Importância do co-cultivo com fibroblastos de camundongo 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo humano

• Autor(a) principal: Cristovam, Priscila Cardoso [UNIFESP]
• Data de Publicação: 2008
• Outros Autores: Glória, Maria Aparecida da [UNIFESP], Melo, Gustavo Barreto de [UNIFESP], Gomes, José Álvaro Pereira [UNIFESP]
• Tipo de documento: Artigo
• Idioma: por
• Título da fonte: Repositório Institucional da UNIFESP
• Texto Completo: http://dx.doi.org/10.1590/S0004-27492008000500015; http://repositorio.unifesp.br/handle/11600/4570
• topic: Epithelial cells; Cell culture techniques; Cell differentiation; Stem cells; Limbus corneae; Células epiteliais; Técnicas de cultura de células; Diferenciação celular; Células-tronco; Limbo da córnea

PURPOSE: To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR). METHODS: Corneo-scleral rims from different donors (n=6) had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the segments was placed with the epithelial side up on the bottom of a 6-well culture plate (Group A). The other two fragments were trypsinized and the obtained cell suspension was cultured with (Group B) or without (Group C) irradiaded 3T3 cells. The cells were cultured in supplemental hormonal epithelial medium (SHEM), the epithelial migration and clone formation in groups A, B and C were evaluated with phase contrast microscopy and rodamine B staining. RESULTS: Epithelial cell growth was observed in 4/6 rims (Group A). All epithelial cell suspensions that were cultured with 3T3 cells (Group B) formed clones. No adhesion or true clone formation (holo- or meroclones) was observed in the cell suspensions that were cultivated without 3T3 (Group C) (p=0.009). CONCLUSIONS: Epithelial cell suspension obtained from corneo-scleral rims in this model needs to be cultivated with 3T3 cells in order to form clones and establish limbal epithelial cell colonies with the potential to be used for ocular surface reconstruction.