Análise teórico-experimental de métodos de vitrificação para aplicações de criopreservação de sêmen

Abstract

The success of a cell cryopreservation procedure is directly related to the method of preparing the biological material, the freezing protocol, the sample volume and the relationship between the cooling rate and the concentration/type of cryoprotective agents (CPA). In order to avoid the formation of ice crystals, the sample cooling rate must be greater than the crystal formation rate. Through the vitrification method, it is possible to achieve high cooling rates, making the direct transition from the liquid state to the vitrified and amorphous state, without the formation of ice crystals. The probability of vitrification increases with higher cooling rates, higher viscosity and smaller sample volume. Vitrification is most used when working with biological materials with a small volume and low concentration of intracellular water, such as semen cryopreservation applications. In this work, two semen cryopreservation methods were developed, the direct immersion vitrification method and the contact droplet vitrification method. An experimental and numerical investigation was conducted, through the design and construction of benches and equipment, computer simulations, evaluation and proposal of methodologies. For the contact droplet vitrification method, solidification advancement behavior, cooling rates, contact angle, geometry and material of the cooling surface were investigated. For the direct immersion vitrification method, the influence of the cooling medium was investigated, comparing immersion in liquid nitrogen (LN2) with nitrogen slush (SN2). The effect of the type of container for sample allocation was also evaluated, comparing cryotubes with PDMS chips, comparing different concentrations of CPA and the impact of volume on the cryopreservation process. Biological tests with semen investigated the influence of the processing method (simple washing and swim-up), and freezing protocol, on the cooling rate, viability and motility. The results in this work support the improvement and development of new methods and protocols for cryopreservation of biological materials, especially for semen vitrification applications.