中国输血杂志2025，Vol.38Issue(2)：181-188,221,9.DOI:10.13303/j.cjbt.issn.1004-549x.2025.02.004
全血来源NK细胞分离纯化技术体系的初步构建
Construction of a system for isolation and purification of NK cells from whole blood donations
曹腾宇 1林华雨 2张璇之 1段翠密 2刘羿 2薛晓楠 2孙莉萍 2于洋1
作者信息
- 1. 解放军医学院,北京 100853||中国人民解放军总医院第一医学中心 输血医学科,北京 100853
- 2. 中国人民解放军总医院第一医学中心 输血医学科,北京 100853
- 折叠
摘要
Abstract
Objective To explore the feasibility of using whole blood as a source of NK cells for allogeneic CAR NK cell therapy and activated NK cell reinfusion therapy,and initially construct a technical system for the separation and purifi-cation of NK cells from whole blood.Methods All peripheral blood mononuclear cells(PBMCs)were enriched from 400 mL of whole blood by manual separation and machine separation,respectively.The erythrocyte loss rate,PBMCs number,NK cell purity of the two methods were compared.NK cells were sorted from PBMCs by three separation and enrichment methods as immunomagnetic bead negative selection method,platelet lysate culture expansion and PERCOLL density gradi-ent separation method,and the purity and yield of NK cells,the activity of NK cells and the tumor-killing ability of the three separation and enrichment methods were compared.Results The proportion of NK cells in the lymphocyte population was higher in the manual separation method than in the machine separation method[(13.16±5.16)%vs(8.56±3.92)%,P<0.05];the number PBMCs was lower in the manual separation method than in the machine separation method[(4.09±1.80)×108 vs(6.49±2.16)×108,P<0.05],and there was no difference in the red blood cell loss between the two meth-ods(P>0.05).The purity of NK cells isolated and enriched from PBMCs by manual separation method using immunomag-netic was(96.77±2.31)%;the yield was(56.27±10.47)%;the inhibition of tumor proliferation was(38.67±14.05)%;and the tumor killing rate was(19.90±8.05)%.The purity of NK cells isolated and enriched from PBMCs by manual separa-tion method using platelet lysis culture expansion method was the highest at day 7,which was(54.84±15.80)%;the cell ex-pansion multiple could reach 16.92±6.28 at day 7;the in vitro tumor killing rate of NK cells was(15.83±5.5)%;the tumor inhibition rate was(44.33±13.5)%;and there was no difference in the toxicity and activity of NK cells between the two methods(P>0.05).The purity of NK cells isolated and enriched by PERCOLL density gradient separation method was(15.83±5.82)%,and the yield was(14±6.25)%,which was significantly lower than the other two methods.Conclusion PBMCs isolated from whole blood by manual separation and NK cells enriched by negative selection with immunomagnetic beads have the potential to provide NK cell materials for CAR-NK cell therapy,and NK cells enriched by platelet lysate-condi-tioned medium have the potential to provide NK cells for large-scale NK cell activation reinfusion therapy.关键词
NK细胞/全血/分离纯化/细胞免疫治疗Key words
NK cells/whole blood/separation and purification/cell immunotherapy分类
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曹腾宇,林华雨,张璇之,段翠密,刘羿,薛晓楠,孙莉萍,于洋..全血来源NK细胞分离纯化技术体系的初步构建[J].中国输血杂志,2025,38(2):181-188,221,9.
