Modeling of the Heat tranfer process in vitrification devices used for oocyte Cryopreservation.

M.V. SANTOS; M. SANSINENA; J. CHIRIFE; N. ZARITZKY

Congreso; Congreso Internacional de Mecanica Computacional MECOM-ENIEF; 2012

Oocyte cryopreservation is of key importance in the preservation and propagation of germplasm, however, the cryopreservation of the female gamete has been met with limited success. Several vitrification devices such as open pulled straws (OPS), fine and ultra fine pipette tips, nylon loops and polyethylene films have been introduced in order to manipulate minimal volumes and achieve high cooling rates. However, experimental comparison of cooling rates presents difficulties mainly because of the reduced size of these systems. To overcome this limitation, the cooling rates of various vitrification systems immersed in liquid nitrogen, (Cryoloop®, Cryotop® ,Miniflex® and Open Pulled Straw) were mathematically modeled using the finite element method by numerically solving the unsteady-state heat conduction partial differential equations for the appropriate geometries of the devices. The thermo-physical properties, thermal conductivity, density and specific heat, which are involved in the partial differential equations, were considered constant since during vitrification there is no phase change of water into ice crystals. The Cryoloop® system (Hampton Research, USA) consists of a mounted nylon loop where the oocytes are loaded and plunged into liquid nitrogen. The Cryotop® (Kitazato Supply, Inc, JP) is a thin polyethylene film strip that holds an open drop with oocytes in a minimal volume before being plunged directly into liquid nitrogen. The Miniflex® polyethylene tips (Sorenson Bioscience, Inc., USA) used for vitrification have 360 ìm inner diameter and 77 ìm wall thickness. The Open Pulled Straw (OPS) is normally manufactured in the laboratory from 0.25 mL polyethylene French straws (I. M. V., Orsay, France). Results showed that at a constant heat transfer coefficient (h=1000 W/m2K )the highest cooling rate (180000 °C/min) was observed for the Cryoloop® system and the lowest rate (5521°C/min) corresponded to the OPS. The Cryotop® exhibited the second best cooling rate of 37500 °C/min, whereas the Miniflex® only achieved a cooling rate of 6164 °C/min. It can be concluded that in cryopreservation systems, the information obtained using the numerical model enables biotechnologist in reproductive areas to determine the performance of each oocyte vitrification device under different operating conditions. 1 INTRODUCTION Oocyte cryopreservation is of key importance in the preservation and propagation of the female germplasm in farm animals. Many livestock breeds are experiencing a gradual diminishment of genetic diversity; therefore, it is in the interest of the international community to conserve the livestock genetics. In order to maintain cell viability, biological functions must be halted by inducing the cell into a suspended animation state by cooling it into a solid phase (Luyet, 1937; Luyet and Hoddap, 1938; Mazur, 1970; Mazur, 1980). The preservation of cells and tissues by vitrification was first described by Luyet (1937). This principle, which involves the solidification of a sample into an amorphous, glassy-state while maintaining the absence of both intracellular and extracellular ice crystals (Rall and Fahy, 1985), requires high concentrations of cryoprotectants and extremely rapid cooling rates in order to avoid intra and extra-cellular ice formation. The reduction of the volume of the vitrification solution results in an increase in cooling and warming rates as well as a reduction in the probability of ice crystal nucleation and formation (Kuwayama et al., 1997). In order to minimize the working volume of the cryoprotectant solution and achieve very high cooling rates, several minimal volume vitrification systems have been commercially introduced in the last years. Although there are different designs of vitrification systems, the basic physical principle behind all of them is to maximize heat transfer during exposure to liquid nitrogen, therefore achieving ultra high cooling rates. Some of these systems include open pulled straws (OPS, generally manufactured ?in laboratory? from polyethylene French straws), nylon loops (Cryoloop?, Hampton Research, CA, USA), polyethylene films (Cryotop®, Kitazato BioPharma Co., Fuji, Japan and McGill Cryoloeaf® (Medicult, Jyllinge, Denmark) and Miniflex® pipette tips (Sorenson Bioscience Inc., UT, USA). The measurement of actual cooling rates of vitrification devices can only be achieved by a few available systems (Vajta et al., 1998; Vajta and Kuwayama, 2006) because of their reduced size. This limitation has, for the most part, precluded the comparison of actual cooling rates between different oocyte vitrification systems. In addition, there is a large variability and ?technician-dependent? fluctuation in the volume of vitrification solution utilized when loading the oocytes, mainly due to the fact that most systems rely on capillary action. Comparisons between vitrification systems reported in literature show lack of consistency and a wide range in oocyte survival and embryo developmental rates after warming (Jain and Paulson, 2006). To date, there have been no randomized, controlled experimental studies comparing different devices (Chian and Quinn, 2010). A numerical simulation of cooling rates achieved in vitrification would provide valuable information in order to compare the efficiency of available vitrification systems. Therefore, the objective of this study was to compare the efficiency of various oocyte vitrification systems immersed in liquid nitrogen, (Cryoloop®, Cryotop®, Miniflex® and Open Pulled Straw) by using finite element numerical simulation of their cooling rates obtained from the solution of the unsteady-state heat conduction equation for the appropriate geometries of the vitrification systems.