Hviid L, Albeck G, Hansen B, Theander T G, Talbot A
Centre for Medical Parasitology, University of Copenhagen, Denmark.
J Immunol Methods. 1993 Jan 4;157(1-2):135-42. doi: 10.1016/0022-1759(93)90079-m.
Protection of the functional integrity of mononuclear cells stored in liquid N2 requires careful control of the freezing procedure. Consequently, optimal quality of cryopreserved cells is usually assured by freezing according to a specified time-temperature gradient generated by computer-controlled freezing devices. While such equipment offers large capacity and secures maximum survival and functional integrity of the lymphocytes upon thawing, it is quite costly and strictly stationary. We have previously developed and tested an alternative, manual device for controlled-gradient lymphocyte freezing, which has proved suitable for field conditions. We report here the development and testing of a similar micro-controller regulated device, allowing unattended and automatic controlled-gradient cell freezing. The equipment exploits the temperature gradient present between the liquid N2 surface and the neck in an ordinary liquid N2 refrigerator. The lymphocyte samples are placed in a small elevator, which is moved through the N2 gas phase by a stepper motor. Time and temperature are measured at regular intervals, and the position of the samples adjusted to ensure that the actual measurements closely match encoded ideal values. Results of assays of the functional integrity and phenotypic composition of human mononuclear cells frozen by the new system were comparable to those obtained when using cells frozen by a commercially available, stationary cell-freezing equipment, or fresh autologous cell samples tested in parallel. Furthermore, there was a good correlation between functional and phenotypic data obtained using frozen and autologous fresh samples of mononuclear cells. The equipment described is low weight and has low N2 consumption, and is thus suitable for the collection and cryopreservation of lymphocytes under field conditions. Furthermore, the technique provides an inexpensive alternative for researchers with a limited requirement for the simultaneous freezing of large quantities of cells.
保护液氮中储存的单核细胞的功能完整性需要严格控制冷冻程序。因此，通常通过按照计算机控制的冷冻设备产生的特定时间 - 温度梯度进行冷冻来确保冷冻保存细胞的最佳质量。虽然这种设备容量大，能确保解冻后淋巴细胞的最大存活率和功能完整性，但成本很高且固定不动。我们之前开发并测试了一种用于控制梯度淋巴细胞冷冻的替代手动设备，已证明它适用于野外条件。我们在此报告一种类似的微控制器调节设备的开发和测试情况，该设备可实现无人值守和自动控制梯度细胞冷冻。该设备利用普通液氮冰箱中液氮表面与颈部之间存在的温度梯度。淋巴细胞样本放置在一个小升降器中，由步进电机驱动其在氮气气相中移动。定期测量时间和温度，并调整样本位置以确保实际测量值与编码的理想值紧密匹配。用新系统冷冻的人单核细胞的功能完整性和表型组成的检测结果，与使用市售固定细胞冷冻设备冷冻的细胞或同时平行测试的新鲜自体细胞样本所获得的结果相当。此外，使用冷冻和自体新鲜单核细胞样本获得的功能和表型数据之间存在良好的相关性。所描述的设备重量轻且液氮消耗低，因此适用于野外条件下淋巴细胞的采集和冷冻保存。此外，该技术为对同时冷冻大量细胞需求有限的研究人员提供了一种廉价的替代方案。
