Effects of different anticoagulant preservation solutions on the proliferation and killing effects of NK cells cultured in umbilical cord blood

doi:10.3877/cma.j.issn.2095-3232.2023.05.019

Abstract

Abstract:

Objective

To evaluate the effects of different anticoagulant preservation solutions on the proliferation of natural killer (NK) cells cultured in umbilical cord blood and their killing effects on liver cancer cells.

Methods

A portion of 20 ml umbilical cord blood was aseptically collected from 10 healthy pregnant women and transferred into anticoagulation tubes containing heparin sodium and blood preservation solution, respectively. Cord blood mononuclear cells (CBMCs) were isolated by using lymphocyte solutions. NK cells were proliferated and cultured in vitro, and cell count was carried out at 0, 3, 6, 9, 12, 15, 18 and 21 d after culture, respectively. The percentage of CD3^-CD56⁺, CD16⁺NK cells and the apoptosis rate were detected by flow cytometry at 15 and 21 d. At 15 d, CCK8 assay was utilized to detect the cytotoxic effects of CBMCs collected from two anticoagulant solutions and NK cells obtained from in vitro proliferation on human liver cancer cell lines (SMMC7721, BEL7402, Sk-hep1, HepG2, Hep3B, and Huh7) according to the effect-target ratio of 5∶1, 10∶1 and 20∶1, respectively. Cell count between two groups was compared by t test. Multi-group comparison was performed by one-way ANOVA. Two-group comparison was conducted by LSD-t test.

Results

NK cells grew logarithmically from 9 d of in vitro culture, and the proliferation capability was declined and the number of cells was decreased at 15 d. Cell growth curve showed that the number of NK cells at 9, 15 and 21 d in the heparin sodium group was (1.8±0.8)×10⁸, (6.2±1.9)×10⁸ and (4.9±1.3)×10⁸, significantly higher than (1.0±0.5)×10⁸, (4.3±2.0)×10⁸ and (3.4±1.9)×10⁸ in the blood preservation solution group (t=2.797, 2.198, 2.177; P<0.05). The cytotoxic effects of NK cells on 6 human liver cancer cells were strengthened with the increase of effect-target ratio, and the killing efficiency was the highest at an effect-target ratio of 20∶1, especially for SMMC7721. However, no significant difference was observed in the killing efficiency between two groups (P>0.05).

Conclusions

Both heparin sodium and blood preservation solution can be used as anticoagulants for NK cells cultured in umbilical cord blood. Heparin sodium can enhance the proliferation of NK cells, whereas it does not increase the killing effect on liver cancer cells.

Key words: NK cells, Cell killing effect, Cytotoxicity, Liver cancer cells, Anticoagulant, Heparin sodium, Blood preservation solution

Cuiping Li, Xiaoyan Chen, Shiyu Qian, Huizhu Lin, Caihui Zeng, Li Yang, Jianxi Lu. Effects of different anticoagulant preservation solutions on the proliferation and killing effects of NK cells cultured in umbilical cord blood[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2023, 12(05): 572-576.

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References 20

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检测时间	肝素钠组			血液保存液组
检测时间	CD3^-CD56⁺占比	CD16⁺占比	凋亡率	CD3^-CD56⁺占比	CD16⁺占比	凋亡率
第15天	86.1±7.5	91.1±2.5	8.6±5.4	85.5±9.2	92.0±3.2	13.5±6.2
第21天	64.9±10.2	68.3±8.3	26.0±13.9	70.7±8.0	72.5±9.8	27.3±9.1
t值	5.297	8.328	-3.690	3.821	5.974	-3.968
P值	<0.001	<0.001	0.002	0.001	<0.001	<0.001

检测时间	肝素钠组			血液保存液组
检测时间	CD3^-CD56⁺占比	CD16⁺占比	凋亡率	CD3^-CD56⁺占比	CD16⁺占比	凋亡率
第15天	86.1±7.5	91.1±2.5	8.6±5.4	85.5±9.2	92.0±3.2	13.5±6.2
第21天	64.9±10.2	68.3±8.3	26.0±13.9	70.7±8.0	72.5±9.8	27.3±9.1
t值	5.297	8.328	-3.690	3.821	5.974	-3.968
P值	<0.001	<0.001	0.002	0.001	<0.001	<0.001

肝癌细胞株	5∶1		10∶1		20∶1
肝癌细胞株	肝素钠组	血液保存液组	肝素钠组	血液保存液组	肝素钠组	血液保存液组
SMMC7721	56.5±3.4	57.6±4.0	74.6±4.8	74.0±3.3	84.7±3.7^a,b	84.6±3.2^a,b
BEL7402	35.2±3.0^c	35.7±3.7^c	54.7±5.6^a,c	57.6±5.0^a,c	71.3±5.2^a,c	80.7±3.5^a,b
SK-hep1	33.5±2.8^c	37.4±1.7^c	48.9±4.2^a,c	56.6±3.4^a,c	70.1±4.6^a,b,c	78.0±3.8^a,b
HepG2	30.3±3.9^c	36.8±3.2^c	49.1±4.1^a,c	50.9±3.3^a,c	64.7±4.2^a,b,c	67.3±4.1^a,b,c
HEP3B	32.0±3.1^c	31.4±2.7^c	47.8±3.0^a,c	46.0±3.6^a,c	68.2±4.5^a,b,c	64.0±4.1^a,b,c
Huh7	26.4±2.2^c	29.5±2.4^c	41.8±4.2^a,c	43.7±4.8^c	57.4±4.1^a,b，c	59.4±5.2^a,b,c
F值	11.740	10.950	6.877	7.677	4.183	6.317
P值	<0.001	<0.001	<0.001	<0.001	0.003	<0.001

肝癌细胞株	5∶1		10∶1		20∶1
肝癌细胞株	肝素钠组	血液保存液组	肝素钠组	血液保存液组	肝素钠组	血液保存液组
SMMC7721	56.5±3.4	57.6±4.0	74.6±4.8	74.0±3.3	84.7±3.7^a,b	84.6±3.2^a,b
BEL7402	35.2±3.0^c	35.7±3.7^c	54.7±5.6^a,c	57.6±5.0^a,c	71.3±5.2^a,c	80.7±3.5^a,b
SK-hep1	33.5±2.8^c	37.4±1.7^c	48.9±4.2^a,c	56.6±3.4^a,c	70.1±4.6^a,b,c	78.0±3.8^a,b
HepG2	30.3±3.9^c	36.8±3.2^c	49.1±4.1^a,c	50.9±3.3^a,c	64.7±4.2^a,b,c	67.3±4.1^a,b,c
HEP3B	32.0±3.1^c	31.4±2.7^c	47.8±3.0^a,c	46.0±3.6^a,c	68.2±4.5^a,b,c	64.0±4.1^a,b,c
Huh7	26.4±2.2^c	29.5±2.4^c	41.8±4.2^a,c	43.7±4.8^c	57.4±4.1^a,b，c	59.4±5.2^a,b,c
F值	11.740	10.950	6.877	7.677	4.183	6.317
P值	<0.001	<0.001	<0.001	<0.001	0.003	<0.001

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