Personne : Carrier, Patrick
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Carrier
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Patrick
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Départements de chirurgie et d'ophtalmologie, Université Laval
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- PublicationRestreintTissue Engineering of Cornea(Marcel Dekker, 2004-06-23) Giasson, Claude-J.; Guérin, Sylvain; Salesse, Christian; Germain, Lucie; Auger, François A.; Carrier, PatrickThe cornea is the transparent barrier between the eye and the environment. Tissue-engineered corneas are currently developed to replace wounded or diseased corneas. Various experimental applications are also foreseen for these tissues reconstructed in vitro by tissue engineering. This article covers the first human corneas reconstructed by tissue engineering from normal human cells and the different models used for the production of human and animal corneas in vitro. Corneal injury and the activation of the complex wound«hea]ing mechanisms are also addressed. Finally, we will attempt to provide the reader with a brief look toward the future of corneal tissue engineering, including the challenges that lie ahead as well as the potential experimental and clinical applications of this field.
- PublicationRestreintTissue engineering of skin and cornea : Development of new models for in vitro studies(Academy of Sciences, 2010-06-02) Guérin, Sylvain; Germain, Lucie; Larouche, Danielle; Bisson, Francis; Paquet, Claudie; Robitaille, Hubert; Auger, François A.; Gaudreault, Manon.; Martel, Israël; Duranceau, Louise; Proulx, Stéphanie; Carrier, Patrick; Simard-Bisson, Carolyne; Fradette, JulieHuman beings are greatly preoccupied with the unavoidable nature of aging. While the biological processes of senescence and aging are the subjects of intense investigations, the molecular mechanisms linking aging with disease and death are yet to be elucidated. Tissue engineering offers new models to study the various processes associated with aging. Using keratin 19 as a stem cell marker, our studies have revealed that stem cells are preserved in human skin reconstructed by tissue engineering and that the number of epithelial stem cells varies according to the donor's age. As with skin, human corneas can also be engineered in vitro. Among the epithelial cells used for reconstructing skin and corneas, significant age-dependent variations in the expression of the transcription factor Sp1 were observed. Culturing skin epithelial cells with a feeder layer extended their life span in culture, likely by preventing Sp1 degradation in epithelial cells, therefore demonstrating the pivotal role played by this transcription factor in cell proliferation. Finally, using the human tissue-engineered skin as a model, we linked Hsp27 activation with skin differentiation.
- PublicationAccès libreAutologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction(Éditeur non identifié, 2006-02-01) Guérin, Sylvain; Germain, Lucie; Giasson, Claude J.; Giroux-Talbot, Mariève; Auger, François A.; Bazin, Richard; Carrier, Patrick; Deschambeault, AlexandrePurpose: Regeneration of the corneal epithelium could be severely impaired in patients suffering from limbal stem cell deficiency. The purpose of this study was to evaluate the restoration of the corneal epithelium by grafting onto denuded corneas autologous limbal cells cultured on fibrin gels. The rabbit model was chosen to allow the microscopic evaluation over time after grafting. Methods: Rabbit limbal epithelial cells (RLECs) were isolated and cultured from small limbal biopsies (3 mm2). The epithelium was separated from stroma after dispase digestion and put in culture on lethally irradiated fibroblasts used as a feeder layer. At the first passage, RLECs were cultured on a fibrin gel matrix. At confluence, the cultured epithelia were grafted in vivo on denuded autologous rabbit corneas. At different postoperative times, grafted and control (without graft or grafted with fibrin gels only) rabbit corneas were compared in vivo with a slit lamp microscope, and in situ by histological and immunohistological microscopy of harvested biopsies. Results: A small limbal biopsy was sufficient to generate enough RLECs to prepare several grafts and to perform cell analysis. Only two weeks were required to produce a cultured epithelium suitable for autologous transplantation. One month after grafting, a normal corneal phenotype was observed on the ocular surface of grafted rabbits in contrast to the control rabbits (ungrafted or grafted with fibrin gel only) where histological signs of conjunctivalization were found. The absence of goblet cells and negative staining for keratin 4 confirmed that the cultured cells persisted and that the epithelium regenerated after grafting was not from conjunctival origin. Conclusions: Our results demonstrate that an autologous epithelium cultured on a physiologically biodegradable matrix can be prepared from a small biopsy and grafted on denuded cornea. The autologous graft allows epithelial regeneration from cultured cells and promotes corneal healing of unilateral total stem cell deficiency.
- PublicationRestreintThe tissue-engineered human cornea as a model to study expression of matrix metalloproteinases during corneal wound healing(Elsevier BV, 2015-11-22) Guérin, Sylvain; Germain, Lucie; Couture, Camille; Zaniolo, Karine; Lake, Jennifer; Patenaude, Julien; Carrier, PatrickCorneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing.
- PublicationAccès libreOptimization of culture conditions for porcine corneal endothelial cells(2007-04-03) Giasson, Claude-J.; Germain, Lucie; Martel, Sophie; Bourget, Jean-Michel; Gagnon, Nicolas; Auger, François A.; Proulx, Stéphanie; Carrier, Patrick; Brunette, Isabelle; Deschambeault, AlexandrePurpose : To optimize the growth condition of porcine corneal endothelial cells (PCEC), we evaluated the effect of coculturing with a feeder layer (irradiated 3T3 fibroblasts) with the addition of various exogenous factors, such as epidermal growth factor (EGF), nerve growth factor (NGF), bovine pituitary extract (BPE), ascorbic acid, and chondroitin sulfate, on cell proliferation, size, and morphology. Methods : PCEC cultures were seeded at an initial cell density of 400 cells/cm2 in the presence or absence of 20,000 murine-irradiated 3T3 fibroblast/cm2 in the classic media Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal bovine serum (FBS). Mean cell size and bromodeoxyuridine incorporation was assessed at various passages. Growth-promoting factors were studies by seeding PCEC at 8,000 cells/cm2 in DMEM with 20% FBS or Opti-MEM I supplemented with 4% FBS and one of the following additives: EGF (0.5, 5, 25 ng/ml), NGF (5, 20, 50 ng/ml), BPE (25, 50, 100, 200 μg/ml), ascorbic acid (10, 20, 40 μg/ml) and chondroitin sulfate (0.03, 0.08, 1.6%), alone or in combination. Cell number, size and morphology of PCEC were assessed on different cell populations. Each experiment was repeated at least twice in three sets. In some cases, cell cultures were maintained after confluence to observe post-confluence changes in cell morphology. Results : Co-cultures of PCEC grown in DMEM 20% FBS with a 3T3 feeder layer improved the preservation of small polygonal cell shape. EGF, NGF, and chondroitin sulfate did not induce proliferation above basal level nor did these additives help maintain a small size. However, chondroitin sulfate did help preserve a good morphology. BPE and ascorbic acid had dose-dependent effects on proliferation. The combination of BPE, chondroitin sulfate, and ascorbic acid significantly increased cell numbers above those achieved with serum alone. No noticeable changes were observed when PCEC were cocultured with a 3T3 feeder layer in the final selected medium. Conclusions : Improvements have been made for the culture of PCEC. The final selected medium consistently allowed the growth of a contact-inhibited cell monolayer of small, polygonal-shaped cells.
- PublicationAccès libreCharacterization of wound reepithelialization using a new human tissue–engineered corneal wound healing model(Association for Research in Vision and Ophthalmology, 2008-04-01) Giasson, Claude-J.; Guérin, Sylvain; Germain, Lucie; Giroux-Talbot, Mariève; Auger, François A.; Carrier, Patrick; Deschambeault, AlexandrePurpose. The reepithelialization of the corneal surface is an important process for restoring the imaging properties of this tissue. The purpose of the present study was to characterize and validate a new human in vitro three-dimensional corneal wound healing model by studying the expression of basement membrane components and integrin subunits that play important roles during epithelial cell migration and to verify whether the presence of exogenous factors could accelerate the reepithelialization. Methods. Tissue-engineered human cornea was wounded with a 6-mm biopsy punch, and the reepithelialization from the surrounding margins was studied. Biopsy samples of the reepithelialized surface were harvested 3 days after wounding and were processed for histologic, electron microscopic, and immunofluorescence analyses. The effects of fibrin and epithelial growth factor (EGF) on wound reepithelialization were also studied. Results. Results demonstrated that this in vitro model allowed the migration of human corneal epithelial cells on a natural extracellular matrix. During reepithelialization, epithelial cell migration followed a consistent wavelike pattern similar to that reported for human corneal wound healing in vivo. This model showed a histologic appearance similar to that of native tissue as well as expression and modulation of basement membrane components and the integrin subunits known to be main actors during the wound healing process. It also allowed quantification of the reepithelialization rate, which was significantly accelerated in the presence of fibrin or EGF. The results indicated that αvβ6 integrin expression was increased in the migrating epithelial cells compared with the surrounding corneal tissue. Conclusions. The similarity observed with the in vivo wound healing process supports the use of this tissue-engineered model for investigating the basic mechanisms involved in corneal reepithelialization. Moreover, this model may also be used as a tool to screen agents that affect reepithelialization or to evaluate the effect of growth factors before animal testing.
- PublicationAccès libreImpact of cell source on human cornea reconstructed by tissue engineering(IOVS, 2009-06-01) Giasson, Claude-J.; Guérin, Sylvain; Germain, Lucie; Audet, Caroline; Giroux-Talbot, Mariève; Auger, François A.; Gauvin, Robert; Carrier, Patrick; Deschambeault, AlexandrePurpose: To investigate the effect of the tissue origin of stromal fibroblasts and epithelial cells on reconstructed corneas in vitro. Methods: Four types of constructs were produced by the self-assembly approach using the following combinations of human cells: corneal fibroblasts/corneal epithelial cells, corneal fibroblasts/skin epithelial cells, skin fibroblasts/corneal epithelial cells, skin fibroblasts/skin epithelial cells. Fibroblasts were cultured with ascorbic acid to produce stromal sheets on which epithelial cells were cultured. After 2 weeks at the air-liquid interface, the reconstructed tissues were photographed, absorption spectra were measured, and tissues were fixed for histologic analysis. Cytokine expression in corneal- or skin-fibroblast-conditioned media was determined with the use of protein array membranes. The effect of culturing reconstructed tissues with conditioned media, or media supplemented with a cytokine secreted mainly by corneal fibroblasts, was determined. Results: The tissue source from which epithelial and mesenchymal cells were isolated had a great impact on the macroscopic and histologic features (epithelium thickness and differentiation) and the functional properties (transparency) of the reconstructed tissues. The reconstructed cornea had ultraviolet-absorption characteristics resembling those of native human cornea. The regulation of epithelial differentiation and thickness was mesenchyme-dependent and mediated by diffusible factors. IL-6, which is secreted in greater amounts by corneal fibroblasts than skin fibroblasts, decreased the expression of the differentiation marker DLK in the reconstructed epidermis. Conclusions: The tissue origin of fibroblasts and epithelial cells plays a significant role in the properties of the reconstructed tissues. These human models are promising tools for gaining a thorough understanding of epithelial-stromal interactions and regulation of epithelia homeostasis.
- PublicationAccès libreInfluence of Sp1/Sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues(Association for Research in Vision and Ophthalmology, 2003-04-01) Guérin, Sylvain; Germain, Lucie; Giasson, Marcelle; Gaudreault, Manon.; Leclerc, Steeve; Carrier, Patrick; Larouche, KathyPURPOSE : Primary cultured epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. However, as such cells are passaged in culture, they often lose their ability to proliferate by progressing toward terminal cell differentiation, a process likely to be determined by altered expression of transcription factors that have functions critical for cell adhesion and differentiation. This study was designed to determine whether the variable life span of primary cultured human corneal epithelial cells (HCECs) might be the consequence of varying expression levels of the well-known transcription factors Sp1 and Sp3 (Sp1/Sp3). METHODS : HCECs were obtained from donor eyes and cultured on irradiated Swiss-3T3. Sp1/Sp3 expression was monitored by Western blot and electrophoretic mobility shift assay (EMSA). The Sp1/Sp3 regulatory influence was evaluated by transfection of HCECs with a recombinant plasmid bearing the Sp1/Sp3-dependent poly(ADP-ribose) polymerase (rPARP) promoter fused to the CAT reporter gene. HCECs that expressed various levels of Sp1/Sp3 were also used for the production of corneal substitutes. RESULTS : Expression of Sp1/Sp3 was dramatically inconsistent between HCECs isolated from the eyes of different donors. Both factors were highly expressed during one passage and then totally disappeared as cells terminally differentiated. Proper stratification of HCECs on reconstructed tissue substitutes could be obtained only with cells that also had a delayed peak of Sp1/Sp3 expression when cultured in vitro. CONCLUSIONS : Expression of Sp1/Sp3 may represent a good predictor for selecting HCECs that are most likely to proliferate, stratify, and differentiate properly when used for the production of reconstructed corneal substitutes.
- PublicationRestreintLa médecine régénératrice : les cellules souches, les interactions cellulaires et matricielles dans la reconstruction cutanée et cornéenne par génie tissulaire(Elsevier Masson, 2008-06-02) Germain, Lucie; Larouche, Danielle; Paquet, Claudie; Auger, François A.; Proulx, Stéphanie; Carrier, Patrick; Lavoie, Amélie; Beauparlant, Annie.Le génie tissulaire vise à produire des tissus ou organes in vitro pour le remplacement permanent des tissus endommagés. À cette fin, la production de tissus autologues possède l’avantage d’éviter tout risque de rejet suite à leur transplantation sur un patient. La maîtrise des conditions de culture des cellules souches humaines postnatales est essentielle à la réalisation de tels tissus. Il est aussi souhaitable que leur organisation histologique et leur fonctionnalité se rapprochent de celles des tissus natifs. De plus, les cellules souches jouent un rôle essentiel au niveau du remplacement des cellules épithéliales différenciées dans les tissus qui doivent constamment se renouveler, tels que la peau et la cornée. Nous avons décrit une méthode qui permet de produire des organes vivants in vitro à partir de cellules humaines postnatales sans ajouter de biomatériaux. Cette méthode d’auto-assemblage repose sur la capacité qu’ont les cellules mésenchymateuses de s’organiser en tissu en présence des conditions de culture adéquates. Grâce à différentes techniques, ces tissus peuvent ensuite être assemblés en organes plus complexes tels que les vaisseaux sanguins, les valves cardiaques, la peau ou encore la cornée. Ces divers tissus pourront éventuellement être utilisés pour le remplacement d’organes malades ou endommagés et fourniront de nouvelles alternatives pour la médecine régénératrice de demain. Cet article de revue sera concentré sur la peau et la cornée. L’importance d’utiliser des conditions d’isolement et de culture qui permettent de conserver les cellules souches et de contrôler l’organisation des tissus afin d’assurer la qualité et la fonctionnalité des organes reconstitués par génie tissulaire sera discutée.
- PublicationAccès libreReconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types(Éditeur non identifié, 2010-10-29) Giasson, Claude-J.; Guérin, Sylvain; Germain, Lucie; Audet, Caroline; Auger, François A.; Uwamaliya, Jeanne d'Arc; Proulx, Stéphanie; Carrier, Patrick; Deschambeault, AlexandrePurpose: The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach. Methods: Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed. Results: Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K+-ATPase α1. Conclusions: This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial.