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Статті в журналах з теми "In vitro platform":
1
Perenkov, Alexey D., Alena D. Sergeeva, Maria V. Vedunova, and Dmitri V. Krysko. "In Vitro Transcribed RNA-Based Platform Vaccines: Past, Present, and Future." Vaccines 11, no. 10 (October 16, 2023): 1600. http://dx.doi.org/10.3390/vaccines11101600.
Анотація:
mRNA was discovered in 1961, but it was not used as a vaccine until after three decades. Recently, the development of mRNA vaccine technology gained great impetus from the pursuit of vaccines against COVID-19. To improve the properties of RNA vaccines, and primarily their circulation time, self-amplifying mRNA and trans-amplifying mRNA were developed. A separate branch of mRNA technology is circular RNA vaccines, which were developed with the discovery of the possibility of translation on their protein matrix. Circular RNA has several advantages over mRNA vaccines and is considered a fairly promising platform, as is trans-amplifying mRNA. This review presents an overview of the mRNA platform and a critical discussion of the more modern self-amplifying mRNA, trans-amplifying mRNA, and circular RNA platforms created on its basis. Finally, the main features, advantages, and disadvantages of each of the presented mRNA platforms are discussed. This discussion will facilitate the decision-making process in selecting the most appropriate platform for creating RNA vaccines against cancer or viral diseases.
2
Gupta, Priyanka, Aline Miller, Adedamola Olayanju, Thumuluru Kavitha Madhuri, and Eirini Velliou. "A Systematic Comparative Assessment of the Response of Ovarian Cancer Cells to the Chemotherapeutic Cisplatin in 3D Models of Various Structural and Biochemical Configurations—Does One Model Type Fit All?" Cancers 14, no. 5 (March 1, 2022): 1274. http://dx.doi.org/10.3390/cancers14051274.
Анотація:
Epithelial Ovarian Cancer (EOC) is a silent, deadly and aggressive gynaecological disease with a relatively low survival rate. This has been attributed, to some extent, to EOC’s high recurrence rate and resistance to currently available platinum-based chemotherapeutic treatment methods. Multiple groups have studied and reported the effect of chemotherapeutic agents on various EOC 3D in vitro models. However, there are very few studies wherein a direct comparative study has been carried out between the different in vitro 3D models of EOC and the effect of chemotherapy within them. Herein, we report, for the first time, a direct comprehensive systematic comparative study of three different 3D in vitro platforms, namely (i) spheroids, (ii) synthetic PeptiGels/hydrogels of various chemical configurations and (iii) polymeric scaffolds with coatings of various extracellular matrices (ECMs) on the cell growth and response to the chemotherapeutic (Cisplatin) for ovary-derived (A2780) and metastatic (SK-OV-3) EOC cell lines. We report that all three 3D models are able to support the growth of EOC, but for different time periods (varying from 7 days to 4 weeks). We have also reported that chemoresistance to Cisplatin, in vitro, observed especially for metastatic EOC cells, is platform-dependent, in terms of both the structural and biochemical composition of the model/platform. Our study highlights the importance of selecting an appropriate 3D platform for in vitro tumour model development. We have demonstrated that the selection of the best platform for producing in vitro tumour models depends on the cancer/cell type, the experimental time period and the application for which the model is intended.
3
Gadde, Manasa, Melika Mehrabi-Dehdezi, Bisrat G. Debeb, Wendy A. Woodward, and Marissa Nichole Rylander. "Influence of Macrophages on Vascular Invasion of Inflammatory Breast Cancer Emboli Measured Using an In Vitro Microfluidic Multi-Cellular Platform." Cancers 15, no. 19 (October 8, 2023): 4883. http://dx.doi.org/10.3390/cancers15194883.
Анотація:
Inflammatory breast cancer (IBC) is an aggressive disease with a poor prognosis and a lack of effective treatments. It is widely established that understanding the interactions between tumor-associated macrophages (TAMs) and the tumor microenvironment is essential for identifying distinct targeting markers that help with prognosis and subsequent development of effective treatments. In this study, we present a 3D in vitro microfluidic IBC platform consisting of THP1 M0, M1, or M2 macrophages, IBC cells, and endothelial cells. The platform comprises a collagen matrix that includes an endothelialized vessel, creating a physiologically relevant environment for cellular interactions. Through the utilization of this platform, it was discovered that the inclusion of tumor-associated macrophages (TAMs) led to an increase in the formation of new blood vessel sprouts and enhanced permeability of the endothelium, regardless of the macrophage phenotype. Interestingly, the platforms containing THP-1 M1 or M2 macrophages exhibited significantly greater porosity in the collagen extracellular matrix (ECM) compared to the platforms containing THP-1 M0 and the MDA-IBC3 cells alone. Cytokine analysis revealed that IL-8 and MMP9 showed selective increases when macrophages were cultured in the platforms. Notably, intravasation of tumor cells into the vessels was observed exclusively in the platform containing MDA-IBC3 and M0 macrophages.
4
McRae, Michael P., Kritika S. Rajsri, Timothy M. Alcorn, and John T. McDevitt. "Smart Diagnostics: Combining Artificial Intelligence and In Vitro Diagnostics." Sensors 22, no. 17 (August 24, 2022): 6355. http://dx.doi.org/10.3390/s22176355.
Анотація:
We are beginning a new era of Smart Diagnostics—integrated biosensors powered by recent innovations in embedded electronics, cloud computing, and artificial intelligence (AI). Universal and AI-based in vitro diagnostics (IVDs) have the potential to exponentially improve healthcare decision making in the coming years. This perspective covers current trends and challenges in translating Smart Diagnostics. We identify essential elements of Smart Diagnostics platforms through the lens of a clinically validated platform for digitizing biology and its ability to learn disease signatures. This platform for biochemical analyses uses a compact instrument to perform multiclass and multiplex measurements using fully integrated microfluidic cartridges compatible with the point of care. Image analysis digitizes biology by transforming fluorescence signals into inputs for learning disease/health signatures. The result is an intuitive Score reported to the patients and/or providers. This AI-linked universal diagnostic system has been validated through a series of large clinical studies and used to identify signatures for early disease detection and disease severity in several applications, including cardiovascular diseases, COVID-19, and oral cancer. The utility of this Smart Diagnostics platform may extend to multiple cell-based oncology tests via cross-reactive biomarkers spanning oral, colorectal, lung, bladder, esophageal, and cervical cancers, and is well-positioned to improve patient care, management, and outcomes through deployment of this resilient and scalable technology. Lastly, we provide a future perspective on the direction and trajectory of Smart Diagnostics and the transformative effects they will have on health care.
5
Park, Seonghyuk, Youngtaek Kim, Jihoon Ko, Jiyoung Song, Jeeyun Lee, Young-Kwon Hong, and Noo Li Jeon. "One-step achievement of tumor spheroid-induced angiogenesis in a high-throughput microfluidic platform: one-step tumor angiogenesis platform." Organoid 3 (February 25, 2023): e3. http://dx.doi.org/10.51335/organoid.2023.3.e3.
Анотація:
Research on the development of anti-cancer drugs has progressed, but the low reliability of animal experiments due to biological differences between animals and humans causes failures in the clinical process. To overcome this limitation, 3-dimensional (3D) in vitro models have been developed to mimic the human cellular microenvironment using polydimethylsiloxane (PDMS). However, due to the characteristics and limitations of PDMS, it has low efficiency and is not suitable to be applied in the preclinical testing of a drug. High-throughput microfluidic platforms fabricated by injection molding have been developed, but these platforms require a laborious process when handling spheroids. We recently developed an injection-molded plastic array 3D culture tissue platform that integrates the process from spheroid formation to reconstruction of an in vitro model with spheroids (All-in-One-IMPACT). In this study, we implemented a 3D tumor spheroid angiogenesis model in the developed platform. We analyzed the tendency for angiogenesis according to gel concentration and confirmed that angiogenesis occurred using cancer cell lines and patient-derived cancer cells (PDCs). We also administered an anti-cancer drug to the PDC tumor spheroid angiogenesis model to observe the drug’s effect on angiogenesis according to its concentration. We demonstrated that our platform can be used to study the tumor microenvironment (TME) and drug screening. We expect that this platform will contribute to further research on the complex mechanisms of the TME and predictive preclinical models.
6
Brocklehurst, Sean, Neda Ghousifam, Kameel Zuniga, Danielle Stolley, and Marissa Nichole Rylander. "Multilayer In Vitro Human Skin Tissue Platforms for Quantitative Burn Injury Investigation." Bioengineering 10, no. 2 (February 17, 2023): 265. http://dx.doi.org/10.3390/bioengineering10020265.
Анотація:
This study presents a multilayer in vitro human skin platform to quantitatively relate predicted spatial time–temperature history with measured tissue injury response. This information is needed to elucidate high-temperature, short-duration burn injury kinetics and enables determination of relevant input parameters for computational models to facilitate treatment planning. Multilayer in vitro skin platforms were constructed using human dermal keratinocytes and fibroblasts embedded in collagen I hydrogels. After three seconds of contact with a 50–100 °C burn tip, ablation, cell death, apoptosis, and HSP70 expression were spatially measured using immunofluorescence confocal microscopy. Finite element modeling was performed using the measured thermal characteristics of skin platforms to determine the temperature distribution within platforms over time. The process coefficients for the Arrhenius thermal injury model describing tissue ablation and cell death were determined such that the predictions calculated from the time–temperature histories fit the experimental burn results. The activation energy for thermal collagen ablation and cell death was found to be significantly lower for short-duration, high-temperature burns than those found for long-duration, low-temperature burns. Analysis of results suggests that different injury mechanisms dominate at higher temperatures, necessitating burn research in the temperature ranges of interest and demonstrating the practicality of the proposed skin platform for this purpose.
7
Kim, Tae Hee, Ji-Jing Yan, Joon Young Jang, Gwang-Min Lee, Sun-Kyung Lee, Beom Seok Kim, Justin J. Chung, Soo Hyun Kim, Youngmee Jung, and Jaeseok Yang. "Tissue-engineered vascular microphysiological platform to study immune modulation of xenograft rejection." Science Advances 7, no. 22 (May 2021): eabg2237. http://dx.doi.org/10.1126/sciadv.abg2237.
Анотація:
Most of the vascular platforms currently being studied are lab-on-a-chip types that mimic capillary networks and are applied for vascular response analysis in vitro. However, these platforms have a limitation in clearly assessing the physiological phenomena of native blood vessels compared to in vivo evaluation. Here, we developed a simply fabricable tissue-engineered vascular microphysiological platform (TEVMP) with a three-dimensional (3D) vascular structure similar to an artery that can be applied for ex vivo and in vivo evaluation. Furthermore, we applied the TEVMP as ex vivo and in vivo screening systems to evaluate the effect of human CD200 (hCD200) overexpression in porcine endothelial cells (PECs) on vascular xenogeneic immune responses. These screening systems, in contrast to 2D in vitro and cellular xenotransplantation in vivo models, clearly demonstrated that hCD200 overexpression effectively suppressed vascular xenograft rejection. The TEVMP has a high potential as a platform to assess various vascular-related responses.
8
Vasconez Martinez, Mateo Gabriel, Eva I. Reihs, Helene M. Stuetz, Astrid Hafner, Konstanze Brandauer, Florian Selinger, Patrick Schuller, et al. "Using Rapid Prototyping to Develop a Cell-Based Platform with Electrical Impedance Sensor Membranes for In Vitro RPMI2650 Nasal Nanotoxicology Monitoring." Biosensors 14, no. 2 (February 18, 2024): 107. http://dx.doi.org/10.3390/bios14020107.
Анотація:
Due to advances in additive manufacturing and prototyping, affordable and rapid microfluidic sensor-integrated assays can be fabricated using additive manufacturing, xurography and electrode shadow masking to create versatile platform technologies aimed toward qualitative assessment of acute cytotoxic or cytolytic events using stand-alone biochip platforms in the context of environmental risk assessment. In the current study, we established a nasal mucosa biosensing platform using RPMI2650 mucosa cells inside a membrane-integrated impedance-sensing biochip using exclusively rapid prototyping technologies. In a final proof-of-concept, we applied this biosensing platform to create human cell models of nasal mucosa for monitoring the acute cytotoxic effect of zinc oxide reference nanoparticles. Our data generated with the biochip platform successfully monitored the acute toxicity and cytolytic activity of 6 mM zinc oxide nanoparticles, which was non-invasively monitored as a negative impedance slope on nasal epithelial models, demonstrating the feasibility of rapid prototyping technologies such as additive manufacturing and xurography for cell-based platform development.
9
Xu, Liangcheng, Xin Song, Gwennyth Carroll, and Lidan You. "Novel in vitro microfluidic platform for osteocyte mechanotransduction studies." Integrative Biology 12, no. 12 (December 2020): 303–10. http://dx.doi.org/10.1093/intbio/zyaa025.
Анотація:
Abstract Osteocytes are the major mechanosensing cells in bone remodeling. Current in vitro bone mechanotransduction research use macroscale devices such as flow chambers; however, in vitro microfluidic devices provide an optimal tool to better understand this biological process with its flexible design, physiologically relevant dimensions and high-throughput capabilities. This project aims to design and fabricate a multi-shear stress, co-culture platform to study the interaction between osteocytes and other bone cells under varying flow conditions. Standard microfluidic design utilizing changing geometric parameters is used to induce different flow rates that are directly proportional to the levels of shear stress, with devices fabricated from standard polydimethylsiloxane (PDMS)-based softlithography processes. Each osteocyte channel (OCY) is connected to an adjacent osteoclast channel (OC) by 20-μm perfusion channels for cellular signaling molecule transport. Significant differences in RANKL levels are observed between channels with different shear stress levels, and we observed that pre-osteoclast differentiation was directly affected by adjacent flow-stimulated osteocytes. Significant decrease in the number of differentiating osteoclasts is observed in the OC channel adjacent to the 2-Pa shear stress OCY channel, while differentiation adjacent to the 0.5-Pa shear stress OCY channel is unaffected compared with no-flow controls. Addition of zoledronic acid showed a significant decrease in osteoclast differentiation, compounding to effect instigated by increasing fluid shear stress. Using this platform, we are able to mimic the interaction between osteocytes and osteoclasts in vitro under physiologically relevant bone interstitial fluid flow shear stress. Our novel microfluidic co-culture platform provides an optimal tool for bone cell mechanistic studies and provides a platform for the discovery of potential drug targets for clinical treatments of bone-related diseases.
10
Foong, Charlene Shu-Fen, Edwin Sandanaraj, Harold B. Brooks, Robert M. Campbell, Beng Ti Ang, Yuk Kien Chong, and Carol Tang. "Glioma-Propagating Cells as an In Vitro Screening Platform." Journal of Biomolecular Screening 17, no. 9 (August 27, 2012): 1136–50. http://dx.doi.org/10.1177/1087057112457820.
Анотація:
Gliomas are the most devastating of primary adult malignant brain tumors. These tumors are highly infiltrative and can arise from cells with extensive self-renewal capability and chemoresistance, frequently termed glioma-propagating cells (GPCs). GPCs are thus the plausible culprits of tumor recurrence. Treatment strategies that eradicate GPCs will greatly improve disease outcome. Such findings support the use of GPCs as in vitro cellular systems for small-molecule screening. However, the nuances in using GPCs as a cellular screening platform are not trivial. These slow-growing cells are typically cultured as suspension, spheroid structures in serum-free condition supplemented with growth factors. Consequently, replenishment of growth factors throughout the screening period must occur to maintain cells in their undifferentiated state, as the more lineage-committed, differentiated cells are less tumorigenic. We present a case study of a small-molecule screen conducted with GPCs and explain how unique sphere activity assays were implemented to distinguish drug efficacies against the long-term, self-renewing fraction, as opposed to transient-amplifying progenitors, the latter of which are detected in conventional viability assays. We identified Polo-like kinase 1 as a regulator of GPC survival. Finally, we leveraged on public glioma databases to illustrate GPC contribution to disease progression and patient survival outcome.
Дисертації з теми "In vitro platform":
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.
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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.
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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.
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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.
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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.
Книги з теми "In vitro platform":
1
Brevini, Tiziana A. L., Alireza Fazeli, and Kursad Turksen, eds. Next Generation Culture Platforms for Reliable In Vitro Models. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1246-0.
2
Mohammad-Hadi, Layla, and Marym Mohammad-Hadi. Applications of Minimally Invasive Nanomedicine-Based Therapies in 3D in vitro Cancer Platforms. Cham: Springer International Publishing, 2021. http://dx.doi.org/10.1007/978-3-031-02388-0.
3
Godier-Furnemont, Amandine Florence Ghislaine. Development of high fidelity cardiac tissue engineering platforms by biophysical signaling: In vitro models and in vivo repair. [New York, N.Y.?]: [publisher not identified], 2015.
4
Turksen, Kursad, Alireza Fazeli, and Tiziana A. L. Brevini. Next Generation Culture Platforms for Reliable in Vitro Models: Methods and Protocols. Springer, 2022.
5
Turksen, Kursad, Alireza Fazeli, and Tiziana A. L. Brevini. Next Generation Culture Platforms for Reliable in Vitro Models: Methods and Protocols. Springer, 2021.
6
Mohammad-Hadi, Layla, and Marym Mohammad-Hadi. Applications of Minimally Invasive Nanomedicine-Based Therapies in 3D in Vitro Cancer Platforms. Springer International Publishing AG, 2020.
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Mohammad-Hadi, Layla, and Marym Mohammad-Hadi. Applications of Minimally Invasive Nanomedicine-Based Therapies in 3D in Vitro Cancer Platforms. Morgan & Claypool Publishers, 2020.
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Mohammad-Hadi, Layla, and Marym Mohammad-Hadi. Applications of Minimally Invasive Nanomedicine-Based Therapies in 3D in Vitro Cancer Platforms. Morgan & Claypool Publishers, 2020.
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Sivarasu, Sudesh. Medical Devices Innovation for Africa: enabling industrialisation. University of Cape Town Libraries, 2022. http://dx.doi.org/10.15641/uctlib40.
Анотація:
It is with great pleasure to recognise all our partners in the merSETA Viro-Vent Innovation Skills Challenge who contributed to this publication: University of Cape Town, Cape Peninsula University of Technology, University KwaZulu Natal, University of Witwatersrand and National Technologies Implementation Platform. Thank you, Professor Sivarasu, for your leadership of the University of Cape Town for supporting these efforts to find new forms of collaboration that focus on “Skills for localisation” and “Skills for re-industrialisation”. This publication comes at a time when South Africa and the world are still recovering from the devastating effects of the covid-19 pandemic complicated by an emerging war in Ukraine. This is expected to continue disrupting social and economic activities, including education, training, and work. The merSETA and its stakeholders are working tirelessly to ensure that training and other skills development activities continue despite these challenges. This innovation project, among others at the merSETA, utilises existing research and Higher Education Institution (HEI) Infrastructure to stimulate rapid response technology innovation aimed at the development, design and prototype production of a medical device in response not only to the COVID-19 pandemic, but also to an economic sector dominated by imports. To serve the skills development mandate of the merSETA, the project investigates the technology management capabilities or future skills required to accelerate South Africa’s post-covid recovery. The concept of innovation, as vested in this program, is aligned to the merSETA’s strategic intentions, that include: i. Supporting skills for Economic Reconstruction, Recovery and Growth, ii. Supporting skills for the changing world of work, iii. Supporting skills for the growth and sustainability of the green and circular economies and iv. Exploring and supporting the role of the mer-sector in the digital economy, as well as v. Continuing to strengthen the role of the SETA as an intermediary body Making informed sector skills planning decisions is the objective of this program. – that is, to understand those future jobs that would drive the localisation of components in a model that could stimulate expanded manufacturing opportunities through relevant skills supply. The merSETA’s Viro-Vent Innovation Skills Challenge anticipates a contribution towards closing the skills gap through a job generation model. The merSETA remains committed and is looking forward to engaging on how this initiative sees a pipeline of new product innovations expanding the manufacturing sector. We owe it to the citizens of South Africa to find innovative ways of harnessing our young talent into industrial expansion.
Частини книг з теми "In vitro platform":
1
York, S. L., J. D. King, A. S. Pietros, B. Zhang Newby, P. Sethu, and M. M. Saunders. "Development of a Microloading Platform for In Vitro Mechanotransduction Studies." In Mechanics of Biological Systems and Materials, Volume 7, 53–59. Cham: Springer International Publishing, 2014. http://dx.doi.org/10.1007/978-3-319-06974-6_8.
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Blasiak, Agata, Sudip Nag, and In Hong Yang. "Subcellular Optogenetic Stimulation Platform for Studying Activity-Dependent Axon Myelination In Vitro." In Methods in Molecular Biology, 207–24. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7862-5_16.
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Rockel, Sylvie, Marcel Geertz, and Sebastian J. Maerkl. "MITOMI: A Microfluidic Platform for In Vitro Characterization of Transcription Factor–DNA Interaction." In Methods in Molecular Biology, 97–114. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-292-2_6.
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Jeong, Sangmoo, and Kayvan R. Keshari. "Hyperpolarized Micro-NMR Platform for Sensitive Analysis of In Vitro Metabolic Flux in Living Cells." In Methods in Molecular Biology, 561–69. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1803-5_29.
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Jankowicz-Cieslak, Joanna, Ivan L. Ingelbrecht, and Bradley J. Till. "Mutation Detection in Gamma-Irradiated Banana Using Low Coverage Copy Number Variation." In Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana, 113–27. Berlin, Heidelberg: Springer Berlin Heidelberg, 2022. http://dx.doi.org/10.1007/978-3-662-64915-2_8.
Анотація:
AbstractMutagenesis of in vitro propagated bananas is an efficient method to introduce novel alleles and broaden genetic diversity. The FAO/IAEA Plant Breeding and Genetics Laboratory previously established efficient methods for mutation induction of in vitro shoot tips in banana using physical and chemical mutagens as well as methods for the efficient discovery of ethyl methanesulphonate (EMS) induced single nucleotide mutations in targeted genes. Officially released mutant banana varieties have been created using gamma rays, a mutagen that can produce large genomic changes such as insertions and deletions (InDels). Such dosage mutations may be particularly important for generating observable phenotypes in polyploids such as banana. Here, we describe a Next Generation Sequencing (NGS) approach in Cavendish (AAA) bananas to identify large genomic InDels. The method is based on low coverage whole genome sequencing (LC-WGS) using an Illumina short-read sequencing platform. We provide details for sonication-mediated library preparation and the installation and use of freely available computer software to identify copy number variation in Cavendish banana. Alternative DNA library construction procedures and bioinformatics tools are briefly described. Example data is provided for the mutant variety Novaria and cv Grande Naine (AAA), but the methodology can be equally applied for triploid bananas with mixed genomes (A and B) and is useful for the characterization of putative Fusarium Wilt TR4 resistant mutant lines described elsewhere in this protocol book.
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Gomes, Luciana C., Rita Teixeira-Santos, Maria J. Romeu, and Filipe J. Mergulhão. "Bacterial Adhesion and Biofilm Formation: Hydrodynamics Effects." In Urinary Stents, 225–43. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-031-04484-7_19.
Анотація:
AbstractThe effectiveness of biomedical surfaces may be highly affected by the hydrodynamic condition. Surfaces releasing antimicrobial substances when exposed to flow may exhibit shorter lifetimes than at static conditions. Likewise, depending on the fluid flow surrounding the surface, contact-killing surfaces that are adhesive for bacterial cells may be covered by bacterial debris, which decreases their antimicrobial activity. To evaluate the anti-adhesive and antimicrobial performance of novel biomedical materials, a number of flow devices have been designed to recreate in vivo flow conditions. Shear stress and flow rate can be accurately controlled and varied in these in vitro flow systems, which requires prior knowledge of the flow dynamics inside the platform. After limiting their operational range, modified Robbins devices, flow chambers and microfluidic devices are suggested as experimental setups to mimic the flow behavior in urinary catheters and stents.
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Bock, Nathalie. "Bioengineered Microtissue Models of the Human Bone Metastatic Microenvironment: A Novel In Vitro Theranostics Platform for Cancer Research." In Methods in Molecular Biology, 23–57. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9769-5_2.
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Hau, Kwan-Leong, Amelia Lane, Rosellina Guarascio, and Michael E. Cheetham. "Eye on a Dish Models to Evaluate Splicing Modulation." In Methods in Molecular Biology, 245–55. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2010-6_16.
Анотація:
AbstractInherited retinal dystrophies, such as Leber congenital amaurosis, Stargardt disease, and retinitis pigmentosa, are characterized by photoreceptor dysfunction and death and currently have few treatment options. Recent technological advances in induced pluripotent stem cell (iPSC) technology and differentiation methods mean that human photoreceptors can now be studied in vitro. For example, retinal organoids provide a platform to study the development of the human retina and mechanisms of diseases in the dish, as well as being a potential source for cell transplantation. Here, we describe differentiation protocols for 3D cultures that produce retinal organoids containing photoreceptors with rudimentary outer segments. These protocols can be used as a model to understand retinal disease mechanisms and test potential therapies, including antisense oligonucleotides (AONs) to alter gene expression or RNA processing. This “retina in a dish” model is well suited for use with AONs, as the organoids recapitulate patient mutations in the correct genomic and cellular context, to test potential efficacy and examine off-target effects on the translational path to the clinic.
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Xu, Jianfeng, Melissa Towler, and Pamela J. Weathers. "Platforms for Plant-Based Protein Production." In Bioprocessing of Plant In Vitro Systems, 1–40. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/978-3-319-32004-5_14-1.
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Fraker, C., C. L. Stabler, K. Asfura-Gattas, and J. Dominguez-Bendala. "A Novel Cell Culture Platform for In-Vitro Enhancement of Oxygen Delivery Leads to Improved Physiological Function of Isolated Islets of Langerhans." In IFMBE Proceedings, 163–64. Berlin, Heidelberg: Springer Berlin Heidelberg, 2009. http://dx.doi.org/10.1007/978-3-642-01697-4_59.
Тези доповідей конференцій з теми "In vitro platform":
1
Brittain, S. B., J. L. Hajjar, and S. S. Nidadavolu. "In-vitro hemostasis test platform." In 2011 37th Annual Northeast Bioengineering Conference (NEBEC). IEEE, 2011. http://dx.doi.org/10.1109/nebc.2011.5778553.
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Imfeld, K., A. Garenne, S. Neukom, A. Maccione, S. Martinoia, M. Koudelka-Hep, and L. Berdondini. "High-resolution MEA platform for in-vitro electrogenic cell networks imaging." In 2007 29th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE, 2007. http://dx.doi.org/10.1109/iembs.2007.4353737.
3
Lichtenfels, Martina, Camila Alves da Silva, Alessandra Borba Anton de Souza, Heloisa Rezende, Luiza Kobe, Isabela Miranda, Antônio Luiz Frasson, and Caroline Brunetto de Farias. "VALIDATION OF A NOVEL IN VITRO BREAST CANCER CHEMORESISTANCE PLATFORM IN NEOADJUVANT SETTING." In Brazilian Breast Cancer Symposium 2022. Mastology, 2022. http://dx.doi.org/10.29289/259453942022v32s2013.
Анотація:
Objective: The aim of this study was to validate a novel in vitro chemoresistance platform for two drugs commonly used in the neoadjuvant setting for breast cancer (BC). Methods: Three BC cell lines (MCF-7 (luminal); SKBR3 (HER2+); and MDA-MB-231 (triple-negative) were used to confirm the efficacy of the platform. Patients with invasive BC and partial response to neoadjuvant chemotherapy were included in this initial report. Fresh tumor samples were collected during surgery and dissociated to obtain the tumor cells. The tumor cells were cultured in the chemoresistance platform with doxorubicin and paclitaxel and after 72-h cell viability was evaluated. The test result is defined based on cell viability as low (60%) resistance. Results: The three BC cell lines presented low resistance to doxorubicin, MCF-7 and SKBR3 cells also presented low resistance to paclitaxel, whereas MDA-MB-231 has intermediate resistance. Samples from 10 BC patients with partial response to neoadjuvant chemotherapy were tested in the novel chemoresistance platform. All the patients received doxorubicin and paclitaxel as part of the treatment. The overall rate of assay success was 100%. Regarding molecular subtypes, 40% were Luminal, 20% Luminal HER2, 10% HER2, and 30% triple-negative. The 10 samples presented 100% high resistance to paclitaxel. High resistance to doxorubicin was observed in 70% of the samples, intermediate in 10%, and low in 20%. The chemoresistance platform demonstrated that samples already treated with paclitaxel and doxorubicin in a neoadjuvant setting presented more high resistance to the drugs compared to the BC cell lines. Conclusion: This preliminary result demonstrated more high resistance in tumors previously treated with doxorubicin and paclitaxel compared to BC cell lines without previous treatment and highlighted the success of the in vitro chemoresistance platform to test tumor samples after neoadjuvant treatment.
4
"Microchannel-based platform for the study of neural circuit development in vitro." In 2009 4th International IEEE/EMBS Conference on Neural Engineering. IEEE, 2009. http://dx.doi.org/10.1109/ner.2009.5109229.
5
Rizou, M. E., and T. Prodromakis. "A planar micro-magnetic platform for stimulation of neural cells in vitro." In 2016 IEEE Biomedical Circuits and Systems Conference (BioCAS). IEEE, 2016. http://dx.doi.org/10.1109/biocas.2016.7833718.
6
Pathi, S. P., C. J. Kowalczewski, R. Tadipatri, and C. Fischbach. "Biomineralized scaffolds as an in vitro platform for studying metastatic bone disease." In 2009 IEEE 35th Annual Northeast Bioengineering Conference. IEEE, 2009. http://dx.doi.org/10.1109/nebc.2009.4967705.
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Zaccaria, Clara, Asiye Malkoç, Beatrice Vignoli, Marco Canossa, and Lorenzo Pavesi. "A Platform for Single Cell Optogenetics to Study Synaptic Engrams in Vitro." In Bio-Optics: Design and Application. Washington, D.C.: Optica Publishing Group, 2023. http://dx.doi.org/10.1364/boda.2023.dtu2a.5.
Анотація:
Memory relies on the firing of simultaneously activated neurons (engram), whose synapses are strengthened by long-term potentiation mechanisms. Optogenetic tools and a fluorescence probe to map synaptic engrams, were combined with a digital light processor device (DLP), to create in-vitro engrams and study populations of potentiated spines.
8
Hagiwara, Masaya, Rina Nobata, and Tomohiro Kawahara. "In vitro 3D culture platform for large-scale imaging by hybrid gel cube." In 2017 IEEE 12th International Conference on Nano/Micro Engineered and Molecular Systems (NEMS). IEEE, 2017. http://dx.doi.org/10.1109/nems.2017.8017066.
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Brackenbury, Louise S., S. Rhiannon Jenkinson, Shilina Roman, Robert D. Nunan, Sylvie D. Hunt, Anna Willox, Neil A. Williams, Omar Aziz, and Ian Waddell. "Abstract 2811: A translational immuno-oncology platform to model the tumor microenvironmentin vitro." In Proceedings: AACR Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/1538-7445.sabcs18-2811.
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Brackenbury, Louise S., S. Rhiannon Jenkinson, Shilina Roman, Robert D. Nunan, Sylvie D. Hunt, Anna Willox, Neil A. Williams, Omar Aziz, and Ian Waddell. "Abstract 2811: A translational immuno-oncology platform to model the tumor microenvironmentin vitro." In Proceedings: AACR Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/1538-7445.am2019-2811.
Звіти організацій з теми "In vitro platform":
1
Khani, Joshua, Lindsay Prescod, Heather Enright, Sarah Felix, Joanne Osburn, Elizabeth Wheeler, and Kris Kulp. Characterizing Rat PNS Electrophysiological Response to Electrical Stimulation Using in vitro Chip-Based Human Investigational Platform (iCHIP). Office of Scientific and Technical Information (OSTI), August 2015. http://dx.doi.org/10.2172/1236739.
2
Committee on Toxicology. COT FSA PBPK for Regulators Workshop Report 2021. Food Standards Agency, April 2024. http://dx.doi.org/10.46756/sci.fsa.tyy821.
Анотація:
The future of food safety assessment in the UK depends on the Food Standards Agency’s (FSA) adaptability and flexibility in responding to and adopting the accelerating developments in science and technology. The Tox21 approach is an example of one recent advancement in the development of alternative toxicity testing approaches and computer modelling strategies for the evaluation of hazard and exposure (New Approach Methodologies (NAMs). A key aspect is the ability to link active concentrations in vitro to likely concentrations in vivo, for which physiologically based pharmacokinetic (PBPK) modelling is ideally suited. The UK FSA and the Committee on Toxicity of Chemicals in Food, Consumer Products, and the Environment (COT) held an “PBPK for Regulators” workshop with multidisciplinary participation, involving delegates from regulatory agencies, government bodies, academics, and industry. The workshop provided a platform to enable expert discussions on the application of PBPK to health risk assessment in a regulatory context. Presentations covered current application of PBPK modelling in the agrochemical industry for in vitro to in vivo extrapolation (IVIVE), pharmaceutical industry for drug absorption related issues (e.g., the effect of food on drug absorption) and drug-drug interaction studies, as well as dose extrapolations to special populations (e.g., those with a specific disease state, paediatric/geriatric age groups, and different ethnicities), environmental chemical risk assessment, an overview of the current regulatory guidance and a PBPK model run-through. This enabled attendees to consider the wide potential and fitness for purpose of the application of PBPK modelling in these fields. Attendees considered applicability in the context of future food safety assessment for refining exposure assessments of chemicals with narrow margins of exposure and/or to fill data gaps from more traditional approaches (i.e., data from animal testing). The overall conclusions from the workshop were as follows: PBPK modelling tools were applicable in the areas of use covered, and that expertise was available (though it is in small numbers). PBPK modelling offers opportunities to address questions for compounds that are otherwise not possible (e.g., considerations of human variability in kinetics) and allows identification of “at risk” subpopulations. The use of PBPK modelling tends to be applied on a case-by-case basis and there appears to be a barrier to widespread acceptance amongst regulatory bodies due to the lack of available in-house expertise (apart from some medical and environmental agencies such as the European Medicines Agency, United States Food and Drug Administration, and the US Environmental Protection Agency, respectively). Familiarisation and further training opportunities on the application of PBPK modelling using real world case studies would help in generating interest and developing more experts in the field, as well as furthering acceptance. In a regulatory context, establishing fitness for purpose for the use of PBPK models requires transparent discussion between regulatory agencies, government bodies, academics, and industry and the development of a harmonised guidance such as by the Organisation for Economic Co-operation and Development (OECD) would provide a starting point. Finally, PBPK modelling is part of the wider “new approach methodologies” for risk assessment, and there should be particular emphasis in modelling both toxicodynamics and toxicokinetics.
3
Bar-Joseph, Moshe, William O. Dawson, and Munir Mawassi. Role of Defective RNAs in Citrus Tristeza Virus Diseases. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575279.bard.
Анотація:
This program focused on citrus tristeza virus (CTV), the largest and one of the most complex RNA-plant-viruses. The economic importance of this virus to the US and Israeli citrus industries, its uniqueness among RNA viruses and the possibility to tame the virus and eventually turn it into a useful tool for the protection and genetic improvement of citrus trees justify these continued efforts. Although the overall goal of this project was to study the role(s) of CTV associated defective (d)-RNAs in CTV-induced diseases, considerable research efforts had to be devoted to the engineering of the helper virus which provides the machinery to allow dRNA replication. Considerable progress was made through three main lines of complementary studies. For the first time, the generation of an engineered CTV genetic system that is capable of infecting citrus plants with in vitro modified virus was achieved. Considering that this RNA virus consists of a 20 kb genome, much larger than any other previously developed similar genetic system, completing this goal was an extremely difficult task that was accomplished by the effective collaboration and complementarity of both partners. Other full-length genomic CTV isolates were sequenced and populations examined, resulting in a new level of understanding of population complexities and dynamics in the US and Israel. In addition, this project has now considerably advanced our understanding and ability to manipulate dRNAs, a new class of genetic elements of closteroviruses, which were first found in the Israeli VT isolate and later shown to be omnipresent in CTV populations. We have characterized additional natural dRNAs and have shown that production of subgenomic mRNAs can be involved in the generation of dRNAs. We have molecularly cloned natural dRNAs and directly inoculated citrus plants with 35S-cDNA constructs and have shown that specific dRNAs are correlated with specific disease symptoms. Systems to examine dRNA replication in protoplasts were developed and the requirements for dRNA replication were defined. Several artificial dRNAs that replicate efficiently with a helper virus were created from infectious full-genomic cDNAs. Elements that allow the specific replication of dRNAs by heterologous helper viruses also were defined. The T36-derived dRNAs were replicated efficiently by a range of different wild CTV isolates and hybrid dRNAs with heterologous termini are efficiently replicated with T36 as helper. In addition we found: 1) All CTV genes except of the p6 gene product from the conserved signature block of the Closteroviridae are obligate for assembly, infectivity, and serial protoplast passage; 2) The p20 protein is a major component of the amorphous inclusion bodies of infected cells; and 3) Novel 5'-Co-terminal RNAs in CTV infected cells were characterized. These results have considerably advanced our basic understanding of the molecular biology of CTV and CTV-dRNAs and form the platform for the future manipulation of this complicated virus. As a result of these developments, the way is now open to turn constructs of this viral plant pathogen into new tools for protecting citrus against severe CTV terms and development of virus-based expression vectors for other citrus improvement needs. In conclusion, this research program has accomplished two main interconnected missions, the collection of basic information on the molecular and biological characteristics of the virus and its associated dRNAs toward development of management strategies against severe diseases caused by the virus and building of novel research tools to improve citrus varieties. Reaching these goals will allow us to advance this project to a new phase of turning the virus from a pathogen to an ally.