034 Survival of mouse oocytes after cooling in lower cryoprotectant concentrations by ultra rapid warming using an IR laser pulse

Abstract

Seki and Mazur have recently shown [PLoS One. 2012;7(4)] that about 90% of MII mouse oocytes survive vitrification in solutions of EAFS 10/10 that contain only half the usual concentration of solutes, provided that the warming rate is extremely high (117,000 °C/min). They reported that fewer than 10% survived if the concentration of EAFS were further reduced to 33% of normal. We are currently investigating whether high survivals could be obtained in 0.33× and 0.25× EAFS if the warming rates were still higher. For this purpose we have cooled MII oocytes in these media on Cryotops to −196 °C and warmed them 5 times more rapidly at ∼600,000 °C/min by subjecting them to a 13 joule pulse of 15 msec duration from a Nd:YAG laser. Kleinhans et al in a companion paper at this meeting discuss the physics attributes of the laser and the interactions of the pulse with the sample, as well as the jig he developed to permit the manipulation of the Cryotop before and during its exposure to the laser pulse. The osmotic/morphological survival of 31 samples suspended in 0.25× EAFS was 15.2 ± 22% (standard deviation). The survival of two samples in 0.33× EAFS was 30%. The total molality of these solutions is 1.27 and 1.72, respectively. These are in the range used in standard slow-freeze cryopreservation. For comparison, note that the total molality of full strength 1x EAFS is 7.37 molal, 5.8 and 4.3-fold higher. Although these preliminary results are encouraging, we have two caveats. One is that the variability of the former is high as evidenced by the large standard errors. The other is that the survival of controls subjected to the same cooling procedure but warmed at 117,000 °C/min (no laser) was close to the same (13.3% vs. 15.2%). Source of funding: NIH Grant 8R01 OD 011201, Peter Mazur, PI. Conflict of interest: None declared. pmazur@utk.edu