[Importance of 3T3 feeder layer to establish epithelial cultures from cell suspension obtained from corneo-scleral rims].
OBJECTIVE
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.
Cristovam
Priscila Cardoso
PC
Instituto da Visão, Centro Avançado de Superfície Ocular, Universidade Federal de São Paulo, São Paulo, Brasil.
Glória
Maria Aparecida da
MA
Melo
Gustavo Barreto
GB
Gomes
José Alvaro Pereira
JA
D016475:3T3 Cells; D000328:Adult; D000818:Animals; D049109:Cell Proliferation; D018920:Coculture Techniques; D004847:Epithelial Cells / Q000166:cytology*; D019573:Epithelium, Corneal / Q000166:cytology*; D005260:Female; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D008297:Male; D051379:Mice; D008875:Middle Aged; D009924:Organ Culture Techniques; D010641:Phenotype; D013997:Time Factors