Ujihira M, Aizawa N, Tanishita K
Department of Mechanical Engineering, Keio University, Kanagawa, Japan.
Biomed Mater Eng. 1994;4(2):115-25.
The purpose of this study is to find the feasibility of preservation of large cell and tissue by maintaining the undercooled state in a freezing process, leading to avoiding the growth of ice crystals in the intracellular space, which causes destruction of cell and tissue. The fertilized killifish egg was employed to test biological tissue. The cooling system was equipped with Peltier devices and able to decrease the temperature of the test section to -50 degrees C. The cooling rate could be regulated by the electric current supplied to the Peltier devices. In the temperature range 0 to -40 degrees C, the morphology of fertilized killifish egg was observed under a microscope with a cooling rate from 0.1 to 10 degrees C/min. The damage rate to the egg in the intracellular undercooled state was evaluated by hatching rate. As a result, intracellular undercooled states were observed in the freezing process with the extracellular undercooling and the extracellular freezing. Extracellular undercooling proves to preserve the egg, and extracellular freezing frequently damages the egg. Thus the cryopreservation of biological material is achieved by maintaining the undercooled state until the temperature of -40 degrees C, then is instantly frozen by the liquid nitrogen to avoid the growth of ice crystals. The maintaining of the stable undercooled state of biological material is requisite for the initial phase in the freezing process. Therefore, dehydration or maintaining the extracellular stable undercooled state should be desirable to maintain the intracellular undercooled state for cryopreservation of biological material.
本研究的目的是通过在冷冻过程中维持过冷状态来探寻保存大细胞和组织的可行性，从而避免细胞内空间中冰晶的生长，因为冰晶生长会导致细胞和组织的破坏。使用受精的鳉鱼卵来测试生物组织。冷却系统配备了珀耳帖装置，能够将测试区域的温度降至-50℃。冷却速率可通过供应给珀耳帖装置的电流进行调节。在0至-40℃的温度范围内，以0.1至10℃/分钟的冷却速率在显微镜下观察受精鳉鱼卵的形态。通过孵化率评估处于细胞内过冷状态下的鱼卵的损伤率。结果表明，在冷冻过程中观察到了细胞内过冷状态，同时伴有细胞外过冷和细胞外结冰现象。细胞外过冷被证明可保存鱼卵，而细胞外结冰则经常会损伤鱼卵。因此，通过维持过冷状态直至-40℃，然后立即用液氮速冻以避免冰晶生长，从而实现生物材料的冷冻保存。在冷冻过程的初始阶段，维持生物材料的稳定过冷状态是必要的。因此，脱水或维持细胞外稳定过冷状态对于生物材料冷冻保存中维持细胞内过冷状态应该是可取的。
