Mathematical prediction of cooling rates during cryopreservation of reproductive cells in liquid nitrogen. Chapter 3

M.V. SANTOS, ; M. SANSINENA,; J. CHIRIFE; ZARITZKY NOEMI

Liquid Nitrogen: Characteristics, Uses and Safety Concerns .Chemistry Research and Applications

Nova Science Publishers

Lugar: New York; Año: 2015; p. 39 - 92

Liquid nitrogen is one of the cryogenic fluids most used for cryopreservation of animal germoplasm; low temperature preservation of oocytes, sperm and embryos is a fundamental cornerstone of assisted reproductive technologies. The cryopreservation protocol for sperm is important because cooling rates that are too high or too low can be detrimental for reproductive cells. A widespread practice is to freeze plastic straws containing sperm in static nitrogen vapor over liquid nitrogen for variable periods of time before plunging into liquid nitrogen for indefinite storage. Regarding oocytes, the cryopreservation of this gamete has increased in recent years due to the application of in vitro fertilization, nuclear transfer and the need to establish ova/gene banks worldwide. Current advances in cryopreservation of oocytes indicate that high cooling methods (vitrification) achieve the best results since there is a minimum risk of intra/extra-celullar ice-formation. Vitrification is a process in which liquid water is converted into a glass-like amorphous solid without any ice formation; it can be achieved by an extremely rapid cooling of a small sample and/or the introduction of cryoprotectant agents that suppress the formation of ice crystals. In order to minimize the working volumes of solutions, several minimal volume devices have been commercially introduced in the last years, such as open pulled straws (OPS), fine and ultra fine pipette tips, nylon loops and polyethylene films. The usual practice is to plunge the devices containing biological samples directly into the liquid nitrogen.Due to the reduced size of these systems the comparison of the cooling rates for each device becomes very difficult. When dealing with liquid nitrogen the initial temperature difference between the fluid and the sample, is large enough to cause boiling of the liquid entering into the film boiling regime (also called Leidenfrost effect); this determines a heat flux from the object to liquid nitrogen creating a pocket of nitrogen vapor around the solid which acts as an insulator retarding further heat transfer. The sample will cool down rather slowly until reaching the Leidenfrost temperature, i.e. the transition point where the nucleate boiling regime begins increasing the heat transfer rate. There is a lack of experimental information concerning which boiling regimes occur during plunging of devices, such as plastic straws containing biological samples.The present chapter describes the mathematical modeling of heat transfer process by solving the partial differential equations by the finite element method in order to analyze the freezing, cooling and vitrification processes that characterize different cryopreservation procedures for reproductive cells. In order to accurately describe the system, thermophysical properties were measured and specific mathematical equations based on the composition of the sample were applied. The surface heat transfer coefficients of the system when submerged either in liquid nitrogen or nitrogen vapor were calculated by comparison of the experimental time-temperature curves and the numerically predicted temperatures. This information is useful to determine the performance of devices used for cryopreservation as well as to optimize the techniques currently applied. Furthermore, it can lead to improvements in the design of cooling devices in the cryobiology field.