Cryopreservation of biological tissues

我们已经成功地冷冻保存了具有非常复杂的细胞结构的水蚤。(1)血管低温保存技术的发展；(2)长期低温保存后生物组织生死判断方法的发展；(3)生物组织冷冻过程的数值模拟。受试者按照非常精确的温度控制历史被冷冻在程序化的冷冻机中。冷冻后的血管在液氮中保存2周~16个月，然后在冰柜中仔细解冻。与水蚤的冷冻保存不同的是，判断冷冻保存的血管是相当困难的。在对几种方法进行了试验后，我们采用了两种判断方法：解冻后从组织中提取的细胞培养，以及血管移植到大鼠身上。结果表明，低温保存后的血管生命功能基本保持完好，32只大鼠的腹主动脉移植后，有8只存活1周以上。术后2周至4个月对这些大鼠进行检查，证实了细胞的活性和移植物的生长是由于大鼠的生长而导致的。对于(3)，本文对冷冻过程进行了数值模拟，考虑了水、细胞液和低温保护剂溶液在细胞膜中的传输和相变过程中的热传递。

We had succeeded in cryopreservation of daphnias which has very complex cellular structure. Then we focused our efforts on the subjects as follow ; (1) Development of the technique for cryopreservation of blood vessels, (2) Development of the method of judging life or death of the biological tissues after long-term cryopreservation, and (3) Numerical simulation of the process of freezing of biological tissues.As for (1) aortas of rats were used in most cases. The subjects were frozen in a programd freezer following a very precisely controlled temperature history. The frozen blood vessels were preserved in liquefied nitrogen as long as 2 weeks to 16 months, and then thawed carefully again in the freezer. Success or failure of the entire process was judged by the procedures as described in the following.Different from cryopreservation of daphnia, judging life or death of the cryopreserved blood vessels was found considerably difficult. After trials on several methods, we adopted two method of judgment ; cultivation of cells taken from the tissue after thawing, and trasplantation of the vessel to a rat. As the result we found that the life function of the blood vessel was kept almost intact after cryopreservation.The cryopreserved grafts were transplanted to abdominal aortas of 32rats, and 8 rats were survived longer than a week. Inspection of these rats, from 2 weeks to 4 months after the operation, revealed that viability of the cells and growth of the grafts due to growth of the grafts due to growth of the rats were confirmed. These results suggest that the tecunique of cryopreservation developed in this study has been successful.As for (3) , numerical simulation of the freezing process was carried our taking into consideration the transport of water, cellular fluids and cryoprotectant solution through cell membrane and transfer of heat with phase change.