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L Ang, DT H Tan, RW Beuerman, RM Lavker; Serum-free Cultivation Of Conjunctival Epithelial Cells And Its Use In An In Vitro Reconstructed Human Conjunctival Equivalent . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3158.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To compare the cultivation, serial growth and differentiation of human conjunctival epithelial (HCjE) cells under serum-free conditions, with conditions that use serum and 3T3 feeder layers, and to use this in the reconstruction of a human conjunctival tissue equivalent. Method: Normal HCjE cells were cultivated in SFM (Modified MCDB 153) in the absence and presence of lethally treated 3T3 feeder cells. Another group of HCjE cells were grown in serum-containing media (DMEM / Ham's F12 with 10% FBS) with 3T3 feeder cells. Conjunctival explants were cultivated using similar SFM on human amniotic membranes (AM) till confluence. The colony forming efficiency (CFE), number of cell generations and number of serial passages were compared. The cultured HCjE cells were examined by light and electron microscopy. Keratin expression was analysed by immunostaining with the AE-5 monoclonal antibody to keratin K3 and antibody to keratin K4. Results: In SFM, the CFE of HCjE cells was 15.8%5% and the cells underwent 8-10 population doublings (6-7 passages) before senescence. The use of feeder layers with SFM did not confer any significant advantage. In the presence of serum and feeder layers, the CFE was 26.1%4%. Under all of the different culture conditions, the cells expressed the K4 keratin that is associated with conjunctival epithelium, and did not express the corneal specific keratin, K3. Under serum-free conditions, the HCjE cells migrated from the explants to form stratified epithelial sheets on the AM. The HCjE cells formed desmosomal contacts, and numerous microvillus infoldings characterized the apical surface of the superficial cells. Conclusions: HCjE cells were serially cultivated under serum-free conditions, and formed stratified cell layers on AM. The keratin expression of the cultivated cells was similar to that of normal human conjunctival epithelium. These reconstructed human conjunctival tissue-equivalents may provide an alternative method of treating ocular surface diseases.
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