<p> Cell encapsulation aims at the delivery of a therapeutic protein to a patient from transplanted cells. Conventional approaches involve immune-isolating cell lines that have been genetically modified to express a therapeutic protein, in alginate-based microcapsules. The long-term success of this approach hinges on the structural stability of the microcapsules, as well as their ability to maintain an environment suitable for the long-term survival of encapsulated cells. The most commonly studied type of microcapsule is the alginate-poly-Llysine-alginate (APA) microcapsule. However, the main concern with AP A microcapsules is the Joss of structural integrity during long-term implantation due to the exchange of calcium ions with other physiological ions, as well as the loss of the polyelectrolyte overcoats. </p> <p> In order to increase the structural stability of the microcapsules, we developed and characterized a number of synthetic polyelectrolytes that undergo phase separation upon complexation, and which are capable of forming covalent cross-links. These reactive polyelectrolytes are designed to take the place of poly-L-lysine and the outer alginate layer. We also explored combining cross-linkable synthetic polyanions with sodium alginate to strengthen the Ca Alginate core, by forming a core cross-linked network extending throughout the microcapsules. The polyelectrolyte complexes, encapsulation processes and microcapsule properties were studied in detail using extensive characterization techniques, including collaborative work on cell viability and host-immune response. </p> <p> Overall, this thesis describes a novel approach and prom1smg materials for cell encapsulations that offer enhanced microcapsule resistance to chemical and mechanical stresses, while preserving the desired biocompatibility. These materials may ultimately be useful for clinical immunosuppressive therapies. </p> / Thesis / Doctor of Philosophy (PhD)

O diabetes mellitus do tipo 1 (DM1) é uma doença causada pela destruição autoimune das células-&#946; produtoras de insulina do pâncreas. O transplante de ilhotas pancreáticas é um procedimento tecnicamente simples sendo uma alternativa terapêutica interessante para o DM1. Entretanto, a oferta limitada de pâncreas de doadores falecidos e a necessidade de imunossupressão crônica são fatores que limitam a aplicabilidade dessa modalidade de transplante. Neste trabalho foram estudadas duas estratégias que visam oferecer soluções aos fatores limitantes do transplante de ilhotas pancreáticas. Na primeira parte do trabalho, o mecanismo molecular que dirige o processo de diferenciação de células-tronco embrionárias murinas (murine embryonic stem cells, mESCs) em células produtoras de insulina (insulin producing cells, IPCs) foi analisado visando otimizar o processo de diferenciação. Nós selecionamos o gene Thioredoxin interacting protein (Txnip), diferencialmente expresso ao longo da diferenciação &#946;-pancreática, para realizar um estudo funcional através da modificação genética de mESCs. Os resultados obtidos permitiram verificar que a inibição de Txnip na diferenciação &#946;-pancreática pode induzir a diferenciação de IPCs com maior expressão de marcadores de células- e mais responsivas ao estímulo de glicose. Além disso, o modelo de zebrafish permitiu elucidar in vivo o papel de Txnip durante a organogênese pancreática, revelando que a inibição desse gene é capaz de aumentar a massa de células-&#946; através do estimulo de células presentes no ducto extra-pancreático. Dessa forma, a inibição de Txnip pode aprimorar os protocolos para obtenção de IPCs a partir de células-tronco pluripotentes. A exposição crônica a agentes imunossupressores diabetogênicos e a perda de componentes de matriz extracelular durante o isolamento de ilhotas pancreáticas são causas para a perda de funcionalidade do enxerto. Dessa forma, na segunda parte do trabalho, um biomaterial inovador foi desenvolvido, contendo um polímero de laminina (polilaminina, PLn) para o encapsulamento e a imunoproteção de ilhotas pancreáticas. As cápsulas produzidas com o biomaterial desenvolvido, Bioprotect-Pln, são térmica- e mecanicamente estáveis, além de serem biocompatíveis e capazes de imunoproteger ilhotas pancreáticas humanas in vitro. O encapsulamento com Bioprotect-Pln preserva a funcionalidade de ilhotas pancreáticas. Além disso, quando cápsulas vazias de Bioprotect-Pln foram implantadas em camundongos imunocompetentes, houve atenuação da resposta inflamatória ao implante, uma das principais causas para perda de funcionalidade de enxertos encapsulados. Os resultados obtidos indicam que a presença de polilaminina na malha capsular induz uma resposta anti-inflamatória que pode beneficiar a preservação do enxerto de ilhotas pancreáticas encapsuladas. Atualmente, o transplante de ilhotas pancreáticas é visto como a terapia celular mais promissora para atingir a independência de insulina em pacientes de DM1, porém, a aplicabilidade desse transplante ainda é limitada. Este trabalho contribuiu para a elucidação dos mecanismos moleculares que podem aprimorar o processo de diferenciação de célulastronco pluripotentes em IPCs, estabelecendo uma fonte alternativa de células para a terapiade reposição, e, também, estabeleceu um biomaterial inovador, capaz de diminuir a resposta inflamatória ao implante de microcápsulas e de imunoproteger células microencapsuladas. Desta forma, este trabalho contribui para o estabelecimento da terapia de reposição celular para pacientes de DM1. / Type 1 diabetes mellitus (DM1) is a disease caused by the autoimmune destruction of insulin-producing pancreatic &#946;-cells. Pancreatic islet transplantation is a technically simple procedure and an interesting alternative therapy for DM1, however, the limited supply of cadaveric donated pancreas and the need of life-long immunosuppression are factors which limit its applicability. In the present work, two strategies were employed aiming at establishing viable solutions for the factors limiting pancreatic islet transplantation. In the first part of this study, the molecular mechanism which drives differentiation of murine embryonic stem cells (mESCs) into insulin producing cells (IPCs) was analyzed in order to optimize the differentiation process. The Thioredoxin interacting protein (Txnip) gene, which is differentially expressed along -pancreatic differentiation, was selected to undergo a functional analysis by genetically modifying mESCs. The results allowed us to verify that Txnip inhibition during the &#946;-pancreatic differentiation process can induce differentiation of IPCs displaying higher expression of &#946;-cell markers and being more responsive to glucose stimuli. In addition, the zebrafish model allowed us to elucidate in vivo the role of Txnip during pancreatic organogenesis, revealing that its inhibition is able to increase the mass of &#946;-cells through stimulation of extra-pancreatic ductal cells. Therefore, Txnip inhibition may turbinate IPCs differentiation from pluripotent stem cells. The chronic exposure to diabetogenic immunosuppressive agents and the loss of extracellular matrix components during isolation of pancreatic islets are probable causes for the loss of pancreatic islet graft functionality. Therefore, in the second part of this study, an innovative biomaterial was developed by incorporating a laminin polymer (polylaminin, PLn) for the encapsulation and immunoprotection of pancreatic islets. The capsules produced with the novel biomaterial, Bioprotect-Pln, are biocompatible, thermally and mechanically stable and are able to immunoprotect human pancreatic islets in vitro. Encapsulation with Bioprotect-Pln preserves the functionality of pancreatic islets. In addition, when empty Bioprotect-Pln capsules were implanted into immunocompetent mice, an attenuation of the inflammatory response to the implant occurred, this being one of the main causes of encapsulated graft loss. The results indicate that polylaminin addition to the capsular mesh induces an anti-inflammatory response which may favor preservation of the engrafted encapsulated pancreatic islets. Pancreatic islet transplantation is currently seen as the most promising cell therapy to achieve insulin independence in DM1 patients, however, the applicability of this transplant is still limited. This work contributed to the elucidation of the molecular mechanisms which can turbinate the differentiation of pluripotent stem cells into IPCs, establishing an alternative source of cells for the replacement therapy, and, also, established an innovative biomaterial which is able to decrease the inflammatory response to the graft, thereby immunoprotecting the microencapsulated cells. Therefore, this work contributes to the establishment of the cell replacement therapy for DM1 patients.

O diabetes mellitus do tipo 1 (DM1) é uma doença causada pela destruição autoimune das células-&#946; produtoras de insulina do pâncreas. O transplante de ilhotas pancreáticas é um procedimento tecnicamente simples sendo uma alternativa terapêutica interessante para o DM1. Entretanto, a oferta limitada de pâncreas de doadores falecidos e a necessidade de imunossupressão crônica são fatores que limitam a aplicabilidade dessa modalidade de transplante. Neste trabalho foram estudadas duas estratégias que visam oferecer soluções aos fatores limitantes do transplante de ilhotas pancreáticas. Na primeira parte do trabalho, o mecanismo molecular que dirige o processo de diferenciação de células-tronco embrionárias murinas (murine embryonic stem cells, mESCs) em células produtoras de insulina (insulin producing cells, IPCs) foi analisado visando otimizar o processo de diferenciação. Nós selecionamos o gene Thioredoxin interacting protein (Txnip), diferencialmente expresso ao longo da diferenciação &#946;-pancreática, para realizar um estudo funcional através da modificação genética de mESCs. Os resultados obtidos permitiram verificar que a inibição de Txnip na diferenciação &#946;-pancreática pode induzir a diferenciação de IPCs com maior expressão de marcadores de células- e mais responsivas ao estímulo de glicose. Além disso, o modelo de zebrafish permitiu elucidar in vivo o papel de Txnip durante a organogênese pancreática, revelando que a inibição desse gene é capaz de aumentar a massa de células-&#946; através do estimulo de células presentes no ducto extra-pancreático. Dessa forma, a inibição de Txnip pode aprimorar os protocolos para obtenção de IPCs a partir de células-tronco pluripotentes. A exposição crônica a agentes imunossupressores diabetogênicos e a perda de componentes de matriz extracelular durante o isolamento de ilhotas pancreáticas são causas para a perda de funcionalidade do enxerto. Dessa forma, na segunda parte do trabalho, um biomaterial inovador foi desenvolvido, contendo um polímero de laminina (polilaminina, PLn) para o encapsulamento e a imunoproteção de ilhotas pancreáticas. As cápsulas produzidas com o biomaterial desenvolvido, Bioprotect-Pln, são térmica- e mecanicamente estáveis, além de serem biocompatíveis e capazes de imunoproteger ilhotas pancreáticas humanas in vitro. O encapsulamento com Bioprotect-Pln preserva a funcionalidade de ilhotas pancreáticas. Além disso, quando cápsulas vazias de Bioprotect-Pln foram implantadas em camundongos imunocompetentes, houve atenuação da resposta inflamatória ao implante, uma das principais causas para perda de funcionalidade de enxertos encapsulados. Os resultados obtidos indicam que a presença de polilaminina na malha capsular induz uma resposta anti-inflamatória que pode beneficiar a preservação do enxerto de ilhotas pancreáticas encapsuladas. Atualmente, o transplante de ilhotas pancreáticas é visto como a terapia celular mais promissora para atingir a independência de insulina em pacientes de DM1, porém, a aplicabilidade desse transplante ainda é limitada. Este trabalho contribuiu para a elucidação dos mecanismos moleculares que podem aprimorar o processo de diferenciação de célulastronco pluripotentes em IPCs, estabelecendo uma fonte alternativa de células para a terapiade reposição, e, também, estabeleceu um biomaterial inovador, capaz de diminuir a resposta inflamatória ao implante de microcápsulas e de imunoproteger células microencapsuladas. Desta forma, este trabalho contribui para o estabelecimento da terapia de reposição celular para pacientes de DM1. / Type 1 diabetes mellitus (DM1) is a disease caused by the autoimmune destruction of insulin-producing pancreatic &#946;-cells. Pancreatic islet transplantation is a technically simple procedure and an interesting alternative therapy for DM1, however, the limited supply of cadaveric donated pancreas and the need of life-long immunosuppression are factors which limit its applicability. In the present work, two strategies were employed aiming at establishing viable solutions for the factors limiting pancreatic islet transplantation. In the first part of this study, the molecular mechanism which drives differentiation of murine embryonic stem cells (mESCs) into insulin producing cells (IPCs) was analyzed in order to optimize the differentiation process. The Thioredoxin interacting protein (Txnip) gene, which is differentially expressed along -pancreatic differentiation, was selected to undergo a functional analysis by genetically modifying mESCs. The results allowed us to verify that Txnip inhibition during the &#946;-pancreatic differentiation process can induce differentiation of IPCs displaying higher expression of &#946;-cell markers and being more responsive to glucose stimuli. In addition, the zebrafish model allowed us to elucidate in vivo the role of Txnip during pancreatic organogenesis, revealing that its inhibition is able to increase the mass of &#946;-cells through stimulation of extra-pancreatic ductal cells. Therefore, Txnip inhibition may turbinate IPCs differentiation from pluripotent stem cells. The chronic exposure to diabetogenic immunosuppressive agents and the loss of extracellular matrix components during isolation of pancreatic islets are probable causes for the loss of pancreatic islet graft functionality. Therefore, in the second part of this study, an innovative biomaterial was developed by incorporating a laminin polymer (polylaminin, PLn) for the encapsulation and immunoprotection of pancreatic islets. The capsules produced with the novel biomaterial, Bioprotect-Pln, are biocompatible, thermally and mechanically stable and are able to immunoprotect human pancreatic islets in vitro. Encapsulation with Bioprotect-Pln preserves the functionality of pancreatic islets. In addition, when empty Bioprotect-Pln capsules were implanted into immunocompetent mice, an attenuation of the inflammatory response to the implant occurred, this being one of the main causes of encapsulated graft loss. The results indicate that polylaminin addition to the capsular mesh induces an anti-inflammatory response which may favor preservation of the engrafted encapsulated pancreatic islets. Pancreatic islet transplantation is currently seen as the most promising cell therapy to achieve insulin independence in DM1 patients, however, the applicability of this transplant is still limited. This work contributed to the elucidation of the molecular mechanisms which can turbinate the differentiation of pluripotent stem cells into IPCs, establishing an alternative source of cells for the replacement therapy, and, also, established an innovative biomaterial which is able to decrease the inflammatory response to the graft, thereby immunoprotecting the microencapsulated cells. Therefore, this work contributes to the establishment of the cell replacement therapy for DM1 patients.

Islet transplantation has emerged as a promising cell-based therapy for the treatment of diabetes, but its clinical efficacy remains limited by deleterious host responses that underlie islet destruction. In this dissertation, we describe the assembly of cell surface-supported thin films that confer molecular-level control over the composition and biophysicochemical properties of the islet surface with implications for improving islet engraftment. Specifically, the process of layer-by-layer (LbL) polymer self assembly was employed to generate nanothin films of diverse architecture with tunable properties directly on the extracellular surface of individual islets. Importantly, these studies are the first to report in vivo survival and function of nanoencapsulated cells, and have helped establish a conceptual framework for translating the diverse applications of LbL films to cellular interfaces. Additionally, through proper design of film constituents, coatings displaying ligands and bioorthogonally reactive handles may be generated, providing a modular strategy for incorporating exogenously derived regulators of host responses alongside native constituents of the islet surface. Towards this end, a strategy was developed to tether thrombomodulin to the islet surface in a site-specific manner, thereby facilitating local generation of the powerful anti-inflammatory agent, activated protein C. Collectively, this work offers novel biomolecular strategies for cell surface engineering with broad biomedical and biotechnological applications in cell-based therapeutics and beyond.

Skeletal muscle injuries and muscle degenerative diseases pose significant challenges to the healthcare. Surgical interventions are restricted due to tissue availability, donor site morbidity and alterations to tissue biomechanics. Current cell-based therapies are hindered by low survival and long-term engraftment for the transplanted cells due to the lack of appropriate supportive microenvironment (cell niche) in the injured muscle. Therefore, there is a critical need for developing strategies that provide cellular and structural support in the regeneration of functional muscle. In the present work, a bioengineered cell niche mimicking the native skeletal muscle microenvironment has been developed for skeletal muscle regenerative engineering. It is hypothesized that the bioengineered scaffolds with appropriate structural and cell instructive properties will support myoblast alignment and function, as well as promote the myogenic responses in clinically relevant skeletal muscle injuries. The current work utilized a three-pronged approach to design biomaterial scaffolds to aid in skeletal muscle regeneration. In the first part, aligned poly(lactide-co-glycolide) (PLGA) fiber scaffolds mimicking the oriented muscle fiber microenvironment with fiber diameters of 335±154 nm (nanoscale), 1352±225 nm (microscale) and 3013±531 nm (microscale) were fabricated and characterized. Myoblasts were found to respond to fiber diameter as observed from the differences in cell alignment, cell elongation, cell spreading area, proliferation and differentiation. <i>In vivo</i> study demonstrated the potential of using microscale fiber scaffolds to improve myogenic potential in the <i>mdx</i> mouse model. In the second part, we designed, synthesized, and characterized an implantable glycosaminoglycan-based composite hydrogel consisting of hyaluronic acid, chondroitin sulfate and polyethylene glycol (HA-CS) with tailored structural and mechanical properties for skeletal muscle regeneration applications. We demonstrated that HA-CS hydrogels provided a suitable microenvironment for <i>in vitro</i> myoblast proliferation and differentiation. Furthermore, <i>in vivo</i> studies using a volumetric muscle loss model in the mouse quadriceps showed that HA-CS hydrogels integrated with the surrounding host tissue and facilitated <i>de novo</i> myofiber generation, angiogenesis, nerve innervation and minimized scar tissue formation. In the third part, we investigated the effects of PC12 secreted signaling factors in modulating C2C12 myoblast behavior. We showed that PC12 conditioned media modulated myoblast proliferation and differentiation in both 2D culture and 3D aligned electrospun fiber scaffold system in a dose dependent manner. We also demonstrated the biomimetic HA-CS hydrogel system enabled 3D encapsulation of PC12 cells secreting signaling factors and promoted survival and proliferation of myoblasts in co-culture. Further proteomics analysis identified a total of 2088 protein/peptides from the secretome of the encapsulated PC12 cells and revealed the biological role and overlapping functions of nerve secreted proteins for skeletal muscle regeneration, potentially through regulating myoblast behavior, nerve function, and angiogenesis. These set of experiments not only provide critical insight on exploiting the interactions between muscle cells and their microenvironment, but they also open new avenues for developing advanced bioengineered scaffolds for regenerative engineering of skeletal muscle tissues.<br>

No description available.

The use of cell transplantation to treat enzyme deficiency disorders is limited by the immune response targeted against foreign tissue or the use of life-long immunosuppressants. Hiding cells from the immune system in an encapsulation device is promising. Cells encapsulated within an anionic calcium alginate hydrogel bead are protected through a semi-permeable membrane formed by polycation, poly-L-lysine (PLL). A final layer of alginate is added to hide the cationic PLL surface but this has proved to be difficult creating capsules which are prone to fibrotic overgrowth, blocking exchange of nutrients, waste and therapeutic enzymes through the capsule. For long term applications these capsules need to be both biocompatible and mechanically robust. This thesis aims to address the biocompatibility issue of high cationic surface charge by synthesizing polycations of reduced charge using N-(3- aminopropyl)methacrylamide hydrochloride (APM) and N-(2- hydroxypropyl)methacrylamide (HPM) and study the associated mechanical properties of the capsules using micropipette aspiration. Micropipette aspiration was applied and validated for alginate based capsules (gel and liquid core) to quantify stiffness. Varying ratios of APM were used to control the overall charge of the polycations formed while HPM was incorporated as a neutral, hydrophilic, nonfouling comonomer. The molecular weight (MW) was controlled by using reversible addition-fragmentation chain transfer (RAFT) polymerization. The biocompatibility of these polymers was tested by cell adhesion and proliferation of 3T3 fibroblasts onto APM/HPM copolymer functionalized surfaces and by solution toxicity against C2C12 myoblasts. The ability for the APM/HPM copolymers to bind to alginate and form capsules was also assessed, along with the integrity and stiffness of the capsule membrane with or without additional covalent cross-linking by reactive polyanion, poly(methacrylic acid-co-2-vinyl-4,4- dimethylazlactone) (PMV60). Thermo-responsive block copolymers of N-isopropylacrylamide (NIPAM) and 2- hydroxyethylacrylamide (HEA) were also synthesized as potential drug delivery nanoparticles, showing control over micelle morphology with varying NIPAM to HEA ratios. / Thesis / Doctor of Science (PhD) / The treatment of enzyme deficiency disorders by cell transplantation is limited by the immune attack of foreign tissue in absence of immunosuppressants. Cells protected in an encapsulation device has shown promise. Poly-L-lysine, a widely used membrane material in these protective capsules, binds to the anionic gel entrapping living cells because it is highly cationic. The high cationic charge is difficult to hide causing the immune system to build tissue around the capsule, preventing the encapsulated cells from exchanging nutrients and therapeutic enzymes. This thesis aims to replace poly-L-lysine by synthesizing a series of more biocompatible materials of decreasing cationic charge. These materials were studied for the ability to support tissue growth and form stable capsules. The membrane strength was measured using an aspiration method validated for these types of capsules. Reducing the cationic charge of the materials increased the biocompatibility of the capsule membrane but also made for weaker membranes.

This thesis is focused on the design and development of an approach, allowing the fabrication of biocompatible/biodegradable self-rolled polymer tubes, which are sensitive to stimuli at physiological conditions, can be homogenously filled with cells and are able to self-assemble into a complex 3D construct with uniaxially aligned pores. These constructs are aimed to recreate the microstructure of tissues with structural anisotropy, such as of muscles and bones. The approach consists of two steps of self-assembly. As a first step, cells are adsorbed on the top of an unfolded bilayer; triggered rolling results in a parallel encapsulation of cells inside the tubes. As a second step, the formed self-rolled tubes with encapsulated cells can be assembled in a uniaxial tubular scaffold. Three polymer systems were designed and investigated in the present work in order to allow triggered folding of the bilayer. These systems allow either reversible or irreversible tube formation. The possibility to encapsulate microobjects inside self-rolled polymer tubes was demonstrated on the example of silica particles, yeast cells and mammalian cells. At conditions when bilayer film is unfolded, particles or cells were deposited from their aqueous dispersion on the top of bilayer. An appropriate change of conditions triggers folding of the bilayer and results in encapsulation of particles or cells inside the tubes. One way swelling of an active polymer allows irreversible encapsulation of cells in a way that tubes do not unroll and cells cannot escape. It was demonstrated that encapsulated cells can proliferate and divide inside the tubes for a long period of time. Since used polymers are optically transparent, encapsulated cells can be easily observed using optical and fluorescent microscopy. Reversible swelling of an active polymer provides the possibility to release encapsulated objects. It was demonstrated that in aqueous media microtubes possessing small amount of negatively charged groups on external walls self-assemble in the presence of oppositely charged microparticles that results in a formation of 3D constructs. In obtained aggregates tubes and therefore pores were well-aligned and the orientation degree was extremely high. Moreover, the approach allows the design of porous materials with complex architectures formed by tubes of different sorts. The assembly of cell-laden microtubes results in a formation of uniaxial tubular scaffold homogeneously filled with cells. The results presented in this work demonstrate that the proposed approach is of practical interest for biotechnological applications. Self-rolled tubes can be filled with cells during their folding providing the desired homogeneity of filling. Individual tubes of different diameters could be used to investigate cell behaviour in confinement in conditions of structural anisotropy as well as to mimic blood vessels. Due to their directionality tubes could be used to guide the growth of cells that is of interest for regeneration of neuronal tissue. Reversibly foldable films allow triggered capture and release of the cells that could be implemented for controlled cell delivery. In perspective, self-assembled 3D constructs with aligned pores could be used for bottom-up engineering of the scaffolds, mimicking such tissues as cortical bone and skeletal muscle, which are characterized by repeating longitudinal units. Such constructs can be also considered as a good alternative of traditional 2D flat cell culture.

This thesis is focused on the design and development of an approach, allowing the fabrication of biocompatible/biodegradable self-rolled polymer tubes, which are sensitive to stimuli at physiological conditions, can be homogenously filled with cells and are able to self-assemble into a complex 3D construct with uniaxially aligned pores. These constructs are aimed to recreate the microstructure of tissues with structural anisotropy, such as of muscles and bones. The approach consists of two steps of self-assembly. As a first step, cells are adsorbed on the top of an unfolded bilayer; triggered rolling results in a parallel encapsulation of cells inside the tubes. As a second step, the formed self-rolled tubes with encapsulated cells can be assembled in a uniaxial tubular scaffold. Three polymer systems were designed and investigated in the present work in order to allow triggered folding of the bilayer. These systems allow either reversible or irreversible tube formation. The possibility to encapsulate microobjects inside self-rolled polymer tubes was demonstrated on the example of silica particles, yeast cells and mammalian cells. At conditions when bilayer film is unfolded, particles or cells were deposited from their aqueous dispersion on the top of bilayer. An appropriate change of conditions triggers folding of the bilayer and results in encapsulation of particles or cells inside the tubes. One way swelling of an active polymer allows irreversible encapsulation of cells in a way that tubes do not unroll and cells cannot escape. It was demonstrated that encapsulated cells can proliferate and divide inside the tubes for a long period of time. Since used polymers are optically transparent, encapsulated cells can be easily observed using optical and fluorescent microscopy. Reversible swelling of an active polymer provides the possibility to release encapsulated objects. It was demonstrated that in aqueous media microtubes possessing small amount of negatively charged groups on external walls self-assemble in the presence of oppositely charged microparticles that results in a formation of 3D constructs. In obtained aggregates tubes and therefore pores were well-aligned and the orientation degree was extremely high. Moreover, the approach allows the design of porous materials with complex architectures formed by tubes of different sorts. The assembly of cell-laden microtubes results in a formation of uniaxial tubular scaffold homogeneously filled with cells. The results presented in this work demonstrate that the proposed approach is of practical interest for biotechnological applications. Self-rolled tubes can be filled with cells during their folding providing the desired homogeneity of filling. Individual tubes of different diameters could be used to investigate cell behaviour in confinement in conditions of structural anisotropy as well as to mimic blood vessels. Due to their directionality tubes could be used to guide the growth of cells that is of interest for regeneration of neuronal tissue. Reversibly foldable films allow triggered capture and release of the cells that could be implemented for controlled cell delivery. In perspective, self-assembled 3D constructs with aligned pores could be used for bottom-up engineering of the scaffolds, mimicking such tissues as cortical bone and skeletal muscle, which are characterized by repeating longitudinal units. Such constructs can be also considered as a good alternative of traditional 2D flat cell culture.

In novel drug delivery system, the encapsulation of therapeutic cells in microparticles has great promises for the treatment of a range of health con- ditions. Therefore, the encapsulation material and technology are of great importance to the validity and efficiency of the advanced medical therapy. Several unsolved challenges in regards to versatile microparticle synthesis ma- terials and methods form the main obstacle for a translation of novel cell therapy concepts from research to clinical practice. Thiol-ene based polymer systems have emerged and gained great popular- ity in material development in general and in biomedical applications specif- ically. The thiol-ene platform is broad and therefore of interest for a variety of applications. At the same time, many aspects of this material platform are largely unexplored, for example material and manufacturing technology developments for microfluidic applications . In this Ph.D. thesis, thiol-ene materials are explored for use in cell encap- sulation. The marriage of these two technology fields breeds the possibility for a novel microfluidic cell encapsulation approach using a novel encapsulation material. To this end, several new manufacturing technologies for thiol-ene and thiol-ene-epoxy droplet microfluidic devices were developed. Moreover, core-shell microparticle synthesis for cell encapsulation based on a novel co- synthesis concept using a thiol-ene based material was developed and inves- tigated. Finally, a thiol-ene-epoxy system was also used for the formation of microwells and microchannels that improve protein analysis on microarrays. The first part of the thesis presents the background and state-of-the-art technologies in regards to cell therapy, microfluidics, and thiol-ene based ma- terials. In the second part of the thesis, a novel manufacturing approach of thiol-ene-epoxy material as well as core-shell particle co-synthesis in micro- fluidics using thiol-ene based material are presented and characterized. The third part of the thesis presents the cell viability studies of encapsulated cells using the novel encapsulation material and method. In the final part of the thesis, two applications of thiol-ene-epoxy gaskets for protein detection mi- croarrays are presented. / Inkapsling av levande celler i mikrokapslar för terapeutiska ändamål är mycket lovande för frmatida behandling av många olika sjukdomar. Emeller- tid är en behandlings effektivitet i hög grad beroende av vilka material som används för inkapsling och vilken teknisk lösning som används för att ska- pa mikrokapslarna. För närvarande återstår det många utmaningar för att omvandla grundforskningresultat till klinisk verklighet, vilken kräver mer än- damålsenliga tillvägagångssätt för att tillverka mikrokapslar i material som är kompatibla med användningsområdena. De senaste åren har tiol-en baserade polymerer har blivit mycket använda för materialutveckling i stort och för biomedicinska tillämpningar i synnerhet. Med tiol-en kemi kan en mycket stor mängd helt olika syntetiska material framställas, vilket gör tiol-ener intressanta för en mängd applikationer. För närvarande är dock mycket inom denna materialklass outforskat, t.ex. inom material och tillverkningmetodik för mikrofluidiktillämpningar. I denna avhandling används tiol-ener för cellinkapsling. Sammanslagning av dessa teknologier möjliggör en ny typ av cellinkapsling med nya materi- alegenskaper. En mängd olika tillverkningssätt där tiol-en eller tiol-en-epoxi används för droplet-mikrofluidiksystem utvecklades. Core-shell mikrokapsel- syntes för cell-inkapsling baserat på en ny metod för samtidig syntes av både core och shell utvecklades och karaktäriserades. Slutligen utvecklades ett tiol- en-epoxi system för enkel integrering med proteinmikroarrayer på objektsglas. I avhandlingens första del presenteras bakgrund och dagens bästa teknolo- gier för terapeutisk cellinkapsling, mikrofluidik och tiol-en baserade material. I avhandlingens andra del presenteras en ny tillverkningsmetod för mikro- strukturerade tiol-en-epoxi artiklar och samtidig syntes av core och shell för mikrokapslar med användande av mikrofluidik. I den tredje delen presenteras cellöverlevandsstudier för de celler som inkapslats med de nya materialen och de nyutvecklade metoderna. I den avslutande delen beskrivs två specifika fall där tiol-en-epoxi komponenter används för proteindetektion och mikroarrayer. / <p>QC 20171122</p>