Fujita Eriko, Yamamoto Syunsuke, Hanada Takeshi, Jogasaki Shingo, Koga Yoshiyuki, Yatsuda Yukinori, Kakizaki Yoshiyuki, Jo Yoshinori, Asano Yuya, Yonezawa Koichi, Moriya Yuu, Nakayama Miyu, Arimura Yukiko, Okawa Yurie, Komatsu Hiroyuki, Ito Masahiko, Suzuki Syunsuke, Kuroda Takuya, Yasuda Satoshi, Kamiyama Yoshiteru, Sato Yoji
Applied Research & Operations, Astellas Pharma Inc., Tsukuba-shi, Ibaraki, Japan.
Center of Excellence for Drug Metabolism, Pharmacokinetics and Modeling, Preclinical and Translational Sciences, Research, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan.
Cytotherapy. 2025 Jan;27(1):51-65. doi: 10.1016/j.jcyt.2024.09.003. Epub 2024 Oct 5.
Regenerative therapies employing cell therapy products (CTPs) have attracted considerable attention. Biodistribution (BD) evaluation of CTPs is mainly performed to clarify the cell survival time, engraftment, and distribution site. This evaluation is crucial for predicting the efficacy and safety profiles of clinical studies based on non-clinical BD study outcomes. However, no internationally unified method has been established for assessing cell BD after administration. Here, we aimed to standardize the BD assay method used for CTPs, conducting the following evaluations using the same protocol across multiple study facilities: (1) in vitro validation of quantitative polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) analyses using the primate-specific Alu gene, and (2) in vivo BD studies after the intravenous administration of human mesenchymal stem cells (hMSCs) to immunodeficient mice, commonly used in non-clinical tumorigenicity studies.
Quality control samples were prepared and analyzed by adding a fixed number of human-derived cells to several mouse tissues. The respective quantitative performances of the qPCR and ddPCR methods were compared for accuracy and precision. hMSCs were intravenously administered to immunodeficient mice, and tissues were collected at 1, 4, and 24 h after administration.
Both methods demonstrated an accuracy (relative error) generally within ±50% and a precision (coefficient of variation) generally less than 50%. While differences in calibration curve ranges were observed between qPCR and ddPCR, no significant differences in quantification were found among the assay facilities. The BD of hMSCs in mice was evaluated at seven facilities (qPCR at three facilities; ddPCR at four facilities), revealing similar tissue distribution profiles in all facilities, with the lungs showing the highest cell distribution among the tissues tested.
Quantitative evaluation of qPCR and ddPCR using Alu sequences was conducted, demonstrating that the test method can be adapted for BD evaluation.
采用细胞治疗产品（CTP）的再生疗法已引起广泛关注。对CTP进行生物分布（BD）评估主要是为了明确细胞存活时间、植入情况及分布部位。基于非临床BD研究结果预测临床研究的疗效和安全性，该评估至关重要。然而，目前尚未建立用于评估给药后细胞BD的国际统一方法。在此，我们旨在规范用于CTP的BD检测方法，在多个研究机构使用相同方案进行以下评估：（1）使用灵长类特异性Alu基因对定量聚合酶链反应（qPCR）和液滴数字PCR（ddPCR）分析进行体外验证，以及（2）在常用于非临床致瘤性研究的免疫缺陷小鼠静脉注射人间充质干细胞（hMSC）后进行体内BD研究。
制备质量控制样本，通过向几种小鼠组织中添加固定数量的人源细胞进行分析。比较qPCR和ddPCR方法各自的定量性能，评估准确性和精密度。将hMSC静脉注射到免疫缺陷小鼠体内，并在给药后1、4和24小时收集组织。
两种方法的准确性（相对误差）一般在±50%以内，精密度（变异系数）一般小于50%。虽然qPCR和ddPCR在校准曲线范围上存在差异，但各检测机构在定量方面未发现显著差异。在七个机构评估了hMSC在小鼠体内的BD（三个机构采用qPCR；四个机构采用ddPCR），所有机构均显示出相似的组织分布情况，在所检测的组织中，肺部的细胞分布最高。
采用Alu序列对qPCR和ddPCR进行了定量评估，结果表明该检测方法可用于BD评估。
