Efficient cryopreservative conditions for cultivated limbal and conjunctival epithelial cells

Abstract

Purpose: To examine the effects of cryopreservation on the viability of cultivated corneal limbal and conjunctival epithelial cells and to evaluate the optimal conditions for cryopreservation. Methods: The cultivated human limbal epithelial cells (HLECs) were stored in media including 20%, 50%, and 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) at - 196 degrees C for 1 week. The cultivated rabbit conjunctival epithelial cells were stored in 10%, 20%, and 50% FBS with 10% glycerol or DMSO as a cryoprotectant at - 196 degrees C for 1 week. After thawing, cell viability was assessed using the trypan blue vital staining and 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazoliurn bromide (MTT) assay. Immunofluorescent staining was performed with cytokeratin 3/12 antibody. Colony-forming efficiency (CFE) was evaluated 2 weeks after culture. Results: HLECs cryopreserved with 50% FBS showed the highest cell viability, whereas those with 20% FBS revealed the lowest survival rate (87.1% +/- 0.8% and 79.8% +/- 4.01%, respectively; P = 0.030). CFE of HLECs was 2.13 +/- 1.35%, 2.31 +/- 2.23%, and 1.94 +/- 0.72% in cells with 20%, 50%, and 90% FBS, respectively (P > 0.05). For conjunctival epithelial cells, the cell viability was the highest with 50% FBS and 10% glycerol (95.0% +/- 4.27%), and the lowest survival rate was observed in the condition of 10% FBS and 10% DMSO (80.0% +/- 5.49%). CFE of cryopreserved conjunctival epithelial cells was 14.1% +/- 1.9% in cells with 20% FBS and glycerol and 13.5% +/- 2.0% in those with 20% FBS and DMSO (P > 0.05). HLECs expressed CK3/12 after cryopreservation in all conditions examined. Conclusions: The best results were yielded by 50% FBS for cell viability in HLECs. Glycerol seems to be superior to DMSO in cell viability of the rabbit conjunctival epithelium after cryopreservation.