45. Effect of heat transfer coefficient on cooling rates during oocyte vitrification in liquid and slush nitrogen

Abstract

Advances in cryopreservation of gametes indicate that slow cooling protocols will soon be replaced by fast cooling methods. Vitrification, a process in which liquid water is converted into a glass-like amorphous solid without any ice formation, normally requires plunging directly into liquid nitrogen in order to achieve high cooling rates. This leads to strong nitrogen vaporization around the sample surface forming a vapor film that acts as a heat insulation layer (Leidenfrost effect) resulting in limited heat transfer coefficient (h) between the surface of the sample and liquid nitrogen. Recently, slush nitrogen (SN2), a mixture of solid and liquid nitrogen obtained by applying negative pressure (average −207 °C) has been introduced in an attempt to minimize this effect. The objective of this study was to conduct a comparison of numerically calculated cooling rates of two small-volume devices plunged in liquid nitrogen versus slush nitrogen, and to analyze the individual effects of a lower temperature (−207 °C) and higher h values. A survey of literature h values for film boiling of small metal objects with different geometries plunged in liquid nitrogen, revealed a range between 125 and 1000 W/(m2 K). These h values were applied to a numerical simulation of cooling rates of two oocyte vitrification devices (open-pulled straw and Cryotop®), plunged in conditions representative of vitrification processes in liquid and slush nitrogen. The heat conduction equation with convective boundary condition was numerically solved using the finite element method to simulate the cooling process (Comsol Multiphysics® software). The cooling rate (°C/min) was defined as the time needed to reduce initial core temperature (warmest point of the system) of the liquid from 20 °C to −150 °C while avoiding ice formation (vitrification); therefore the thermophysical properties of the vitrifying solution and the plastic device were considered independent of temperature. Numerical simulations were carried out with different h values likely to represent the stagnant and slush nitrogen conditions. The thermal properties for supercooled water at −23 °C were used; thermal conductivity (k) 0.50 W/m K, specific heat (Cp) 4218 J/kg K, and density (ρ) 983 kg/m3. For plastics (polyethylene) the following values at 23–25 °C were used: k = 0.22 W/m K, Cp = 1680 J/kg K, and ρ = 900 kg/m3. Predicted cooling rates for open-pulled straw and Cryotop® when cooled at −196 °C (liquid nitrogen) or −207 °C (slush nitrogen) indicated that lowering the cooling temperature produces only a maximum 10% increase in cooling rate confirming that the main benefit of plunging in slush over liquid nitrogen does not arise from their temperature difference. Numerical simulations also predicted that the fourfold increase in the cooling rate of vitrification devices when plunging in slush nitrogen would be explained by an increase in h values; this physical condition can be attributed to the fact that there is less or null film boiling when a sample is placed in slush nitrogen because it first melts the solid nitrogen before causing the liquid to boil and form a film. Funding sources: CONICET – Universidad Nacional de La Plata – Agencia de Promocion Cientifica y Tecnológica (ANPCYT) – Universidad Católica Argentina.