Comparison of culture media for ex vivo cultivation of limbal epithelial progenitor cells

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Loureiro, Renata Ruoco [UNIFESP]
Cristovam, Priscila Cardoso [UNIFESP]
Martins, Caio Marques [UNIFESP]
Covre, Joyce Luciana [UNIFESP]
Sobrinho, Juliana Aparecida [UNIFESP]
Ricardo, José Reinaldo da Silva [UNIFESP]
Hazarbassanov, Rossen Myhailov [UNIFESP]
Hoefling-Lima, Ana Luisa [UNIFESP]
Belfort, Rubens Junior [UNIFESP]
Nishi, Mauro [UNIFESP]
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Purpose: To compare the effectiveness of three culture media for growth, proliferation, differentiation, and viability of ex vivo cultured limbal epithelial progenitor cells.Methods: Limbal epithelial progenitor cell cultures were established from ten human corneal rims and grew on plastic wells in three culture media: supplemental hormonal epithelial medium (SHEM), keratinocyte serum-free medium (KSFM), and Epilife. The performance of culturing limbal epithelial progenitor cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for adenosine 5'-triphosphate-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT) and real-time reverse transcription polymerase chain reaction (RT-PCR) for CK3, ABCG2, and p63, and cell viability using Hoechst staining.Results: Limbal epithelial progenitor cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis showed that proliferated cells in the SHEM had lower expression for markers related to progenitor epithelial cells (ABCG2) and putative progenitor cells (p63), and a higher percentage of positive cells for differentiated epithelium (CK3) when compared to KSFM and Epilife. In PCR analysis, ABCG2 expression was statistically higher for Epilife compared to SHEM. Expression of p63 was statistically higher for Epilife compared to SHEM and KSFM. However, CK3 expression was statistically lower for KSFM compared to SHEM.Conclusions: Based on our findings, we concluded that cells cultured in KSFM and Epilife media presented a higher percentage of limbal epithelial progenitor cells, compared to SHEM.
Molecular Vision. Atlanta: Molecular Vision, v. 19, p. 69-77, 2013.