Cryopreservation of human cumulus cells for co-cultures and assessment of DNA damage after thawing using the comet assay

Purpose. Cumulus cells have been shown to be beneficial for blastocysts for mation in co-cultures but information on cumulus cryopreservation is lackin g. The objective was to use the fixed cell comet assay to analyze for DNA d amage in cumulus cells after cryopreservation. Methods. Discarded cumulus cells from follicular aspirates obtained during assisted reproduction procedures (N = 4 cases) were pooled and cryopreserve d 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 sme ared on glass slides, fixed, stained with acridine orange, embedded in a mi ni-agarose layer, and electrophoresis carried out. Fluorescent images were analyzed. Results. The cumulus tail moment, a calculated index of DNA damage, was sig nificantly lower for each of the three cryoprotectant when compared with th e 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 me dium had the highest cell viability with little or no DNA damage after free ze-thaw.