| Title | Efficient Cryopreservative Conditions for Cultivated Limbal and Conjunctival Epithelial Cells. | | Author(s) | Oh JY, Kim MK, Shin KS, Shin MS, Wee WR, Lee JH, Ko SM, Lee JL | | Institution | From the *Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea; the †Seoul National University Hospital Clinical Research Institute, Seoul, Korea; the ‡Department of Ophthalmology, Chungnam National University Hospital, Taejon, Korea; and the §Valued Eye Clinic, Taejon, Korea. | | Source | Cornea 2007 Aug; 26(7):840-846. | | 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 tetrazolium 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. | | Language | ENG | | Pub Type(s) | JOURNAL ARTICLE
| | PubMed ID | 17667619 |
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