冷冻保存不会改变过表达细胞质治疗转基因的同种异体间充质干细胞的性能和特性，用于癌症治疗。

BACKGROUND: Mesenchymal stem cells (MSCs) driven gene directed enzyme prodrug therapy is a promising approach to deliver therapeutic agents to target heterogenous solid tumours. To democratize such a therapy, cryopreservation along with cold chain transportation is an essential part of the logistical process and supply chain. Previously, we have successfully engineered MSCs by a non-viral DNA transfection approach for prolonged and exceptionally high expression of the fused transgene cytosine deaminase, uracil phosphoribosyl transferase and green fluorescent protein (CD::UPRT::GFP). The aim of this study was to determine the effects of cryopreservation of MSCs engineered to highly overexpress this cytoplasmic therapeutic transgene. METHODS: Modified MSCs were preserved in a commercially available, GMP-grade cryopreservative-CryoStor10 (CS10) for up to 11 months. Performance of frozen-modified MSCs was compared to freshly modified equivalents in vitro. Cancer killing potency was evaluated using four different cancer cell lines. Migratory potential was assessed using matrigel invasion assay and flow cytometric analysis for CXCR4 expression. Frozen-modified MSC was used to treat canine patients via intra-tumoral injections, or by intravenous infusion followed by a daily dose of 5-flucytosine (5FC). RESULTS: We found that cryopreservation did not affect the transgene expression, cell viability, adhesion, phenotypic profile, and migration of gene modified canine adipose tissue derived MSCs. In the presence of 5FC, the thawed and freshly modified MSCs showed comparable cytotoxicity towards one canine and three human cancer cell lines in vitro. These cryopreserved cells were stored for about a year and then used to treat no-option-left canine patients with two different types of cancers and notably, the patients showed progression-free interval of more than 20 months, evidence of the effectiveness in treating spontaneously occurring cancers. CONCLUSION: This study supports the use of cryopreserved, off-the-shelf transiently transfected MSCs for cancer treatment.

背景：间充质干细胞（MSCs）驱动的基因定向酶前体药物疗法是将治疗剂递送到靶向异质实体瘤的有前途的方法。为了使这种治疗方法民主化，冷冻保存以及冷链运输是物流过程和供应链的重要组成部分。先前，我们已经通过非病毒 DNA 转染方法成功地对 MSCs 进行了工程改造，以实现融合转基因胞嘧啶脱氨酶、尿嘧啶磷酸核糖基转移酶和绿色荧光蛋白（CD::UPRT::GFP）的延长和异常高表达。本研究的目的是确定冷冻保存高度过表达这种细胞质治疗性转基因的 MSC 的效果。

方法：使用商业上可获得的 GMP 级冷冻保存剂-CryoStor10（CS10）将修饰后的 MSC 保存长达 11 个月。将冷冻保存的修饰后的 MSC 的性能与新鲜修饰的等效物进行了比较。使用四种不同的癌细胞系评估了癌症杀伤效力。使用基质胶侵袭测定和 CXCR4 表达的流式细胞术分析评估了迁移潜能。通过肿瘤内注射或静脉内输注后每天给予 5-氟胞嘧啶（5FC）将冷冻修饰的 MSC 用于治疗犬患者。

结果：我们发现冷冻保存不会影响转基因表达、细胞活力、粘附、表型特征和基因修饰犬脂肪组织来源的 MSC 的迁移。在 5FC 的存在下，解冻和新鲜修饰的 MSC 在体外对一种犬和三种人癌细胞系显示出相当的细胞毒性。这些冷冻保存的细胞储存了大约一年，然后用于治疗两种不同类型的癌症的无选择余地的犬患者，并且患者表现出超过 20 个月的无进展间隔，证明了治疗自发发生的癌症的有效性。

结论：这项研究支持使用冷冻保存的即用型瞬时转染 MSC 进行癌症治疗。