在中枢神经系统肿瘤中，颅内（IC）局部输送嵌合抗原受体（CAR）T细胞呈现出一种吸引人的输送方法。尽管IC输送在早期临床研究中得到了积极应用，但在IC给药前尚未发表去除神经毒性抗冻保护剂二甲基亚砜（DMSO）的解冻/洗涤方法。因此，本研究的目的是开发和验证一种简单的解冻/洗涤程序。我们开发了一种解冻/洗涤程序，包括：在37°C下解冻产品，用无防腐剂的生理盐水（PFNS）平衡5分钟，将额外8个容量的PFNS稀释，通过洗涤步骤去除DMSO，用2.0 mL PFNS重悬并在20-25°C下储存于注射器中。根据解冻完成后的3小时内的稳定性参数，评估最终制剂产品的质量、安全属性和稳定性。稳定性参数包括CAR T细胞存活率、转基因表面表达和细胞性活性。所开发的程序将计算出的DMSO%减少到小于0.025%。FP细胞存活率和恢复率（与低温保存前相比）在可接受的规范范围内（平均存活率：85.3%，范围：83%-88%；总核细胞恢复率平均值：76.5%，范围：65.4%-82.5%）。所有FP（n=3）也满足了预定的质量保证/质量控制参数，包括外观/完整性、无菌性和内毒素水平（≤ 1.0 EU/mL）。确认了解冻/洗涤后3小时的稳定性。与解冻未稀释/洗涤的对照CAR T细胞相比，所有产品的细胞存活率均保持在70%以上（平均80.0%，范围79%-81%），转基因表达或B7-H3-CAR T细胞的细胞性活性变化不显著。我们已经开发了一种简单的解冻/洗涤程序，用于准备B7-H3-CAR T细胞以进行局部区域输送到神经轴。虽然在这里我们关注CAR T细胞，但是这些方法很容易适应其他低温保存的免疫效应器细胞制品。 © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.

Intracranial (IC) locoregional delivery of chimeric antigen receptor (CAR) T cells presents an attractive delivery method to central nervous system tumors. Although IC delivery is actively being employed in early-phase clinical studies, no thaw/wash methods have been published to remove the neurotoxic cryoprotectant dimethyl sulfoxide (DMSO) from CAR T-cell products before IC administration. Thus, the aim of this study was to develop and validate a simple thaw/wash procedure.We developed a thaw/wash procedure that consist of product thaw at 37°C, equilibration for 5 min in 1 volume of preservative-free normal saline (PFNS), dilution with an additional 8 volumes of PFNS, removal of DMSO through a washing step, resuspension in 2.0 mL of PFNS and storage in a syringe at 20-25°C. Final formulated products (FPs) were assessed for quality and safety attributes and stability over 3 h from the completion of the thaw. Stability parameters included CAR T-cell viability, transgene surface expression and cytolytic activity.The developed procedure reduced the calculated % of DMSO to less than 0.025%. FP cell viability and recovery (versus pre-cryopreservation) were within acceptable specifications (mean viability: 85.3%, range: 83%-88%; total nucleated cell recovery mean: 76.5%, range: 65.4%-82.5%). Other prespecified quality assurance/quality control parameters including appearance/ integrity, sterility and endotoxin level (≤1.0 EU/mL), were also met by all FPs (n = 3). Three hours' post thaw/wash stability was confirmed. All products maintained cell viability greater than 70% (mean, 80.0%; range, 79%-81%), with no significant change in transgene expression or cytolytic activity of B7-H3-CAR T cells compared with thawed not diluted/washed control CAR T cells.We have developed a simple thaw/wash procedure to prepare B7-H3-CAR T cells for their locoregional delivery to the neural axis. While we focus here on CAR T cells, the methods could be readily adapted to other cryopreserved immune effector cell products.Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.