CAR-NK细胞疗法不需要HLA匹配，副作用最小。然而，将 CAR 工程化到人体组织衍生的 NK 细胞中的传统方法表现出异质性、转导效率低和制造成本高。在这里，我们提供了一种可靠的方法，用于从人胚胎干细胞 (hESC) 作为替代细胞来源生成大规模冷冻间皮素 (MSLN) CAR-NK 细胞。我们首先构建了表达 MSLN CAR 的 hESC 以降低 CAR 工程成本，随后通过高效的类器官诱导系统将这些干细胞分化为 MSLN CAR-NK 细胞。 MSLN CAR-NK细胞表现出NK细胞的激活受体、抑制受体和效应分子的典型表达模式。在肿瘤细胞存在的情况下，与诱导的 NK 细胞相比，MSLN CAR-NK 细胞显示出 IFN-γ 和 TNF-α 分泌增加，以及 CD107a 表达水平升高。我们使用临床级冷冻介质 (CS10) 将 MSLN CAR-NK 细胞在液氮中冷冻保存 6 个多月，以模拟现成的 CAR-NK 细胞产品。解冻的MSLN CAR-NK细胞在培养48-72小时后立即恢复，并有效消除卵巢肿瘤细胞，包括来自患者的人原发性卵巢肿瘤细胞。解冻的 MSLN CAR-NK 细胞可有效抑制体内卵巢肿瘤的发展，并延长荷瘤小鼠的存活时间。我们的研究为 hESC 衍生的 MSLN CAR-NK 细胞作为一种有前途的现成细胞产品的临床转化提供了见解。© 2024 作者。北京干细胞与再生医学研究院和John Wiley联合出版的《细胞增殖》

CAR-NK cell therapy does not require HLA matching and has minimal side effects. However, traditional methods of engineering CARs into human tissue-derived NK cells exhibit heterogeneity, low transduction efficiency, and high manufacturing costs. Here, we provide a reliable approach for generating large-scale and cryopreserved mesothelin (MSLN) CAR-NK cells from human embryonic stem cells (hESCs) as an alternative cell source. We first constructed MSLN CAR-expressing hESCs to reduce CAR engineering costs and subsequently differentiated these stem cells into MSLN CAR-NK cells via an efficient organoid induction system. The MSLN CAR-NK cells exhibit the typical expression patterns of activating receptors, inhibitory receptors, and effector molecules of NK cells. In the presence of tumour cells, the MSLN CAR-NK cells show increased secretion of IFN-γ and TNF-α, as well as elevated CD107a expression level compared with induced NK cells. We cryopreserved the MSLN CAR-NK cells in liquid nitrogen using a clinical-grade freezing medium (CS10) for more than 6 months to mimic an off-the-shelf CAR-NK cell product. The thawed MSLN CAR-NK cells immediately recovered after 48-72-h culture and effectively eliminated ovarian tumour cells, including human primary ovarian tumour cells from patients. The thawed MSLN CAR-NK cells efficiently suppressed ovarian tumour development in vivo and prolonged the survival of tumour-bearing mice. Our study provides insights into the clinical translation of hESC-derived MSLN CAR-NK cells as a promising off-the-shelf cell product.© 2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.