CAJ | 학술논문

树突状细胞(dendritic cells,DCs)作为专职的抗原递呈细胞,已被广泛地应用于临床的肿瘤疫苗治疗.目前的临床方案大多为分次给病人注射＞10 6个细胞/次,不同批次培养的DCs,连续4-6周.为了提高疗效,简化治疗程序及规范疗程,有必要将DCs低温保存,使患者在治疗过程中得到同批次的培养的DCs.本实验从患者外周血采取单个核细胞,经细胞淘洗分离单核细胞,在800 U/ml GM-CSF+100 ng/ml IL-13的培养条件下,将单核细胞于Teflon疏水袋中诱导产生DCs.将DCs以-1℃梯度降温及不控温两种方式将DCs冻存于-80℃及液氮中.1个月后,42℃快速复温.检测其免疫表型(CD1a,CD14,CD40,CD80,CD83,CD86,CD54,CD58,CD16,CD32,CD64,HLA-DR)及其对自体及异体淋巴细胞的刺激作用.结果表明,2种方式的低温保存及复温过程不改变DCs的免疫表型及对淋巴细胞的刺激功能.结论:应用Teflon袋,能够从外周血单核细胞中诱导出大批量的DCs,这些DCs可被低温保存而不改变其功能,为其l临床应用奠定了基础.
수돌상세포(dendritic cells,DCs)작위전직적항원체정세포,이피엄범지응용우림상적종류역묘치료.목전적림상방안대다위분차급병인주사＞10 6개세포/차,불동비차배양적DCs,련속4-6주.위료제고료효,간화치료정서급규범료정,유필요장DCs저온보존,사환자재치료과정중득도동비차적배양적DCs.본실험종환자외주혈채취단개핵세포,경세포도세분리단핵세포,재800 U/ml GM-CSF+100 ng/ml IL-13적배양조건하,장단핵세포우Teflon소수대중유도산생DCs.장DCs이-1℃제도강온급불공온량충방식장DCs동존우-80℃급액담중.1개월후,42℃쾌속복온.검측기면역표형(CD1a,CD14,CD40,CD80,CD83,CD86,CD54,CD58,CD16,CD32,CD64,HLA-DR)급기대자체급이체림파세포적자격작용.결과표명,2충방식적저온보존급복온과정불개변DCs적면역표형급대림파세포적자격공능.결론:응용Teflon대,능구종외주혈단핵세포중유도출대비량적DCs,저사DCs가피저온보존이불개변기공능,위기l림상응용전정료기출.
Dendritic cells are professional antigen presenting cells which are being used as adjuvants in tumor vaccination trials. Most clinical protocols currently include 4 to 10 weekly infusions of doses ＞ 10 6 cells, each inoculum coming from a simple culture of blood monocytes. In the present study, several millions of dendritic cells from a single leukapheresis were produced; monocytes were isolated by elutriation and then cultured in Teflon bags in presence of 800 U/ml GM-CSF+ 100 μg/ml IL-13 + 10% fetal calf serum (FCS). The dendritic cells from this single batch were aliquoted in many doses for potential multiple infusions and cryopreserved in 10% DMSO + 2% human albumin in Teflon- kapton Fresenius bags either at - 1℃/min using a controlled rate freezer, or putting the bags directly in a - 80℃ mechanical freezer without controlling the temperature rate. Six experiments were carried out. After one month of cryopreservation, the cells were thawed in a 40℃ water bath. Before and after freezing, cells were evaluated for immunophenotype (CD1a, CD14, CD40, CD80, CD83, CD86, CD54, CD58, CD16, CD32, CD64 and HLA-DR) and for their capacity to stimulate allogenic (MLR) or autologous (antigen presentation tests) lymphocytes. The results demonstrated that the mean recovery rates after freezing in liquid nitrogen or at - 80℃ were (67 ± 14)% and (71 ±13)% respectively, without any significant difference between the two techniques. The immunophenotype was not modified by the freezing-thawing procedure, as well as the lymphocyte stimulating capacities. In conclusion, our study showed that substantial numbers of functional DCs can be derived from peripheral blood monocytes using Teflon bags.DCs can be cryopreserved in a good laboratory practice setting for further clinical trials with an acceptable loss of cells and without modification of their functions.