Trehalose—A nonpermeable cryoprotectant for direct freezing of early stage murine embryos

Abstract

Trehalose, a non-reducing disaccharide of glucose has been found to stabilize non-mammalian cell membranes during cellular dehydration. Trehalose has been shown to be an effective membrane stabilizer when compared to both sucrose and glycerol at low water activities. To our knowledge it has not yet been tested as a cryoprotectant in mammalian embryos. The purpose of this study was to compare trehalose with sucrose as a non-permeable cryoprotectant using a newly developed procedure for freezing embryos by plunging them directly into liquid nitrogen. This new method protects by dehydrating the embryos prior to freezing using glycerol as a permeable and sucrose as a non-permeable cryoprotectant. A total of 94 early day-3 embryos from Swiss Webster mice were divided into two treatment groups which differed in the use of the non-permeable cryoprotectant; treatment I utilized 1.1 M trehalose while treatment II contained 1.1 M sucrose. All embryos were equilibrated in 1.4 M glycerol in phosphate buffered saline plus 1% bovine serum albumin (PBS-BSA) for 25 to 30 min. The embryos were placed into a 0.05 ml drop of 1.4 M glycerol and 1.1 M of the non-permeable cryoprotectant (Part A) at the tip of a 0.5 ml French straw. The drop was separated by a small air space from a larger 0.4 ml volume (Part B) which consisted of Part A mixed with equal parts of PBS. The embryos were equilibrated in Part A for 1 min prior to plunging directly into liquid nitrogen. After a storage period of 2 to 10 days in liquid nitrogen the straws were thawed in a water bath at 37°C for 30 set and Part A and Part B were mixed by emptying the straw into a petri dish. The embryos were kept in this mixture for 3 to 5 min while morphological integrity was assessed under 70X magnification. Embryos were then equilibrated in 1.1 M trehalose (treatment I) or 1.1 M sucrose (treatment II) for 10 min. All embryos were washed in Whittens medium plus 10% fetal calf serum (WM-FCS) and kept at room temperature for 10 min and then cultured in drops of WM-FCS for 48 hrs at 37°C and 5% CO in a humidified air atmosphere. In treatment I, 24 of 42 (57%) embryos and in treatment II, 35 of 52 (67%) embryos developed into blastocysts. In this study trehalose appeared to be as effective (P >.lO) a cryoprotectant as sucrose in supporting the development of 8to 16-cell mouse embryos into blastocysts following direct freezing in liquid nitrogen.