Mesenchymal stem cells (MSCs) have great potential to improve clinical outcomes for many inflammatory and degenerative diseases through delivery of exogenous MSCs via injection or cell-laden scaffolds and through mobilization and migration of endogenous MSCs to injury sites. MSC fate and function is determined by microenvironmental cues, specifically dimensionality, topography, and cell-cell interactions. MSC responses of migration and differentiation are the focus of this dissertation. Cell migration occurs in several physiological and pathological processes; migration mode and cell signaling are determined by the environment and type of confinement in three-dimensional (3D) models. Tendon injury is a common musculoskeletal disorder that occurs through cumulative damage to the extracellular matrix (ECM). Studies combining nanofibrous scaffolds and MSCs to determine an optimal topographical environment have promoted tenogenic differentiation under various conditions. We investigated cellular response of MSCs on specifically designed nanofiber matrices fabricated using a novel spinneret-based tunable engineered parameters production method (STEP). We designed suspended and aligned nanofiber scaffolds to study cellular morphology, tendon marker gene expression, and matrix deposition as determinants for tendon differentiation. The delivery and maintenance of MSCs at sites of inflammation or injury are major challenges in stem cell therapies. Enhancing stem cell homing could improve their therapeutic effects. Homing is a process that involves cell migration through the vasculature to target organs. This process is defined in leukocyte transendothelial migration (TEM); however, far less is known about MSC homing. We investigated two population subsets of MSCs in a Transwell system mimicking the vasculature; migrated cells that initiated transmigration on the endothelium and nonmigrated cells in the apical chamber that failed to transmigrate. Gene and protein expression changes were observed between these subsets and evidence suggests that multiple signaling pathways regulate TEM. The results of these experiments have demonstrated that microenvironmental cues are critical to understanding the cellular and molecular mechanisms of MSC response, specifically in homing and differentiation. This knowledge has identified scaffold parameters required to stimulate tenogenesis and signaling pathways controlling MSC homing. These findings will allow us to target key regulatory molecules and cell signaling pathways involved in MSC response towards development of regenerative therapies. / Ph. D. / Stem cell therapy is one way to improve tissue injury and inflammatory conditions, but to optimize such therapy, we need to study how the environment around cells influence turning them into the injured tissue and how to control their movement to these sites in order for mesenchymal stem cells (MSCs) to exert their therapeutic functions. MSCs move through and detect their environment through the material around them, including organization of the fibers they attach to and neighboring cells. Cell migration is an important cell behavior that occurs in normal and diseased processes. MSCs have great potential to improve clinical outcomes for many inflammatory and degenerative diseases whether through delivery of exogenous MSCs or through mobilization and migration of endogenous MSCs to injury sites. Tendon damage can occur slowly over time and optimal treatment for normal function after injury remains unknown. Equine MSCs were harvested from bone marrow and subjected to scaffolds of different fiber orientation to study whether cells develop characteristics of tendon cells. Cellular responses were similar between scaffolds of aligned fiber orientation. Manipulation of equine bone marrow MSCs through the use of specifically designed nanofiber scaffolds aid in understanding the mechanisms by which the cells respond and function in tendon development, injury, and repair. Inflammation is a necessary process after tissue injury; however, it must progress in a controlled manner and be resolved before it leads to tissue damage and dysfunction. MSCs function in regulating the effects of inflammation and immune cells; however, getting them to these sites and keeping them there remains challenging. MSCs adhere to and migrate through capillaries towards these sites, known as stem cell homing. Human bone marrow MSCs were loaded onto human synovial microvascular endothelial cells to study migration towards an inflammatory stimulus. This stimulus acted on the endothelial cells to produce another stimulus that attracted MSCs to the endothelial cells. These actions resulted in complete MSC migration through the endothelial cells and activated intracellular signals that can be used to increase the number of MSCs that reach the inflammatory sites and stimulate tissue-healing effects.

Due to the limited regenerative capacity of the central nervous system (CNS) upon injury, regenerative medicine and tissue engineering strategies show great promise for treatment. These aim to restore tissue functions by combining principles of cell biology and engineering, using biomaterial scaffolds which can help in recapitulating the 3D environment of the brain and improving cell survival after grafting. Stroke and TBI are severe forms of disruptions of brain architecture, and two of the leading causes of mortality and morbidity worldwide, as no effective treatments are available. Several studies report how neural stem cells (NSCs) are able to improve functional recovery upon transplantation. However, the efficacy of these treatments is limited because of the mortality these cells are subject to after transplantation. In this context, the transplantation of mesenchymal cells (MSCs) has shown beneficial effects by secreting molecules and factors that help in the healing process. In this study, we tested alginate-based hydrogels as candidates to support human NSCs and MSCs transplantation into the brain, in the view of exploiting the beneficial effects of both and analyzing whether their combined use could have a synergistic effect. In the first part, we studied the suitability of alginate-based scaffolds for the three-dimensional encapsulation and culture of hNSCs and hMSCs. We analyzed their ability to support cell survival, and we evaluated whether changes in their concentration or modifications with ECM molecules could influence cell viability. We showed that the best survival conditions are found when using an RGDs-functionalized alginate scaffold at a low concentration (0.5% w/v). We then worked on the identification of the best conditions for MSCs culture and the definition of coculture conditions. Since serum is necessary for MSCs, but it is reported to induce glial differentiation of NSCs, we explored two different experimental setups. On one hand, we investigated the feasibility to exploit biomaterials to create "compartmentalized" cocultures that would at least partially retain serum. In parallel, we positively observed that MSCs can survive, proliferate and maintain their stemness even in absence of serum, supporting the hypothesis that the use of “compartmentalized” coculture systems would likely be exploitable for MSCs culture. Finally, we tested the reported beneficial effects of MSCs in our 3D culture system, in which NSCs do not show a great viability. Encapsulated NSCs were cultured on an MSCs monolayer, and we analyzed cell survival, proliferation, differentiation and stemness retention. Gene expression analyses highlighted that NSCs maintain stemness characteristics, but we were not able to observe any improvement in NSCs survival in coculture, with respect to standard culture. In the last part of the project we decided to test our system for tissue engineering approaches, exploiting axotomized brain organotypic slices (OSCs). We evaluated the presence of cells 7 days after transplantation, their integration in the OSCs and glial response. Preliminary results suggest that the biomaterial does not cause activation of glial cells, although stem cells do not seem to migrate out of scaffold and integrate into the brain slice.

Yes / Interest in adipose tissue is fast becoming a focus of research after many years of being considered as a simple connective tissue. It is becoming increasingly apparent that adipose tissue contains a number of diverse cell types, including adipose-derived stem cells (ASCs) with the potential to differentiate into a number of cell lineages, and thus has significant potential for developing therapies for regenerative medicine. Currently, there is no gold standard treatment for scars and impaired wound healing continues to be a challenge faced by clinicians worldwide. This review describes the current understanding of the origin, different types, anatomical location, and genetics of adipose tissue before discussing the properties of ASCs and their promising applications for tissue engineering, scarring, and wound healing. / The authors thank the Plastic Surgery and Burns Research Unit at the University of Bradford, Bradford, UK for financial support for LVT

Regenerative medicine is a growing interdisciplinary domain within biomedical research that seeks to restore, regenerate, and substitute impaired tissues and cells. Platelet-rich plasma (PRP) is an emerging therapeutic modality in the field of regenerative medicine, which has garnered considerable attention for its capacity to facilitate and expedite tissue healing processes. Despite the availability of various conventional therapeutic approaches focusing on wound healing and growth factors, the use of new treatments remains a clinical challenge. Hence, the current study aims to investigate the field of regenerative skin wound healing, with a specific focus on the use of platelet-rich plasma (PRP) and establish a meaningful connection between clinical outcomes and patient satisfaction, while also exploring the properties and potential advantages of PRP. This qualitative study included fifteen patients from medical spas and clinics in Orlando, Florida, who had received PRP treatments within the last two years. The data, obtained through semi-structured interviews, were analyzed by conventional content analysis using Graneheim and Lundman’s approach (2004). Finally, data analysis identified three major categories: clinical outcomes, patient satisfaction, and quality of life (QoL). Improvement in facial texture and hair quality was found to be a significant predictor of positive clinical outcomes, and higher levels of self-esteem and a positive outlook on the aging process were strong indicators of improved quality of life (QoL). The present study offers empirical evidence that substantiates the positive patient satisfaction scores and the strong safety profile that are commonly linked with platelet-rich plasma (PRP) injections. Therefore, based on our analysis, we consider platelet-rich plasma (PRP) to be a viable and reliable therapeutic approach for addressing conditions such as alopecia, acne scars, and skin rejuvenation.

Degenerative joint disease, or osteoarthritis, is a major cause of lameness and morbidity in horses, humans, and dogs. There are no truly satisfactory cures for this widespread problem and current treatments all have limitations or unwanted side effects. New cell-based strategies to repair joint surface lesions have generated a high level of interest, but have yet to achieve the full restoration of articular cartilage structure and function. Currently used therapy cells include autologous chondrocytes and adult mesenchymal cells such as bone marrow derived cells and adipose derived cells. Unfortunately, the resultant repair tissue is biomechanically inferior fibrocartilage. A critical gap in knowledge in this regard is a limited understanding of the specific cellular phenotype of normal, robust articular chondrocytes. This thesis examines the global mRNA transcriptome of equine articular cartilage to test the hypothesis that adult articular chondrocytes have a unique gene expression profile. In the first part of the study, RNA-sequencing was used to compare the mRNA transcriptome of normal adult articular cartilage with five other cartilaginous tissues. From these comparisons, locus level gene expression and alternative splicing patterns have been identified that clearly distinguish articular cartilage. In the second part of the study, fetal (interzone, cartilage anlagen chondrocytes, dermal fibroblasts) and adult (bone marrow derived, adipose derived, articular chondrocytes, dermal fibroblasts) primary cells were grown in culture and stimulated to differentiate into chondrocytes. The chondrogenic differentiation potential as assessed by matrix proteoglycan and the expression of cartilage biomarker genes was highly variable among cell types. Together, these results advance our understanding of the specific phenotype of articular chondrocytes and the potential of prospective therapeutic progenitor cells to differentiate into articular chondrocytes. This new knowledge will improve efforts to optimize cell-based therapies for osteoarthritis and the repair of joint cartilage lesions.

Le discours sur l’innovation oriente la recherche scientifique médicale publique vers un développement technologique et économique à court terme. À ce titre, la médecine régénératrice est une thérapie innovatrice marquée par une logique d’accumulation spéculative qui porte à la fois sur les cellules humaines et sur la façon de mener la recherche. Or, une réorganisation de la recherche scientifique liée à une nouvelle conception économique de la science et de la technologie ainsi qu’un rôle différent attribué à l’État constituent le cadre institutionnel contemporain qui émerge à la fin des années 1970. Le changement induit par cette idée d’innovation et sur lequel s’attarde ce mémoire porte non pas sur l’usage ou la destination de la science, mais sur l’extension du raisonnement économique. Celui-ci ne survient pas à l’étape du développement, après que la recherche ait été effectuée en vertu du modèle de la « Big Science ». Au contraire, il remonte du marché pour s’installer très tôt au stade de la compréhension des mécanismes biologiques et dans un espace qui relève de la propriété collective : le laboratoire public. Le passage du caractère « exogène » à « endogène » de la recherche scientifique publique vis-à-vis de l’économie est au cœur d’une discussion sur l’hégémonie de la logique de marché. / This dissertation discusses the idea of technological innovation in regenerative medicine in Canada. While this potentially groundbreaking therapy is publicly funded and at an early stage of the understanding of cellular processes, the analysis shows that it is already concerned with the marketing of the scientific work. This raises questions about how public laboratories framed by a scientific research closely tied up with economic concerns and with a shift in the role of the State carry speculation and lead to a technological oriented production of knowledge. This writing doesn’t debate science as a mean or science as an end. It is rather about the extension of the economic reasoning. Indeed, the market’s reading grid as the starting point is the story of the hegemony of a specific logic which turns regenerative medicine research into an economic venture.

Apesar do grande potencial de regeneração do tecido ósseo, em algumas situações a extensão da lesão impede que o tecido se repare completamente. Como uma alternativa em relação aos tratamentos convencionais, a terapia celular tem sido considerada como promissora para o reparo de defeitos ósseos. No entanto, poucos estudos investigaram a terapia celular utilizando osteoblastos, portanto, avaliamos o efeito da injeção direta de osteoblastos na regeneração do tecido ósseo. Como os osteoblastos têm origens embrionárias diferentes, foi comparado in vitro o potencial osteogênico de osteoblastos derivados da crista neural, do mesoderma e de ambas as origens embrionárias. Considerando a necessidade de grande número de células para a terapia, foi comparado o efeito de uma subcultura, como meio de aumentar a quantidade de células, no potencial osteogênico dos osteoblastos. Para avaliação da regeneração dos defeitos, osteoblastos foram injetados diretamente nesses defeitos. Osteoblastos foram obtidos da calvária de ratos recém-nascidos (Wistar), sendo que os derivados da crista neural foram isolados dos ossos frontais (OB-CN); os do mesoderma isolados dos ossos parietais (OB-MS); e de ambas as origens embrionárias isolados de toda a calvária (OB-Cal). O efeito da subcultura no potencial osteogênico foi avaliado em OB-Cal ou na primeira passagem dessa cultura (OB-Cal P1). Após até 14 dias em cultura, foram avaliadas a proliferação celular, atividade de fosfatase alcalina (ALP), formação de matriz extracelular mineralizada e a expressão dos genes marcadores osteoblásticos: fator de transcrição runt-related 2 (RUNX2), ALP, osteocalcina (OC) e sialoproteína óssea (BSP). Para avaliar a regeneração do tecido ósseo, foram criados defeitos de 5 mm de diâmetro na calvária de ratos Wistar, que após 2 semanas foram tratados com 5 x 106 osteoblastos derivados de OB-Cal P1, por meio de injeção local. Ao final de 4 semanas, a formação óssea foi avaliada por microtomografia computadorizada e análise histológica. Os dados foram comparados por ANOVA, seguido do teste de Student-Newman-Keuls, ou teste t, quando apropriado, considerando o nível de significância de 5%. A comparação do potencial osteogênico em relação à origem embrionária mostrou que, os OB-MS apresentaram maior proliferação mas não houve diferença entre as culturas no evento final da diferenciação osteoblástica, que é a formação de matriz mineralizada. No entanto, como as culturas de OB-Cal apresentaram maior expressão gênica de marcadores iniciais, intermediários e finais dessa diferenciação; e considerando que, essas culturas são aquelas nas quais é possível obter o maior número de células, optamos por utilizar essas culturas na avaliação da formação óssea induzida pela terapia celular. Além disso, os resultados mostraram que os OB-Cal P1 tem seu potencial osteogênico reduzido, mas considerando que a subcultura permite a obtenção de maior número de células, são uma boa escolha para a terapia celular. Tanto que, ao avaliar in vivo a capacidade regenerativa das OBCal P1 no reparo dos defeitos ósseos, as análises microtomográficas e histológicas mostraram que que a terapia celular com injeção local de osteoblastos obtidos de fragmentos ósseos da calvária constitui uma estratégia adequada para estimular o reparo ósseo / Despite of the great potential of regeneration of the bone tissue, in some situations the extension of the lesion prevents the tissue from repairing completely. As an alternative to conventional treatments, cell therapy has been considered a promising strategy for the repair of bone defects. However, few studies investigated cell therapy using osteoblasts, therefore, we evaluated the effect of direct injection of osteoblasts on the regeneration of bone tissue. Since osteoblasts have different embryonic origins, the osteogenic potential of osteoblasts derived from neural crest, mesoderm and both embryonic origins was compared in vitro. Considering the need for a large number of cells for the therapy, the effect of a subculture, a common way to increase the amount of cells, on the osteogenic potential was also compared. Then, osteoblasts were injected directly into bone defects to evaluate the regeneration of bone tissue. Osteoblasts were obtained from the calvaria of newborn rats (Wistar), the neural crest derivatives were isolated from the frontal bones (OB-CN); those of the mesoderm isolated from the parietal bones (OB-MS); and from both embryonic origins isolated from the entire calvaria (OB-Cal). The effect of the subculture on the osteogenic potential was evaluated in OB-Cal and in its firstpassage (OB-Cal P1). After up to 14 days, all cultures were assayed for cell proliferation, alkaline phosphatase activity (ALP), mineralized extracellular matrix formation and the expression of the osteoblastic marker genes: runtrelated transcription factor 2 (RUNX2), ALP, osteocalcin (OC) and bone sialoprotein (BSP). To evaluate the effect of cell injection on bone regeneration, defects of 5 mm diameter were created in the calvaria of Wistar rats, that after 2 weeks were injected with 5 x 106 OB-Cal P1-derived osteoblasts. Vehicle injections were used as control. At the end of 4 weeks, the bone formation was evaluated by computerized microtomography and histological analysis. Data were compared by ANOVA, followed by the Student-Newman-Keuls test, or ttest, when appropriate, and the level of significance was set at 5%. The comparison of the osteogenic potential related to the embryonic origin showed that the OB-MS presented a greater proliferation but there was no difference between the cultures in the final event of the osteoblastic differentiation, that is the formation of mineralized matrix. However, as OB-Cal cultures showed greater gene expression of initial, intermediate and final markers of this differentiation; and considering that these cultures are those in which it is possible to obtain the largest number of cells, we selected these cultures for evaluating the bone formation induced by the cell therapy. In addition, the results showed that OB-Cal P1 still holds its osteogenic potential and despite being lower than that of OB-Cal as the subculture allows obtaining more cells, it has been considered as a good choice for cell therapy. In agreement with this, OB-Cal P1-derived osteoblasts injected into the bone defects were capable of inducing more bone formation than control, as revealed by microtomographic and histological analyzes. Therefore, it supports the idea that cell therapy with local injection of osteoblasts obtained from calvarial bone fragments is an adequate strategy to stimulate bone formation

O fator de crescimento transformante beta tipo 1, TGF-β1, é uma proteína extracelular homodimérica secretada por vários tipos celulares, que pode ter ação parácrina ou endócrina. Essa proteína está envolvida em processos celulares de diferenciação, proliferação, mobilidade e formação de matriz extracelular. Além disso, é parte importante dos processos de regeneração tecidual, atuando, de maneira decisiva, no reparo, atraindo macrófagos e fibroblastos para o local da injúria e estimulando a angiogênese. Assim, considerando o papel desse peptídeo no processo regenerativo, o uso de TGF-β1 como proteína terapêutica na área de Bioengenharia Tecidual é bastante promissor. Apesar disso, a venda dessa proteína, para fins terapêuticos, é inexistente no mercado e a proteína recombinante vendida, que só pode ser utilizada em pesquisas científicas, não é produzida nacionalmente e chega a custar R$200.000,00/mg. Nesse contexto, o objetivo do presente trabalho é desenvolver uma metodologia de produção do fator recombinante TGF-β1 em células de ovário de hamster chinês (CHO), visando à obtenção de níveis altos de rendimento, e, futuramente, a transferência da tecnologia de produção para a iniciativa privada, tornando possível seu uso na Medicina Regenerativa, sozinho ou em combinação com outros fatores de crescimento. O cDNA de TGF-β1 foi amplificado a partir de um banco de cDNA humano e clonado no vetor proprietário pNU1 de expressão de mamífero. A construção pNU1/TGF-β1 foi utilizada para transfectar estavelmente células CHO DG44 e uma estratégia de co-amplificação foi utilizada para selecionar células transfectantes com maior número de cópias da sequência correspondente a TGF-β1. Estas culturas foram submetidas ao processo de amplificação gênica com concentrações crescentes de metotrexato. Ensaios de Western Blot e ELISA foram realizados utilizando-se o meio condicionado pelas populações selecionadas e por clones superprodutores. Entre os 41clones obtidos, cinco apresentaram maiores níveis de produção de TGF-β1, entre 1.000 e 2.000 ng/mL. Estes clones foram selecionados para a realização de testes de atividade in vitro utilizando-se células A549, que permitem avaliar a transição epitélio-mesênquima. Um ensaio de cicatrização de feridas em peles do dorso de camundongos foi padronizado e utilizado para avaliar a atividade in vivo do clone que apresentou melhor resultado in vitro. A proteína TGF-β1 foi parcialmente purificada por HPLC em uma coluna de afinidade. Portanto, a proteína TGF-β1 humana recombinante foi produzida, apresentando atividade biológica in vitro e in vivo, sendo capaz de reparar eficientemente feridas cutâneas. Essa iniciativa pode oferecer aos pacientes uma alternativa para o tratamento de lesões teciduais, acelerando a cicatrização de feridas e o reparo de tecidos. / The transforming growth factor beta 1, TGF-β1, is a homodimeric extracellular protein secreted by several cell types, which may have paracrine or endocrine action. This protein is involved in cellular processes of differentiation, proliferation, mobility and formation of extracellular matrix. In addition, it is an important part of the tissue regeneration processes, acting decisively on repair, attracting macrophages and fibroblasts to the site of injury and stimulating angiogenesis. Therefore, considering the role of this peptide in the regenerative process and the use of TGF-β1 as a therapeutic protein in the field of Tissue Bioengineering is very promising. Despite this, the sale of this protein for therapeutic purposes is nonexistent in the market and the recombinant protein available in the market, which can only be used in scientific research, is not produced nationally and the costs are in the order of R$ 200,000.00/mg. In this context, the objective of the present work is to develop a methodology for the production of the TGF-β1 recombinant factor in Chinese hamster ovary (CHO) cells, aiming at obtaining high yields, and, in the future, transfering the production technology to the private initiative, allowing its use in Regenerative Medicine, alone or in combination with other growth factors. The TGF-β1 cDNA was amplified from a human cDNA library and cloned into the proprietary pNU1 mammalian expression vector. The pNU1/TGF-β1 construct was used to stably transfect CHO DG44 cells, and a co-amplification strategy was used to select transfectant cells with the largest number of gene copies. These cultures were subjected to the process of gene amplification with methotrexate. Western Blot and ELISA were used to assay the conditioned medium obtained from the selected cell populations and from overproducing cell clones. Among the 41 clones obtained, five presented higher levels of TGF-β1 production, between 1,000 and 2,000 ng/mL. These clones were selected for in vitro activity testing using A549 cells to evaluate the epithelial-mesenchymal transition. Awound healing assay on mouse dorsal skin was standardized and used to evaluate the in vivo activity of the cell clone which displayed the highest result in vitro. The TGF-β1 protein was partially purified by HPLC on an affinity column. Therefore, the recombinant human TGF-β1 protein was produced and shown to display biological activity both in vitro and in vivo, being able to eficiently repair cutaneous wounds. This initiative may provide patients with an alternative treatment for tissue damage, accelerating wound healing and tissue repair.

Currently, the only cure for liver failure is orthotopic liver transplantation. However, there are insufficient donor organs available to treat every patient on the transplant list and many die before they are able to receive a liver transplant. The bioartificial liver (BAL) device is a potential extracorporeal treatment strategy utilising hepatocytes or hepatocyte-like cells (HLCs) within a bioreactor to recapitulate normal liver function and therefore ‘bridge’ a patient with liver failure until they receive a transplant. The work in this thesis utilised tissue engineering methods to develop novel approaches to BAL device design through development and characterisation of a polymer membrane scaffold (“PX”) for hollow fibre bioreactor (HFB) culture and a HLC source generated from the transdifferentiation of pancreatic AR42J-B13 (B13) cells. A flat sheet membrane model was used for the development of asymmetrical, hydrophobic polystyrene (PS) phase inversion membranes. Oxygen plasma significantly increased PS membrane surface wettability through addition of oxygen functional groups to create an environment conducive for cell culture. The treated membrane was henceforth referred to as “PX”. The culture medium HepatoZYME+ was investigated for its ability to induce transdifferentiation of B13 cells to HLCs and maintain the hepatic phenotype. Overall, HepatoZYME+-cultured cells experienced viability loss. A diluted version, “50:50”, showed induction of the hepatic markers carbamoylphosphate synthetase-1 (CPS-1) and HNF4α, as well as a change towards a HLC morphology. When using 50:50 as a maintenance medium, transdifferentiated HLCs retained loss of pancreatic amylase and also induction of hepatic markers, with comparable serum albumin secretion to the established Dex + OSM treatment. However, culture viability in 50:50 was still compromised. Therefore, HepatoZYME+ based media were deemed unsuitable for induction and maintenance compared to Dex-based protocols. PX flat sheet membranes were able to support culture of B13 cells and also the human osteosarcoma cell line, MG63, demonstrating improved cell attachment over non-surface treated PS membranes. PX membranes supported transdifferentiation of B13 cells to HLCs, presenting with loss of pancreatic amylase, induction of the hepatic markers transferrin, GS and CPS-1 and serum albumin secretion. Furthermore, PX showed no change in mass or loss of culture surface area over 15 days in culture conditions. Together, the novel membrane material and the media formulation and feeding regime developed have strong potential to be translated to a HFB setting and guide future BAL device design.

This thesis presents the findings from an ethnography of stem cell science based on fieldwork with researchers in two connected laboratories in Scotland and the United States. It explores stem cell scientists' complicated interactions with live stem cell cultures within national projects of translational regenerative medicine. This analysis both draws upon and contributes to the social studies of biomedicine, reproductive studies and science and technology studies. I examine how stem cell scientists, involved in an international research initiative, navigate the challenging landscapes of translational regenerative medicine and attempt to transform fragile live cell cultures into successful biotechnical, medical and economic products. By considering translational regenerative medicine as an effort to reformulate the relationship between biology and technology in terms of applicability and utility, I illuminate tensions between the specific practices of care that enable stem cell growth in vitro and the elusive goals of national projects of biotechnological innovation. A major focus of this study is the means by which scientists in the two laboratories manage the inherent uncertainties of both cell culture and translational science. By exploring how researchers react to unstable and unpredictable cellular behaviour in the laboratory, while also managing the expectations of government and external funding bodies, I provide a portrait of the complex sociality of contemporary bioscience. In addition to the international collaboration between the two laboratories, I explore scientists' interdisciplinary work with medical specialists and public engagement with stakeholders in regenerative medicine. In doing so, I pay attention to the ways in which scientists themselves deal with and reflect on the relational and interdependent nature of their endeavours. Drawing on twenty-two months of ethnographic fieldwork and fifty qualitative interviews, I show how stem cell scientists' new engagement practices also inform scientific work and the care of stem cells in the laboratory. In short, I argue that translation of science across different sites at once creates and depends on new social relations between stem cells, people and communities. After providing an overview of the literature, central questions and methodology that frame this thesis, I introduce the opportunities and challenges that translational regenerative medicine goals create for the care of stem cells in vitro. From there, I zoom out beyond the tissue culture flask to demonstrate how the necessity for science applicability creates new responsibilities for scientists to connect with stakeholders in regenerative medicine outside of the laboratory. I conclude that a consideration of scientists' ties and societal links is significant for an understanding of the connection between the biological and the technological.