A Defined Serum-Free Medium CnT-20 for ex vivo Expansion of Human Corneal Epithelial Progenitor Cells
Chuang EY, Pflugfelder SC, Li DQ
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine
ARVO Meeting Abstract
To evaluate a defined serum-free medium CnT-20 that supports ex vivo expansion of human corneal epithelial progenitor cells. Human corneal epithelial cells were cultivated from fresh limbal tissue explants and isolated limbal epithelial single cells in 8 different media: 3 defined serum-free media (CnT-20, CnT-30 and D-KSFM), 4 serum-free media with bovine pituitary extracts (CnT-50, KSFM, KGM2 and EpiLife), and 5% FBS containing SHEM. The cells were passaged when subconfluent. Hemocytometer and MTT assay were used to evaluate cell proliferation. Cell morphology and immunofluorescent staining for corneal epithelial stem cell and differentiation associated markers were used to evaluate cell phenotype. Results: All 8 media supported corneal epithelial primary culture from limbal explants, but only CnT-20 supports primary culture and serial passages from single limbal epithelial cells without 3T3 feeder layer. The cells cultured in CnT-20 and CnT-50 media could serially subcultured at more than 4 passages, cells in CnT-30 could passage 2-3 times, while cells in D-KSFM, KSFM, KGM2, EpiLife and SHEM media could not grow in 2nd passage. MTT assay showed highest proliferative rate of cells in CnT-20 media compared with other serum-free media. The cells grown in CnT-20 displayed significantly higher levels of stem cell associated markers such as p63, integrin b1 and EGFR, but lower levels of differentiation markers, keratin 3, involucrin and connexin 43, than cells in other media. Conclusions: These findings demonstrated that CnT-20 is a promising medium providing niche environment for ex vivo expansion of corneal epithelium progenitor cells that retain less differentiation status and high proliferative capacity. This completely defined serum-free medium would be useful for epithelial stem cell research and corneal epithelial tissue bioengineering. Click here to see the full poster.