Development of a novel non-programmable cryopreservation method capable of accurate cooling rate manipulation

Conventional fixed-height non-programmable cryopreservation methods produce unstable cooling rates (CRs). We developed a method that predicts CRs based on the measurement of the temperatures around the straw, which allows for the adjustment of sample height to obtain accurate CRs. We used spermatozoa from the Japanese pearl oyster as an experimental model. A stainless-steel jar containing liquid nitrogen (LN) with a cork lid was used as the cooling vessel. A slit was made in the lid and a stainless-steel ruler inserted through the slit. A Styrofoam rack holding straws was attached to the lower end of the ruler. With this device, the height of the straws could be adjusted during cooling with the lid closed. The CR was controlled over two temperature ranges from 0 °C to the commencement of solidification heat (CSH) and from the commencement of ice nucleation (CIN) to − 40 °C. CR0-CSH was controlled for by the distance from the LN surface at the beginning of cooling. CRCIN-40 correlated with the temperature around the straw at CIN (R2 = 0.993) and this temperature correlated with the temperature around the straw when the inside of the straw reached − 10 °C (R2 = 0.996). From these relationships, CRCIN-40 could be predicted by the temperature around the straw when the interior of the straw reached − 10 °C. If the predicted CRCIN-40 was not achieved, the temperature around the straw could be adjusted by changing the sample height immediately after CIN.