Construction of a system for isolation and purification of NK cells from whole blood donations
10.13303/j.cjbt.issn.1004-549x.2025.02.004
- VernacularTitle:全血来源NK细胞分离纯化技术体系的初步构建
- Author:
Tengyu CAO
1
,
2
;
Huayu LIN
3
;
Xuanzhi ZHANG
1
,
2
;
Cuimi DUAN
3
;
Yi LIU
3
;
Xiaonan XUE
3
;
Liping SUN
3
;
Yang YU
1
,
2
Author Information
1. Medical School of Chinese PLA
2. Department of Transfusion Medicine, The First Medical Center, Chinese PLA General Hospital, Beijing 100853, China
3. Department of Transfusion Medicine, The First Medical Center, Chinese PLA General Hospital, Beijing 100853, China
- Publication Type:Journal Article
- Keywords:
NK cells;
whole blood;
separation and purification;
cell immunotherapy
- From:
Chinese Journal of Blood Transfusion
2025;38(2):181-188
- CountryChina
- Language:Chinese
-
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 purification 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 gradient 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)×108vs (6.49±2.16)×108, P<0.05], and there was no difference in the red blood cell loss between the two methods (P>0.05). The purity of NK cells isolated and enriched from PBMCs by manual separation method using immunomagnetic 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 separation method using platelet lysis culture expansion method was the highest at day 7, which was (54.84±15.80)%; the cell expansion 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-conditioned medium have the potential to provide NK cells for large-scale NK cell activation reinfusion therapy.
