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N. Gagnon, F.A. Auger, L. Germain; Porcine Corneal Endothelial Cell Culture Improvement: Effect of Initial Seeding Density and Presence of a Feeder Layer . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5006.
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Purpose: To optimize the culture conditions of adult porcine corneal endothelial cells (PCEC) for use in the development of a living tissue–engineered substitute to replace a damaged corneal endothelium. The porcine model was chosen to undertake the pre–clinical studies. The study aims at comparing different culture methods to optimize for the conservation of small cell size with distinctive endothelial morphology found in–vivo and serial cultivation in order to obtain a large quantity of cells from a very small starting quantity, such as would be the case with a biopsy preceding an operation. Methods: Adult porcine eyes were obtained from a local abattoir and their corneal endothelial cells harvested following incubation with EDTA and trypsin. PCEC were cultured in plastic flasks without or with a feeder layer of irradiated murine 3T3 cells. The culture medium was Dulbecco's Modified Eagle's Medium supplemented with 20% foetal bovine serum. Cells were trypsinized near confluence and subcultured in the same conditions to evaluate the number of passages. Cell size and morphology were evaluated at different seeding densities (4, 40, 80 or 120 PCEC per mm2) for optimization. Results: PCEC co–cultured with irradiated 3T3 cells at passages up to 4 still retained their characteristic endothelial morphology while the same cells cultured on plastic started losing their characteristic morphology at passage 3 and became irregularly–shaped and elongated. The size of PCEC cultured with 3T3 cells was on average 1 µm smaller than PCEC cultured alone. The optimal seeding density of PCEC for primary cultures and subcultures was found to be 120 cells per mm2. Conclusions: A high initial seeding density is necessary in order to obtain cultures with the characteristic corneal endothelial cell morphology. Furthermore, PCEC co–cultured with irradiated murine 3T3 cells are smaller in size and retain the characteristic endothelial morphology up to passage 4. Improvement of PCEC cultures is important in view of using these cells as a substitute to replace damaged corneal endothelium.
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