Le procédé de la métastase cancéreuse et sa compréhension sont devenus un des sujets majeurs de recherche en Biologie. En utilisant des modèles in-vitro en culture statique et dynamiques, nous avons étudié la possibilité de reproduire l’environnement physiologique in-vivo avec ces modèles. Nous avons développé un modèle de coculture hiérarchique dans des plaques à fond en PDMS. Composé d’hépatocytes, de pericytes et de cellules endothéliales. Dans différentes conditions, l’influence de ces cellules sur l’adhésion de cellules cancéreuses ou promyéloblastiques a été analysée ainsi que leur effet sur l’état inflammatoire du système. Afin de reproduire le flux sanguin et les forces de cisaillement présents in-vivo, le modèle a été transféré dans un système microfluidique. Le système se compose de trois canaux séparés par des micro-piliers, pouvant être remplis indépendamment. Les pericytes insérés dans du gel, les hépatocytes, les cellules endothéliales et finalement les cellules cancéreuses ont été injectés de façon successive afin de reproduire l’environnement in-vivo. Les cellules ont été trouvées viables durant toute la culture et des marqueurs relatifs au foie et à l’inflammation exprimés. L’influence des hépatocytes et des pericytes a été analysé. Il a été observé que les cellules cancéreuses adhérées dans le canal du haut étaient attirées par les autres cellules dans les diffèrent canaux. Les modèles établis posent de solides bases pour d’autres systèmes plus complexes et d’intérêt pouvant servir de complément aux modèles in-vivo lors de la recherche de nouvelles substances médicamenteuses. / The cancer metastatic process and its understanding have been a major topic of interest for researchers in the past. Using in-vitro models in both standard culture conditions and in microfluidic devices, we investigated the feasibility of such models in the representation of the physiological in-vivo situation. We developed a hierarchical coculture model in PDMS plates, composed of hepatocytes, pericytes and endothelial cells. In different culture conditions, the influence of the different cells composing the model on the adhesion of cancer cells and promyeloblastic cells was investigated as well as the influence on the inflammatory state of the culture. To reproduce the in-vivo blood flow and shear stress to which the endothelial cells and the adhering cells are subjected, the model was then transferred into a microfluidic biochip. The device was composed of three channels, separated by micropillars and which could be filled independently one from another. Pericytes embedded in a hydrogel, hepatocytes, endothelial cells and finally pancreatic cancer cells could be inserted successively to reproduce the in-vivo hierarchical situation. Cells were found to viable after the culture and markers related to the liver and inflammation to be expressed. The influence of the presence of hepatocytes and pericytes was investigated by varying the culture conditions. It was found that pancreatic cancer cells were attracted by the cells in other channels in coculture. The established models lay the bases for more complex and relevant systems that could complement their in-vivo counterparts in the drug discovery process.

Liver failure, which is the result of liver injury and pathological inflammation, is currently only successfully treated by organ transplantation. However donor organ shortages preclude transplantation for many patients in need. Thus, bioartificial liver systems (BALS) are being developed as a bridge to transplantation, or to create an environment conducive to liver regeneration. Hepatocytes, the main functional cells of the liver, are the cells of choice for BALSs, but in standard conditions ex vivo, they rapidly suffer from a reduction of their functionality and viability. Coculture with stromal cells, for example bone marrow mesenchymal stromal cells (BM-MSC), has been shown to improve, and extend, hepatocyte function ex vivo up to 21 days. But, only small numbers of BM-MSCs can be harvested from adult volunteers. We have previously described an alternative, more plentiful, source of MSCs — human umbilical cord perivascular cells (HUCPVC) — that are easily expanded and non-alloreactive. Our hypothesis was that HUCPVCs are putative stromal cells for hepatocytes. Our results show that HUCPVCs improved hepatocyte albumin secretion, urea synthesis and maintained hepatocyte cytochrome activity and the expression of hepato-specific genes. Furthermore, there was a net proliferation of hepatocytes, which were polarized in coculture with HUCPVCs, as judged by functional bile canaliculi that were present for up to 40 days. We found that both soluble and non-soluble factors contributed to these effects, while neither was able to allow net proliferation individually. Moreover, HUCPVCs expressed both hepato-trophic and anti-inflammatory factors, at different levels to BM-MSCs, indicating the potential for differential hepato-therapeutics. We conclude that HUCPVCs are putative stromal cells for hepatocytes; they improve hepatocyte functionality, polarity, morphology and net proliferation, and thus present an opportunity for the improvement of both BALS function and liver therapy.

Liver failure, which is the result of liver injury and pathological inflammation, is currently only successfully treated by organ transplantation. However donor organ shortages preclude transplantation for many patients in need. Thus, bioartificial liver systems (BALS) are being developed as a bridge to transplantation, or to create an environment conducive to liver regeneration. Hepatocytes, the main functional cells of the liver, are the cells of choice for BALSs, but in standard conditions ex vivo, they rapidly suffer from a reduction of their functionality and viability. Coculture with stromal cells, for example bone marrow mesenchymal stromal cells (BM-MSC), has been shown to improve, and extend, hepatocyte function ex vivo up to 21 days. But, only small numbers of BM-MSCs can be harvested from adult volunteers. We have previously described an alternative, more plentiful, source of MSCs — human umbilical cord perivascular cells (HUCPVC) — that are easily expanded and non-alloreactive. Our hypothesis was that HUCPVCs are putative stromal cells for hepatocytes. Our results show that HUCPVCs improved hepatocyte albumin secretion, urea synthesis and maintained hepatocyte cytochrome activity and the expression of hepato-specific genes. Furthermore, there was a net proliferation of hepatocytes, which were polarized in coculture with HUCPVCs, as judged by functional bile canaliculi that were present for up to 40 days. We found that both soluble and non-soluble factors contributed to these effects, while neither was able to allow net proliferation individually. Moreover, HUCPVCs expressed both hepato-trophic and anti-inflammatory factors, at different levels to BM-MSCs, indicating the potential for differential hepato-therapeutics. We conclude that HUCPVCs are putative stromal cells for hepatocytes; they improve hepatocyte functionality, polarity, morphology and net proliferation, and thus present an opportunity for the improvement of both BALS function and liver therapy.

Despite microbial degradation being the primary route of degradation of PAHs in soils, high molecular weight polycyclic aromatic hydrocarbons (such as benzo[a]pyrene) have consistently proven to e resistant to microbial attack. However, recent research has demonstrated the potential for bacterial-fungal co-cultures to achieve biodegradation of high molecular weight PAHs. The aim of this research was to determine the efficacy of co-culture bioaugmentation for the remediation of high molecular weight PAHcontaminated soils. PAH degrading bacteria were enriched on multiple PAHs and isolated on pyrene from both contaminated (soil from a former manufactured gas plant) and uncontaminated (agricultural soil, termite mound matrix and kangaroo faeces) sources. The bacterial isolates were identified using 16SrRNA analysis as Mycobacterium sp. Strain BS5, Mycobacterium sp. Strain KA5 and Mycobacterium sp. Strain KF4 or fatty acid methyl ester (FAME) analysis as Ralstonia pickettii and Stenotrophomonas maltophilia. The initial phase of assessment of PAH degradation by fungal and bacterial coculture components was undertaken using liquid media. Two fungal isolates from a previous investigation into the coculture process (Penicillium janthinellum) and the American Type Culture Collection (Phanerochaete chrysosporium) were assessed for their ability to degrade benzo[a]pyrene in minimal media and MYPD. The fungal isolates were found to be able to degrade benzo[a]pyrene cometabolically in MYPD. The bacterial isolates and two others from previous investigations were assessed for their ability to degrade single PAHs (fluorene, phenanthrene, fluoranthene, pyrene and benzo[a]pyrene) in liquid culture. This process was used as an initial screen to select the best bacterial isolates for further investigation of PAH degradation by axenic cultures and cocultures with the fungal isolates using a PAH mixture. Based on the results of these experiments four bacterial isolates (VUN 10,010, Mycobacterium 1B, Mycobacterium sp. Strain BS5 and Mycobacterium sp. Strain KA5) and the two fungal isolates were selected to investigate further using a PAH mixture composed of the previously mentioned PAHs. It was found that the use of a fungal bacterial coculture increased the degradation of the PAH mixture beyond that of axenic bacterial cultures. Based on these experiments, the coculture composed of P. janthinellum and VUN 10,010 was selected for assessment of its ability to degrade the same PAH mixture in spiked soil microcosm experiments. Natural attenuation, axenic P. janthinellum, axenic VUN 10,010 and a coculture of these two organisms were assessed for PAH degradation in soil microcosms over a 100 day period. Inoculation of microcosms with the coculture resulted in the removal of benzo[a]pyrene by 11 mg/kg (± 1.21 mg/kg) (30%) over the 100 day incubation period. Substantial PAH degradation was also observed in the microcosms assesing natural attenuation. Using an alternative sequential inoculation method, initially inoculating with P. janthinellum then 50 days later with VUN 10,010 significantly enhanced the removal of benzo[a]pyrene. After 100 days incubation, benzo[a]pyrene was degraded below detection limits in two of three microcosms, compared to a 4.95 mg/kg (± 4.64 mg/kg) (14.7 %) reduction in soil microcosms inoculated using an alternative inoculation process of VUN 10,010 followed by P. janthinellum. Attempts were made to optimise the process using sequential inoculation and soil amendments intended to enhance the performance of the fungal component using distilled water and 1% glucose. The addition of distilled water was not observed to substantially influence the ability of the coculture to degrade PAHs, whereas the addition of 1% glucose was found to inhibit PAH degradation.

La modulation phénotypique des cellules musculaires lisses (CML) artérielles d’un phénotype contractile vers un phénotype synthétique en réponse à des stimuli pathogènes est impliquée dans le développement de l’artériosclérose. Il est clairement établi que l'homocystéine (HCY) induit un remodelage de type artériosclérose au niveau des artères élastiques. Ce remodelage pathologique induit par l'HCY est dominé par l'accumulation de CML présentant un phénotype synthétique associée à une dysfonction endothéliale. Cependant, l'effet de l'HCY sur le phénotype des CML n’a pas été clairement décrit. Compte tenu du rôle déterminant des cellules endothéliales dans la modulation du phénotype des CML artérielles, l’objectif de notre travail est de déterminer dans quelle mesure l’effet de l’HCY sur le phénotype des CML est direct ou est dépendant d’une dysfonction endothéliale. Nous avons montré que des concentrations pathologiques d’HCY augmentent la prolifération de CML d’artères ombilicales humaines en culture et augmentent leur potentiel protéolytique par un mécanisme dépendant des espèces réactives de l'oxygène. Dans le même temps, l’HCY ne modifie pas l'expression des collagènes de type I et III et de la fibronectine, ni l'expression de protéines contractiles (SM a-actine, calponine hl, SMMHC et smoothéline B). Que ce soit sur des CML pré-conditionnées ou non par du milieu de culture conditionné (MC) de cellules endothéliales de veine ombilicales (HUVEC), nous avons montré que le MC d’HUVEC stimulées par 100µM d'HCYne lève pas l'inhibition de la prolifération des CML induite par les HUVEC et ne modifie pas l'expression des collagènes de type I et III et de la fibronectine, ni l'expressìon de protéines contractiles. Nos résultats sont en faveur d’un effet modulateur direct de l'HCY orientant le phénotype des CML vers un phénotype synthétique. S’il est connu que l'HCY induit une dysfonction endothéliale, cette dysfonction ne semble pas avoir de conséquences sur le phénotype des CML. / Phenotype switching of arterial smooth muscle cells (SMC) from a contractile phenotype through a synthetic phenotype in response to pathogenic stimuli is involved in the development of arteriosclerosis. It is well established that homocysteine (HCY) induces arteriosclerosis in elastic arteries. This pathologic arterial remodelling is characterized by the accumulation of SMC showing synthetic phenotype associated with an endothelial dysfunction. However, the effects of HCY on SMC phenotype are unclear. Since endothelial cells play a key role in the regulation of SMC phenotype, the goal of our study is to determine whether HCY modulates the SMC phenotype through a direct effect or through the induction of an endothelial dysfunction. We found that pathologic concentrations of HCY increase the proliferation of human arterial umbilical SMC in culture and increase their proteolytic potential through a reactive oxygen species dependant mechanism. We also found that HCY has not effect on the expression of type I and III collagen and fibronectin, as well as contractile proteins (SM a-actine, calponine hl, SMMHC et smootheline B). Using pre-conditionned or not pre-conditionned SMC by conditionned culture medium (MC) from human umbilical vein endothelial cells (HUVEC), we found that MC from HUVEC stimulated with 100µM of HCY prevent the inhibition of proliferation induced HUVEC and did not altered the expression of type I and III collagen and fibronectin, as well as contractile proteins. Our results suggest that HCY can directly modulate the SMC phenotype towards a synthetic phenotype. Although HCY as been previously shown to induce an endothelial dysfunction, this latter does not seem to be involve in the modulation of SMC phenotype associated with hyper homocysteinemia.

Un substitut osseux hybride, composé d’un biomatériau support (scaffold) et de cellules vivantes, a été étudié, développé par la méthode d’ingénierie tissulaire et caractérisé. Il devait répondre aux attentes spécifiques de la chirurgie maxillofaciale : un protocole standard pouvant s’adapter aux géométries complexes des défauts osseux de chaque patient, une forme souple et manipulable, une pré-vascularisation et une cohésion mécanique suffisante. Une forme de feuillet fin et plat a ainsi été définie et développée au sein d’une chambre de culture parallélépipédique spécifique, en utilisant une monocouche de granules de phosphate de calcium comme support. Après une caractérisation biologique et mécanique complète à partir d’une lignée cellulaire, le procédé a été validé puis transposé à une coculture de cellules primaires humaines (cellules souches et endothéliales). La bonne différenciation et la pré-vascularisation ont été constatées mais le maintien mécanique pouvait être considéré comme insuffisant pour assurer une manipulation en cours d’opération chirurgicale. La dernière partie de ce travail de thèse a donc consisté dans la mise en place d’un montage de production de fibres électrospinnées et leur utilisation comme nouveau support de culture. La formation de ces matériaux a été rendue opérationnelle de façon optimale pour différents polymères. Leur potentiel en tant que scaffold favorisant la différenciation en os ou en tendon a été vérifié et comparé à d’autres matériaux fibreux obtenus dans le cadre de collaborations nationales et internationales. La faisabilité de l’application de sollicitations mécaniques aux substituts en cours de culture a également été étudiée. / An hybrid bone substitute, based on a specific biomaterial (scaffold) and living cells, was studied, developed with a tissue engineered method and characterized. It should meet the expectations of the maxillofacial surgery : a standard process which could fit with the complex geometries of each patient’s bone mass loss, a flexible shape with an easy handling, a prevascularization and a sufficient mechanical cohesion. A sheet-like shape was thus designed and developed in a specific flat cell culture chamber, with a monolayer of calcium phosphate granules as a scaffold. After both biological and mechanical full characterizations with a cell line, the process was adapted to a coculture of human primary cells (stem and endothelial cells). Relevant differentiation and prevascularization were highlighted but the mechanical cohesion could be noticed as too low to ensure an easy handling during the surgery. The last part of this thesis project was thus the set-up of a device for electrospun polymer fibers in order to use them as a new scaffold. The production of these materials was efficiently performed for several polymers. The differentiation potential for bone and tendon lineages was studied and compared to other scaffolds from national and international collaborations. The application of mechanical solicitations to the substitutes during cellculture was also studied.

La ciguatéra est une forme d’intoxication faisant suite à l’ingestion de poissons contaminés par des toxines appelées « ciguatoxines ». Cette intoxication endémique des régions tropicales est un problème économique et de santé non négligeable qui tend à prendre de plus en plus d’ampleur. L’essor du tourisme, le réchauffement climatique et la hausse des exportations internationales de poissons tropicaux favorisent l’expansion de la ciguatéra aux parties du globe au climat tempéré, jusqu’alors peu concernées par cette maladie. Les enjeux thérapeutiques et économiques de la ciguatéra sont de taille puisqu’il n’existe aucun moyen rapide et fiable de détecter un poisson contaminé et qu’aucun traitement efficace permettant sa prise en charge n’a actuellement été établi.Le prurit, terme médical désignant les démangeaisons est un symptôme notamment associé aux maladies de peau qui impacte grandement la qualité de vie des patients qui en souffrent.Ces dernières décennies, de nombreuses études scientifiques ont permis de mieux comprendre sa physiopathologie. Le prurit est un symptôme fréquemment observé chez les personnes atteintes de ciguatéra, d’où l’appellation « la Gratte », souvent employée pour faire référence à la pathologie.Dans le but d’étudier les mécanismes cellulaires à l’origine du prurit et des autres troubles sensoriels cutanés survenant lors de la ciguatéra, nous avons étudié l’effet des ciguatoxines sur un modèle in vitro de neurones sensoriels cocultivés avec des kératinocytes. Nous avons mis en évidence la libération dans le surnageant des neuropeptides de l'inflammation neurogène, SP et CGRP. L'effet d'antagonistes sélectionnés a été testé afin mettre en évidence les médiateurs impliqués dans la libération de neuropeptides. Par ailleurs, la signalisation cellulaire sous-jacente de cette sécrétion de neuropeptides a été étudiée par des expériences d’imagerie calcique réalisées sur neurones et kératinocytes.Les résultats obtenus valident notre coculture en tant que modèle de choix pour l’étude in vitro des mécanismes cellulaires, et plus généralement, dans les troubles neurocutanés impliqués dans le prurit ciguatérique. Parmi les antagonistes testés, une molécule s’est avérée particulièrement intéressante pour inhiber les effets constatés de la toxine. Ces résultats originaux, obtenus avec l’antagoniste d’un médiateur du prurit, présentent une perspective thérapeutique nouvelle et prometteuse, pour répondre à un enjeu de santé publique futur. / Ciguatera fish poisoning (CFP) is a seafood poisoning occurring after contaminated fish fleshes ingestion containing toxins called « ciguatoxines » (CTXs). This illness, originating from tropical and subtropical areas, is an economic and health problems which becomes substantial in relation to international tropical fishes export and tourism development, as well as global warming rise. Those factors contribute to CFP sprouting in non-endemic temperate climate regions which were not until then concerned by CFP. Economic and health stakes of CFP are important since no reliable and ready-to-use detection system for CTXs in fishes have been developed, along with no relevant cure has been established to treat CFP.Pruritus, medical term refer to itch, is a clinical sign usually associated to skin diseases which strongly alter patients’ quality of life. Last decades, several studies allowed to better understand pruritus pathophysiology. Interestingly, people suffering from CFP frequently present pruritus, hence designation “La Gratte” or “La Gratel (le)” employed in endemic areas.The aim of these works was to study cellular and molecular mechanisms of CTXs at the root of neurological cutaneous troubles occurring in CFP. Here, we evaluated CTXs effects on in vitro model composed of sensory neurons cocultived with primary keratinocytes, quantifying neuropeptides known to be involved in pruritus. Compiling knowledges about CFP and pruritus pathophysiology, some antagonists were tested to neutralize CTXs-mediated neuropeptide release. To deal with signaling pathways in depth of neuropeptide exocytosis induced by CTXs, mechanism known to be accurately regulated by calcium homeostasis, calcium imaging experiments were performed.Results obtained in this project confirm the use of such a model to elucidate cellular mechanism of CFP pruritus, but also constitute an alternative in vitro tool to study chemicals inducing abnormal cutaneous senses. Among antagonists tested, one stands out from the crowd and was proved to be effective to inhibit CTXs-evoked effects studied. Those originals results, collected with antagonist of pruritus mediator, show new and promiscuous therapeutic prospects for future health concern.

No description available.

L’ingénierie du tissu musculaire squelettique vise à reconstituer in vitro un tissu fonctionnel aussi physiologique que possible dans le but de mieux comprendre la myogenèse, l’impact de mutations génétiques et tester des médicaments. Ces dernières années, différents modèles de tissus musculaires tridimensionnels ont été développés. Toutefois, l’utilisation prépondérante de cellules murines et la taille de ces modèles restreint leur pertinence pour les études de pathologies humaines et le criblage pharmacologique. Dans le cadre de ce travail de thèse, nous avons donc développé différents modèles de tissus musculaires humains micrométriques pour répondre à ces limitations. Dans un premier temps, nous avons conçu et optimisé par microfabrication une plateforme caractérisée par la présence de microcanaux. Nous avons ainsi généré des tissus musculaires multicouches alignés présentant une organisation proche du muscle natif à partir de myoblastes murins immortalisés C2C12 puis de myoblastes humains immortalisés. Nous avons ainsi montré l’influence de la topographie et de la concentration cellulaire sur l’alignement des myotubes et la maturation du tissu musculaire. Dans un second temps, nous avons développé une plateforme constituée de micropuits contenant chacun deux micropiliers permettant d’analyser la contractilité des tissus. Des microtissus musculaires 3D standardisés ont ainsi été générés avec cette plateforme à partir de myoblastes murins, et de myoblastes C2C12 électroporés avec un gène muté ou non de la desmine. Par la suite, des microtissus ont été générés à partir de myoblastes humains. L’importance du choix de la matrice dans la formation des microtissus et les bénéfices d’une coculture de myoblastes et fibroblastes dans la stabilité des tissus ont ainsi été mis en évidence. La géométrie de micropiliers a aussi été optimisée afin de générer et comparer des microtissus composés de myoblastes isolés de patients sains et malades (dystrophie musculaire de Duchenne). Une preuve de concept démontrant la possibilité d’utiliser cette technologie pour tester des thérapies chimiques et géniques a été établie. Nous avons en effet suivi en temps réel les effets de l’inhibiteur de la kinase Rho-associée Y-27632 sur la contractilité des microtissus, ainsi que la transduction d’un gène rapporteur fluorescent modèle par les cellules composant les microtissus. Les résultats de ce travail de thèse démontrent le potentiel de cette technologie pour l’étude des processus fondamentaux de la myogenèse, l’évaluation des effets fonctionnels de mutations patient-spécifique et le criblage de thérapies chimiques et géniques. / Skeletal muscle tissue engineering aims to build functional and physiological tissues in vitro in order to better understand myogenesis, to investigate the impact of genetic mutations and to screen potential therapies. Over the past few years, bi- and tridimensional models of muscle tissue have been developed, but most of these models are based on the use of murine cells and require large amounts of cells, thus limiting their relevance to study pathologies of human muscles and drug screening assays. Here we aimed at developing different models of human muscle microtissues to address these issues. By using microfabrication techniques, we first engineered a microgrooved platform we used to generate aligned multilayered skeletal muscle tissues from murine C2C12 myoblasts and human immortalized myoblasts. We showed the impact of topography and cell density on the maturation and myotube alignment. We then fabricated a microdevice, consisting of microwells containing two micropillars allowing an easy access to the contractility of muscle tissues. We engineered microtissues from C2C12 and C2C12 myoblasts electroporated with a mutated gene of desmin, and showed some limitation of this technique of transduction. Finally, we generated microtissues from human myoblasts. We investigated the role of the extracellular matrix in the tissue formation and evidenced the benefits of coculturing myoblasts and fibroblasts on the stability of muscle microtissues. Furthermore, we optimized the geometry of the micropillars to engineer and compare microtissues composed of human myoblasts isolated from healthy and diseased (Duchenne muscular dystrophy) patients. A proof of concept of the potential of this technology for screening chemical and gene therapies was established. We were indeed able to analyze in real time the effects of the Rho-associated kinase-inhibitor Y-27632 on the tissue contractility, as well as the transduction of a model fluorescent reporter gene. Altogether, the results of this work demonstrate the potential of this technology to study fundamental muscle biology, examine functional effects of patient-specific mutations or screen chemical and gene therapies.

<div>Vocal fold scarring is the fibrotic manifestation of most common pathological voice disorders. Voice disorders lead to direct healthcare costs of over $200 million annually and significantly reduce quality of life for patients. Despite advances in understanding the pathophysiology of vocal fold scarring, effective treatments for scarring and fibrosis remain elusive. The wound-healing cascade associated with vocal fold injury involves complex signaling interactions between cells and their extracellular matrix (ECM), which remain largely unexplored due to the lack of a physiologically relevant preclinical model to study them. Traditional preclinical models do not capture the complex 3D microenvironment of the vocal folds, and the use of stem cells or fibroblasts alone in models has resulted in poor reproducibility and predictability of in vitro models. Toward this end, this work describes the development of a preclinical model that strives to take into account cellular interactions between fibroblasts and epithelial cells and achieve a balance in the native vocal fold 3D environment to function as an in vitro model.</div><div><br></div><div>Since a major shortcoming of current in vitro models is the lack of a standardized epithelial fibroblast coculture, initial work focused on developing a coculture system between commercially available tracheal epithelial cells and vocal fold fibroblasts in an in vitro setting that would provide more accurate information about the disease pathophysiology and help design better targeted treatments. We designed a healthy and disease state coculture model that can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1. We also demonstrated that both cell types maintained phenotype in the healthy and disease state coculture models.</div><div><br></div><div>To further transfer this model in a physiologically relevant 3D system, follow-up research characterized 3D matrices to mimic the native ECM of the vocal folds by using natural biomimetic materials found in the vocal folds such as hyaluronic acid, type I collagen, and type III collagen. We hypothesized that the ability to control the viscoelastic and structuralcharacteristics of the scaffold in combination with presenting relevant biological cues to cells will result in a better biomimetic scaffold. This research is expected to lay effective groundwork for developing a functional tissue engineered 3D coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform.</div>