Document Detail

Efficient cryopreservative conditions for cultivated limbal and conjunctival epithelial cells.
MedLine Citation:
PMID:  17667619     Owner:  NLM     Status:  MEDLINE    
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.
Joo Youn Oh; Mee Kum Kim; Kyung Seon Shin; Mi Sun Shin; Won Ryang Wee; Jin Hak Lee; Seon Mi Ko; Jae Lim Lee
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Cornea     Volume:  26     ISSN:  0277-3740     ISO Abbreviation:  Cornea     Publication Date:  2007 Aug 
Date Detail:
Created Date:  2007-08-01     Completed Date:  2007-10-16     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8216186     Medline TA:  Cornea     Country:  United States    
Other Details:
Languages:  eng     Pagination:  840-6     Citation Subset:  IM    
Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea.
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MeSH Terms
Cell Differentiation / physiology
Cell Survival / physiology
Cells, Cultured
Conjunctiva / cytology*,  metabolism
Cryopreservation / methods*
Cryoprotective Agents
Epithelial Cells / cytology*,  metabolism
Fluorescent Antibody Technique, Indirect
Keratin-12 / metabolism
Keratin-3 / metabolism
Limbus Corneae / cytology*,  metabolism
Organ Preservation*
Reg. No./Substance:
0/Cryoprotective Agents; 0/Keratin-12; 0/Keratin-3; 56-81-5/Glycerol

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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