Supplementary cryoprotective effect of carboxylated ε-poly-l-lysine during vitrification of rat pancreatic islets.

This study was designed to investigate whether cryosurvival of rat pancreatic islets can be improved by carboxylated ε-poly-l-lysine (CPLL). Islets isolated from Wistar × Brown-Norway F1 rats (101-200 µm in diameter) were cryopreserved in three vitrification solutions containing ethylene glycol (EG; 30%, v/v) and CPLL (0%, 10%, or 20%, v/v) by Cryotop® protocol (10 islets per device). The post-warm survival rate of the islets vitrified in the presence of 20% CPLL (74%), assessed by FDA/PI double staining, was higher than those in 0% and 10% CPLL (65% and 66%, respectively). Decreased EG concentrations (10% and 20%) in the presence of 20% CPLL resulted in impaired post-warm islet survival rates (50% and 64%, respectively). Value of stimulus index (SI) for 20 mM/3 mM glucose-stimulated insulin secretion was 4.1 in islets vitrified-warmed in the presence of 30% EG and 20% CPLL, which was comparable with those in fresh control islets and vitrified islets in 30% EG alone (4.1 and 4.4, respectively). A large number of islets (50 islets per device) could be cryopreserved in the presence of 30% EG and 20% CPLL by using nylon mesh as the device, without considerable loss of post-warm survival (68%) and SI value (3.7). In conclusion, supplementation of antifreeze 20% CPLL was effective in improving the post-warm survival of isolated rat pancreatic islets when vitrification solution containing 30% EG was used.

Nylon mesh cryodevice for bovine mature oocytes, easily removable excess vitrification solution.

The aim of this study was to determine pore size of nylon mesh (NM) device suitable for cryosurvival of bovine mature oocytes and to apply the device to vitrification of large quantities of the oocytes. Ten to twelve oocytes were loaded onto an NM device (a square opening 37-, 57- or 77-µm on a side length). After removal of the excess volume of vitrification solution by paper absorption, the oocytes were vitrified-warmed, fertilized and cultured in vitro. Oocyte recovery and morphological survival were comparable among the three groups. However, blastocyst yield in the 37-µm group (39%) was higher than that in the 77-µm group (28%), and the yield in the 57-µm group (31%) was the intermediate. The 37-µm NM device was applicable for increased oocyte number >40 (blastocyst yield, 33%). These results suggest that 37-µm-pore sized NM can serve as cryodevice to vitrify large quantities of in vitro-matured bovine oocytes.

Silk fibroin sheet multilayer suitable for vitrification of in vitro-matured bovine oocytes.

Minimum volume cooling (MVC) procedure has been successfully applied to vitrify mammalian oocytes, but high skill of capillary pipetting is required to load the oocytes on a cryodevice with a minimal volume (<1 µL) of vitrification solution (VS). Here we report a novel cryodevice for bovine oocyte vitrification, silk fibroin (SF) sheet multilayer, of which spontaneous absorption property can eliminate pipette operation for removal of excess VS. Based on physical stability and scanning electron microscopic observation, the SF sheet prepared from 1.5% (wt/vol) fibroin solution was selected and layered around a polypropylene strip (0.1-mm thickness, 0.7-mm width, 10-mm depth). Ten denuded bovine mature oocytes were loaded onto the SF sheet multilayer with 2-3 µL of the VS, and then cooled rapidly by plunging into liquid nitrogen. Nylon mesh (NM) device with square opening 37-µm length of a side and commercially available Cryotop® (CT) device were used as controls, and the minimization of VS volume was performed by paper towel absorption and capillary aspiration, respectively. In SF, NM and CT groups, post-warming oocyte recovery rates were 99.5, 99.1 and 100%, and the morphological survival rates were 99.7, 94.5 and 99.0%, respectively. Subsequent IVF and 8-days IVC resulted in comparable blastocyst yields among the three groups (25.5, 25.0 and 26.1% in SF, NM and CT groups, respectively). These results suggest that SF sheet multilayer is a useful cryodevice for bovine matured oocytes in MVC vitrification because VS volume surrounding the oocytes can be easily minimized through its absorption property.