玻璃化冷凍過程提高冷凍液溫度可能會提高其毒性
玻璃化冷凍過程提高時間>15min可能會提高其毒性
PROH 毒性> DMSO毒性> or = EG毒性
合併使用多種抗凍液&下降其濃度可減少其毒性

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3217997/

PLoS One. 2011;6(11):e27604. doi: 10.1371/journal.pone.0027604. Epub 2011 Nov 16.

Comparison and avoidance of toxicity of penetrating cryoprotectants.

Szurek EA1, Eroglu A.

Author information

Abstract

The objective of this study was to elucidate the toxicity of widely used penetrating cryoprotective agents (CPAs) to mammalian oocytes. To this end, mouse metaphase II (M II) oocytes were exposed to 1.5 M solutions of dimethylsulfoxide (DMSO), ethylene glycol (EG), or propanediol (PROH) prepared in phosphate buffered saline (PBS) containing 10% fetal bovine serum. To address the time- and temperature-dependence of the CPA toxicity, M II oocytes were exposed to the aforementioned CPAs at room temperature (RT, ∼23°C) and 37°C for 15 or 30 minutes. Subsequently, the toxicity of each CPA was evaluated by examining post-exposure survival, fertilization, embryonic development, chromosomal abnormalities, and parthenogenetic activation of treated oocytes. Untreated oocytes served as controls. Exposure of MII oocytes to 1.5 M DMSO or 1.5 M EG at RT for 15 min did not adversely affect any of the evaluated criteria. In contrast, 1.5 M PROH induced a significant increase in oocyte degeneration (54.2%) and parthenogenetic activation (16%) under same conditions. When the CPA exposure was performed at 37°C, the toxic effect of PROH further increased, resulting in lower survival (15%) and no fertilization while the toxicity of DMSO and EG was still insignificant. Nevertheless, it was possible to completely avoid the toxicity of PROH by decreasing its concentration to 0.75 M and combining it with 0.75 M DMSO to bring the total CPA concentration to a cryoprotective level. Moreover, combining lower concentrations (i.e., 0.75 M) of PROH and DMSO significantly improved the cryosurvival of MII oocytes compared to the equivalent concentration of DMSO alone. Taken together, our results suggest that from the perspective of CPA toxicity, DMSO and EG are safer to use in slow cooling protocols while a lower concentration of PROH can be combined with another CPA to avoid its toxicity and to improve the cryosurvival as well.

Figure 2

Time- and temperature-dependence of CPA toxicity.

Ovulated mouse oocytes were exposed to a 1.5 M solution of each CPA at 37°C for 0, 15, and 30 minutes and evaluated for their (A) post-exposure survival, (B) fertilization, (C) embryonic development, (D) parthenogenetic activation, and (E) ploidy. Data shown are mean±SEM except for the ploidy rates, which represent percentage of total number of euploid eggs. The total number of oocytes (n) used in each group was also shown. The differences between the control, DMSO and EG groups were not significant while only a few oocytes survived exposure to 1.5 M PROH at 37°C. Therefore, parthenogenetic activation and ploidy were not evaluated in the PROH group. N/A: not applicable.