CN114958771B - 一种ipsc-car-nk细胞的制备方法 - Google Patents

一种ipsc-car-nk细胞的制备方法 Download PDF

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CN114958771B
CN114958771B CN202210735797.4A CN202210735797A CN114958771B CN 114958771 B CN114958771 B CN 114958771B CN 202210735797 A CN202210735797 A CN 202210735797A CN 114958771 B CN114958771 B CN 114958771B
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CN114958771A (zh
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齐国光
彭大为
湛振键
刘世豪
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Hainan Kangdun Biopharmaceutical Co ltd
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Abstract

本发明提供一种IPSC‑CAR‑NK细胞的制备方法，包括：S1：将带有嵌合抗原受体CAR基因的慢病毒载体感染诱导性多能干细胞iPSC，得表达嵌合抗原受体CAR的iPSC细胞；S2：定向分化诱导，得造血祖细胞HPC；S3：转入含有P/S双抗、TGF‑β转化生长因子、细胞因子、磷脂酰肌醇3‑激酶抑制剂和拮抗剂的无血清的DMEM培养基培养，调整细胞密度，依次转入含有不同浓度的氨甲环酸的扩增培养基中培养，得抗原特异性的iPSC‑CAR‑NK细胞；本发明实现更加稳定高产率地由IPSC诱导CAR‑NK细胞，且IPSC‑CAR‑NK细胞具备高细胞毒性和优异增殖能力，促进CAR‑NK细胞更广泛的临床应用。

Description

一种IPSC-CAR-NK细胞的制备方法
技术领域
本发明涉及生物医学领域，特别涉及一种IPSC-CAR-NK细胞的制备方法。
背景技术
嵌合抗原受体(chimeric antigen receptor，CAR)，能够增强淋巴细胞对多种恶性肿瘤细胞的靶向识别和杀伤。CAR基因主要包括细胞外识别域和细胞内信号转导结构域：胞外区主要负责靶向特定的肿瘤抗原，而信号转导区则负责介导淋巴细胞的活化。但CAR-T存在本身的缺陷，存在细胞因子风暴的过度免疫应答，而且CAR-T属于个性化治疗，需要从患者体内提取T细胞，在体外进行基因修饰，扩增培养后再回输人体，制造过程复杂，成本高，无法大量普及。
自然杀伤细胞(NK)是一类重要的免疫细胞,其作为人体抵御病原体入侵和细胞恶变第一道防线的重要组成部分。NK细胞因其无需预先致敏并且不会导致移植物抗宿主病，在CAR细胞疗法中受到越来越多的关注。近年来，对CAR-NK细胞疗法靶点的临床前研究结果表明，其在肿瘤治疗中显示出较好的疗效。
虽然NK细胞可以作为T细胞的替代细胞来进行过继免疫治疗，但现有过继性免疫疗法往往受限于抗原特异性的CAR-NK细胞的缺乏，通过诱导的多能干细胞(IPSC)经过诱导分化获得表达CAR的NK细胞，是抗原特异性NK细胞来源的途径之一，但在多能干细胞定向分化CAR-NK细胞的过程中，细胞产率低，因此，提出一种高产率的IPSC诱导CAR-NK细胞的制备方法，以实现大规模生产均一、稳定的细胞产品，促进CAR-NK细胞更广泛的临床应用。
发明内容
鉴于此，本发明提出一种IPSC诱导CAR-NK细胞的制备方法，实现稳定高产率的CAR-NK细胞获取，且CAR-NK细胞具有良好癌细胞杀伤能力。
本发明的技术方案是这样实现的：
本发明提供一种IPSC-CAR-NK细胞的制备方法，包括如下步骤：
S1：将带有嵌合抗原受体CAR基因的慢病毒载体感染诱导性多能干细胞iPSC，得到表达嵌合抗原受体CAR的iPSC细胞；
S2：将表达嵌合抗原受体CAR的iPSC细胞，定向分化诱导，得到造血祖细胞HPC；
S3：将造血祖细胞HPC转入含有0.3-1.5％P/S双抗、50-70ng/mlTGF-β转化生长因子、10-20ng/ml细胞因子、3-6μM磷脂酰肌醇3-激酶抑制剂和3-4μM拮抗剂的无血清的DMEM培养基中，连续培养5-6d后，并调整细胞密度1-3×102个/mL，依次转入含有浓度为0.1-0.3g/L氨甲环酸的第一扩增培养基和含有浓度为0.5-1g/L的氨甲环酸第二扩增培养基中扩增培养，得到抗原特异性的iPSC-CAR-NK细胞。
进一步说明，所述iPSC细胞为选择来自皮肤、尿液或血液的细胞经过重编程得到的iPSC细胞。
进一步说明，步骤2中，所述定向分化诱导为：将表达嵌合抗原受体CAR的iPSC细胞在含有干细胞因子SCF、GSK3β抑制剂、骨形态发生蛋白BMP、FGF2和VEGF的培养基中培养，并添加FLT-3L配体、血小板生成素TPO和IL7，进行定向分化诱导，得到造血祖细胞HPC。
进一步说明，步骤3中，所述拮抗剂为血管紧张素II 1型受体AT1拮抗剂、Mek/Erk拮抗剂和TGFβ拮抗剂的组合。
进一步说明，步骤3中，所述细胞因子为SCF、TPO、IL3、IL-7、IL-11、IL-15和IL-18的任意一种或多种组合。
进一步说明，步骤4中，所述第一扩增培养基为含有5-8％体积比自体灭活血清、95-105ng/mL IL-2、2-4ng/mL FGF2、0.005-0.01g/L i-肌醇、0.04-0.05g/L叶酸、0.1-0.2g/L羊胎盘素、0.6-0.8g/L盐酸吡哆醇和0.1-0.3g/L氨甲环酸的DMEM培养基。
进一步说明，步骤4中，于第一扩增培养基中扩增培养24h后，更换相同扩增培养基，并加入0.2-0.5g/L的谷胱甘肽，继续培养24h，添加一定浓度谷胱甘肽，进一步延长细胞增殖能力，提升iPSC-CAR-NK细胞后期的稳定增殖作用。
进一步说明，步骤4中，经过第一扩增培养基培养48h后转入第二扩增培养基，所述第二扩增培养基为含有2-3％体积比自体灭活血清、100-120ng/mL IL-2、3-5ng/mL FGF2、0.01-0.15g/L i-肌醇、0.04-0.05g/L叶酸、0.1-0.3g/L羊胎盘素、1.0-1.5g/L盐酸吡哆醇和0.5-1g/L氨甲环酸的DMEM培养基；继续培养至72h，得到抗原特异性的IPSC-CAR-NK细胞。
与现有技术相比，本发明的有益效果是：
本发明通过以表达嵌合抗原受体CAR的iPSC细胞进行定向分化诱导，获得造血祖细胞HPC的基础上，利用含有P/S双抗、TGF-β转化生长因子、细胞因子、磷脂酰肌醇3-激酶抑制剂和拮抗剂的培养基作为造血祖细胞HPC的分化培养基，对造血祖细胞HPC的培养，诱导分化获得iPSC-CAR-NK细胞，并经过了对iPSC-CAR-NK细胞依次经过不同扩增培养扩增培养，既实现了高产率地IPSC诱导CAR-NK细胞，而且iPSC-CAR-NK细胞具有优异癌细胞杀能力，实现了高效稳定地定向诱导诱导性多能干细胞(iPSC)分化成CAR-NK细胞。
本发明在对iPSC-CAR-NK细胞进行双次扩增培养的过程中，采用i-肌醇、叶酸、羊胎盘素和盐酸吡哆醇组合，提升细胞活力，还通过添加氨甲环酸，不仅促进iPSC-CAR-NK细胞的扩增效率，并且依次提升氨甲环酸的浓度，有效增强了iPSC-CAR-NK细胞的肿瘤裂解能力，使iPSC-CAR-NK细胞取代原代NK细胞，具备高细胞毒性和优异增殖能力，有效实现大规模生产均一、稳定的IPSC-CAR-NK细胞，操作简便，促进CAR-NK细胞更广泛的临床应用。
附图说明
图1为本发明iPSC细胞的来源示意图；
图2为本发明由iPSC定向分化成CAR-NK细胞的流程示意图；
图3为本发明实施例不同IPSC-CAR-NK细胞的产率效果示意图；
图4为本发明实施例不同IPSC-CAR-NK细胞的扩增倍数效果示意图。
具体实施方式
为了更好理解本发明技术内容，下面提供具体实施例，对本发明做进一步的说明。
本发明实施例所用的实验方法如无特殊说明，均为常规方法。
本发明实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1-制备IPSC-CAR-NK细胞，包括如下步骤：
S1：将带有嵌合抗原受体CAR基因的慢病毒载体感染由血液细胞重编程得到的诱导性多能干细胞iPSC，得到表达嵌合抗原受体CAR的iPSC细胞；
S2：将表达嵌合抗原受体CAR的iPSC细胞在含有50ng/ml干细胞因子SCF、2μm/mlGSK3β抑制剂、25ng/ml骨形态发生蛋白BMP、50ng/ml FGF2和、20mg/ml VEGF的培养基中培养，并添加20ng/ml FLT-3L配体、50ng/ml血小板生成素TPO和10ng/ml IL7，进行定向分化诱导，得到造血祖细胞HPC；
S3：将造血祖细胞HPC转入含有0.3％P/S双抗、50ng/mlTGF-β转化生长因子、10ng/ml细胞因子、3μM磷脂酰肌醇3-激酶抑制剂和3μM拮抗剂的无血清的DMEM培养基中；拮抗剂为1μM血管紧张素II 1型受体AT1拮抗剂(沙坦)、1μM Mek/Erk拮抗剂(PD0325901)和1μMTGFβ拮抗剂(SB431542)；细胞因子为3ng/ml SCF、2ng/ml TPO、2ng/ml IL3、2ng/ml IL-11和1ng/ml IL-15的组合；每18h更换新培养基，培养5d；
调整细胞密度1×102个/mL，依次转入含有浓度为0.1g/L氨甲环酸的第一扩增培养基和含有浓度为0.5g/L的氨甲环酸第二扩增培养基中扩增；其中，第一扩增培养基为含有8％体积比自体灭活血清、95ng/mL IL-2、2ng/mL FGF2、0.005g/L i-肌醇、0.04g/L叶酸、0.1g/L羊胎盘素、0.6g/L盐酸吡哆醇和0.1g/L氨甲环酸的DMEM培养基；24h后更换相同扩增培养基。
经过第一扩增培养基培养48h后转入第二扩增培养基，第二扩增培养基为含有2％体积比自体灭活血清、100ng/mL IL-2、3ng/mL FGF2、0.01g/L i-肌醇、0.04g/L叶酸、0.1g/L羊胎盘素、1.0g/L盐酸吡哆醇和0.5g/L氨甲环酸的DMEM培养基；继续培养至72h，得到抗原特异性的IPSC-CAR-NK细胞。
实施例2-制备IPSC-CAR-NK细胞，包括如下步骤：
S1：将带有嵌合抗原受体CAR基因的慢病毒载体感染由血液细胞重编程得到的诱导性多能干细胞iPSC，得到表达嵌合抗原受体CAR的iPSC细胞；
S2：将表达嵌合抗原受体CAR的iPSC细胞在含有60ng/ml干细胞因子SCF、2μm/mlGSK3β抑制剂、30ng/ml骨形态发生蛋白BMP、55ng/ml FGF2和、50mg/ml VEGF的培养基中培养，并添加40ng/ml FLT-3L配体、80ng/ml血小板生成素TPO和30ng/ml IL7，进行定向分化诱导，得到造血祖细胞HPC；
S3：将造血祖细胞HPC转入含有1.5％P/S双抗、70ng/mlTGF-β转化生长因子、20ng/ml细胞因子、6μM磷脂酰肌醇3-激酶抑制剂和4μM拮抗剂的无血清的DMEM培养基中；拮抗剂为1μM血管紧张素II 1型受体AT1拮抗剂(沙坦)、2μM Mek/Erk拮抗剂(PD0325901)和1μMTGFβ拮抗剂(SB431542)；细胞因子为5ng/ml SCF、2ng/ml TPO、3ng/mlIL3、5ng/ml IL-11、5ng/mI lL-15的组合；每18h更换新培养基，培养6d；
调整细胞密度3×102个/mL，依次转入含有浓度为0.3g/L氨甲环酸的第一扩增培养基和含有浓度为1g/L的氨甲环酸第二扩增培养基中扩增；其中，第一扩增培养基为含有5％体积比自体灭活血清、105ng/mL IL-2、4ng/mL FGF2、0.01g/Li-肌醇、0.05g/L叶酸、0.2g/L羊胎盘素、0.8g/L盐酸吡哆醇和0.3g/L氨甲环酸的DMEM培养基；24h后更换相同扩增培养基。
经过第一扩增培养基培养48h后转入第二扩增培养基，第二扩增培养基为含有3％体积比自体灭活血清、120ng/mL IL-2、5ng/mL FGF2、0.15g/L i-肌醇、0.05g/L叶酸、0.3g/L羊胎盘素、1.5g/L盐酸吡哆醇和1g/L氨甲环酸的DMEM培养基；继续培养至72h，得到抗原特异性的IPSC-CAR-NK细胞。
实施例3-制备IPSC-CAR-NK细胞，包括如下步骤：
S1：将带有嵌合抗原受体CAR基因的慢病毒载体感染由血液细胞重编程得到的诱导性多能干细胞iPSC，得到表达嵌合抗原受体CAR的iPSC细胞；
将表达嵌合抗原受体CAR的iPSC细胞在含有55ng/ml干细胞因子SCF、2μm/ml GSK3β抑制剂、28ng/ml骨形态发生蛋白BMP、50ng/ml FGF2和、35mg/ml VEGF的培养基中培养，并添加30ng/ml FLT-3L配体、60ng/ml血小板生成素TPO和20ng/ml IL7，进行定向分化诱导，得到造血祖细胞HPC；
S3：将造血祖细胞HPC转入含有1.0％P/S双抗、60ng/mlTGF-β转化生长因子、15ng/ml细胞因子、5μM磷脂酰肌醇3-激酶抑制剂和3μM拮抗剂的无血清的DMEM培养基中；拮抗剂为1μM血管紧张素II 1型受体AT1拮抗剂(沙坦)、1μM Mek/Erk拮抗剂(PD0325901)和1μMTGFβ拮抗剂(SB431542)；细胞因子为3ng/ml SCF、4ng/mlTPO、3ng/ml IL3、3ng/ml IL-11、2ng/ml IL-15的组合；每18h更换新培养基，培养6d；
调整细胞密度2×102个/mL，依次转入含有浓度为0.2g/L氨甲环酸的第一扩增培养基和含有浓度为0.8g/L的氨甲环酸第二扩增培养基中扩增；其中，第一扩增培养基为含有6％体积比自体灭活血清、100ng/mL IL-2、3ng/mL FGF2、0.008g/L i-肌醇、0.04g/L叶酸、0.15g/L羊胎盘素、0.7g/L盐酸吡哆醇和0.2g/L氨甲环酸的DMEM培养基；24h后更换相同扩增培养基。
经过第一扩增培养基培养48h后转入第二扩增培养基，第二扩增培养基为含有2.5％体积比自体灭活血清、110ng/mL IL-2、4ng/mL FGF2、0.013g/L i-肌醇、0.05g/L叶酸、0.2g/L羊胎盘素、1.3g/L盐酸吡哆醇和0.8g/L氨甲环酸的DMEM培养基；继续培养至72h，得到抗原特异性的IPSC-CAR-NK细胞。
实施例4-制备IPSC-CAR-NK细胞，包括如下步骤：
S1：将带有嵌合抗原受体CAR基因的慢病毒载体感染由血液细胞重编程得到的诱导性多能干细胞iPSC，得到表达嵌合抗原受体CAR的iPSC细胞；
S2：将表达嵌合抗原受体CAR的iPSC细胞在含有55ng/ml干细胞因子SCF、2μm/mlGSK3β抑制剂、28ng/ml骨形态发生蛋白BMP、50ng/ml FGF2和、35mg/ml VEGF的培养基中培养，并添加30ng/ml FLT-3L配体、60ng/ml血小板生成素TPO和20ng/ml IL7，进行定向分化诱导，得到造血祖细胞HPC；
S3：将造血祖细胞HPC转入含有1.0％P/S双抗、60ng/ml TGF-β转化生长因子、15ng/ml细胞因子、5μM磷脂酰肌醇3-激酶抑制剂和3μM拮抗剂的无血清的DMEM培养基中；拮抗剂为1μM血管紧张素II 1型受体AT1拮抗剂(沙坦)、1μM Mek/Erk拮抗剂(PD0325901)和1μMTGFβ拮抗剂(SB431542)；细胞因子为3ng/ml SCF、4ng/ml TPO、3ng/ml IL3、3ng/ml IL-11、2ng/ml IL-15的组合；每18h更换新培养基，培养6d；
调整细胞密度2×102个/mL，依次转入含有浓度为0.2g/L氨甲环酸的第一扩增培养基和含有浓度为0.8g/L的氨甲环酸第二扩增培养基中扩增；
其中，第一扩增培养基为含有6％体积比自体灭活血清、100ng/mL IL-2、3ng/mLFGF2、0.008g/L i-肌醇、0.04g/L叶酸、0.15g/L羊胎盘素、0.7g/L盐酸吡哆醇和0.2g/L氨甲环酸的DMEM培养基；于第一扩增培养基中扩增培养24h后，更换相同扩增培养基，并加入0.3g/L的谷胱甘肽，继续培养24h；
经过第一扩增培养基培养48h后转入第二扩增培养基，第二扩增培养基为含有2.5％体积比自体灭活血清、110ng/mL IL-2、4ng/mL FGF2、0.013g/L i-肌醇、0.05g/L叶酸、0.2g/L羊胎盘素、1.3g/L盐酸吡哆醇和0.8g/L氨甲环酸的DMEM培养基；继续培养至72h，得到抗原特异性的IPSC-CAR-NK细胞。
对照例1-如实施例3IPSC-CAR-NK细胞的制备方法，设定不同的造血祖细胞HPC的分化培养基配方，具体为：
将造血祖细胞HPC转入含有3％P/S双抗、30ng/ml TGF-β转化生长因子、30ng/ml细胞因子、8μM磷脂酰肌醇3-激酶抑制剂和3μM拮抗剂的无血清的DMEM培养基中；拮抗剂为1μM血管紧张素II 1型受体AT1拮抗剂(沙坦)、1μM Mek/Erk拮抗剂(PD0325901)和1μMTGFβ拮抗剂(SB431542)；细胞因子为3ng/mlSCF、4ng/mlTPO、3ng/ml IL3、3ng/ml IL-11、2ng/ml IL-15的组合。
对照例2-如实施例3IPSC-CAR-NK细胞的制备方法，设定对造血祖细胞HPC的不同扩增培养条件，具体为：
采用的第一扩增培养基和第二扩增培养基中，添加同一浓度氨甲环酸0.5g/L。
其中，第一扩增培养基为含有6％体积比自体灭活血清、100ng/mL IL-2、3ng/mLFGF2、0.008g/L i-肌醇、0.04g/L叶酸、0.15g/L羊胎盘素、0.7g/L盐酸吡哆醇和0.5g/L氨甲环酸的DMEM培养基；经过第一扩增培养基培养48h后转入第二扩增培养基，第二扩增培养基为含有2.5％体积比自体灭活血清、110ng/mL IL-2、4ng/mL FGF2、0.013g/L i-肌醇、0.05g/L叶酸、0.2g/L羊胎盘素、1.3g/L盐酸吡哆醇和0.5g/L氨甲环酸的DMEM培养基；继续培养至72h，得到抗原特异性的IPSC-CAR-NK细胞。
对照例3-如实施例3IPSC-CAR-NK细胞的制备方法，采用相同的扩增培养基对造血祖细胞HPC进行扩增培养，具体为：
扩增培养基为含有6％体积比自体灭活血清、100ng/mL IL-2、4ng/mL FGF2、0.01g/L i-肌醇、0.045g/L叶酸、0.2g/L羊胎盘素、1.0g/L盐酸吡哆醇、0.5g/L氨甲环酸和0.2g/L的谷胱甘肽的DMEM培养基；每24h更换相同的扩增培养基，连续培养至72h，得到抗原特异性的IPSC-CAR-NK细胞。
实施例5-IPSC-CAR-NK细胞的产率
采用细胞计数法统计上述实施例和对照例所制备的IPSC-CAR-NK细胞的产率；IPSC-CAR-NK细胞产率＝输出IPSC-CAR-NK细胞数/初始iPSC细胞数；
统计上述对上述实施例和对照例的制备方法所扩增培养72h后的IPSC-CAR-NK细胞扩增倍数，IPSC-CAR-NK细胞产率＝输出IPSC-CAR-NK细胞数/初始IPSC-CAR-NK细胞数，其流式检测结果和扩增倍数，如图1和2所示。
如图1所示不同实验组中的IPSC-CAR-NK细胞的产率，由实施例1-4中可以看出，本发明由诱导性多能干细胞iPSC可有效获得高产率的IPSC-CAR-NK细胞，且其明显高于对照例1的IPSC-CAR-NK细胞产率，表明本发明通过利用一定配比的含有P/S双抗、TGF-β转化生长因子、细胞因子、磷脂酰肌醇3-激酶抑制剂和拮抗剂的培养基作为造血祖细胞HPC的分化培养基，对造血祖细胞HPC的培养，可高效获得诱导分化获得iPSC-CAR-NK细胞。如图2所示，实施例1-4中对IPSC-CAR-NK细胞经过含不同浓度的氨甲环酸的扩增培养进行扩增培养，iPSC-CAR-NK细胞的扩增效率提高，其中，实施例4中的扩增效率最优，而对照例2和3中则明显低于实施例3的iPSC-CAR-NK细胞扩增效率，表明本发明在对iPSC-CAR-NK细胞进行双次扩增培养的过程中，采用i-肌醇、叶酸、羊胎盘素和盐酸吡哆醇组合的基础上，通过依次提升氨甲环酸的浓度的分阶段组合培养，有利于使iPSC-CAR-NK细胞优异增殖能力，提高其增殖效率。
实施例6-IPSC-CAR-NK细胞的对体外癌细胞杀伤能力
对上述实施例1-4的制备方法所扩增培养72h后的IPSC-CAR-NK细胞进行细胞杀伤活性检测。采用MTT法，调整细胞浓度为105个/ml，分别按效应细胞：靶细胞＝1:1和5：1的效靶比例放入96孔板，靶细胞为人淋巴瘤细胞(Raji靶细胞)，另设单独靶细胞和效应细胞组，每组设3个复孔。孵育24h，每孔加入MTT溶液，继续培养孵育4h，平板离心机去掉悬液，每孔加入二甲亚砜溶液150mL，振荡，在酶标仪上测定吸收值(结果用均值表示)A，计算靶细胞杀伤活性。靶细胞杀伤活性％＝[靶细胞A值-(效应细胞A值-实验组A值]/靶细胞A值×100。靶细胞为人淋巴瘤细胞(Raji靶细胞)，检测结果如表1所示。
表1IPSC-CAR-NK细胞的对体外癌细胞杀伤活力
由上表1可知，本发明由诱导性多能干细胞iPSC可有效获得高产率的IPSC-CAR-NK细胞具有较高的癌细胞杀能力，在效靶比例为5:1时，IPSC-CAR-NK细胞对Raji靶细胞的杀伤活力最高可达到95.15％，在不同的造血祖细胞HPC的分化培养基配方下，对照例1中的IPSC-CAR-NK细胞活细胞杀伤活力低于实施例3；对照例2和3的IPSC-CAR-NK细胞也低于实施例3，表明在对IPSC-CAR-NK细胞的扩增培养过程中，依次经过含有不同浓度的氨甲环酸的扩增培养基下，有利于更好地保持IPSC-CAR-NK细胞的肿瘤裂解能力，使iPSC-CAR-NK细胞取代原代NK细胞，不仅实现大规模生产均一、稳定的IPSC-CAR-NK细胞，而且能够具有优异癌细胞杀能力。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (6)

1.一种IPSC-CAR-NK细胞的制备方法，其特征在于：包括如下步骤：
S1：将带有嵌合抗原受体CAR基因的慢病毒载体感染诱导性多能干细胞iPSC，得到表达嵌合抗原受体CAR的iPSC细胞；
S2：将表达嵌合抗原受体CAR的iPSC细胞，定向分化诱导，得到造血祖细胞HPC；
S3：将造血祖细胞HPC转入含有0.3-1.5％P/S双抗、50-70ng/mlTGF-β转化生长因子、10-20ng/ml细胞因子、3-6μM磷脂酰肌醇3-激酶抑制剂和3-4μM拮抗剂的无血清的DMEM培养基中，连续培养5-6d后，并调整细胞密度1-3×102个/mL，依次转入含有浓度为0.1-0.3g/L氨甲环酸的第一扩增培养基和含有浓度为0.5-1g/L的氨甲环酸第二扩增培养基中扩增培养，得到抗原特异性的iPSC-CAR-NK细胞；
拮抗剂为1μM血管紧张素II 1型受体AT1拮抗剂、1μM Mek/Erk拮抗剂和1μM TGFβ拮抗剂；
细胞因子为3ng/ml SCF、2ng/ml TPO、2ng/ml IL3、2ng/ml IL-11和1ng/ml IL-15的组合。
2.如权利要求1所述的一种IPSC-CAR-NK细胞的制备方法，其特征在于：所述iPSC细胞为选择来自皮肤、尿液或血液的细胞经过重编程得到的iPSC细胞。
3.如权利要求1所述的一种IPSC-CAR-NK细胞的制备方法，其特征在于：步骤2中，所述定向分化诱导为：将表达嵌合抗原受体CAR的iPSC细胞在含有干细胞因子SCF、GSK3β抑制剂、骨形态发生蛋白BMP、FGF2和VEGF的培养基中培养，并添加FLT-3L配体、血小板生成素TPO和IL7，进行定向分化诱导，得到造血祖细胞HPC。
4.如权利要求1所述的一种IPSC-CAR-NK细胞的制备方法，其特征在于：步骤3中，所述第一扩增培养基为含有5-8％体积比自体灭活血清、95-105ng/mL IL-2、2-4ng/mL FGF2、0.005-0.01g/Li-肌醇、0.04-0.05g/L叶酸、0.1-0.2g/L羊胎盘素、0.6-0.8g/L盐酸吡哆醇和0.1-0.3g/L氨甲环酸的DMEM培养基。
5.如权利要求1所述的一种IPSC-CAR-NK细胞的制备方法，其特征在于：步骤3中，于第一扩增培养基中扩增培养24h后，更换相同扩增培养基，并加入0.2-0.5g/L的谷胱甘肽，继续培养24h。
6.如权利要求5所述的一种IPSC-CAR-NK细胞的制备方法，其特征在于：步骤3中，经过第一扩增培养基培养48h后转入第二扩增培养基，所述第二扩增培养基为含有2-3％体积比自体灭活血清、100-120ng/mL IL-2、3-5ng/mL FGF2、0.01-0.15g/Li-肌醇、0.04-0.05g/L叶酸、0.1-0.3g/L羊胎盘素、1.0-1.5g/L盐酸吡哆醇和0.5-1g/L氨甲环酸的DMEM培养基；继续培养至72h，得到抗原特异性的IPSC-CAR-NK细胞。
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