冻存密度对外周血单个核细胞冻存效果的影响

doi:10.13560/j.cnki.biotech.bull.1985.2018-0646

生物技术通报 ›› 2019, Vol. 35 ›› Issue (6): 119-124.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0646

冻存密度对外周血单个核细胞冻存效果的影响

林科佳^1,3, 刘赴平², 马冬磊¹, 胡锐¹, 魏宗科¹, 谭毅²

1. 深圳市润科生物科技有限公司,深圳 518000;
2. 东莞市南城医院,东莞 523000;
3. 中国人民大学统计学院,北京 100872

收稿日期:2018-07-18 出版日期:2019-06-26 发布日期:2019-07-08
作者简介:林科佳,男,硕士研究生,研究方向：细胞学;E-mail：bluekejia@126.com
基金资助:
东莞市科学技术局科技项目（201650715001456）

Effect of Cryopreservation Density on Cryopreservation Effect of Peripheral Blood Mononuclear Cells

LIN Ke-jia^1,3, LIU Fu-ping², MA Dong-lei¹, HU Rui¹, WEI Zong-ke¹, TAN Yi²

1. Shenzhen Rekindle Biology Science and Technology Co.,Ltd.,Shenzhen 518000;
2. Dongguan Nancheng Hospital,Dongguan 523000;
3. School of Statistics, Renmin University of China,Beijing 100872

Received:2018-07-18 Published:2019-06-26 Online:2019-07-08

摘要/Abstract

摘要： 探讨冻存密度对外周血单个核细胞（Peripheral blood mononuclear cell,PBMC）冻存效果的影响。设定新鲜PBMC组（F组）及3个PBMC冻存密度组即2.0×10⁷/mL（A组）,4.0×10⁷/mL（B组）,6.0×10⁷/mL（C组）。通过对冻存前后的PBMC活率及复苏后多种细胞因子诱导的杀伤细胞（Cytokine-induced killer,CIK）的扩增倍数、淋巴细胞亚群、体外杀伤效率进行比较,验证其冻存效果。结果显示,冻存前F组与冻存后A组、B组、C组的细胞活率分别为（96.0±0.3）%、（95.6±0.4）%、（94.7±0.2）%和（94.9±0.4）%,B组与C组显著低于A组,P<0.05;细胞扩增14 d后,F组与A组、B组、C组细胞扩增倍数分别为（156.4±18.2）倍、（160.2±28.4）倍、（126.1±19.8）倍和（110.4±11.3）倍,B组与C组显著低于F组（P<0.05）;PBMC冻存前复苏后淋巴细胞亚群结果无显著差异。与F组相比,A组、B组、C组细胞扩增14 d后,CD3+、CD3+ CD4+、CD3+ CD8+、CD3-CD56+、CD3+CD56+淋巴细胞亚群无显著差异（P>0.05）,体外杀伤效率无显著差异（P>0.05）。过高的冻存密度会影响PBMC冻存效果而间接影响细胞的复苏应用,而过低的冻存密度则增加冻存体积而大大提高冻存成本,因此,选择合适的细胞冻存密度是细胞库或细胞银行必须要考虑的问题。

关键词: 冻存, 外周血单个核细胞, 活率, 细胞密度

Abstract: The objective of this study is to investigate the effect of cryopreservation density on the cryopreservation of peripheral blood mononuclear cell（PBMC）. Fresh PBMC group（Group F）and 3 PBMC cryopreserved cell density groups：2×10⁷cells/mL（group A）,4×10⁷ cells/mL（group B）,6×10⁷cells/mL（group C）were set up. By comparing these data,such as the survival rate of PBMC before and after cryopreservation,as well as the amplification multiples,lymphocyte subsets,and in vitro killing efficiency of multiple cytokines-induced killer（CIK）cells after resuscitation,the cryopreservation effect was validated. Results shows as,the cell viability of group F,group A,group B and group C before and after cryopreservation were（96 + 0.3）%,（95.6 + 0.4）%,（94.7 + 0.2）% and（94.9 + 0.4）%,respectively,i.e.,the cell viability of group B and group C were significantly lower than that of group A（P<0.05）. At 14 days after cell amplification,the cell amplification of group F and group A,group B,and group C were（156.4 + 18.2）times,（160.2 + 28.4）times,（126.1 + 19.8）times,and（110.4 + 11.3）times,respectively,i.e.,that cell amplification of group B and group C was significantly lower than that of group F（P < 0.05）. There was no significant difference in the results of lymphocyte subsets before PBMC freezing and after PBMC resuscitation. Compared with group F,at 14 d after cell amplification of group A,group B,and group C,the lymphocyte subsets and the in vitro killing efficiency of CD3+,CD3+CD4+,CD3+ CD8+,CD3-CD56+ and CD3+CD56+ demonstrated no significant difference（P>0.05）. In conclusion,too high cryopreservation density may affect the cryopreservation effect of PBMC so as to indirectly affect the cell resuscitation. However,the too low cryopreservation density increases the cryopreservation volume so as to greatly raise cost. Therefore,choosing appropriate cell cryopreservation density is an issue that must be considered for a cell library or cell bank.

Key words: cryopreservation, peripheral blood mononuclear cell, survival rate, cell density

林科佳, 刘赴平, 马冬磊, 胡锐, 魏宗科, 谭毅. 冻存密度对外周血单个核细胞冻存效果的影响[J]. 生物技术通报, 2019, 35(6): 119-124.

LIN Ke-jia, LIU Fu-ping, MA Dong-lei, HU Rui, WEI Zong-ke, TAN Yi. Effect of Cryopreservation Density on Cryopreservation Effect of Peripheral Blood Mononuclear Cells[J]. Biotechnology Bulletin, 2019, 35(6): 119-124.

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冻存密度对外周血单个核细胞冻存效果的影响

Effect of Cryopreservation Density on Cryopreservation Effect of Peripheral Blood Mononuclear Cells

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