Comparação de diferentes meios para o cultivo ex vivo de células epiteliais do limbo
Data
2013
Autores
Loureiro, Renata Ruoco 
Orientadores
Nishi, Mauro
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: Comparar a eficacia de tres diferentes meios de cultura para o crescimento, diferenciacao, proliferacao e viabilidade de celulas-tronco epiteliais do limbo cultivadas ex vivo. Metodos: As culturas de celulas epiteliais do limbo foram estabelecidas a partir de 10 rimas corneo-esclerais cultivadas em tres diferentes meios de cultura: Supplemental Hormonal Epithelial Medium (SHEM), Keratinocyte Serum-Free Medium (KSFM) e Epilife. O desempenho das culturas de celulas epiteliais do limbo em cada meio foi avaliado de acordo com os seguintes parametros: area de crescimento e migracao epitelial; imunocitoquimica para ATP-binding cassette member 2 (ABCG2), p63, Ki67, citoqueratina 3 (CK3) e vimentina (VMT); reacao em cadeia da polimerase via transcriptase reversa (RT-PCR) em tempo real para CK3, ABCG2 e p63, e viabilidade celular utilizando a coloracao Hoechst. Resultados: As celulas epiteliais do limbo cultivadas em SHEM apresentaram migracao mais rapida, em comparacao com KSFM e Epilife. A analise imunocitoquimica evidencia que as celulas cultivadas em meio SHEM apresentaram menor expressao de marcadores relacionados com celulas-tronco epiteliais (ABCG2), e celulas indiferenciadas (p63), e maior porcentagem de celulas positivas para epitelio diferenciado (CK3), quando comparado com KSFM e Epilife. Na analise da RT-PCR, a expressao de ABCG2 foi estatisticamente maior para Epilife em comparacao ao SHEM. A expressao de p63 foi estatisticamente maior para Epilife quando comparada com SHEM e KSFM. Por outro lado, expressao de CK3 foi significativamente menor em KSFM quando comparada com SHEM. Conclusao: Com base em nossos resultados, concluimos que as celulas cultivadas nos meios KSFM e Epilife apresentaram maior porcentagem de celulas-tronco epiteliais limbicas, quando comparadas com as celulas cultivadas no meio SHEM
Purpose: To compare the effectiveness of three different culture media for growth, proliferation, differentiation and viability of ex vivo cultured limbal epithelial cells. Methods: Limbal epithelial cells cultures were established from 10 human corneal rims in three different culture media: Supplemental Hormonal Epithelial Medium (SHEM), Keratinocyte Serum-Free Medium (KSFM) and Epilife. The performance of cultured limbal epithelial cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for ATP-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT); real-time reverse transcription polimerase chain reaction (RT-PCR) for CK3, ABCG2 and p63, and cell viability using Hoechst staining. Results: Limbal epithelial cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis pointed that proliferated cells at SHEM medium had lower expression for markers related to progenitor epithelial cells (ABCG2), and putatitve cells (p63); and 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. On the other hand, 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 higher percentage of limbal epithelial stem cells, compare to SHEM.
Purpose: To compare the effectiveness of three different culture media for growth, proliferation, differentiation and viability of ex vivo cultured limbal epithelial cells. Methods: Limbal epithelial cells cultures were established from 10 human corneal rims in three different culture media: Supplemental Hormonal Epithelial Medium (SHEM), Keratinocyte Serum-Free Medium (KSFM) and Epilife. The performance of cultured limbal epithelial cells in each medium was evaluated according to the following parameters: growth area of epithelial migration; immunocytochemistry for ATP-binding cassette member 2 (ABCG2), p63, Ki67, cytokeratin 3 (CK3), and vimentin (VMT); real-time reverse transcription polimerase chain reaction (RT-PCR) for CK3, ABCG2 and p63, and cell viability using Hoechst staining. Results: Limbal epithelial cells cultivated in SHEM showed a tendency to faster migration, compared to KSFM and Epilife. Immunocytochemical analysis pointed that proliferated cells at SHEM medium had lower expression for markers related to progenitor epithelial cells (ABCG2), and putatitve cells (p63); and 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. On the other hand, 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 higher percentage of limbal epithelial stem cells, compare to SHEM.
Descrição
Citação
LOUREIRO, Renata Ruocco. Comparação de diferentes meios para o cultivo ex vivo de células epiteliais do limbo. 2013. 39f. Dissertação (Mestrado em Oftalmologia) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2013