Maximizing the retention of antigen specific lymphocyte function after cryopreservation
Maximizing the retention of antigen specific lymphocyte function after cryopreservation
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DOI：
10.1016/j.jim.2005.09.011
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发表时间：
2006-01-20
影响因子：
2.2
通讯作者：
Maecker, HT
中科院分区：
文献类型：
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作者：
Disis, ML;dela Rosa, C;Maecker, HT
冷冻保存外周血单核细胞 (PBMC) 中的淋巴细胞以在解冻后保留其功能的能力对于癌症免疫治疗研究的分析至关重要。我们评估了多种冷冻保存策略，旨在开发一种优化的冷冻和解冻 PBMC 方案，以保留活力和功能。我们确定了冷冻保存后不影响细胞活力的几个因素，例如在干冰上运输冷冻样品、解冻后洗涤和离心样品的时间长度和速度，以及每个容器冷冻的细胞数量。然而，不同的培养基添加剂确实会影响解冻后细胞的活力。与测试的所有其他添加剂相比，使用人 AB 血清时冷冻后细胞的活力显着降低 (p < 0.000)。第二个关键参数是从冷冻管中取出后用于清洗细胞的介质的温度。当洗涤前将培养基冷却至 4 摄氏度时，平均活力为 69.7 +/- 12.5%，25 摄氏度时为 92.55 +/- 3.1%，37 摄氏度时为 95.11 +/- 2.5%。最后，我们使用具有不同培养基添加剂的优化冷冻保存方案来确定是否可以保留对破伤风类毒素的功能性 T 细胞反应。与其他添加剂（如葡聚糖或胎牛血清）相比，非冷冻保存的 PBMC 的破伤风特异性刺激指数 (S.I.) 与从用含有人血清白蛋白的培养基冷冻的细胞获得的 SI 之间存在统计学上显着的相关性。 (c) 2005 Elsevier B.V. 保留所有权利。
The ability to cryopreserve lymphocytes in peripheral blood mononuclear cells (PBMC) to retain their function after thawing is critical to the analysis of cancer immunotherapy studies. We evaluated a variety of cryopreservation strategies with the aim of developing an optimized protocol for freezing and thawing PBMC to retain viability and function. We determined several factors which do not affect cell viability after cryopreservation such as shipping frozen samples on dry ice, the length of time and speed at which samples are washed and centrifuged after thawing, and the number of cells frozen per container. Different media additives, however, did impact the viability of the cells after thawing. There was a significant reduction in the viability of the cells after freezing when using human AB serum compared to all other additives tested (p < 0.000). A second critical parameter was the temperature of the media used to wash the cells after removal from the cryotubes. When the media was cooled to 4 degrees C prior to washing, the mean viability was 69.7 +/- 12.5%, at 25 degrees C 92.55 +/- 3.1%, and at 37 degrees C 95.11 +/- 2.5%. Finally, we used an optimized cryopreservation protocol with different media additives to determine if functional T cell responses to tetanus toxoid could be preserved. There was a statistically significant correlation between the tetanus specific stimulation index (S.I.) of the noncryopreserved PBMC and SI obtained from cells frozen with media containing human serum albumin as compared to other additives such as dextran or fetal bovine serum. (c) 2005 Elsevier B.V. All rights reserved.