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Hideyuki Miyashita, Shigeto Shimmura, Kazuo Tsubota; Combination Of ROCK Inhibitor And KGF Improves The Quality Of Epithelial Cell Sheets. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3511.
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ROCK inhibitor improves the colony forming efficiency (CFE) of human embryonic stem cells and human corneal endothelial cells. Keratinocyte growth factor (KGF) improves expression of undifferentiated markers in epithelial cell sheets in serum and feeder-free system. We confirmed whether combining ROCK inhibitor and KGF improves the quality of epithelial cell sheets even in traditional feeder co-culture system with serum.
F12/DMEM supplemented with 4% FBS, insulin, tri-iodo-thyronine, hydrocortisone, isoproterenol, antibiotics, and EGF or KGF (10 ng/mL) were used as media. ROCK inhibitor Y-27632 (final 10 µM) was supplemented in some cases. Human corneal limbal epithelial cells (HLE) were isolated from US eyebank eyes. HLE were directly inoculated on mitomycin C (MMC)-treated NIH/3T3 at clonal density, or inoculated at 2-4x104 / cm2 on cell culture inserts coated with fibrin glue and co-cultured with MMC-treated human mesenchymal stem cells for 2 weeks.
Y-27632 increased CFE of HLE in both EGF medium and KGF medium (3.8±2.2 and 2.7±0.4 fold, respectively). Fibroblast contamination was rare in Y-27632 supplemented culture. Although Y-27632 is reported to inhibit the stratification of epidermal keratinocytes, Y-27632 supplemented HLE sheets were stratified. Expression of differentiation marker K12 and undifferentiation marker K15 and p63, and CFE of HLE sheet was higher in KGF culture in compared to EGF culture, as reported in serum and feeder-free culture. Y-27632 did not interfere the KGF effect.
Y-27632 may increase CFE and inhibit the appearance of fibroblast during culture, and KGF may maintain undifferentiated cells during culture. Combination of ROCK inhibitor Y-27632 and KGF seems to improve the quality of epithelial cell sheets even in traditional feeder co-culture system with serum.
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