Feuerstein B, Berger T G, Maczek C, Röder C, Schreiner D, Hirsch U, Haendle I, Leisgang W, Glaser A, Kuss O, Diepgen T L, Schuler G, Schuler-Thurner B
Department of Dermatology University of Erlangen-Nuremberg, D-91052, Erlangen, Germany.
J Immunol Methods. 2000 Nov 1;245(1-2):15-29. doi: 10.1016/s0022-1759(00)00269-6.
Dendritic cells (DC) are increasingly used as a vaccine. Unfortunately, a satisfactory cryopreservation of DC in the absence of FCS is not yet available, so that laborious repeated generation of DC from fresh blood or frozen peripheral blood mononuclear cells for each vaccination has been required to date. We now aimed at developing an effective cryopreservation method, and by testing several variables found that it was crucial to combine the most advantageous maturation stimulus with an improved freezing procedure. We generated monocyte-derived DC from leukapheresis products by using GM-CSF and IL-4 and showed that amongst several known maturation stimuli the cocktail consisting of TNF-alpha+IL-1 beta+IL-6+PGE(2) achieved the highest survival of mature DC. We then systematically explored cryopreservation conditions, and found that freezing matured DC at 1 degrees C/min in pure autologous serum+10% DMSO+5% glucose at a cell density of 10x10(6) DC/ml gave the best results. Using this approach 85-100% of the frozen DC could be recovered in a viable state after thawing (Table 1). The morphology, phenotype, survival as well as functional properties (allogeneic mixed leukocyte reaction, induction of influenza matrix or melan A peptide-specific cytotoxic T cells) of these thawed DC were equivalent to freshly prepared ones. The addition of CD40L or TRANCE/RANKL further improved DC survival. Importantly, we demonstrate that DC can effectively be loaded with antigens (such as Tetanus Toxoid, influenza matrix and melan A peptides) before cryopreservation so that it is now possible to generate antigen-preloaded, frozen DC aliquots that after thawing can be used right away. This is an important advance as both the generation of a standardized DC vaccine under GMP conditions and the carrying out of clinical trials are greatly facilitated.
树突状细胞（DC）越来越多地被用作疫苗。不幸的是，目前尚无在无胎牛血清（FCS）情况下对DC进行令人满意的冷冻保存方法，因此迄今为止每次接种疫苗都需要从新鲜血液或冷冻外周血单核细胞中费力地反复生成DC。我们现在旨在开发一种有效的冷冻保存方法，通过测试几个变量发现，将最有利的成熟刺激与改进的冷冻程序相结合至关重要。我们使用粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-4（IL-4）从白细胞分离产物中生成单核细胞衍生的DC，并表明在几种已知的成熟刺激中，由肿瘤坏死因子-α（TNF-α）+白细胞介素-1β（IL-1β）+白细胞介素-6（IL-6）+前列腺素E2（PGE2）组成的混合物能使成熟DC的存活率最高。然后我们系统地探索了冷冻保存条件，发现以1℃/分钟的速度在纯自体血清+10%二甲基亚砜（DMSO）+5%葡萄糖中、细胞密度为10×10⁶个DC/ml的条件下冷冻成熟的DC效果最佳。使用这种方法，85% - 100%的冷冻DC在解冻后能够以存活状态恢复（表1）。这些解冻后的DC的形态、表型、存活率以及功能特性（同种异体混合淋巴细胞反应、诱导流感基质或黑色素A肽特异性细胞毒性T细胞）与新鲜制备的DC相当。添加CD40L或肿瘤坏死因子相关激活诱导细胞因子（TRANCE）/核因子κB受体活化因子配体（RANKL）可进一步提高DC的存活率。重要的是，我们证明DC在冷冻保存前能够有效地负载抗原（如破伤风类毒素、流感基质和黑色素A肽），这样现在就有可能生成预加载抗原的冷冻DC等分试样，解冻后可立即使用。这是一项重要进展，因为它极大地促进了在药品生产质量管理规范（GMP）条件下标准化DC疫苗的生产以及临床试验的开展。
