52 CRYOPRESERVATION OF BOVINE SOMATIC CELL USING ANTIFREEZE POLYAMINO-ACID (CARBOXYLATED POLY-L-LYSINE)

Abstract

Cryopreservation is used for long-term preservation of biological materials containing cell and tissue, somatic cells are important to commercial application in gene bank and medical engineer. For cell cryopreservation, dimethyl sulfoxide (DMSO) have been used widely as cryoprotective material for long time. Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. It is reported that the addition of CPLL into cryopreservation medium improves the post-thaw survival rate of murine L929 cells, human induced pluripotent stem cells and murine embryo. In this research, the effectiveness of CPLL as bovine somatic cell cryoprotective material was examined. In experiment 1, using bovine fibroblast cells from skin, the cryopreservation media were prepared as follows: 10% (vol/vol) DMSO, 5% (vol/vol) DMSO + 5% (wt/vol) CPLL, 5% (wt/vol) CPLL, 7.5% (wt/vol) CPLL, 10% (wt/vol) CPLL and 20% (wt/vol) CPLL were dissolved in DMEM and pH was adjusted to 7.4. Cells were resuspended in 1 mL of cryopreservation medium at a cell density of 5 × 10⁵ and stored in a −80°C freezer. After storage for at least 1 wk, cells were thawed. The 5% CPLL showed significantly higher post-thaw survival rate than 10% DMSO (96.5% v. 86.7%; P < 0.05), and 7.5% CPLL and 20% CPLL showed significantly higher proliferation rates than 10% DMSO at 72 h after thawing (10% DMSO v. 7.5% CPLL v. 20% CPLL ; 99.4% v. 173.6% v. 207.4%; P < 0.05). Although it is necessary to remove the cryoprotective material after thawing, the proliferation rate at 72 h after thawing of 5% CPLL and 10% CPLL without removal of cryoprotective material were significantly higher than that when removing cryoprotective material (111.2% v. 269.6%, 113.0% v. 210.0%). In addition, there was no difference between the removal treatment of CPLL and the NOT removal treatment of CPLL at the thawing. In experiment 2, using bovine cumulus cells, the cryopreservation media were prepared as follows: 10% (vol/vol) DMSO, 5% (vol/vol) DMSO + 5% (wt/vol) CPLL, 5% (wt/vol) CPLL, 10% (wt/vol) CPLL, 20% (wt/vol) CPLL, 25% (wt/vol) CPLL, and 30% (wt/vol) CPLL were dissolved in DMEM and other conditions were the same as experiment 1. There was no significant difference for post-thaw survival rate (93.7, 91.8, 89.2, 89.2, 95.3, 91.6, and 90.2%). At 72 h after thawing, 10% DMSO, 5% DMSO + 5% CPLL and 25% CPLL showed higher proliferation rate (10% DMSO v. 5% DMSO + 5% CPLL v. 25% CPLL; 258.4% v. 281.4% v. 257.4%). The significant differences in survival rate were determined by chi-squared test, and in proliferation were determined by ANOVA. In bovine somatic cells, it was possible to cryopreserve cells using CPLL without DMSO. The cryoprotective effect of CPLL is equal or superior to that of DMSO, the conventional cryoprotective material. Further, when using CPLL, it was possible to culture without removing the cryopreservation medium after thawing. These results suggest that CPLL has promise as a new cryoprotective material for bovine somatic cells.