In vivo, les cellules souches résident au sein de niches, des microenvironnements extrêmement spécialisés qui leur permettent de réguler leur prolifération ainsi que leur différentiation en cellules matures et fonctionnelles. Ces microenvironnements sont caractérisés par des interactions cellules-cellules, la présence de facteurs de croissance et de cytokines, et une matrice extracellulaire, qui fournissent des signaux qui permettent de contrôler le comportement des cellules souches.Bone morphogenetic protein 2 (BMP-2) est un facteur de croissance ostéoinductif qui est impliqué dans toutes les étapes de la différentiation ostéoblastique, ainsi que dans la transdifférentiation de myoblastes vers une lignée ostéogénique. In vivo, ce facteur existe à la fois en solution et lié à la matrice extracellulaire. Bien que ces deux modes de présentation influencent les comportements cellulaires de façon distincte, leur rôle dans le microenvironnement de la niche et leur pertinence fonctionnelle dans la genèse de la réponse biologique n’a presque pas été étudié à l’échelle cellulaire. Ici nous avons utilisé l’affinité naturelle de la BMP-2 pour la fibronectine afin de créer des micromotifs de BMP-2 de taille cellulaire sur des biomatériaux mous.Cette technique nous a permis de contrôler simultanément la présentation spatiale de la BMP-2 liée à la fibronectine, ainsi que l’étalement cellulaire. Ces micromotifs ont induit une organisation d’actine et d’adhésions focales autour du noyau, et a déclenché la phosphorylation et la translocation nucléaire de SMAD1/5/8 dans des myoblastes murins C2C12 et des cellules souches mésenchymateuses. Ceci est un indicateur précoce de leur transdifférentiation ostéoblastique. Nous avons trouvé que l’étalement cellulaire lui-même facilitait la phosphorylation de SMAD1/5/8 dépendante de la BMP-2, et avons démontré que la signalisation SMAD médiée par la fibronectine et la BMP-2 dépendait de LIM kinase 2 et de ROCK, plutôt que d’une activation de la myosine II.Nous avons également pu utiliser cet outil pour étudier les cinétiques d’adhésion et d’étalement, les changements d’organisation du cytosquelette dépendants du mode de présentation de la BMP-2, et la signalisation entre la matrice et la cellule, médiée par les intégrines. Nous avons obtenu des résultats préliminaires suggérant un effet du mode de présentation de la BMP-2 sur les forces cellulaires, suggérant que le mode de présentation des facteurs de croissance pourrait être pertinent à d’autres mécanismes cellulaires tels que la mécanotransduction.Dans l’ensemble, nous résultats montrent que les patterns de BMP-2 liée à la fibronectine sont un outil utile à l’étude de mécanismes cellulaires. De façon plus large, notre approche pourrait être adaptée à d’autres combinaisons de protéines de la matrice et de facteurs de croissance, ouvrant ainsi une avenue fascinante pour recréer in vitro des niches spécifiques aux tissus. / In vivo, stem cells reside within niches, highly specialized microenvironments which allow them to regulate their self-renewal and differentiation into mature and functional cells. These microenvironments are characterized by cell-cell interactions, the presence of growth factors and cytokines, and an extracellular matrix, all of which provide cues that control stem cell behavior.Bone morphogenetic protein 2 (BMP-2) is an osteoinductive growth factor which is involved in all stages of osteoblastic differentiation, as well as the transdifferentiation of myoblasts toward an osteogenic lineage. In vivo, it exists both in solution and bound to the ECM. While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2 on soft biomaterials.This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic transdifferentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8, and demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation.We were also able to use this tool to investigate adhesion and spreading kinetics, changes in cytoskeletal organization depending on BMP-2 presentation mode, and reciprocal integrin-mediated signaling between the ECM and the cell. We were able to show preliminary results suggesting an effect of BMP-2 presentation mode on cellular forces, suggesting that growth factor presentation may be relevant to other cellular mechanisms like mechanotransduction.Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

The primary research aim in this thesis is to demonstrate the versatility of porous silicon based nanomaterials for biomaterial and photonic applications. In chapter 2 of this thesis, the suitability of porous silicon as a biomaterial was investigated by performing different surface modifications on the porous silicon films and evaluating biocompatibility of these surfaces in vitro. The porous silicon surfaces were characteriszed by means of atomic force microscopy (AFM), scanning electron microscopy (SEM), diffuse reflectance infrared spectroscopy (DRIFT) and interferometric reflectance spectroscopy (IRS). Cell attachment and growth was studied using fluorescence microscopy and cell viability assays. Both fabrication of the porous silicon films and subsequent surface modifications were demonstrated. Polyethylene glycol functionalised porous silicon prevented cell attachment, whilst collagen or fetal bovine serum coating encouraged cell attachment. Surface modifications were also performed on porous silicon films with different pore sizes and the influence of pore size and surface modification on primary hepatocyte growth was recorded over a course of 2 weeks by means of laser scanning confocal microscopy (LSCM), toxicity and metabolic assays. On collagen-coated surfaces with average pore sizes of 30 nm, multilayer cells stacks were formed. This stacking behaviour was not observed on samples with smaller pore sizes (10 nm), or in the absence of collagen. Hepatocytes remained viable and functional (judging by a metabolic assay) for 6 days, after which they generally underwent apoptosis. Collagen-coated porous silicon films showed later onset of apoptosis than porous silicon films not coated with collagen or collagen-coated flat silicon.. In chapter 3 of this thesis, the nitrogen laser of a laser desorption/ionization (LDI) mass spectrometer was used to selectively ablate regions on porous silicon films that had been functionalised with a non-fouling polyethylene oxide layer, affording a microscale patterning of the surface. Surface characterization was performed by means of AFM, SEM, LDI mass spectrometry, DRIFT and IRS. This approach allowed the confinement of mammalian cell attachment exclusively on the laser-ablated regions. By using the more intense and focussed laser of a microdissection microscope, trenches in a porous silicon film were produced of up to 50 micron depth, which allowed the construction of cell multilayers within these trenches, mimicking the organization of liver cords in vivo. Fluorescent staining and LSCM was used to study cell multilayer organization. To gain a better understanding of how surface topography influences cell attachment and behaviour, porous silicon films were fabricated containing a gradient of pore sizes by means of asymmetric anodisation (chapter 4). These gradients allowed the investigation of the effect of subtle changes of pore size on cell behaviour on a single sample. Analysis by means of LSCM and SEM showed that pore size can dictate cell size and area as well as cell density. In addition, a region of pore size where cell attachment and proliferation was strongly discouraged was also identified. This information can prove to be useful for designing non-biofouling surface topographies. Using the same asymmetric anodisation setup, photonic mirrors gradients were produced and overlaid over one another to produce multidirectional lateral photonic mirror gradients that display a series of roving spectral features (photonic stop-bands) from each gradient layer (chapter 4). These multidirectional photonic gradients have the potential to serve as optical barcodes or contributing to the development of graded refractive index devices such as lenses for high quality image relay and graded-index optical fibers.

<p>The role of cardiac tissue structure in both normal and abnormal impulse conduction has been extensively studied by researchers in cardiac electrophysiology. However, much is left unknown on how specific micro- and macroscopic structural features affect conduction and conduction block. Progress in this field is constrained by the inability to simultaneously assess intramural cardiac structure and function, as well as the intrinsic complexity and variability of intact tissue preparations. Cultured monolayers of cardiac cells, on the other hand, present a well-controlled in vitro model system that provides the necessary structural and functional simplifications to enable well-defined studies of electrical phenomena. In this thesis, I developed a novel, reproducible cell culture system that accurately replicates the realistic microstructure of cardiac tissues. This system was then applied to systematically explore the influence of natural structure (e.g. tissue boundaries, expansions, local fiber directions) on normal and arrhythmogenic electrical conduction.</p><p>Specifically, soft lithography techniques were used to design cell cultures based on microscopic DTMRI (diffusion tensor magnetic resonance imaging) measurements of fiber directions in murine ventricles. Protein micropatterns comprised of mosaics of square pixels with angled lines that followed in-plane cardiac fiber directions were created to control the adhesion and alignment of cardiac cells on a two-dimensional substrate. The high accuracy of cell alignment in the resulting micropatterned monolayers relative to the original DTMRI-measured fiber directions was validated using immunofluorescence and image processing techniques.</p><p>Using this novel model system, I first examined how specific structural features of murine ventricles influence basic electrical conduction. (1) Realistic ventricular tissue boundaries, either alone or with (2) microscopic fiber directions were micropatterned to distinguish their individual functional roles in action potential propagation. By optically mapping membrane potentials and applying low-rate pacing from multiple sites in culture, I found that ventricular tissue boundaries and fiber directions each shaped unique spatial patterns of impulse propagation and additively increased the spatial dispersion of conduction velocity.</p><p>To elucidate the roles that natural tissue structure play in arrhythmogenesis, I applied rapid-rate pacing from multiple sites in culture in an attempt to induce unidirectional conduction block remote from the pacing site--a precursor to reentry. The incidence of remote block was found to be highly dependent on the direction of wave propagation relative to the underlying tissue structure, and with a susceptibility that was synergistically increased by both realistic tissue boundaries and fiber directions. Furthermore, all instances of remote block in these micropatterned cultures occurred at the anterior and posterior junctions of the septum and right ventricular free wall. At these sites, rapid excitation yielded more abrupt conduction slowing and promoted wavefront-waveback interactions that ultimately evolved into transmural lines of conduction block. The location and shape of these lines of block was found to strongly correlate with the spatial distribution of the electrotonic source-load mismatches introduced by ventricular structures, such as tissue expansions and sharp turns in fiber direction.</p><p>In summary, the overall objective of the work described in this thesis was to reveal the distinct influences of realistic cardiac tissue structure on action potential conduction and conduction block by engineering neonatal rat cardiomyocyte monolayers that reproducibly replicated the anatomical details of murine ventricular cross-sections. In the future, this novel model system is expected to further our understanding of structure-function relationships in normal and structurally diseased hearts, and possibly enable the development of novel gene, cell, and ablation therapies for cardiac arrhythmias.</p> / Dissertation

No description available.

No description available.

Self assembled monolayers (SAMs) are widely used for surface modification. Alkylsilane monolayers are one of the most widely deposited and studied SAMs. My work focuses on the preparation, patterning, and application of alkysilane monolayers. 3-aminopropyltriethoxysilane (APTES) is one of the most popular silanes used to make active surfaces for surface modification. To possibly improve the surface physical properties and increase options for processing this material, I prepared and studied a series of amino silane surfaces on silicon/silicon dioxide from APTES and two other related silanes by chemical vapor deposition (CVD). I also explored CVD of 3-mercaptopropyltrimethoxysilane on silicon and quartz. Several deposition conditions were investigated. Results show that properties of silane monolayers are quite consistent under different conditions. For monolayer patterning, I developed a new and extremely rapid technique, which we termed laser activation modification of semiconductor surfaces or LAMSS. This method consists of wetting a semiconductor surface with a reactive compound and then firing a highly focused nanosecond pulse of laser light through the transparent liquid onto the surface. The high peak power of the pulse at the surface activates the surface so that it reacts with the liquid with which it is in contact. I also developed a new application for monolayer patterning. I built a technologically viable platform for producing protein arrays on silicon that appears to meet all requirements for industrial application including automation, low cost, and high throughput. This method used microlens array (MA) patterning with a laser to pattern the surface, which was followed by protein deposition. Stencil lithography is a good patterning technique compatible with monolayer modification. Here, I added a new patterning method and accordingly present a simple, straightforward procedure for patterning silicon based on plasma oxidation through a stencil mask. We termed this method subsurface oxidation for micropatterning silicon (SOMS).

The canonical Wnt/β-catenin signaling pathway is essential for the proper regulation of cell-fate decisions throughout embryogenesis and in adult issues. Activation of the Wnt signaling pathway allows for nuclear localization of the cell adhesion protein β-catenin, which then interacts primarily with members of the T-Cell Factor/Lymphoid Enhancer Factor (TCF/LEF) transcription factor family to modulate gene activity. The TCF/LEF family includes TCF7, TCF7L1, TCF7L2, and LEF1. While all four family members share a common DNA binding consensus sequence, their expression throughout embryogenesis and adult stem cell populations is unique, with their misexpression commonly occurring in Wnt related cancers and correlating strongly with metastasis and poor patient outcomes. TCF/LEF exchange at target gene loci is a key feature of mediating context-specific cellular responses to Wnt signaling and can be observed to occur in a variety of populations throughout development and in adult stem cell populations. To model TCF/LEF exchange in vitro we have optimized a micropatterning fabrication and culture protocol capable of identifying and isolating discrete LEF1-only and TCF7L1-only populations during gastrulation-like processes. To characterize how complements of TCF/LEFs change during cellular divisions we have developed a novel mitotic chromatin proteomic technique. This method identifies LEF1 as the only TCF/LEF to remain associated with mitotic chromatin in Wnt-activated conditions in mouse embryonic stem cells that are transitioning out of pluripotency as a consequence of removing leukemia inhibitory factor from their culture medium. Additionally, gene targeting techniques were used to label endogenous LEF1 and TCF7L1 with different fluorescent proteins in a single mouse embryonic stem cell line, allowing us to use TCF/LEF protein expression as a reporter of Wnt/β-catenin pathway status, which we found to be capable of identifying a unique set of compounds that are undetected by traditional Wnt activity (TOP-Flash) reporter screens. By using gene editing technology, and novel applications of proteomic and cell culture techniques, we have been able to investigate the mechanisms driving TCF/LEF expression and exchange in mouse embryonic stem cells to identify potentially clinically relevant therapeutic targets for their potential use in addressing TCF/LEF dysregulation in cancer. We have identified a novel mechanism through which TCF/LEFs maintain cell fate over cellular division; presented a novel live-cell drug screening platform capable of identifying compounds missed by existing platforms; and presented an optimized cell culture technique for the isolation of TCF/LEF exchange events. Taken together, the work in this thesis provides new insights into the mechanisms through which TCF/LEFs regulate their gene targets during cell fate transitions and throughout mitosis. / Thesis / Doctor of Science (PhD) / Throughout development and adult life cells are in constant communication, using a variety of cell signaling pathways to maintain adult stem cell populations and to pattern tissues throughout the body. Communication between cells often requires one cell to release a protein molecule (called a ligand) that is recognized by a receptor molecule on the surface of another cell. These cell surface receptors, when bound by the signaling ligand become activated and often set of a cascade of internal cellular events that ultimately result in changes in gene transcription in the nucleus. These transcriptional changes are toggled by proteins known as sequence-specific transcription factors that are able to selectively regulate expression of target genes. The net effect of combinations of extracellular ligands binding cell surface receptors determines the selective recruitment of specific transcription factors that activate a cell’s transcriptional program, in turn defining its fate and function. A very important developmental signaling pathway is the Wnt signaling pathway, which employs a family of secreted Wnt molecules as ligands. The Wnt pathway is critical at all stages of organismal development and plays an essential role in tissue maintenance in mature animals. However, due to its critical role in stem cell maintenance, when mutations occur in Wnt signaling components it can have dire consequences. Wnt signaling has been found to be disrupted in more than 70-80% of all cancers. One major feature among these Wnt-related cancers is the inappropriate expression and mobilization of Wnt transcription factors. While the expression and activity of Wnt transcription factors – known as T-Cell Factor/Lymphoid Enhancer Factors (TCF/LEFs) – changes throughout development and stem cell maintenance, their inappropriate expression is frequently associated with metastasis and poor patient outcomes. We have used mouse embryonic stem cells (mESCs) as a model system with which to study the mechanisms employed by TCF/LEFs to regulate their target genes. Through a number of approaches, which include adding fluorescent tags to TCF/LEF factors to track their intercellular locations and expression levels or enzymatic tags to identify proteins that interact with individual TCF/LEFs during a snapshot of cell activity, we have gained new knowledge about how these critical transcription factors regulate Wnt-regulated transcriptional programs. We also describe a method for generating micropatterned growth surfaces for mESCs that forces clusters of cells to grow within small circular shapes with a diameter of 1 mm or less. We show that mESCs confined to circular micropatterns differentiate in a highly reproducible manner that allows us to study the cell populations undergoing differentiation with a focus on cell fate determination mechanisms.

Le guidage axonal est un processus très important dans le développement du cerveau, permettant de lui donner sa structure et son organisation. La communauté scientifique des neurosciences lui porte un intérêt grandissant ces dernières années. Plusieurs outils appartenant au domaine des microtechnologies, que sont la microfluidique et le micropatterning, sont d’une aide importante pour étudier le guidage axonal in vitro. Ils permettent de confiner les neurones et leurs axones et de leur appliquer des gradients de molécules de guidage. Lors de ce travail de thèse, j’ai voulu développer un système pour étudier l’effet de gradients de molécules de guidage sur le guidage axonal. J’ai pour cela testé plusieurs configurations de dispositifs microfluidiques, de micromotifs (micropatterns) et de combinaisons de ces derniers.Nous avons d’abord utilisé deux approches pour isoler les axones de neurones dissociés de leurs somas afin de pouvoir étudier, à haut débit, l’effet de l’environnement moléculaire sur les cônes de croissance des neurones. La première approche consistait à faire pousser des neurones sur des motifs (patterns) de différentes protéines. Elle a permis de montrer leur capacité d’adhésion spécifique sur ces motifs. La seconde consistait à ensemencer des neurones dans un dispositif microfluidique dans lequel, lors de leur pousse, les axones sont séparés des somas par des microcanaux. Nous avons ensuite étudié l’effet, sur les axones, de gradients de molécules de guidage. Pour commencer, nous avons mesuré l’effet de deux molécules de guidage : l’éphrine et la sémaphorine, en cultivant des neurones en présence de gradients patternés de ces deux molécules. Par la suite, nous avons étudié un autre modèle où les neurones sont plus proches de leur environnement in vivo, des explants poussant sur des motifs de laminine contenant un gradient. Pour aider au positionnement de l’explant, nous avons polymérisé des hydrogels. Ensuite, nous avons mis des explants à côté de gradients patternés d’éphrine. Enfin, nous avons cherché à obtenir un gradient soluble de molécules de guidage entretenu sur des temps longs, plus proche des gradients existant in vivo. Dans ce but, nous avons voulu fabriquer un dispositif microfluidique permettant d’appliquer un gradient soluble de molécules de guidage sur des neurones. Pour obtenir un gradient stable dans le temps, nous avons aussi cultivé des neurones à côté de cellules exprimant la nétrine, une autre molécule de guidage. Pour finir, nous avons cultivé des neurones et des glies dissociés pour étudier leurs interactions.L’ensemble de ces recherches n’a pas permis d’obtenir un dispositif fiable pour étudier l’effet de molécules sur la pousse et le guidage des axones. Néanmoins, la configuration consistant en une coculture de neurones à proximité de cellules relargant de la nétrine nous a permis d’obtenir des premiers résultats encourageants. Nous avons ainsi mis au point un ensemble de méthodes qui pourront nous permettre de finaliser le développement d’un système pour étudier le guidage axonal, fonctionnel et efficace. / Axonal guidance is a very important process during brain development, allowing to give it its structure and organization. The neuroscience scientific community has a growing interest in it during the last years. Several tools belonging to the field of microtechnologies, microfluidics and micropatterning are of important help to study axonal guidance in vitro. They allow to confine neurons and their axons and to apply gradients of guidance molecules. During this thesis, my goal was to develop a system to study the effect of guidance molecules gradients on axonal guidance. For that, I tested several configurations of microfluidic devices, micropatterns and combinations of both.First, we used two approaches to isolate dissociated neurons axons from their somas. Our goal was to study the effect of the molecular environment on neurons growth cones, with a high throughput. The first approach consisted in growing neurons on different proteins patterns. It also allowed to show their capacity to adhere on these patterns. The second one consisted in seeding neurons in a microfluidic device in which, during their growth, axons are separated from somas by microchannels. Then we studied the effect, on the axons, of guidance molecules gradients. To begin, we measured the effect of two guidance molecules: ephrin and semaphorin, by culturing neurons in the presence of patterned gradients of these two molecules. After that, we studied another model where neurons are closer from their environment in vivo, explants growing on laminin patterns containing a gradient. To help the explant positioning, we polymerized hydrogels. Then, we put explants next to patterned gradients of ephrin. Finally, we tried to obtain a soluble gradient of guidance molecules, over a long period of time (days), closer to existing gradients in vivo. In that goal, we wanted to build a microfluidic device enabling the application of a soluble gradient of guidance molecules on neurons. To obtain a constant gradient, we also cultured neurons next to cells expressing netrin, another guidance molecule. Finally, we cultured dissociated neurons and glial cells to study their interactions.All these experiments did not allow to obtain a reliable device to study the effect of molecules on axons growth and guidance. Nevertheless, the configuration consisting in a coculture of neurons next to cells releasing netrin allows us to obtain promising preliminary results. We thus drew up a group of methods that will enable us to finalize the development of a system to study axonal guidance, functional and efficient.

Mon travail de thèse fut de développer différents projets en vue de mieux comprendre la dynamique et l'organisation des réseaux d'actine, ainsi que les mécanismes moléculaires à l'origine de la production de force grâce à différents systèmes reconstitués biomimétiques. Dans un premier temps, je me suis intéressée à l'étude de l'organisation spatiotemporelle des réseaux dynamiques d'actine et de ses protéines associées durant la propulsion de particules recouvertes de promoteurs de nucléation des filaments d'actine (Achard et al, Current Biology, 2010 et Reymann et al, accepté à MBoC). J'ai notamment suivi en temps réel l'incorporation de deux régulateurs de l'actine (Capping protein, protéine de coiffe et ADF/cofilin, protéine de fragmentation) et montré que leurs actions conjuguées assurent un contrôle biochimique de l'assemblage d'un réseau complexe d'actine, mais gouvernent également les propriétés mécaniques de ce réseau. Par ailleurs, afin de mieux caractériser les propriétés mécaniques de ces réseaux d'actine en expansion, j'ai développé un système biomimétique novateur utilisant la procédure de micropatrons ou "micropatterning" qui permet un contrôle spatial reproductible des sites de nucléation d'actine. Cela m'a permis de montrer comment des barrières géométriques, semblables à celles trouvées dans les cellules, peuvent influencer la formation dynamique de réseaux organisés d'actine et ainsi contrôler la localisation de la production de forces. (Reymann et al, Nature Materials, 2010). De plus, l'incorporation de moteurs moléculaires dans ce système versatile, nous a permis d'étudier la contraction induite par des myosines. En particulier, j'ai pu montrer que les myosines VI HMM interagissent de manière sélective avec différentes architectures d'actine (organisation parallèle ou antiparallèle, réseau enchevêtré), aboutissant à un processus en trois phases: tension, puis déformation des réseaux d'actine fortement couplée à un désassemblage massif des filaments. Aussi, ce phénomène de désassemblage massif induit par la myosine est intimement dépendant de l'architecture du réseau d'actine et pourrait, de ce fait, jouer un rôle essentiel dans la régulation spatiale des zones d'expansion et de contraction du cytosquelette in vivo.

Cornea is the most exterior, avascular and transparent layer of the eye and is about 500 &micro / m in thick. It protects the eye from external objects and it is the main optical element of the eye refracting 70 % of the incoming light. After cataract, corneal diseases and wounds are the second leading cause of the blindness that affects more than 4 million people worldwide. For the highly damaged corneas where the corrections with spectacles or contact lenses cannot be achieved, tissue replacement is the only choice, and is done by cornea transplantation or keratoprostheses. However, due to limited number of donor corneas and the risk of infections during transplantation, and development of glaucoma, necrosis and other complications caused by the keratoprostheses, prevent them from meeting expectations. Tissue engineering is a promising field which emerged from biomaterials science and aims to replace, restore or improve the function of the diseased or injured tissues. In this method, after the production of an ideal scaffold that mimics the natural human tissue, cells of the host are isolated, increased in number, and seeded on the scaffold developed to serve as the microenvironment of the cells. In the current study a 3D corneal stroma replacement was designed to mimic the native stroma. It consisted of 4 films of patterned collagen or collagen blended with Elastin Like Recombinamer (ELR) stacked on top of each other and then crosslinked by dehydrothermal (DHT) treatment. The characterization of the films showed that the pattern fidelity was good and they did not deteriorate after crosslinking. Enzymatic and in situ degradation studies showed that the DHT treatment at 150 oC for 24 h (DHT150) was the optimum condition. The transparency of all the films was quite high where uncrosslinked (UXL) films and DHT150 Col:ELR films yielded the best results. The individual films and 3D construct of 4 stacked films were seeded with isolated human corneal keratocytes (HK) and cultured for 21 days. Cells attached and proliferated well on the single Col and Col:ELR films. However, the proliferation was higher on Col multilayer constructs than their Col:ELR counterparts. Cells were aligned along the patterns of the films while no significant alignment was observed for the cells on unpatterned films. Ultimate tensile strength (UTS) and Young&rsquo / s Modulus (E) of Col and Col:ELR films were significantly lower after a 30 day culture than that of unseeded films of Day 1. Transparency of the seeded Col:ELR films was superior to Col films over a 30 days test and quite close to the transmittance of the native human cornea. It was concluded that the Col and Col:ELR patterned films and their 3D constructs have a significant potential for use as a corneal stroma equivalent.