Taylor M J, London N J, Thirdborough S M, Lake S P, James R F
MRC Medical Cryobiology Group, University Department of Surgery, Cambridge, England.
Cryobiology. 1990 Jun;27(3):269-78. doi: 10.1016/0011-2240(90)90026-z.
Dendritic cells (DCs) are now regarded as specialized leucocytes with distinctive morphological and functional characteristics as accessory or stimulator cells for many lymphocyte responses. While knowledge of the response of other leucocytes (e.g., lymphocytes, macrophages, and granulocytes) to freezing and thawing has been established for some years, an understanding of the cryobiological properties of DCs has not, hitherto, been determined specifically. Such information is important both for establishing procedures for the long-term storage of these cells for use in immunological procedures and for defining freezing conditions that might selectively kill DCs in attempts to modulate the immunogenicity of transplantable tissues during cryopreservation. Preparations of rat and human spleen cells enriched for DCs were frozen to -60 degrees C at one of six cooling rates (0.3, 1.5, 10, 20, 70, or 150 degrees C/min) using a procedure that was established for pancreatic islets with 2 M dimethyl sulfoxide (Me2SO) as the cryoprotectant. Following storage at -196 degrees C the survival of thawed cells was assessed by evaluating both the numbers of cells recovered after the complete process and the membrane integrity of the recovered cells using a supravital fluorescent probe assay. Survival profiles for DCs showed a dependence upon cooling rate similar to other lymphoid cells but DCs were more sensitive to freezing injury than either lymphocytes or macrophages: Optimum survival (75% recovery of numbers and 57% membrane integrity) of rat DCs was achieved by slow cooling (0.3 degrees C/min). Optimal recovery of human DCs was significantly higher (83% recovery of numbers and 72% membrane integrity) after cooling at either 0.3 or 1.5 degrees C/min. The viable yield of DCs from both species declined abruptly as cooling rate was increased, with less than 10% survival after cooling at 20 degrees C/min and negligible survival after cooling at 70 degrees C/min or greater. Analysis of variance of the survival data showed that the response of DCs to freezing and thawing was significantly different (P less than 0.005) from that of either lymphocytes or macrophages, thus providing additional evidence that DCs are distinct from other leucocytes, especially macrophages. This study defines conditions that either will provide effective cryopreservation of DCs for immunological purposes or are most likely to bring about their inactivation in cryobiological approaches to modulating tissue immunogenicity.
树突状细胞（DCs）现在被视为具有独特形态和功能特征的特殊白细胞，是许多淋巴细胞反应的辅助或刺激细胞。虽然关于其他白细胞（如淋巴细胞、巨噬细胞和粒细胞）对冻融反应的认识已经确立多年，但迄今为止，尚未专门确定DCs的低温生物学特性。这些信息对于建立这些细胞的长期储存程序以用于免疫学程序，以及确定在冷冻保存过程中可能选择性杀死DCs以调节可移植组织免疫原性的冷冻条件都很重要。使用为胰岛建立的程序，以2M二甲基亚砜（Me2SO）作为冷冻保护剂，将富含DCs的大鼠和人脾细胞制剂以六种冷却速率（0.3、1.5、10、20、70或150℃/分钟）之一冷冻至-60℃。在-196℃储存后，通过评估整个过程后回收的细胞数量以及使用超活荧光探针测定法评估回收细胞的膜完整性来评估解冻细胞的存活率。DCs的存活曲线显示出对冷却速率的依赖性，类似于其他淋巴细胞，但DCs比淋巴细胞或巨噬细胞对冷冻损伤更敏感：大鼠DCs通过缓慢冷却（0.3℃/分钟）实现最佳存活（细胞数量回收率75%，膜完整性57%）。在0.3或1.5℃/分钟冷却后，人DCs的最佳回收率显著更高（细胞数量回收率83%，膜完整性72%）。随着冷却速率的增加，两种物种的DCs活产率急剧下降，在20℃/分钟冷却后存活率低于10%，在70℃/分钟或更高冷却后存活率可忽略不计。对存活数据的方差分析表明，DCs对冻融的反应与淋巴细胞或巨噬细胞的反应显著不同（P小于0.005），从而提供了额外证据表明DCs与其他白细胞不同，尤其是巨噬细胞。本研究确定了要么将为免疫学目的提供DCs有效冷冻保存，要么在调节组织免疫原性的低温生物学方法中最有可能导致其失活的条件。
