Viability evaluation using the effective thermal conductivity during extracellular freezing in killifish embryos

The purpose of this study is to evaluate the correlation between the viability of milliscale biological materials and their effective thermal conductivity during extracellular freezing in order to achieve successful cryopreservation. The killifish embryo (Oryzias latipes, orange-red variety, φ1.3 mm sphere) was employed as a sample biological material and the viability was evaluated by the hatching rate. Dimethylsulfoxide-water was selected as the cryoprotectant. The thermal conductivity was measured using a self-heated thermistor technique developed by T.A. Balasubramaniam (1977). The thermistor (φ2.5 mm sphere) was installed at the bottom of a test section 8×8×12 mm, and exposed to biological material. The cooling system was equipped with Peltier devices and was capable of reducing the temperature of the test section to -30°C. Between 150 and 200 embryos were loaded in the test section with 5, 15 and 20% w/w Dimethylsulfoxide-water. The effective thermal conductivity during extracellular freezing was measured over the temperature range -20 to 6°C with a cooling rate of 0.1 to 10°C/min. The effective thermal conductivity increased from 0.5 to 1 W/ (m·K) with decreasing temperature and increasing the cooling rate. The decrease in the hatching rate and the variation in the effective thermal conductivity were correlated. Thus the viability could be controlled using their effective thermal conductivity during the extracellular freezing.