Taylor M J, Bank H L
Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston 29425.
Cryobiology. 1988 Feb;25(1):1-17. doi: 10.1016/0011-2240(88)90014-4.
The survival of tissue allografts can be extended by pretreating the tissue to remove the stimulatory leucocytes that populate the graft; with this in mind, we have recently begun to explore a cryobiological approach to modulating tissue immunogenicity by using the differential susceptibility of different cells to freezing injury. The sensitivity of leucocytes to fast cooling rates, which were used in procedures that have been reported to yield viable pancreatic islets of Langerhans, was examined. The loss of both cell numbers and the ability of peripheral blood lymphocytes to undergo blastogenic transformation in response to the mitogen concanavalin A after freezing and thawing was determined over a range of cell concentrations using the "curve-shift" method. Lymphocytes frozen at 1 degree C/min by a control procedure that was designed to yield maximum survival of lymphocytes showed that although there was a decrease in the number of responding cells, there was no reduction in the ability of the recovered cells to undergo blastogenesis when compared with the response of nonfrozen cells. However, cooling at 1 degree C/min in the experimental procedures resulted in both the loss of cells as well as a marked reduction in the ability of recovered cells to incorporate 125I-deoxyuridine into nucleic acid. Cells cooled at either 20 or 75 degrees C/min by any of the procedures showed total inability to respond to stimulation. Lysozyme is produced continuously by all types of macrophages in culture. The large net increase in total lysozyme content of macrophage cultures is therefore a useful measure of the viability of these accessory cells. Cooling at 1 degree C/min by a control, optimized procedure yielded 91% survival of viable peritoneal exudate cells. Cooling at either 1 or 20 degrees C/min in the experimental procedures resulted in 72-75% survival of cells frozen by one method and 33% survival when frozen by an alternative procedure. Negligible recovery of viable cells was obtained after cooling at 75 degree C/min. The preservation protocols employed in this study differ significantly in the variables known to influence the survival of the cells; these include the concentration of cryoprotectant (CPA), the length and temperature of exposure to CPA, the dilution regimen, and the optimum cooling rate for survival of pancreatic islets. This study therefore defines clearly those conditions most likely to effect a depletion of "passenger" lymphoid cells by freezing during the cryopreservation of islets of Langerhans.
通过对组织进行预处理以去除移植组织中的刺激性白细胞，可以延长组织同种异体移植物的存活时间；考虑到这一点，我们最近开始探索一种低温生物学方法，即利用不同细胞对冷冻损伤的不同敏感性来调节组织免疫原性。我们研究了白细胞对快速冷却速率的敏感性，这种冷却速率在据报道能产生存活的胰岛细胞的操作中被使用。使用“曲线位移”方法，在一系列细胞浓度范围内，测定了冷冻和解冻后外周血淋巴细胞的细胞数量损失以及对促有丝分裂原刀豆球蛋白A发生增殖转化的能力。通过一种旨在使淋巴细胞获得最大存活率的对照程序，以每分钟1摄氏度的速度冷冻淋巴细胞，结果表明，尽管应答细胞数量有所减少，但与未冷冻细胞的应答相比，复苏细胞进行增殖的能力并未降低。然而，在实验程序中以每分钟1摄氏度的速度冷却，导致细胞损失以及复苏细胞将125I - 脱氧尿苷掺入核酸的能力显著降低。通过任何一种程序以每分钟20或75摄氏度的速度冷却的细胞，完全无法对刺激作出反应。培养中的所有类型巨噬细胞都会持续产生溶菌酶。因此，巨噬细胞培养物中溶菌酶总含量的大幅净增加是这些辅助细胞活力的一个有用指标。通过一种对照的优化程序以每分钟1摄氏度的速度冷却，可使存活的腹腔渗出细胞存活率达到91%。在实验程序中以每分钟1或20摄氏度的速度冷却，通过一种方法冷冻的细胞存活率为72 - 75%，通过另一种程序冷冻时存活率为33%。以每分钟75摄氏度的速度冷却后，活细胞的回收率可忽略不计。本研究中采用的保存方案在已知影响细胞存活的变量方面存在显著差异；这些变量包括冷冻保护剂（CPA）的浓度、接触CPA的时间和温度、稀释方案以及胰岛存活的最佳冷却速率。因此，本研究明确界定了在胰岛冷冻保存过程中，最有可能通过冷冻使“过客”淋巴细胞耗竭的那些条件。
