In recent years, adoptive cell therapy of immune effector cells, such as chimeric antigen receptor-T (CAR-T) cells, natural killer (NK) cells, and epitope-specific cytotoxic T lymphocyte (CTL) cells have been employed in clinical trials. In addition, CD19 CAR-T cells have been approved by the FDA for treatment of non-Hodgkin lymphoma and diffuse large B-cell lymphoma. In this context, it is vital to detect cellular cytotoxicity and monitor the quality of ex vivo expanded immune cells before product release and patient infusion. Target cells could proliferate in parallel with effector cells during the cytotoxicity assay, making it difficult to estimate the death ratio using conventional approaches. Meanwhile, non-specific dyes or non-homogeneous biomarkers for target cells may interfere with the final readout post addition of effector cells. Here, we modified a component of the coincubation medium to suppress the spontaneous release of bis(acetoxymethyl)2,2′:6′,2″-terpyridine-6,6″-dicarboxylate and sustained the window at a stable range (~70%). Further, the optimized Eu-TDA method presented reliable outcomes compared with lactate dehydrogenase detection and was compatible with cytotoxicity tests for NK cells and specific CTLs. Finally, the reported assay can accurately detect death of target cells depending on the amount of hydrophilic complex and can be reliably applied in quality control and cell activity evaluation tests on co-suspended effector and target cells.

Summary

A medium component for cellular coincubations (and associated protocols) have been optimized and validated for cytotoxicity assays, which can reliably evaluate the potency of engineered CD19 CAR-T cells, NK cells, and specific CTLs. In particular, the reported method can be applied widely in routine assays for bi-suspended effector and target cells with a stable window.

中文翻译：

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优化共悬浮效应细胞和靶细胞的细胞毒性测定

近年来，免疫效应细胞的过继细胞疗法，如嵌合抗原受体-T（CAR-T）细胞、自然杀伤（NK）细胞和表位特异性细胞毒性T淋巴细胞（CTL）细胞已应用于临床试验。 . 此外，CD19 CAR-T细胞已被FDA批准用于治疗非霍奇金淋巴瘤和弥漫性大B细胞淋巴瘤。在这种情况下，在产品发布和患者输注之前检测细胞毒性并监测体外扩增免疫细胞的质量至关重要。在细胞毒性测定过程中，靶细胞可能与效应细胞平行增殖，因此很难使用传统方法估计死亡率。同时，靶细胞的非特异性染料或非均质生物标志物可能会干扰效应细胞添加后的最终读数。这里，我们修改了共培养培养基的一个成分，以抑制双（乙酰氧基甲基）2,2':6',2"-terpyridine-6,6"-dicarboxylate 的自发释放，并将窗口维持在稳定范围 (~70%) . 此外，与乳酸脱氢酶检测相比，优化的 Eu-TDA 方法提供了可靠的结果，并且与 NK 细胞和特定 CTL 的细胞毒性测试兼容。最后，报告的测定可以根据亲水复合物的量准确检测靶细胞的死亡，并且可以可靠地应用于共悬浮效应细胞和靶细胞的质量控制和细胞活性评估测试。与乳酸脱氢酶检测相比，优化的 Eu-TDA 方法提供了可靠的结果，并且与 NK 细胞和特定 CTL 的细胞毒性测试兼容。最后，报告的测定可以根据亲水复合物的量准确检测靶细胞的死亡，并且可以可靠地应用于共悬浮效应细胞和靶细胞的质量控制和细胞活性评估测试。与乳酸脱氢酶检测相比，优化的 Eu-TDA 方法提供了可靠的结果，并且与 NK 细胞和特定 CTL 的细胞毒性测试兼容。最后，报告的测定可以根据亲水复合物的量准确检测靶细胞的死亡，并且可以可靠地应用于共悬浮效应细胞和靶细胞的质量控制和细胞活性评估测试。

概括

用于细胞共培养（和相关协议）的培养基成分已针对细胞毒性测定进行了优化和验证，可以可靠地评估工程化 CD19 CAR-T 细胞、NK 细胞和特定 CTL 的效力。特别是，所报告的方法可广泛应用于具有稳定窗口的双悬浮效应细胞和靶细胞的常规检测。