人外周血树突状细胞低温保存的优化研究

Study on the optimal conditions for the cryopreservation peripheral blood derived dendritic cells

目的探寻冻存树突状细胞(DCs)优化的冷冻保护剂组合。方法配制含不同浓度二甲基亚砜(DM-SO)的3种冷冻保护剂组合,A组:5%DMSO+6%羟乙基淀粉(HES)+4%人血清白蛋白(HAS);B组:10%DMSO+40%FCS;C组:12%DMSO+40%FCS,比较3组冷冻保护剂对人外周血CD14+单个核细胞诱导产生的成熟树突状细胞(mDCs)的冻存效果:采用两步法将mDCs冻存于-80℃冰箱过夜后转移至-196℃液氮气相中放置24 h,再将冻存的mDCs复苏后继续培养,并检测、比较冻存前后DC的形态、存活率、细胞表型及其对同种异体T细胞刺激活性的差异。结果 3组不同组合冷冻保护剂冷冻保存的mDCs复苏后其存活的细胞的形态没有发生明显改变,仍保留其成熟表型,并具备对T细胞的刺激活性。结论 3种不同浓度的DMSO冷冻保存mDCs,5%DMSO+6%HES+4%HSA组合更适宜。

Objective To establish a practical and efficient concentration of cryoprotective agents for the cryopreser-ration of dendritic cells （DCs） derived from peripheral blood. Methods Cryoproteetive agents,dimethylsuffoxide（DMSO） , hydroxyethyl starch （HES） , human serum albumin （HSA） and fetal cow serum （FCS） were mixed in different concentrations. Group A consisted of 5% DMSO ＋ 6% HES ＋ 4% HSA ; group B 10% DMSO ＋ 40% FCS ; group C 12% DM- SO ＋ 40% FCS. Mature DCs induced from human CD14^＋ monoeytes were cooled with each of the three groups of different cryoproteetive mixtures,in a -80℃ refrigerator overnight and then the eryovials were directly transferred into the vapourphase of liquid nitrogen and stored for 24 hours. Then the DCs were thawed and cultured. The morphology,viability,phenotype, and mixed lymphocyte reaction of the cryopreserved DCs were studied and compared to those before eryopreservation. Results Compared with control mDCs,the thawed mDCs showed no distinct change in morphological characteristics,mature phenotype and SI of DCs to T cells. Conclusion Biological properties of DCs eryopreserved by uncontrolled-rate freezing technique and two-step freezing method are not changed when they are compared with fresb DCs, indicating that this method is appropriate. Group C cryopreservatives,which is 5 % DMSO ＋ 6% HES ＋ 4% HAS,is more efficient for clinical application.