Efficient cryopreservative conditions for cultivated limbal and conjunctival epithelial cells.
OBJECTIVE
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.
Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea.
Joo Youn
JY
Oh
Mee Kum
MK
Kim
Kyung Seon
KS
Shin
Mi Sun
MS
Shin
Won Ryang
WR
Wee
Jin Hak
JH
Lee
Seon Mi
SM
Ko
Jae Lim
JL
Lee
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005990:Glycerol; D006801:Humans; D053551:Keratin-12 / Q000378:metabolism; D053558:Keratin-3 / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D008297:Male; D009926:Organ Preservation; D011817:Rabbits; D044967:Serum
D003451:Cryoprotective Agents; D053551:Keratin-12; D053558:Keratin-3; D005990:Glycerol