80 research outputs found

    Chemically diverse polyacrylate and polyacrylamide surfaces for human cardiomyocyte culture and their effect on phenotype

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    Human pluripotent stem cell (hPSC) derived cardiomyocytes can provide robust in vitro models for pharmaceutical drug screening and modelling cardiac disease. To fully realise these potentials, hPSC-cardiomyocytes must be reproducibly cultured to a more mature state than has thus far been achieved. Defined and well controlled culture conditions underpin the ability to derive hPSC-cardiomyocytes of consistent quality. These include cell source, culture medium and substrate. This thesis is concerned with the latter; culture substrates are currently biological in nature creating inherent variability in culture conditions. There is limited knowledge on the interaction between cardiomyocytes and synthetic, non-biological substrates making rational design of materials impractical. To aid discovery of novel culture substrates, 115 polyacrylate and polyacrylamide substrates were microarrayed and investigated using a parallel screening approach. The use of polyacrylates and polyacrylamides as biomaterials has been demonstrated previously, including contact lenses, bone cements and hydrogels to support cells. The diverse chemistry exhibited across the range of polymers enables modelling of structure-activity relationships between substrate chemistry and cardiomyocyte behaviour. Cardiomyocytes derived from human embryonic stem cells (hESC) were cultured on the microarrays in the presence and absence of serum. Attachment density (nuclei count via DAPI staining) and cardiomyocyte spread (surface area using sarcomeric Ξ±-actinin immunostaining) on each substrate was acquired by automated fluorescence microscopy and image analysis software. From this primary screen, 70 % of polymers were found to support cardiomyocytes adhesion in serum conditioned arrays, BM34 (fufuryl methacrylate) supported the highest relative cell density (0.59 Β±0.28) and also the largest cell size (1364 Β±937 Β΅m2), which was comparable to gelatin control. In serum free conditions, only 10 % of polymers supported cardiomyocyte attachment. The highest relative cell density was on AM38 (Dimethylamino-propyl acrylate) and largest cell size on BM49 (Tert-butylamino-ethyl methacrylate), 274 Β±75 Β΅m2, which was significantly lower than cell density and cell area (2019 Β±596 Β΅m2) on gelatin control. To investigate if synergy exists between polymers that enable adhesion in serum free conditions and those that support larger cell areas in serum conditioned arrays, 24 polymers were mixed pair-wise to form second generation microarrays comprising of 576 co-polymers. This diverse library enabled unique combinations of chemical moieties to be explored and co-polymers were found to have a greater proportion (74 %) that supported cardiomyocyte attachment in serum free conditions, largest average cell size was now 1089 Β±260 Β΅m2 on BM80/AM64 (Methacryloyloxy)ethyl acetoacetate/ Hexadecafluoro-9-(trifluoromethyl)decyl acrylate). Five co-polymers were chosen to perform more detailed characterisation of cardiomyocytes cultured on them for 5 and 25 days. Electrophysiological profiling and quantification of myofibril organisation identified co-polymers AD17/BM54 (Hexanediol ethoxylate diacrylate/ Ethoxyethyl methacrylate) and BM80/AD17 to be comparable to control gelatin. Partial least squares multivariate regression analysis correlated chemical species from the polymeric substrate, identified using time of flight secondary ion mass spectrometry, with cardiomyocyte response and identified moieties beneficial or detrimental for cardiomyocyte adhesion and cell area. This may aid rational design of tailor-made non-biological substrates for cell culture. In summary, the parallel screening of co-polymers of acrylates and acrylamides has been the first step in a discovery process of lead materials capable of progressing the culture of cardiomyocytes in more reproducible, economical and defined conditions. Only five substrates were analysed in detail, leaving a large library of co-polymers worthy of further investigation, including the physical properties of the polymers that need to be considered for practical use of the polymers in culture

    The early career researcher's toolkit: translating tissue engineering, regenerative medicine and cell therapy products

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    Although the importance of translation for the development of tissue engineering, regenerative medicine and cell-based therapies is widely recognized, the process of translation is less well understood. This is particularly the case among some early career researchers who may not appreciate the intricacies of translational research or make decisions early in development which later hinders effective translation. Based on our own research and experiences as early career researchers involved in tissue engineering and regenerative medicine translation, we discuss common pitfalls associated with translational research, providing practical solutions and important considerations which will aid process and product development. Suggestions range from effective project management, consideration of key manufacturing, clinical and regulatory matters and means of exploiting research for successful commercialization

    A defined synthetic substrate for serum-free culture of human stem cell derived cardiomyocytes with improved functional maturity identified using combinatorial materials microarrays

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    Cardiomyocytes from human stem cells have applications in regenerative medicine and can provide models for heart disease and toxicity screening. Soluble components of the culture system such as growth factors within serum and insoluble components such as the substrate on which cells adhere to are important variables controlling the biological activity of cells. Using a combinatorial materials approach we develop a synthetic, chemically defined cellular niche for the support of functional cardiomyocytes derived from human embryonic stem cells (hESC-CMs) in a serum-free fully defined culture system. Almost 700 polymers were synthesized and evaluated for their utility as growth substrates. From this group, 20 polymers were identified that supported cardiomyocyte adhesion and spreading. The most promising 3 polymers were scaled up for extended culture of hESC-CMs for 15 days and were characterized using patch clamp electrophysiology and myofibril analysis to find that functional and structural phenotype was maintained on these synthetic substrates without the need for coating with extracellular matrix protein. In addition, we found that hESC-CMs cultured on a co-polymer of isobornyl methacrylate and tert-butylamino-ethyl methacrylate exhibited significantly longer sarcomeres relative to gelatin control. The potential utility of increased structural integrity was demonstrated in an in vitro toxicity assay that found an increase in detection sensitivity of myofibril disruption by the anti-cancer drug doxorubicin at a concentration of 0.05 ?M in cardiomyocytes cultured on the co-polymer compared to 0.5 ?M on gelatin. The chemical moieties identified in this large-scale screen provide chemically defined conditions for the culture and manipulation of hESC-CMs, as well as a framework for the rational design of superior biomaterials

    The early career researcher's toolkit:translating tissue engineering, regenerative medicine and cell therapy products

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    Although the importance of translation for the development of tissue engineering, regenerative medicine and cell-based therapies is widely recognized, the process of translation is less well understood. This is particularly the case among some early career researchers who may not appreciate the intricacies of translational research or make decisions early in development which later hinders effective translation. Based on our own research and experiences as early career researchers involved in tissue engineering and regenerative medicine translation, we discuss common pitfalls associated with translational research, providing practical solutions and important considerations which will aid process and product development. Suggestions range from effective project management, consideration of key manufacturing, clinical and regulatory matters and means of exploiting research for successful commercialization

    Doing implementation research on health governance: a frontline researcher’s reflexive account of field-level challenges and their management

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    BACKGROUND: Implementation Research (IR) in and around health systems comes with unique challenges for researchers including implementation, multi-layer governance, and ethical issues. Partnerships between researchers, implementers, policy makers and community members are central to IR and come with additional challenges. In this paper, we elaborate on the challenges faced by frontline field researchers, drawing from experience with an IR study on Village Health Sanitation and Nutrition Committees (VHSNCs). METHODS: The IR on VHSNC took place in one state/province in India over an 18-month research period. The IR study had twin components; intervention and in-depth research. The intervention sought to strengthen the VHSNC functioning, and concurrently the research arm sought to understand the contextual factors, pathways and mechanism affecting VHSNC functions. Frontline researchers were employed for data collection and a research assistant was living in the study sites. The frontline research assistant experienced a range of challenges, while collecting data from the study sites, which were documented as field memos and analysed using inductive content analysis approach. RESULTS: Due to the relational nature of IR, the challenges coalesced around two sets of relationships (a) between the community and frontline researchers and (b) between implementers and frontline researchers. In the community, the frontline researcher was viewed as the supervisor of the intervention and was perceived by the community to have power to bring about beneficial changes with public services and facilities. Implementers expected help from the frontline researcher in problem-solving in VHSNCs, and feedback on community mobilization to improve their approaches. A concerted effort was undertaken by the whole research team to clarify and dispel concerns among the community and implementers through careful and constant communication. The strategies employed were both managerial, relational and reflexive in nature. CONCLUSION: Frontline researchers through their experiences shape the research process and its outcome and they play a central role in the research. It demonstrates that frontline researcher resilience is very crucial when conducting health policy and systems research.Scopu

    High throughput screening for discovery of materials that control stem cell fate

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    Insights into the complex stem cell niche have identified the cell–material interface to be a potent regulator of stem cell fate via material properties such as chemistry, topography and stiffness. In light of this, materials scientists have the opportunity to develop bioactive materials for stem cell culture that elicit specific cellular responses. To accelerate materials discovery, high throughput screening platforms have been designed which can rapidly evaluate combinatorial material libraries in two and three-dimensional environments. In this review, we present screening platforms for the discovery of material properties that influence stem cell behavior

    Drug delivery across length scales

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    Over the last century, there has been a dramatic change in the nature of therapeutic, biologically active molecules available to treat disease. Therapies have evolved from extracted natural products towards rationally designed biomolecules, including small molecules, engineered proteins and nucleic acids. The use of potent drugs which target specific organs, cells or biochemical pathways, necessitates new tools which can enable controlled delivery and dosing of these therapeutics to their biological targets. Here, we review the miniaturisation of drug delivery systems from the macro to nano-scale, focussing on controlled dosing and controlled targeting as two key parameters in drug delivery device design. We describe how the miniaturisation of these devices enables the move from repeated, systemic dosing, to on-demand, targeted delivery of therapeutic drugs and highlight areas of focus for the future

    A Gain-of-Function Germline Mutation in Drosophila ras1 Affects Apoptosis and Cell Fate during Development

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    The RAS/MAPK signal transduction pathway is an intracellular signaling cascade that transmits environmental signals from activated receptor tyrosine kinases (RTKs) on the cell surface and other endomembranes to transcription factors in the nucleus, thereby linking extracellular stimuli to changes in gene expression. Largely as a consequence of its role in oncogenesis, RAS signaling has been the subject of intense research efforts for many years. More recently, it has been shown that milder perturbations in Ras signaling during embryogenesis also contribute to the etiology of a group of human diseases. Here we report the identification and characterization of the first gain-of-function germline mutation in Drosophila ras1 (ras85D), the Drosophila homolog of human K-ras, N-ras and H-ras. A single amino acid substitution (R68Q) in the highly conserved switch II region of Ras causes a defective protein with reduced intrinsic GTPase activity, but with normal sensitivity to GAP stimulation. The ras1R68Q mutant is homozygous viable but causes various developmental defects associated with elevated Ras signaling, including cell fate changes and ectopic survival of cells in the nervous system. These biochemical and functional properties are reminiscent of germline Ras mutants found in patients afflicted with Noonan, Costello or cardio-facio-cutaneous syndromes. Finally, we used ras1R68Q to identify novel genes that interact with Ras and suppress cell death

    TLR1/2 Activation during Heterologous Prime-Boost Vaccination (DNA-MVA) Enhances CD8+ T Cell Responses Providing Protection against Leishmania (Viannia)

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    Leishmania (Viannia) are the predominant agents of leishmaniasis in Latin America. Given the fact that leishmaniasis is a zoonosis, eradication is unlikely; a vaccine could provide effective prevention of disease. However, these parasites present a challenge and we do not fully understand what elements of the host immune defense prevent disease. We examined the ability of vaccination to protect against L. (Viannia) infection using the highly immunogenic heterologous prime-boost (DNA-modified vaccinia virus) modality and a single Leishmania antigen (TRYP). Although this mode of vaccination can induce protection against other leishmaniases (cutaneous, visceral), no protection was observed against L. (V.) panamensis. However, we found that if the vaccination was modified and the innate immune response was activated through Toll-like receptor1/2(TLR1/2) during the DNA priming, vaccinated mice were protected. Protection was dependent on CD8 T cells. Vaccinated mice had higher CD8 T cell responses and decreased levels of cytokines known to promote infection. Given the long-term persistence of CD8 T cell memory, these findings are encouraging for vaccine development. Further, these results suggest that modulation of TLR1/2 signaling could improve the efficacy of DNA-based vaccines, especially where CD8 T cell activation is critical, thereby contributing to effective and affordable anti parasitic vaccines

    Beyond form and functioning: Understanding how contextual factors influence village health committees in northern India

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    Health committees are a common strategy to foster community participation in health. Efforts to strengthen committees often focus on technical inputs to improve committee form (e.g. representative membership) and functioning (e.g. meeting procedures). However, porous and interconnected contextual spheres also mediate committee effectiveness. Using a framework for contextual analysis, we explored the contextual features that facilitated or hindered Village Health, Sanitation and Nutrition Committee (VHSNC) functionality in rural north India. We conducted interviews (n = 74), focus groups (n = 18) and observation over 1.5 years. Thematic content analysis enabled the identification and grouping of themes, and detailed exploration of sub-themes. While the intervention succeeded in strengthening committee form and functioning, participant accounts illuminated the different ways in which contextual influences impinged on VHSNC efficacy. Women and marginalized groups navigated social hierarchies that curtailed their ability to assert themselves in the presence of men and powerful local families. These dynamics were not static and unchanging, illustrated by pre-existing cross-caste problem solving, and the committee's creation of opportunities for the careful violation of social norms. Resource and capacity deficits in government services limited opportunities to build relationships between health system actors and committee members and engendered mistrust of government institutions. Fragmented administrative accountability left committee members bearing responsibility for improving local health without access to stakeholders who could support or respond to their efforts. The committee's narrow authority was at odds with widespread community needs, and committee members struggled to involve diverse government services across the health, sanitation, and nutrition sectors. Multiple parallel systems (political decentralization, media and other village groups) presented opportunities to create more enabling VHSNC contexts, although the potential to harness these opportunities was largely unmet. This study highlights the urgent need for supportive contexts in which people can not only participate in health committees, but also access the power and resources needed to bring about actual improvements to their health and wellbeing.IS
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