Personne : Giasson, Claude-J.
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Laboratoire d'organogénèse expérimentale (LOEX) du Centre de recherche du Centre hospitalier affilié universitaire de Québec, Université Laval
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- 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 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.
- PublicationAccès libreAmnios et problèmes de surface oculaire(EDK, 2006-06-15) Giasson, Claude-J.; Bouchard, Cindy; Germain, Lucie; Boisjoly, HélèneLa membrane amniotique, enclave interne de la vie naissante, presente diverses proprietes exploitees en ophtalmologie. Elle est utile pour : (1) limiter la formation d’adhesions fibreuses entre la paupiere et le globe oculaire (symblepharon) ou la progression d’excroissances fibrovasculaires vers la cornee (pterygion) ; (2) contribuer a la guerison d’ulceres corneens, de keratites bulleuses et des deficiences en cellules souches de la cornee dues a des brulures thermiques, chimiques ou d’autre origine. L’amnios, alors greffe avec des cellules limbiques saines, favoriserait la proliferation de cellules moins differenciees, aptes a reconstruire l’epithelium corneen. La membrane amniotique contient des cytokines, reduit l’acuite des reactions immunologiques et possede des proprietes antalgiques, anti-bacteriennes et anti-inflammatoires ; de plus, elle favorise, comme le fait la peau foetale, une guerison avec un minimum de cicatrices. La connaissance des mecanismes d’action de la membrane amniotique obtenue grâce a la recherche pourrait fournir de nouvelles avenues pharmacologiques afin de traiter des maladies de la surface oculaire.
- PublicationRestreintTissue Engineering of Feline Corneal Endothelium Using a Devitalized Human Cornea as Carrier(Mary Ann Liebert, Inc. Publishers, 2009-01-05) Giasson, Claude-J.; Germain, Lucie; Audet, Caroline; Uwamaliya, Jeanne d'Arc; Proulx, Stéphanie; Carrier, Patrick; Brunette, Isabelle; Deschambeault, AlexandreThe difficulties in obtaining good quality tissue for the replacement of corneas of patients suffering from endothelial dysfunctions have prompted us to evaluate the feasibility of producing a tissue-engineered (TE) corneal endothelium using devitalized human stromal carriers. Thus, corneal substitutes were produced by seeding cultured feline corneal endothelial cells on top of previously frozen human corneal stromas. After two weeks of culture to allow attachment and spreading of the seeded cells, the TE corneal endothelium was stained with alizarin red for endothelial cell count and fixed for histology, immunofluorescence labeling, scanning and transmission electron microscopy. Histology and Hoechst staining showed that there were no remaining cells in the devitalized stroma. After seeding, histology and transmission electron microscopy showed that the TE corneal endothelium formed a monolayer of tightly packed cells that were well adhered to Descemet’s membrane. Scanning electron microscopy corroborated that the cells covered the entire posterior corneal surface and had an endothelial morphology. Alizarin staining showed that mean cell counts were 2272 ± 344 cells=mm2 , indicating that the cell density was appropriate for grafting. The TE feline corneal endothelium also expressed the function-related proteins Na+ =HCO3 , ZO-1, and Na+ =K+ -ATPase a1, and could easily be marked with a fluorescent tracker. This study demonstrates the feasibility of reconstructing a highly cellular and healthy corneal endothelium on devitalized human corneal stromas.
- 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 human cornea(CRC Press, 2014-03-27) Guillemette, Maxime.; Giasson, Claude-J.; Guérin, Sylvain; Germain, Lucie; Auger, François A.; Gaudreault, Manon.; Proulx, Stéphanie; Carrier, Patrick; Chirila, TraianThe cornea is a well-organized tissue composed of three cell types (epithelial, stromal and endothelial cells), each having an important role for its functionality. This chapter will address different tissue engineering approaches to the reconstruction of either partial or full-thickness living corneal substitutes that can be used either as in vitro models for woundhealing studies, or in vivo, eventually replacing the donor cornea for transplantation in humans. Isolation of the proper cells, followed by appropriate culture conditions, and assembly into a three-dimensional tissue construct, are the first steps required for producing a functional corneal substitute.