Estudo experimental de criopreservação de espermatozoides usando evaporação de filme fino de nitrogênio

Abstract

The cryopreservation of biological material, specifically semen, allows for the long-term storage of samples while preserving their viability and function after thawing, contributing to increased success rates in assisted reproductive technologies. Various freezing protocols and thawing techniques have been developed and evaluated, utilizing new cryoprotective agents (CPAs) aimed at vitrifying biological material and preventing the formation of crystals that can damage cells. In this study, the objective was to evaluate vitrification through thin film evaporation of liquid nitrogen (LN2). For this purpose, an experimental apparatus was developed, comprising a vacuum chamber and cryoprobes containing liquid nitrogen. The sample was stored in a PDMS device between two copper plates with a surface of microporous metal foam (nickel or copper). Jets of LN2 were applied, promoting the evaporation and freezing or vitrification of the samples. The pressure in the chamber and the sample temperatures were monitored throughout the experiments. Initially, cryoprotective agents such as glycerol in PBS (Phosphate-buffered saline) and a solution containing 1 mol of sucrose, 35% v/v glycerol, 1% v/v human albumin, and 64% v/v HTF (Human Tubal Fluid) were used. The evaluated variables included sample volume, material and thickness of the metal foam, and the thickness of the PDMS reservoir. Subsequently, tests with human semen were conducted using different combinations of sucrose (0.25 mol/l ,0.5 mol/l and 1 mol/l), human albumin (1% v/v), glycerol (35% v/v), HTF (Human Tubal Fluid) (64%), and TYB (test yolk buffer). The EFF experiments demonstrated that the concentration of cryoprotectants, volume, material and thickness of the metal foam, and the thickness of the reservoir significantly influence cooling rates and vitrification. Glycerol at 50% v/v favored vitrification, while copper foams provided higher cooling rates due to their thermal conductivity. However, the presence of residual water in the biological samples hindered vitrification, highlighting the need for optimizations in preparation techniques and studies on new cryoprotective solutions.