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L.P. Ang, C. Seah, R. Beuerman, D.T. Tan; The Use of Human Serum in the Cultivation of Human Conjunctival Epithelial Cells on Amniotic Membranes and Plastic . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3788.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate the use of human serum (HS) in the cultivation, serial propagation and differentiation of human conjunctival epithelial (HCjE) cells on human amniotic membranes (HAM) and plastic. Methods: Normal HCjE cells were cultivated in basal media (MCDB 153) supplemented with 5% HS on HAM and on plastic. This was compared with cultivation in the presence of 5% fetal bovine serum (FBS), as well as in basal media alone. The cellular morphology, rate of explant outgrowth, colony forming efficiency, number of serial passages and number of cell generations were analyzed. Cultured HCjE cells underwent light microscopic examination. Keratin expression was analyzed by immunostaining with monoclonal antibodies to cytokeratins, K3 and K4. Results: Primary explant cultures on HAM demonstrated a slightly slower rate of cellular proliferation in HS compared to FBS. The relative ratios of the outgrowth areas in the presence of HS or FBS, as compared to basal medium alone, were 0.84±0.15 and 1.21±0.16 respectively. Cells cultivated in both serum conditions were more densely organized and formed stratified sheets. Prolonged exposure to HS and FBS for cells cultured on plastic resulted in differentiated colonies by passage 2. In the absence of serum, cells remained less differentiated and more proliferative. Significant fibroblast contamination was noted in both serum-containing conditions. The number of serial passages that the cells could undergo before senescence for HS, FBS, or basal media alone,was 2, 3 and 4, respectively. HCjE cells grown on HAM and plastic expressed the K4 keratin, but did not express the cornea-specific keratin, K3, which was consistent with the normal conjunctival phenotype. Conclusions: Although both HS and FBS support the growth of HCjE cells on amniotic membranes, prolonged exposure results in terminal differentiation and decreased proliferation. The presence of significant fibroblast overgrowth further limits the use of either HS or FBS in the selective serial propagation of HCjE cells.
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