CRYOPRESERVATION OF MACAQUE OVARIAN TISSUES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background: We have previously reported that the rate at which equine and macaque ovarian tissue sections are first cooled from +25 ¿C to +4 ¿C has a significant effect on the measured subzero freezing response when these tissues are frozen in 0.85 M solutions of glycerol, dimethylsulfoxide (DMSO) or ethylene glycol (EG) (Devireddy et al., 2006, Mol. Reprod. Dev. 73:330-341; Li et al. 2006, Mol. Reprod. Dev. 73:1600-1611). We have measured the effect of mildly pre-heating the ovarian tissues before being frozen in mixtures of DMSO and EG. Methods: Sections of freshly collected equine and macaque ovarian tissue sections were suspended in a mixture of 0.9 M EG plus 0.7 M DMSO (total:1.6 M, equivalent to a mixture of ~5% v/v of EG and DMSO) and heated to 37 ¿C from RT (25 ¿C) at a heating rate of either 1 or 10 ¿C/min. The tissue sections were equilibrated at 37 ¿C for 2 minutes before being cooled from +37 ¿C to +4 ¿C at 40 ¿C/min and then frozen to a subzero temperature of -50 ¿C at 5 ¿C/min in the presence of extracellular ice. As the tissues were being frozen, a shape-independent Differential Scanning Calorimeter (DSC) technique was used to measure water loss from the tissues and consequently, the best-fit subzero membrane permeability parameters (Lpg and ELp) of ovarian tissues during freezing. Results: The water transport response of ovarian tissue samples pre-heated to +37 ¿C at 1 ¿C/min from +25 ¿C was significantly different (confidence level 95%) from that of tissue samples heated at 10 ¿C/min from +25 ¿C to +37 ¿C; possibly due to the inducement of heat shock proteins in the ovarian tissue membranes and the associated changes in the water permeability). We fitted a model of water transport to the experimentally-derived volumetric shrinkage data and determined the best-fit membrane permeability parameters (Lpg and ELp) of macaque ovarian tissue during freezing. Conclusions: Calculations made of the theoretical response of tissue at subzero temperatures suggest that the optimal cooling rates to cryopreserve ovarian tissue are significantly dependent upon suprazero heating conditions.