicouche lipidique supportée sur une lamelle de verre (SLB) portant une monocouche de streptavidine (SAv), qui peut lier des molécules biotinylées par l'intermédiaire de liaisons biotine-SAv. Je présente ici un contrôle de la mobilité dans le plan des molécules ancrées à la bicouche lipidique fluide en utilisant le glutaraldéhyde (GTA) comme agent de réticulation pour la SAv. Des densités de greffage contrôlées de chaînes d'HA biotinylées à une extrémité et de différentes longueurs permettent de créer des brosses aux propriétés mécaniques différentes. Je présente également une nouvelle méthodologie permettant d'ajuster quantitativement la densité de greffage de molécules biotinylées plus petites, qui est utilisée ici pour contrôler la densité de greffage d'une "protéine adaptatrice" pour l'ancrage de la P-sélectine. Le nouveau modèle in vitro du glycocalyx permet ainsi de contrôler la mobilité latérale, la densité de surface et l'orientation de deux molécules fonctionnelles distinctes.Le deuxième élément clé de la nouvelle plateforme consiste en des modèles de globules blancs, développés sur la base de microbilles disponibles dans le commerce ayant la taille d'une cellule et une fonctionalisation de surface avec de la SAv. Je présente une méthodologie pour le greffage simultané de deux types de protéines à la surface des billes : CD44 biotinylé (un ligand exprimé à la surface des leucocytes, interagissant spécifiquement avec l'HA) et PSGL-1 (un ligand de la P-sélectine). En outre, je présente une méthode permettant de contrôler la densité de surface de chacune de ces protéines.J'utilise une combinaison de méthodes comme outils de quantification et de contrôle de la qualité de la formation du modèle de glycocalyx et de la fonctionnalisation des billes : microbalance à quartz avec mesure de dissipation (QCM-D) ; ellipsométrie spectroscopique (SE), microscopie à contraste interférentiel par réflexion (RICM) ; microscopie confocale avec redistribution de fluorescence après photoblanchiment (FRAP), et cytométrie en flux.Cette plateforme nouvellement établie offre des conditions contrôlées pour l'étude de l'adhésion des cellules sanguines, reliant les interactions chimiques cellule-glycocalyx et les aspects mécaniques de la migration cellulaire sous flux. Elle est facilement complexifiable ou adaptable, permettant une compréhension de plus en plus fine de l'adhésion des cellules aux vaisseaux sanguinsCell adhesion to the blood vessel wall is a complex, highly regulated physiological process. Red blood cells must repel from the blood vessel wall to prevent blood clotting while immune cells can be recruited from the vascular system to migrate into surrounding tissues. Cell adhesion hinges on the critical role played by the glycocalyx, a soft gel-like layer coating the vascular wall. However, how glycocalyx mechanical (softness, thickness) and biochemical (the composition and the density of surface receptors) properties affect this regulation is still poorly understood. Our hypothesis is that selective cell adhesion requires an intricate interplay of mechanical and biochemical cues. Elucidating the physical and molecular mechanisms that underpin selective adhesion directly in real blood vessels is challenging owing to the complexity and lack of control in in vivo systems. In my research, I aimed to construct an in vitro molecular interaction platform to facilitate mechanistic analyses. The platform combines a molecularly-defined model of the glycocalyx with mimetics of white blood cells under flow. While developing such a platform posed challenges, it offers the advantage of precise control over the physical and biochemical parameters of both the glycocalyx mimetic and cell mimetics.The newly developed glycocalyx model includes several key ingredients with tightly controlled properties: a brush of hyaluronan (HA, an essential component of the endothelial glycocalyx) is combined with P-selectin (an adhesion molecule on the endothelial cell surface critical for the homing of leukocytes). Building on previous experience in my research group, I employed a silica-supported lipid bilayer (SLB) bearing a monolayer of streptavidin (SAv), that can bind biotinylated molecules via biotin-SAv bonds. I introduce here a control of the in-plane mobility of molecules anchored to the fluid lipid bilayer using glutaraldehyde (GTA) as a cross-linking agent for SAv. Controlled grafting densities of one-end biotinylated HA chains of various lengths then create brushes of different mechanical properties. I also present a new methodology for quantitatively tuning the grafting density of smaller biotinylated molecules, which is deployed here to control the grafting density of an ‘adapter protein’ for anchoring P-selectin. The new in vitro model of the glycocalyx thus affords control over the lateral mobility, the surface density and the orientation of two distinct functional molecules.The second key component of the newly developed platform consists of white blood cell mimetics, developed based on commercially available microbeads with the size of a cell and a SAv coating. I introduce a methodology for simultaneous grafting of two types of proteins onto the bead surface: biotinylated CD44 (a ligand expressed on leukocyte surfaces, interacting specifically with HA) and PSGL-1 (a ligand of P-selectin). Additionally, I present a method for controlling the surface density of each of these proteins.I use a combination of methods as monitoring and quality control tools of glycocalyx model formation and bead functionalization: quartz crystal microbalance with dissipation monitoring (QCM-D); spectroscopic ellipsometry (SE), reflection interference contrast microscopy (RICM); confocal microscopy with fluorescence recovery after photobleaching (FRAP) capabilities, and flow cytometry.This newly established platform provides a controlled environment for studying blood cell adhesion, effectively bridging the divide between cell-glycocalyx chemical interactions and the mechanical aspects of cell migration under flow, including attachment and repulsion from the vascular wall. This platform holds the potential for expansion to encompass other surface adhesion molecules or to integrate multiple adhesion molecules, to gradually advance from the bottom up our understanding of the mechanisms governing cell adhesion to blood vessels
26
Guan, Shan [Verfasser], and Carsten [Akademischer Betreuer] Rudolph. "Peptide-based platform enabling amphiphilic block copolymers to acquire in vitro transfection ability and more potent in vivo lung gene transfer for cystic fibrosis / Shan Guan ; Betreuer: Carsten Rudolph." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/1207270016/34.
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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.
Анотація:
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.
28
Júnior, Mário Roberto Moraes. "Formação in vitro de biofilme de Candida albicans em materiais usados no preenchimento dos acessos aos parafusos das próteses sobre implantes." Universidade do Estado do Rio de Janeiro, 2012. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=6132.
Анотація:
Os orifícios de acesso aos parafusos de retenção devem ser preenchidos para que o parafuso não seja danificado caso seja necessária a remoção da prótese. Dentre os materiais mais utilizados estão o algodão, a fita de politetrafluoretileno e a guta percha. O objetivo deste estudo é avaliar a formação de biofilme de Candida albicans nos materiais anteriormente descritos, buscando estabelecer um parâmetro que contribua para a escolha do tipo de material mais adequado a ser utilizado clinicamente. Foram utilizados UCLAs, análogos e parafusos sextavados, todos de titânio. Os conjuntos foram montados com torque de 32N. Os materiais foram condensados no interior dos UCLAs e colocados em meio de cultura com uma suspensão de 3x106 células/ml de Candida albicans. O sistema foi armazenado à 37C com agitação, por 15 dias e o meio foi renovado a cada 48 horas. A quantificação de biofilme foi realizada pelo ensaio de MTT e leitura à 490nm, resultando em diferentes valores de densidade óptica. A normalidade (p=0,304 - Kolmogorov-Smirnov) e a igualdade de variâncias (p=0,721 - Scheffe) foram testadas primeiramente. O teste de análise de variância demonstrou diferença significativa entre os grupos (p<0,001) e com o Holm-Sidak foi observada diferença significativa entre os grupos algodão e guta (p<0,05) e algodão e fita de politetrafluoetileno (p<0,05); não houve diferença significativa entre os grupos guta e fita de politatrafluoretileno (p>0,05), apesar dos valores da fita de politetrafluoetileno terem sido maiores. Considerando-se as limitações deste estudo in vitro, podemos concluir que tanto a guta-percha quanto a fita de politetrafluoretileno apresentaram menor formação de biofilme, não havendo diferença estatisticamente significativa entre os materiais. O algodão apresentou um nível de formação de biofilme significativamente maior que a fita de politetrafluoretileno e a guta percha. Diante disso, serão necessários novos estudos para confirmar as limitações que este tipo de material pode apresentar quando usado como material de preenchimento do acesso do parafuso da prótese sobre implante.The access holes for fixing screws in prostheses on implants must be completed so that the screw is not damage if is necessary to remove the prostheses. Among the most common materials used are cotton, gutta percha and polytetrafuoroetylene tape. The aim of this study is to evaluate the biofilm formation in the materials described above, in order to establish a parameter that contributes to the choice of most suitable material. We used UCLAs, similar (4,1 mm) and titanium screws. The set was assembled with a torque of 32 N.cm. The materials were condensed inside the UCLAs and placed in culture medium with suspension of 3x 106 cells/ml of Candida albicans. The system was stored at 37 o C with shaking for 15 days and renewed every 48 hours. The quantification of biofilm was performed by MTT assay at 490 nm and reading. Analyzed the normal (p=0,304-Kolmogorov-Smirnov) and equal variances (p= 0,721- Scheffe). The ANOVA showed significant difference between groups (p<0,001) and Holm- Sidak significant difference was observed between the cotton and gutta groups (p<0,05) and cotton and PTFE (p<0,05), not significant difference between groups gutta and polytetrafuoroetylene tape (p>0,05), although values were higher polytetrafuoroetylene tape. Considering the limitations of this in vitro study, we conclude that:1. Both gutta-percha and polytetrafluorethylene tape showed less biofilm formation, with no statistically significant difference between the materials. 2. Cotton showed a level of biofilm formation significantly higher than the polytetrafluoroethylene tape and gutta percha. Therefore, further studies are necessary to confirm the limitations that this type of material can have when used as filling material of the screw access on the prosthetic implant.
29
Madiedo-Podvršan, Sabrina. "Development of a lung-liver in vitro coculture model for the risk assessment of inhaled xenobiotics." Electronic Thesis or Diss., Compiègne, 2022. http://www.theses.fr/2022COMP2703.
Анотація:
L’urbanisation et la mondialisation sont des phénomènes de société qui multiplient et complexifient les sources de pollution. Parmi elles, la pollution atmosphérique impacte notablement la santé humaine à l’échelle mondiale de par son caractère transfrontière. L’appareil respiratoire est une voie d’absorption de nombreux xénobiotiques, sous forme de gaz, d’aérosols ou de nanoparticules. Une fois dans les voies respiratoires, les substances inhalées sont susceptibles d’interagir avec les cellules pulmonaires. Les mécanismes par lesquels des xénobiotiques inhalés induisent des dommages pulmonaires sont complexes, notamment en raison de l’hétérogénéité cellulaire des poumons. En raison de cette complexité, les modèles animaux constituent un outil de référence pour les études toxicologiques prédictives, cependant, dans le contexte européen de réduction de l’expérimentation animale (REACH, et les règles 3R), le développement de méthodes alternatives fiables est devenu une nécessité. Les modèles in vitro sont de bons candidats car plus simple et moins couteux à mettre en oeuvre que les modèles vivo et permettent de travailler avec des cellules ou des tissus d’origine humaine ce qui contribue à améliorer la pertinence des résultats. Cependant, l’extrapolation limitée du vitro au vivo est souvent liée à un manque de complexité des modèles, notamment en raison de l’absence de communication inter-organes. Les technologies des multi-organes sur puce cherchent à surmonter ces limitations en connectant plusieurs organoïdes métaboliquement actifs au sein d’un même circuit de culture afin de reproduire des interactions de type systémiques. Dans ce contexte, nous décrivons un modèle permettant de connecter in vitro, par le biais de la microfluidique, une barrière pulmonaire (voie d’entrée des xénobiotiques inhalés) à un organe détoxifiant tel que le foie, afin d’évaluer la toxicité liée à un stress inhalatoire de façon plus systémique. Cette approche permet de considérer la biotransformation des composés inhalés et l’interaction inter-organes comme possible modulateurs de la toxicité. Le projet étant dans les premières phase de développement, la robustesse expérimentale était au coeur du projet. L’objectif principal était de prouver qu’une substance modèle était capable de transiter dans le dispositif, au travers des deux compartiments tissulaires, afin de pouvoir étudier la dynamique inter-organes poumon/foie en condition de stress xénobiotique. Le projet a été articulé en trois phases expérimentales : - Caractérisation des réponses biologiques spécifiques aux tissus pulmonaire et hépatique en réponse à un stress. La viabilité, la fonctionnalité et les activités métaboliques des monocultures ont été évaluées après exposition à une substance modèle. - Adaptation et préparation des monocultures aux conditions de co-culture afin de préserver la viabilité et la fonctionnalité des tissus. - Les compartiments pulmonaire et hépatique ont été cultivés jointement dans un circuit de culture microfluidique fermé. La co-culture a été exposée à une substance modèle à travers la barrière pulmonaire afin d’imiter un mode d’exposition inhalatoire. Les paramètres de viabilité et de fonctionnalité des tissus ont été évalué post-culture afin de mettre en évidence quelconque phénomène d’interaction inter-organe. La caractérisation du modèle de co-culture a été réalisé grâce à l’exposition d’un agent hépatotoxique de référence, largement étudié dans la littérature : l’acétaminophène aussi connu sous le nom de paracétamol (APAP). L’exposition à la barrière pulmonaire n’est pas physiologique mais permet d’observer quantitativement le passage et la circulation du xénobiotique à travers le dispositif car l’APAP interfère avec la viabilité et les performances métaboliques hépatique, permettant ainsi de vérifier que le compartiment hépatique peut avoir accès à l’exposition effectuée à travers la barrière pulmonaireUrbanization and globalization are prevailing social phenomena that multiply and complexify the sources of modern pollution. Amongst others, air pollution has been recognized as an omnipresent life-threatening hazard, comprising a wide range of toxic airborne xenobiotics that expose man to acute and chronic threats. The defense mechanisms involved in hazardous exposure responses are complex and comprise local and systemic biological pathways. Due to this complexity, animal models are considered prime study models. However, in light of animal experimentation reduction (3Rs), we developed and investigated an alternative in vitro method to study systemic-like responses to inhalationlike exposures. In this context, a coculture platform was established to emulate interorgan crosstalks between the pulmonary barrier, which constitutes the route of entry of inhaled compounds, and the liver, which plays a major role in xenobiotic metabolism. Both compartments respectively comprised a Calu-3 insert and a HepG2/C3A biochip which were jointly cultured in a dynamically-stimulated environment for 72 hours. The present model was characterized using acetaminophen (APAP), a well-documented hepatotoxicant, to visibly assess the passage and circulation of a xenobiotic through the device. Two kinds of models were developed: (1) the developmental model allowed for the technical setup of the coculture, and (2) the physiological-like model better approximates a vivo environment. Based on viability, and functionality parameters the developmental model showed that the Calu-3 bronchial barrier and the HepG2/C3A biochip can successfully be maintained viable and function in a dynamic coculture setting for 3 days. In a stress-induced environment, present results reported that the coculture model emulated active and functional in vitro crosstalk that seemingly was responsive to high (1.5 and 3 mM) and low (12 and 24 μM) xenobiotic exposure doses. Lung/liver crosstalk induced modulation of stress response dynamics, delaying cytotoxicity, proving that APAP fate, biological behaviors and cellular stress responses were modulated in a broader systemic-like environment
30
Frederico, Éric Heleno Freire Ferreira. "Influência de um extrato aquoso de Coriandrum sativum na marcação in vitro de constituintes sanguíneos com tecnécio-99m e de sua associação com vibração gerada por plataforma oscilante na biodistribuição do radiofármaco Na99mTcO4 e na concentração de biomarcadores em ratos Wistar." Universidade do Estado do Rio de Janeiro, 2014. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=7550.
Анотація:
Coriandrum sativum, conhecido popularmente como coentro, é um vegetal usado na alimentação humana. Também é utilizado como planta medicinal para tratamento de diabetes, complicações gastrintestinais, e como um antiedêmico, antisséptico e emenagogo. Em investigações acerca dos efeitos do extrato de plantas, é importante a determinação de alguns parâmetros físico-químicos. Diversos modelos experimentais têm sido usados, inclusive com o emprego de radionuclídeos. Em procedimentos da Medicina Nuclear que auxiliam o diagnóstico de doenças, o tecnécio-99m (99mTc) é o radionuclídeo mais utilizado. Hemácias marcadas com 99mTc estão entre as várias estruturas celulares que podem ser marcadas com este radionuclídeo e usadas como radiofármaco. Para a marcação com 99mTc é necessária a presença de um agente redutor, e o mais utilizado é o cloreto estanoso (SnCl2). As terapias com drogas e condições de dieta além de doenças podem alterar a marcação de constituintes sanguíneos, bem como a biodistribuição de diferentes radiofármacos. A exposição às vibrações geradas por plataforma oscilatória produz exercícios de corpo inteiro. O objetivo deste estudo foi caracterizar a preparação de um extrato do Coriandrum sativum, através de parâmetros físico-químicos, verificar os efeitos desse produto natural na radiomarcação de constituintes sanguíneos e em associação à vibração gerada pela plataforma na biodistribuição de Na99mTcO4 e na concentração de alguns biomarcadores. O extrato de coentro teve a o pico de absorbância em 480 nm. O extrato de coentro foi inversamente correlacionado com a concentração na condutividade elétrica. Foi encontrado o maior valor de pH na menor concentração do extrato (0,5 mg/mL). Não houve uma alteração significativa na marcação de constituintes sanguíneos com 99mTc. E a associação do extrato de coentro e vibração gerada por plataforma com frequência de 12 Hz teve efeito no baço, como observado na fixação do radiofármaco nesse órgão e ação em alguns órgãos alternando a concentração de alguns biomarcadores. Em conclusão, parâmetros físico-químicos podem ser úteis para caracterizar o extrato estudado. Provavelmente, as propriedades redox associadas com substâncias desse extrato podem ser os responsáveis pela ausência do efeito na radiomarcação de constituintes sanguíneos. A determinação da captação do Na99mTcO4 em diferentes órgãos permite verificar que o extrato de coentro sozinho não foi capaz de interferir na biodistribuição do radiofármaco. Contudo o tratamento de animais com vibração gerada pela plataforma alterou significativamente a fixação do pertecnetato de sódio no baço e a concentração do colesterol, triglicerídeo, CK e bilirrubina.Coriandrum sativum, popularly known as coriander, is a vegetable used in human alimentation. It is also used as a medicinal plant for the treatment of diabetes, gastrointestinal complications, and as an antiedemic, antiseptic and emenagogue. In the investigations about the effects of a plant extract, it is important to determine some of its physicochemical parameters. Several experimental models have been used, including the use of radionuclides. In Nuclear Medicine procedures that assist the diagnosis of diseases, technetium-99m (99mTc) is the most used radionuclide. Red blood cells labeled with 99mTc are among the various cellular structures that can be labeled with this radionuclide and used as a radiopharmaceutical. The labeling process involving 99mTc requires the presence of a reducing agent and the most used for this purpose is the stannous chloride (SnCl2). The drugs therapy, diet conditions and diseases can alter the labeling of blood constituents, as well the biodistribution of several radiopharmaceuticals. The exposure to vibrations generated in oscillating platform produces whole body vibration (WBV) exercise. The aim of this study was to characterize the preparation of an extract of Coriandrum sativum, through physicochemical parameters, verify the effects of this natural product in radiolabeling of blood constituents and in association with vibration generated by platform on the Na99mTcO4 biodistribution, and in concentrations of some biomarkers. The extract of coriander had a pick absorbance at 480 nm. The coriander extract was inversely correlated with the concentration in electric conductivity. Was founded the highest value of pH at the lower concentration of the extract (0.5 mg/mL). There was no significant alteration on the labeling of blood constituents with 99mTc. The association between the coriander extract and vibration generated in platform with frequency of 12 Hz had effect in spleen, as observed in the fixation of the radiopharmaceutical in the organ and action in some organs altering the concentration of some biomarkers. In conclusion, physicochemical parameters can be useful to characterize the studied extract. Probably, the redox properties associated with the substances of this extract could be responsible by the absence of effect on the radiolabeling of blood constituents. The determination of the uptake of the radiopharmaceutical sodium pertechnetate in different organs permits to verify that extract of coriander alone was not capable in interfering on the biodistribution of the radiopharmaceutical. However, the treatment of the animals with vibration generated in the platform alters significantly the fixation of the sodium pertechnetate in the spleen and the concentrations of Cholesterol, triglyceride, CK and bilirubin.
31
Viray, Christina Marie. "Developing Innovative Bioengineering Platforms to Recapitulate Cell Microenvironments In Vitro." Thesis, The University of Sydney, 2021. https://hdl.handle.net/2123/24955.
Анотація:
Bioprinting has recently emerged as a promising fabrication technique for developing 3D biologically functional, tissue-like constructs. Amongst the various bioprinting approaches, stereolithography (SLA) offers the highest feature resolutions that can be utilised to mimic the complex hierarchical architectures found within human tissues and organs. While significant efforts have been directed towards harnessing digital light processing (DLP) SLA techniques, less attention has been given towards the use of laser-based SLA and the corresponding development of cytocompatible bioresins for such setups. In this work, a novel hydrogel material system to be used as a bioresin was developed based on the biosynthetic polypeptide poly(L-glutamic acid) functionalised with tyramine moieties (PLGA-Tyr), and crosslinked using a visible light photoinitiator system. The gelation ability, swelling characteristics, degradation profiles, mechanical properties, and encapsulated cell viability were elucidated by varying the concentrations of PLGA-Tyr and the co-photoinitiator.
This work also introduces a custom-built, cost-effective, single-photon laser SLA bioprinting system named the ‘MicroNC’. Methods to improve the diversity of scaffold geometries to be printed, feature resolution, and shape fidelity were systematically investigated using commonly used bioresins, achieving feature resolutions ranging between 8 to 20 µm. Most notably, it was shown that this bioprinting system could fabricate well-resolved hydrogel filaments (less than 8 µm in width) using the newly developed visible light hydrogel material system, which were capable of manipulating 2D single-cell morphology whilst supporting high cell viability (>90%) and proliferation up to 14 days. Overall, these experiments have underscored the exciting potential of using the visible light photoinitiated PLGA-Tyr material system for developing physiologically relevant in vitro hydrogel scaffolds for use in a wide range of biomedical applications such as tissue engineering, drug screening, disease modelling, and fundamental mechanobiological studies.
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Recha, Sancho Lourdes Georgina. "Development of biomaterial self-assembling based platforms to obtain human cartilage tissue in vitro." Doctoral thesis, Universitat Ramon Llull, 2016. http://hdl.handle.net/10803/394009.
Анотація:
El cartílag articular té una capacitat limitada de creixement i regeneració i, els tractaments per restaurar la funció del teixit, després d’una lesió, són limitats i poc entesos per la comunitat mèdica. Existeix, per tant, un gran interès en trobar una solució pràctica i agradable pel pacient que aconsegueixi la reparació del cartílag. La enginyeria de teixits va sorgir per restablir teixits danyats usant noves plataformes terapèutiques basades en cèl·lules i/o biomaterials. Aquestes noves teràpies pretenen crear estructures similars al cartílag que imiten les propietats mecàniques i biològiques que trobem in vivo. En aquest context, l’ús de matrius biomimètiques que reprodueixin estructural i funcionalment el microambient natiu han despertat gran interès en aquest camp. Els pèptids auto-ensamblants representen candidats ideals per crear nínxols cel·lulars, ja que les seves nanofibres i propietats biomecàniques son similars a les de la matriu extracel·lular.
En aquesta tesi, s’ha desenvolupat nous biomaterials sintètics amb gran potencial per la reparació de cartílag. Aquests estan basats en el pèptid auto-ensamblant RAD16-I decorat amb motius bioactius, amb l’objectiu de reproduir la matriu del cartílag. Donada la versatilitat del hidrogel RAD16-I, les noves matrius es van formar per simple mescla del pèptid RAD16-I amb molècules d’heparina, condroitin sulfat i decorina. Aquestes matrius bi-composades presenten bona estabilitat química i estructural a pH fisiològic i son capaces d’unir i alliberar, gradualment, factors de creixement. L’avaluació d’aquestes matrius es va dur a terme mitjançant dues estratègies in vitro diferents: la rediferenciació de condròcits articulars humans i la inducció del llinatge condrogènic en cèl·lules mare derivades de teixit adipós. Ambdós tipus cel·lulars son considerats una bona font cel·lular per obtenir constructes que reparin defectes al cartílag. Els resultats presentats en aquest treball mostren diferencies a nivell de comportament cel·lular, patrons d’expressió i propietats mecàniques entre els dos tipus cel·lulars i les diferents condicions de cultiu (matrius i medis). Cal destacar que els dos tipus cel·lulars es diferencien a un llinatge condrogènic en medi d’inducció i que els constructes presenten propietats mecàniques compatibles amb un sistema condrogènic. A més s’ha determinat que la presencia de molècules d’heparina a la matriu promou la supervivència de les cèl·lules mare derivades de teixit adipós. En conjunt, les noves matrius bi-composades representen un material fàcil de preparar i prometedor per promoure la diferenciació condrogènica.
Finalment, part d’aquesta tesi s’ha centrat en el desenvolupament d’una nova matriu composta mitjançant la infiltració del pèptid RAD16-I amb cèl·lules en microfibres de policaprolactona (PCL). S’ha demostrat que aquesta nova combinació ofereix una estructura funcional i biomimètica, ja que proporciona suport mecànic per les fibres de PCL i a la vegada, facilita l’adhesió i el creixement cel·lular per l’hidrogel RAD16-I. El cultiu in vitro de condròcits humans desdiferenciats demostra que la nova matriu composada promou la supervivència cel·lular i el restabliment del llinatge condrogènic. En general, les propietats sinèrgiques de la nova matriu composada proporcionen una plataforma terapèutica ideal per ajudar a la reparació del cartílag.El cartílago articular tiene una capacidad limitada de crecimiento y regeneración y, los tratamientos para restaurar la función del tejido, después de una lesión, son limitados y poco entendidos por la comunidad médica. Existe, por tanto, un gran interés en encontrar una solución práctica y agradable para el paciente que consiga la reparación del cartílago. La ingeniería de tejidos surgió para restaurar tejidos dañados usando nuevas plataformas terapéuticas basadas en células y/o biomateriales. Estas nuevas terapias pretenden crear estructuras similares al cartílago que imiten las propiedades mecánicas y biológicas que se dan in vivo. En este sentido, el uso de matrices biomiméticas que reproduzcan estructural y funcionalmente el microambiente nativo ha generado gran interés en este campo. Los péptidos auto-ensamblantes representan candidatos ideales para crear nichos celulares dado que, sus nanofibras y propiedades biomecánicas son similares a las de la matriz extracelular. En esta tesis, se han desarrollado nuevos biomateriales sintéticos con gran potencial para la reparación de cartílago. Éstos, están basados en el péptido auto-ensamblante RAD16-I decorado con motivos bioactivos, tratando de reproducir la matriz del cartílago. Dada la versatilidad del hidrogel RAD16-I, las nuevas matrices se formaron por simple mezcla del péptido RAD16-I con moléculas de heparina, condroitin sulfato y decorina. Estas matrices bi-compuestas presentan buena estabilidad química y estructural a pH fisiológico y son capaces de unir y liberar, gradualmente, factores de crecimiento. La evaluación de estas matrices se llevó a cabo mediante dos estrategias in vitro diferentes: la rediferenciación de condrocitos articulares humanos y, la inducción del linaje condrogénico en células madre derivadas de tejido adiposo. Ambos tipos celulares son considerados una buena fuente de células para obtener constructos que reparen defectos en el cartílago. Los resultados presentados en este trabajo muestran diferencias a nivel de comportamiento celular, patrones de expresión y propiedades mecánicas entre los dos tipos celulares y las diferentes condiciones de cultivo (matrices y medios). Cabe destacar que, ambos tipos celulares se diferencian a un linaje condrogénico en medio de inducción y que los constructos presentan propiedades mecánicas compatibles con un sistema condrogénico. Además, se ha determinado que la presencia de moléculas de heparina en la matriz promueve la supervivencia de las células madre derivadas de tejido adiposo. En conjunto, las nuevas matrices bi-compuestas representan un material fácil de preparar y prometedor para promover la diferenciación condrogénica. Por último, parte de esta tesis se ha centrado en el desarrollo de una nueva matriz compuesta mediante la infiltración del péptido RAD16-I con células en microfibras de policaprolactona (PCL). Se ha demostrado que esta nueva combinación ofrece una estructura funcional y biomimética, dado que, proporciona soporte mecánico por las fibras PCL y a su vez, facilita la adhesión y el crecimiento celular debido al hidrogel RAD16-I. El cultivo in vitro de condrocitos humanos desdiferenciados demuestra que la nueva matriz compuesta promueve la supervivencia celular y el restablecimiento del linaje condrogénico. En general, las propiedades sinérgicas de la nueva matriz compuesta proporcionan una plataforma terapéutica ideal para ayudar a la reparación del cartílago.
Adult articular cartilage has a limited capacity for growth and regeneration and, after injury, treatments to restore tissue function remain poorly understood by the medical community. Therefore, there is currently great interest in finding practical and patient-friendly strategies for cartilage repair. Tissue engineering has emerged to restore damaged tissue by using new cellular or biomaterial-based therapeutic platforms. These approaches aim to produce cartilage-like structures that reproduce the complex mechanical and biological properties found in vivo. To this end, the use of biomimetic scaffolds that recreate structurally and functionally the native cell microenvironment has become of increasing interest in the field. Self-assembling peptides are attractive candidates to create artificial cellular niches, because their nanoscale network and biomechanical properties are similar to those of the natural extracellular matrix (ECM). In the present thesis, new composite synthetic biomaterials were developed for cartilage tissue engineering (CTE). They were based on the non-instructive self-assembling peptide RAD16-I and decorated with bioactive motifs, aiming to emulate the native cartilage ECM. We employed a simple mixture of the self-assembling peptide RAD16-I with either heparin, chondroitin sulfate or decorin molecules, taking advantage of the versatility of RAD16-I. The bi-component scaffolds presented good structural and chemical stability at a physiological pH and the capacity to bind and gradually release growth factors. Then, these composite scaffolds were characterized using two different in vitro assessments: re-differentiation of human articular chondrocytes (ACs) and induction of human adipose derived stem cells (ADSCs) to a chondrogenic commitment. Both native chondrocytes and adult mesenchymal stem cells (MSCs), either bone marrow or adipose-tissue derived, are considered good cell sources for CTE applications. The results presented in this work revealed differences in cellular behavior, expression patterns and mechanical properties between cell types and culture conditions (scaffolds and media). Remarkably, both cell types underwent into chondrogenic commitment under inductive media conditions and 3D constructs presented mechanical properties compatible to a system undergoing chondrogenesis. Interestingly, as a consequence of the presence of heparin moieties in the scaffold cell survival of ADSCs was enhanced. Altogether, the new bi-component scaffolds represent a promising "easy to prepare" material for promoting chondrogenic differentiation. Finally, part of this thesis was focus on developing a composite scaffold by infiltrating a three-dimensional (3D) woven microfiber poly (ε-caprolactone) (PCL) scaffold with the RAD16-I self-assembling peptide and cells. This new combination resulted into a multi-scale functional and biomimetic tissue-engineered structure providing mechanical support by PCL scaffold and facilitating cell attachment and growth by RAD16-I hydrogel. The in vitro 3D culture of dedifferentiated human ACs evidenced that the new composite supports cell survival and promotes the reestablishment of the chondrogenic lineage commitment. Overall, the synergistic properties of the novel composite scaffold may provide an ideal therapeutic platform to assist cartilage repair.
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Rohde, Rosemary Dyane Blake Geoffrey A. Heath James R. "Developing high-affinity protein capture agents and nanotechnology-based platforms for in vitro diagnostics /." Diss., Pasadena, Calif. : California Institute of Technology, 2009. http://resolver.caltech.edu/CaltechETD:etd-06032009-230415.
34
Cimetta, Elisa. "Design and development of microscale technologies and microfluidic platforms for the in vitro culture of stem cell." Doctoral thesis, Università degli studi di Padova, 2009. http://hdl.handle.net/11577/3426444.
Анотація:
The impelling needs related to the processes of drug and therapy development for the cure of diseases such as Duchenne Muscular Dystrophy or myocardial infarction, led to an increasing demand for the development of innovative methods and strategies. It is also clear how stem cells could represent a fundamental source for the production of artificial human tissues to be employed in such processes. This perspective, would require both sophisticated tools for the control of stem cells differentiation and their integration within procedures apt to satisfy the fundamental requirements for obtaining tissue-model on which perform pharmacological or therapeutic studies. Among the fundamental requisites are: micronization, versatility, low-cost and highthroughput.
The main aims of this thesis have been the design, development and fabrication of microscale technologies capable of both reproducing a biomimetic stimulation inspired to the in vivo cell microenvironment and responding to the above mentioned technological requirements.
We performed a semi-quantitative analysis of the characteristic times of microscale phenomena that lead to the generation of operative diagrams that would prove useful in the design and development phases of experimental strategies. We developed microfluidic microbioreactors that have been used for biological studies involving cell cultures. In particular, we investigated the role and effect of concentration gradients on the fundamental Wnt signaling pathway.
We developed techniques for obtaining a topological control at the microscale of cell cultures on hydrogels with tunable mechanical properties. These techniques were successfully applied and interfaced with relevant biological systems such as primary human myoblasts from dystrophic patients and human embryonic stem cells-derived cardiomyocytes.
We finally proposed a prototype of an integrated microfluidic platform capable of coupling the topological stimulations to the control of the soluble microenvironment on cell cultures.
The obtained results open new and interesting perspectives for both de efficient development of drugs or therapies for defined diseases and for gaining deeper insights into the complexity of biological systems.Le impellenti necessità legate allo sviluppo di nuovi farmaci e di terapie innovative per la cura di malattie dell’apparato muscolare, quali ad esempio la Distrofia Muscolare di Duchenne o l’infarto miocardico, hanno portato alla crescente domanda di nuovi metodi e tecnologie. E’ chiaro inoltre come le cellule staminali possano costituire una risorsa fondamentale per la generazione di tessuti umani artificiali da impiegare in tali processi. Tale prospettiva, richiederebbe da un lato strumenti sofisticati per il controllo e il differenziamento delle cellule staminali e dell’altro, l’integrazione di questi all’interno di procedure in grado di soddisfare i requisiti fondamentali dei modelli sui quali operare per lo sviluppo di nuovi farmaci o strategie terapeutiche. Tra i requisiti fondamentali da rispettare si citano quindi: micronizzazione, versatilità, basso costo e highthroughput. Obiettivo fondamentale di questa tesi sono stati la progettazione, sviluppo e fabbricazione di tecnologie su scala micrometrica in grado di riprodurre una stimolazione biomimetica ispirata al microambiente cellulare in vivo e, nello stesso tempo, di rispondere ai requisiti tecnologici descritti sopra. E’ stata effettuata un’analisi semi quantitativa basata sull’analisi dei tempi caratteristi dei fenomeni su microscala, che ha portato alla produzione di diagrammi operativi da impiegarsi nelle fasi di progettazione e sviluppo di tali strategie sperimentali. Sono stati sviluppati microbioreattori all’interno di piattaforme microfluidiche applicate poi allo studio di sistemi cellulari. In particolare, sono stati effettuati studi biologi sull’effetto di gradienti di concentrazione all’interno dell’importante signaling cellulare del Wnt. E’ stata realizzata una tecnica per l’organizzazione topologica su microscala di colture cellulari su substrati in hydrogel dalle proprietà meccaniche definite. Le tecnologie sviluppate sono state impiegate per casi studio dall’elevato valore scientifico e sono state interfacciate con colture di particolare interesse quali mioblasti umani distrofici e cellule cardiache derivate da staminali embrionali umane. Infine, è stato proposto un prototipo di piattaforma microfluidica in grado di accoppiare le stimolazioni di tipo topologico al controllo dell’ambiente solubile su colture cellulari. I risultati ottenuti aprono nuove ed interessanti prospettive sia per lo sviluppo di nuovi farmaci che di strategie terapeutiche volti alla cura di patologie ed allo studio approfondito della complessità dei sistemi biologici.
35
Fatanat, Didar Tohid. "Lab-on-Chip platforms: enabling technology for label-free detection, separation, patterning and «in vitro» culture of cells." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=117116.
Анотація:
Biomedical devices developed for detection, sorting and in vitro culture of cells are important tools in both clinical diagnostics and fundamental research. Recently, with the advances in miniaturization, Lab-on-Chip (LOC) devices have started to play an important role in detection and enrichment of rare cells. Since they do not alter the properties of target cells, label-free methods are a suitable option for cell separation. This dissertation focused on two main label-free approaches, namely adhesion-based and size-based and several novel microchips were introduced for separation of target cells using both size-based and adhesion-based approaches. The first device a multilayered, fully thermoplastic-based microfluidic chip was designed and fabricated for high-throughput size-based separation of micro/nano particles and cells. High-throughput (100 μl/min) separation of micro/nano particles and rare primary cells, with greater than 95% separation efficiency, was successfully demonstrated (Chapters 4 and 5). The second series of microchips were based on adhesion-based separation; multiplex covalently attached microarrays and gradients of biomolecules were produced and embedded inside a single microfluidic chip (chapters 7 and 8). The developed bio-functional interfaces were embedded in a multi-purpose adhesion-based microchip to simultaneously capture, separate, pattern and culture primary and rare cells in vitro. Using this chip, oligodendrocyte progenitor cells and cardiomyocytes were successfully separated from rat brain and heart tissues, respectively with greater than 95% separation efficiency in 10min (Chapter 9). Separation of two dissimilar primary cells, in terms of biological properties and initial population, demonstrated the universality of the developed chip towards efficient cell separation. More importantly separated cells can be cultured on the same chip for different subsequent applications such as proliferation for cell therapy or drug testing.Les dispositifs biomédicaux développés pour la détection, le triage et la culture cellulaire sont des outils importants en diagnostic clinique et en recherche fondamentale. Récemment, avec les progrès dans le domaine de la miniaturisation et les microfluidiques, les 'laboratoires-sur-puces' (LOC) ont commencé à jouer un rôle significatif dans la détection et l'enrichissement des différents types de cellules. Parmi les multitudes LOC développés pour ces applications, ceux n'impliquant pas de marquage représentent une option attrayante, car ils ne modifient pas les propriétés des cellules ciblées par le triage. Séparation à base d'adhérence cellulaire ou en utilisant la différence entre la taille physique des cellules sont les deux approches principales qui ne nécessitent pas de prétraitement des cellules et leur marquage. Dans cette thèse, des micropuces proposées emploient des méthodes sans marquage préalable des cellules ciblées tout en exploitent, soit leurs propriétés d'adhésion et leur affinité pour la biointerface, soit leur différence de taille par rapport la taille des autres cellules dans le mélange initial. En ce qui concerne la première micropuce, nous avons développé un dispositif microfluidique original et multicouche à base de thermoplastique pour la séparation de micro/nanoparticules et de cellules de tailles différentes. Une efficacité de séparation supérieure à 95% a pu être réalisée à haut débit (150 μl/min),Quant à la séparation basée sur l'adhérence, tout d'abord, nous avons introduit des LOCs pour produire des gradients de concentration de biomolécules dans un seul canal microfluidique. Dans un deuxième temps, ce concept a été utilisé pour fabriquer une puce multifonctionnelle sur laquelle, il était possible de simultanément capturer, séparer des cellules rares de source primaire, paver la surface avec des motifs des cellules et les cultiver sur la même puce. En utilisant cette puce, des cellules progénitrices d'oligodendrocytes (OPC) et des cardiomyocytes provenant respectivement du cerveau et du cœur du rat, ont pu être séparés en 10 min avec une efficacité de séparation supérieure à 95% des autres cellules dans le mélange tissulaire. La séparation de ces deux types de cellules primaires démontre l'efficacité et l'universalité de cette puce multifonctionnelle pour la séparation d'une gamme de mélanges cellulaires avec différentes concentrations initiales des cellules ciblées par le triage.
36
Pliquett, Jacques. "Development of fluorescent platforms for the design of multifunctional compounds for in vitro and in vivo applications in molecular imaging." Thesis, Bourgogne Franche-Comté, 2018. http://www.theses.fr/2018UBFCK067.
Анотація:
Cette thèse s’inscrit dans le développement et l’évaluation de nouvelles plateformesmoléculaires pour une application en imagerie optique par fluorescence. Nous avons cherché àdévelopper de nouveaux outils multifonctionnels et modifiables à façon. Cette approche estnécessaire car l’introduction d’un fluorophore peut fortement influencer les propriétés ducomposé final. Cela signifie que l’introduction du fluorophore sur l’agent sélectionné doit avoirêtre réalisé dès le départ. Pour cela deux axes principaux ont été étudiés; le premier consiste àutiliser des BODIPY pour le développement d’agents thérapeutiques traçables pour uneapplication principalement in vitro; le deuxième cible sur la conception de plateformes à based’AzaBODIPY compatibles avec l’imagerie in vivo.Dans la première partie deux fluorophores à base de 3,5-dichloro-BODIPY ont été identifiéscomme plateformes prometteurs. Ils ont été fonctionnalisés sélectivement par un agent or(I)-phosphine, un thiosucre et un phosphonium afin de pouvoir étudier l’influence du positionnementde chaque substituant sur les propriétés finales. Nous avons pu démontrer qu’unefonctionnalisation sélective et spécifique est possible avec ces substituants fragiles ; cela nous apermis de développer 12 agents théranostiques à base d’or(I). Les propriétés photophysiques etbiologiques ont ensuite été évaluées; pour cela nous avons déterminé leurs propriétés antiprolifératives (3 lignés cellulaires), la balance hydrophile, l’accumulation d’or dans les cellules etla localisation des composés des composés par microscopie confocale. Cette stratégie deplateforme multifonctionnelle nous a permis de développer un panel de composés traçables ayantdes activités mixtes ainsi que des distributions cellulaires distinctes. Cette étude a permisl’identification et la sélection de trois ou quatre composés qui feront l’objet d’une étudeapprofondie.Dans la deuxième partie de cette thèse nous avons développé des plateformes multifonctionnellescompatibles avec l’imagerie in vivo; pour cela nous avons poursuivi deux approches différentes.La première était l’utilisation de 1,7-di(phenol)3,5-di(phenyl)-azaBODIPY, suivi par safonctionnalisation sur les groupements OH afin de développer un traceur bioconjugablefluorescent dans le proche infrarouge (NIR-I). Malheureusement ce traceur possède despropriétés optiques très défavorables. Nous avons alors développé une approche innovante baséesur la fonctionnalisation de l’atome de bore. En s’appuyant sur cette approche deux traceursfortement fluorescents dans le proche infrarouge et solubles dans l’eau ont été développés. Cesfluorophores ont été conjugués sur un anticorps innovateur afin de permettre l’imagerie optiquedu ligand PD-L1. Les traceurs se sont montrés stables pour au moins 48h dans le plasma murin etpossèdent de très bonnes propriétés optiques. Comme preuve de concept nous avons conduitune étude préclinique in vivo. Cette étude a montré que les traceurs sont fortement fluorescents(NIR-I) et ne possèdent pas de toxicité imminente.La méthodologie développée pendant cette thèse présente un grand potentiel pour des étudesallant plus loin et des futures applications ; il est possible d’appliquer les principes et outilsdéveloppés sur d’autre fluorophores ; la méthodologie permet une fonctionnalisation très richeavec une grande variété de substituants d’intérêt. Son utilisation n’est pas limitée aux applicationsbiologiques, biochimiques et médicinalesThe objective of this thesis was the development and evaluation of new molecular platformsfor optical fluorescence imaging applications. This work sought to develop new tools that caneasily be modified and adapted to the specific needs of the intended use. This is required asthe fluorophore will influence the final properties and should thus be incorporated beforestructural optimization of the selected agent rather than at the very end. Two main axes wereexplored; the use of BODIPYs for the development of trackable therapeutic agents that areprimarily intended for in vitro applications and the use of azaBODIPYs for the design of an invivo compatible fluorescent platform.In the first part two fluorophores on the basis of a 3,5-dichloro-BODIPY were identified aspromising platforms. These platform molecules were selectively functionalized using a gold(I)-phosphine moiety, a thiosugar and a phosphonium to explore their selective functionalizationand investigate the influence of each substitutents position on the final properties. Weshowed that a site-specific, selective functionalization with these fragile substituents ispossible and developed 12 gold(I)-bearing therapeutic agents. We evaluated thephotophysical properties of all obtained compounds which was followed by a characterizationof their biological properties (antiproliferative properties on 3 cancer cell lines, lipophilicbalance and cellular gold accumulation as well as fluorescence imaging on 3 cell lines for upto 24h). We succeeded in developing a panel of closely related trackable compounds thatdisplay mixed activity in cells and distinct cellular localization. This investigation permitted theselection of three to four hits that will be studied further.In the second part we developed an in vivo-compatible multifunctional platform following twostrategies: the first was the use of 1,7-di(phenol)-3,5-di(phenyl)-azaBODIPY and thefunctionalization of the hydroxy groups for the development of a bioconjugable NIR-I probe.Unfortunately the developed probe displayed very unfavourable optical properties; wetherefore developed a new strategy that is entirely based on the functionalization of the boronatom. Using this approach we successfully synthesized 2 watersoluble, strongly fluorescent(NIR-I) molecular platforms that were conjugated to an innovative antibody to image the PD-L1 ligand. The developed probes displayed excellent optical properties, are stable for at least48h in mice plasma and were validated in a preclinical study on mice. The developed probesdisplayed strong fluorescence in vivo and showed no acute toxicity.The developed methodology shows great potential for further investigations and futurestudies; it can be transposed onto other closely related fluorophores and permits versatilefunctionalization with a large variety of compounds of interest. Its use is thus not limited tobiological, biochemical and medical applications
37
Griffiths, Natalie Helen. "Mechanisms of GABAA and glycine receptor analgesia in the spinal dorsal horn : in vitro models as translational platforms for drug discovery." Thesis, University of Leeds, 2015. http://etheses.whiterose.ac.uk/12560/.
Анотація:
Available analgesics do not always provide adequate pain relief and are often associated with a significant side-effect profile. Following reports of a loss of inhibitory signalling in the spinal dorsal horn (DH) network in persistent pain conditions, γ-amino butyric acid type A (GABAA) and glycine receptors have been identified as promising targets. The present study assesses two in vitro models for their suitability for screening novel analgesics targeting GABAA and glycine receptors. Firstly, the embryonic cultured spinal DH cell model and secondly an acute rat spinal cord slice model. Immunofluorescence characterisation of the spinal DH culture illustrated that this model displays many similarities with the in vivo spinal DH. Immunofluorescence and RT-PCR demonstrated the presence of GABAA and glycine receptor subunits in the spinal DH culture. Calcium imaging and extracellular multi-electrode array (MEA) recording techniques were utilised to study the effect of GABAA, GABAB and glycine receptor drugs on the spinal DH culture network activity. All drugs tested significantly modulated the culture’s spontaneous firing. A further study assessed whether lentivirus and Accell siRNA mediated glycine receptor α subunit gene silencing modulates calcium responses in the DH culture model. The lentiviruses had low transfection efficiencies and caused cell death, however Accell siRNA transfection was successful and significantly decreased baseline spontaneous activity compared to untreated cultures. Single electrode and MEA extracellular recordings were performed with the acute spinal cord slice model. GABAA, GABAB and glycine receptor drugs modulated 4-aminopyridine-induced hyperexcitability in the substantia gelatinosa lamina of the slices. The MEA recordings illustrated that 4-aminopyridine-induced activity manifested more prominently in the DH than the ventral horn (VH) and that the DH network activity was highly synchronous. Taken together, these findings demonstrate that these in vitro models provide suitable platforms to test novel analgesics targeting GABAA and glycine receptors in the spinal DH network.
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Srinivasan, Rajesh. "Platform Technologies for In vitro Point-of- Care Diagnostics." Thesis, 2022. https://etd.iisc.ac.in/handle/2005/5737.
Анотація:
In vitro diagnostic (IVD) tests are critical for making informed clinical decisions. These
tests are performed outside the body using specimens such as blood, sputum, saliva, and
urine. IVD tests are usually performed at centralized labs with complex and expensive
instrumentation, and associated protocols. There are only a few commercially available
point-of-care (POC) devices for performing IVD Tests. This thesis presents platform
technologies for POC testing of three categories of IVDs: Clinical Biochemistry,
Immunodiagnostics, and Molecular diagnostics. Additionally, a Biosafety Level 2+ (BSL-
2+) Mobile Laboratory was designed & developed for testing of infectious diseases at the
point-of-need.
Clinical biochemistry tests are quantitative tests that measure the concentration of
biochemicals in body fluids. Most clinical biochemistry tests involve the testing of serum.
A POC serum separation device from micro-volumes of blood using an air-displacement
pipette and a chemical-preloaded disposable pipette-tip cartridge is introduced in Chapter-
2. To address the issue of reagent stabilization and room-temperature storage, trehalosebased
enzyme reagent matrix (TERM) cartridge with dried reagents was developed. The
cartridge was tested for triglycerides measurement in blood and found to offer good shelflife.
In addition, the development of reagent kits for conducting sickle-cell disease
confirmatory test at point-of-need was reported. Finally, as an alternative to conventional
biochemical analyzers, a portable multi-spectral absorbance reader capable of detecting
the results at POC of a wide range of clinical biochemical assays was developed and
validated.
Immunodiagnostics includes tests that can detect and quantify antigens and
antibodies in a sample. Chapter-3 of the thesis proposes the development of platform
technologies in this category based on introducing innovative (i) protocols and (ii)
processes: (i) Counting target CD4+ T-Cells in blood was demonstrated using anti-CD4
antibodies conjugated to superparamagnetic iron oxide nanoparticles (SPIONs) and a
commercially available haematology analyser. (ii) A bare fibre Bragg grating (bFBG)
sensor-based technique is developed for pathogen detection, and demonstrated its ability in
detecting E. coli in water by coating anti-E. Coli antibodies onto the bFBG sensor.
Molecular diagnostics comprises of techniques that can detect the presence or
modification of nucleic acid (DNA/RNA). These tests usually require sophisticated and
iv
expensive instruments. To address this issue, Chapter-4 reports the development of
Portable PCR System: i) Isothermal Amplification Device (LAMP assays), ii) Handheld
Thermal Cycler (tested for HCV & Dengue), and iii) Fluorescence Reader (tested for
CoViD-19). In Chapter-5, a mobile laboratory for infectious diseases testing and reporting
(MITR Lab) was conceptualized, designed, fabricated, and found to offer 100% match
with static lab results in testing CoViD-19. The MITR lab is the first ICMR approved
Mobile BSL2+ lab for COVID-19 testing and aided in conducting few lakh tests during the
past one year.
All the technologies developed as part of the thesis are validated with clinical
samples and showed sensitivity and specificity above 90%. These innovative, affordable
platform technologies are expected to create huge social impact by providing timely
diagnoses at the point-of-need.
39
Lai, Tzu-Hsiang, and 賴子詳. "An antibiotic delivery platform using in vitro packed AP205 VLPs." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/9ww673.
Анотація:
碩士國立臺灣大學
生化科學研究所
107
Nowadays, small molecular drugs are used for chemotherapy to treat cancer diseases despite that these small molecules are non-targeting drugs. However, all efficacious drugs have adverse side effects. In order to avoid these unwanted hazards, scientists dedicate efforts to develop a carrier, which is capable of delivering drugs to target cancer cells precisely instead of anonymously. VLPs (Virus like particles) is a nanoscale self-assembled protein-based cage that is composed of only capsid from virus. The surface of capsid is tolerated with chemical modifications or genetic engineering to present the targeting peptides and epitopes. By far, the Qβ, MS2, and HBVc VLPs have been extensively studied in application of vaccine and drug delivery. In this study, the AP205 VLPs are used to encapsulate two small molecular drugs, doxorubicin (dox) and neomycin (neo), through the RNA hairpin-capsomere interactions. The encapsulated RNA hairpin is phosphorothioated by T4 PNK reaction, and then covalently coupled with dox or neo through amine-to-sulfhydryl crosslinker. The AP205 VLPs dox and AP205 VLPs neo are packed successfully in vitro. However, both the in vitro packed VLPs show low cytotoxicity towards cancer cells, which indicates that low coupling efficiency of dox/neo to the RNA hairpin, contributing to a low level of compound loading within the VLPs. The dox/neo and RNA hairpin coupling require further investigations in future works.
40
Lin, Meng Bo, and 林孟柏. "Design and Development of An In-Vitro Ultrasonic Stimulation Testing Platform." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/20041466419041108065.
41
Pereira, Ana Rita Oliveira Alves. "Modelling breast cancer metastatic bone niche: a novel in vitro 3D microfluidic platform." Master's thesis, 2016. https://hdl.handle.net/10216/90164.
42
Mei, Hsiang-I., and 梅湘怡. "In-vitro Er:YAG laser treatment platform construction with the implant for peri-implantitis." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/6w6p59.
Анотація:
碩士國立陽明大學
生物醫學工程學系
105
Peri-implantitis, which is induced by dental biofilm leading to the periodontal immune reaction, subsequently causes peri-implantitis inflammation and bone loss. The peri-implantitis is usually treated by using conservative mechanical debridement, air abrasives, antiseptic treatment, laser etc. The Er: YAG laser is a 2940nm wavelength laser with the highest energy insertion by closest connection to water among the existing lasers. This dental laser has high potentials for peri-implantitis treatment. Unfortunately, there has been no in-vitro laser experiment platform that considers different implant positions and the levels of peri-implantitis diseases using a real dental implant with a threaded surface. The standard clinical adult tooth jaw model was scanned to construct the digital model with 2mm and 6mm bone loss depth mimicking serious peri-implantitis at the incisor, first premolar, and first molar. This study constructs a standard in-vitro laser treatment platform with dental implant surface on which bacterial adhesion for peri-implantitis at different tooth positions. An Er: YAG laser, working with a chisel type glass tip, was moved from the buccal across the implant thread to the lingual for about 60 seconds per sample to verify the in-vitro laser treatment platform. The result showed that the sterilization rate can reach up to 90% and the jaw model was not damaged after laser irradiation testing.This study concluded that using integrated image processing, reverse engineering, CAD system and a 3D printer to construct a peri-implantitis model replacing the implant on bacterial adhesion and acceptable sterilization rate proved the feasibility of the proposed laser treatment platform.
43
Pereira, Ana Rita Oliveira Alves. "Modelling breast cancer metastatic bone niche: a novel in vitro 3D microfluidic platform." Dissertação, 2016. https://hdl.handle.net/10216/90164.
44
Scamardella, Sara. "A NEW HUMAN SKIN EQUIVALENT MODEL AS IN VITRO TESTING PLATFORM FOR BIOACTIVE MOLECULES." Tesi di dottorato, 2015. http://www.fedoa.unina.it/10401/1/Scamardella_Sara.pdf.
Анотація:
The aim of this PhD thesis is the realization of an in vitro human skin model and its validation as platform for testing bioactive molecules. Due to its endogenous nature the human skin equivalent realized was able to recapitulate both physiological and pathological status of the human skin. As consequence it has been used for recapitulating the typical UVA-induced skin alterations and to test the efficacy of photoprotectants. The thesis has been organized as follow: chapter 1 deals with a critical analysis of the literature reporting an overview of the existing human skin equivalent model highlighting their importance as alternative to animal model for testing the irritancy and toxicity of several bioactive compounds. This is a very important point, since the EU Directive prohibits the use of animals in cosmetic testing for certain endpoints, and in general strongly dictate the reduction of animal testing.
In the chapter 2 the realization of 3D dermis equivalent completely made up of endogenous extracellular matrix has been reported. The dermis is the connective part of the skin, we realized it by assembling functional microtissues precursor and demonstrate the presence of all dermis matrix components by histological and immunohistochemical analyses.
In the chapter 3 the 3D human dermis equivalent model has been used to quantify photoaging damage induced by UVA irradation to cells and extra-cellular matrix (ECM) and to evaluate bioactivity of cosmetic compounds. To this aim, matrix metalloproteinases (MMPs), collagen and hyaluronic acid (HA) synthesis -as well as collagen organization remodeling- have been investigated by immunofluorescence, histological and second harmonic generation (SHG) imaging. The results indicate that, despite to the existing model consisting in fibroblasts-populated exogenous matrix, the endogenous dermal equivalent developed is a unique model to investigate and quantify in vitro, the alterations in the structure and assembly of the ECM due to photoaging.
In chapter 4 is reported the bio-fabrication of a skin full thickness in vitro model. After a first morphological characterization, through histological and immunofluorescence analysis, the model has been used as in vitro screening tool. A photodamage due to UVA has been induced as reported in the chapter 4, and the damage on both dermal and epidermal part of the skin has been assessed. Finally the effect of a cosmetic compound has been tested. Our results reported that the model is able to recapitulate several events typical of in vivo photoaging (collagen reduction, MMP overexpression and cellular damage such as reduction of Ki67 and P63 expression). We speculated that due to its endogenous nature the skin model realized is able to recapitulate a more physiological microenviroment for cell and tissue development compared to existing fibroblasts-populated exogenous matrix.
Moreover we hypothesized that the human skin model realized could be used not only in vitro but also in vivo as skin substitute in clinical application
45
Langella, Angela. "Fabrication of in vitro epithelial tissues as a testing platform for drug delivery systems." Tesi di dottorato, 2017. http://www.fedoa.unina.it/12097/1/Langella_Angela_phd_thesis.pdf.
Анотація:
In the last years, tissue engineering (TE) has advanced offering the potential for regenerating almost every tissue and organ of the human body. Three dimensional (3D) human tissue models can provide a good platform for pharmacological screening and drug delivery rather than traditional bidimensional (2D) cell culture or animal models. In native tissues the extracellular matrix (ECM) promotes the crosstalk between cells, and is responsible of several fundamental patophysiological processes. For this reason, tissue engineering is moving forward the development of in vitro models in which endogenous ECM is present. In the perspective of using in vitro tissues as testing platform. Nowadays there is an overlapping between tissue engineering and nanomedicine since they share the common application to improve the new molecules screening in vitro and the pre-clinical testing. Nanomedicine and nanofabrication allow to create miniaturized release-based systems such as nano-biocapsulates while novel tissue engineering processess allow to building up in vitro tissues and organ mimicking their in vivo counterpart. Nanomedicine relies on the fundamental principles of tissue engineering as well as the tissue engineering field favors from continuous progress in nanomedicine. Indeed, due to the complexity of these tissues, studying and analyzing possible strategies to implement specific nanocarriers is becoming more and more challenging. The big challenge is engenneering capsules able to specifically target a certain organ of interest and, then, ensure an effective in situ controlled release. 9 The aim of my PhD work is to effectively bridge nanomedicine and tissue engineering. Particularly, on the one hand-side, this work focused on the realization of functionalized nanoparticles that are able to transport lipophilic drug preventing their early degradation. On the other side, a 3D intestinal diseased model as a platform for drug screening was realized. We created nanocapsules starting from an oil-in-water (O/W) nanoemulsion coated with a polysaccharide layer film, i.e. a glycolmodified chitosan, and subsequently curcumin and paclitaxel were loaded. These compounds are usually unlikely water-soluble. In particular, curcumin can be involved as a factor for preventive therapy while Paclitaxel is included in farmaceuticals for treatment of colorectal carcinoma. The final nano-carriers are completely biocompatible and biostable. In the first part, I investigated the enhancement of the effect of curcumin loaded in our system across monolayers of intestinal epithelial cells (CaCo-2) in transwell culture. Such in vitro platform is suitable for evaluating the functionality of the nano-carrier and its specificity towards the mucosal epithelial layer. As an applicative example, the investigation of the anti-inflammatory effects exerted by the encapsulation of curcumin was carried out. In the second part, I developed a more complex in vitro cellular model. This is relevant for creating a unique 3D tumoral intestinal model capable of mimic physiological in vivo diseased architecture. Under these conditions, it was possible to precisely evaluate the antitumoral effets of paclitaxel in nanocapsules. In conclusion, I started from a 2D system to test the internalization capability and the efficacy of nanocapsules and then I moved towards 10 the development of a pathological 3D system and implemented the aforementioned internalization of nanocapsules loaded with potential chemioterapeutics (paclitaxel). Effectively, this work contributes to develop a future high throughput platform for drug screening of a variety of nano-carriers against tumoral-like cellular components.
46
Addae-Mensah, Kweku A. "A microfabricated microcantilever array a platform for investigation of cellular biomechanics and microforces in vitro /." Diss., 2008. http://etd.library.vanderbilt.edu/available/etd-08182008-153315/.
47
Chien, Chi-Chen, and 簡基城. "Studies of Immunomodulation Effect of Ganoderma lucidum Using an in vitro Cord Blood MNC-Based Platform Method." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/6kq78m.
Анотація:
博士國立清華大學
生命科學系
92
An in vitro model for testing natural substances is developed in this study. The model used mononuclear cells (MNCs) from human umbilical cord blood (hUCB) for phenotypic expression induction platform. Natural substances, such as Ganoderma or grape seed proanthocyanidins extract, were added to the medium and then cultured for seven days. The stem cells/progenitors contained in the mononuclear cells were modulated by the active ingredients and changed in the course of differentiation and maturation. Flow cytometry was used for phenotypic analysis. Meanwhile, we utilized a cell-fluid based microchip on Agilent Bioanalyzer, and discovered an alternative assay method for phenotypic expression studies. Such a measurement could analyze the phenotypes of cells in a small population as few as 200 cells with good sensitivity and accuracy. We found that Ganoderma lucidum extract could enhance the NK cells composition in immune cell subpopulation when used to treat hUCB MNCs. After treatment, the enriched NK cells preserved similar cytotoxicity function. Enrichment of specific subsets of immune cells enabled us to explore further studies, such as investigating the optimal effector-to-target cells ratio in this study, or potential future systems biology experiments during differentiation courses. The model was also utilized to screen different sort of natural substances. We found that, for example, wheat grass extract up-regulated CD 56+ NK cell composition, whereas rutin and quercetin down-regulated instead. Different fractions of natural substances could be further testified quickly with this platform. Furthermore, we observed no significant change as the concentration of natural substance was below a critical concentration. The optimal oral consumption dosage of a specific natural health product could be predicted. Taken together, we demonstrated that this technique provides a good platform to study natural substances. The responses of stem cells/progenitors to natural substances, the alteration in phenotypic expression of immune cell subpopulation, and the modulation of human immunity could be further explicated using this technique.
48
Magalhães, Marta Isabel Ricardo de. "Development of an in vitro platform for a 3D microphysiological systems of human iPS-derived endothelial cells." Master's thesis, 2021. http://hdl.handle.net/10362/120566.
Анотація:
ix In this work, we presented the original development of a chip for co-culture of cells to build up a 3D microvascular networks. The chip layout was designed in Auto-desk AutoCAD2020 to obtain three interconnected tissue chambers where the cells will be seeding and growing. The cell compartments are interconnected and fed with culture medium through the hourglass shaped side pores of the chamber (two pores for each chamber). The cell chambers are connected with inlet/outlet pairs for medium, which is delivered by an automatic feeding system. One of the initial goals for this work was to have the vessels grow towards both pores, so a pressure gradient was necessary. A medium delivery system was designed in, built with a 3D printer, and operated by a digital actuator connected to a microcon-troller board. The oscillating system maintains a difference of level (static pressure) be-tween the two culture medium containers, changing their positions automatically by previously defined time intervals, promoting uniform cell growth. The level height difference of culture medium and the time between changes can be adjusted by the programmable control system. Furthermore, the design of the chip layout allows for the growth of cells towards mimicking a 3D network of blood vessels. After seeding and polymerization of the fibrin gel, the cells grew occupying all the chamber space. That can be observed by microscopy without stopping the culture, as the chip support is transparent. These indicate that a suitable platform for biological assays using different cell types in co-culture (e.g. fibro-blast with human endothelial cells) can be carried on.Neste trabalho apresentamos o desenvolvimento original de um chip para co-cultura de células para a construção de redes microvasculares 3D. O layout do chip foi projetado no Autodesk AutoCAD2020 para obter 3 câmaras de tecido interconectadas onde as células serão semeadas e irão crescer. Os compartimentos das células são interligados e alimentados com o meio de cultura através dos poros laterais em forma de ampulheta (dois poros para cada câmara). As câmaras celulares estão conectadas à entrada/saída de meio, que é entregue por um sistema de alimentação automático. Um dos objetivos iniciais deste trabalho era fazer com que os vasos crescessem em direção a ambos os poros, para isso um gradiente de pressão era necessário. Um sistema de entrega de meio foi projetado e construído com uma impressora 3D e operado por um atuador digital com uma placa microcontrolador. O sistema oscilante mantém uma diferença de nível (pressão estática) entre os dois recipientes do meio de cultura, mudando as suas posições automaticamente por intervalos de tempo previamente definidos, promovendo um crescimento celular uniforme. A diferença de altura do meio de cultura e o tempo entre as mudanças podem ser ajustados pelo sistema de controle programável. Para além disso, o design do layout do chip permite o crescimento de células para simular uma rede 3D de vasos sanguíneos. Após a semeação e polimerização do gel de fibrina, as células cresceram ocupando todo o espaço da câmara. Isto pode ser observado por microscopia sem interrupção da cultura, pois o suporte do chip é transparente. Estes indicam que, uma plataforma adequada para ensaios biológicos usando diferentes tipos de células em co-cultura (por exemplo, fibroblasto com células endoteliais humanas) pode ser realizada.
49
Gioiella, Filomena. "A new bioengineered 3D tumor platform in vitro to replicate tumor-stroma interaction and investigate anti-cancer drug delivery." Tesi di dottorato, 2016. http://www.fedoa.unina.it/10856/1/Gioiella_Filomena_28.pdf.
Анотація:
Nowadays, cancer is still a second leading cause of death after cardiovascular disease in the world. One of the main factors that lead the failure of cancer therapy is related to the fact that little is known about the interaction of cancer cells with microenvironment.
Indeed, as hypothesized in the "seed" and "soil" theory by Paget over a century ago, tumor progression is determined not only by tumor cells but also by the surrounding stromal milieu. For these reasons in the last years, increasing attention is focused on the importance of tumor microenvironment in an effort to develop successful strategies in cancer disease treatment. In traditional two-dimensional in vitro models the absence of 3D architecture generates misleading and contradictory results. Hence emerges the need to have an in vitro versatile platform that closely recapitulates pathophysiological features of the native tumor tissue and its surrounding microenvironment. In this PhD thesis a microtissue precursors assembling strategy (µTP), was used and translated to produce 3D tumor engineered models composed by tumor and/or stromal cells. In contrast with the classical spheroid model, the µTP we proposed presents the production of extracellular matrix directly synthesized by stromal cells.
First of all, in the chapter 1 a stat of art overview was presented which highlights the importance of tumor microenvironment in cancer research, the existing models for studying tumor development and the nanotechnology contribution in cancer treatment.
Then the chapter 2 is focalized on the realization of stromal microtissues fabricated seeding normal or activated fibroblasts on microporous beads, in order to monitor their dynamic evolution in terms of metabolic activity, mechanical properties and ECM composition. In particular it is demonstrated how the microtissue configuration is able to keep phenotypic differences between normal and activated fibroblasts in all the aspects investigated compared to the classical 3D spheroidal model.
In the chapter 3 the cross talk between epithelial tumor and the surrounding stroma in a microfluidic device is investigated. Thanks to the combination of 3D microtissues with microfluidic technology,
it is possible to detect in real time the modification occurring at cellular and ECM level during the activation period.
In the second part of work, the tumor microtissue model is validated as a potential drug-screening platform. In particular, in chapter 4 a commonly drug used in chemotherapy (Doxorubicin) is tested in order to detect the difference in chemoresistance between microtissues and spheroid models, both in monoculture and coculture.
Finally, a stimuli-responsive nanoparticles are tested on normal and tumor 3D heterotypic microtissues to demonstrate their significant selectively. At last, the microtissue system may be a useful \emph{in vitro} screening tool for testing innovative approaches of drug delivery, reducing expensive and time-consuming protocol nowadays used in preclinical studies.
50
Ramappa, Nirmala Kuppalu. "A system biology approach to study pancreatic ductal adenocarcinoma (PDAC) cells in in vitro culture." Doctoral thesis, 2018. http://hdl.handle.net/2158/1116922.