Effect of cryopreservation of mesenchymal stromal cells after non-viral transfection on cell viability and transgene expression

Salkhordeh, M.; Tan, Yuan; Altmieme, Z.; Florian, Maria; Wang, Y.; Stewart, Duncan J.; Mei, Shirley H. J.

doi:10.1016/j.jcyt.2020.03.179

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“…To date, the effect of cryopreservation on transient, highly over-expressed gene modified MSCs has yet to be clearly defined. A recent study reported that cell viability and transgene expression of the thawed cells are transfection protocol dependent [23]. Thus, it is crucial to assess the impact of cryopreservation in accordance with the target product profile.…”

Section: Introductionmentioning

confidence: 99%

Cryopreservation does not change the performance and characteristics of allogenic mesenchymal stem cells highly over-expressing a cytoplasmic therapeutic transgene for cancer treatment

Loke

Woo

et al. 2022

Stem Cell Res Ther

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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.

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Section: Introductionmentioning

confidence: 99%