Cryopreservation of human cumulus cells for co-cultures and assessment of DNA damage after thawing using the comet assay.
OBJECTIVE
Cumulus cells have been shown to be beneficial for blastocysts formation in co-cultures but information on cumulus cryopreservation is lacking. The objective was to use the fixed cell comet assay to analyze for DNA damage in cumulus cells after cryopreservation.


METHODS
Discarded cumulus cells from follicular aspirates obtained during assisted reproduction procedures (N = 4 cases) were pooled and cryopreserved in either 40% ethylene glycol and 0.5 M sucrose, 12:20% glycerol-egg yolk medium, 28% glycerol hypoosmolar medium or control medium. The cells were processed and stored in liquid nitrogen for 48 h. The thawed cells were smeared on glass slides, fixed, stained with acridine orange, embedded in a mini-agarose layer, and electrophoresis carried out. Fluorescent images were analyzed.


RESULTS
The cumulus tail moment, a calculated index of DNA damage, was significantly lower for each of the three cryoprotectant when compared with the control. The two cryoprotectants containing glycerol were associated with higher cumulus cell viability. However, the glycerol-egg yolk combination yielded the highest cell viability.


CONCLUSIONS
The cumulus comet assay demonstrated similar DNA integrity in cells frozen in each of the three cryoprotectants. The glycerol-egg yolk medium had the highest cell viability with little or no DNA damage after freeze-thaw. More studies are needed to examine the long-term effect of the cryoprotectants on thawed cumulus cell viability.
Lindley
E M
EM
Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, 11370 Anderson Street Suite 3950, Loma Linda, California 92354, USA.
Jacobson
J D
JD
Corselli
J
J
King
A
A
Chan
P J
PJ
D000165:Acridine Orange / Q000737:chemistry; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods*; D020552:Comet Assay; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004249:DNA Damage; D005260:Female; D005307:Fertilization in Vitro; D005456:Fluorescent Dyes / Q000737:chemistry; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D008297:Male; D006080:Ovarian Follicle / Q000166:cytology*; D011247:Pregnancy
D003451:Cryoprotective Agents; D005456:Fluorescent Dyes; D000165:Acridine Orange; D005990:Glycerol