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

Abstract

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.

Optimizing cryopreservation conditions for use of fucosylated human mesenchymal stromal cells in anti-inflammatory/immunomodulatory therapeutics

“…Trilineage differentiation ability of MSCs (i.e., adipogenic, osteogenic, and chondrogenic) is another criterion of the International Society for Cellular Therapy (ISCT) guide for defining MSCs ( 61 ). In agreement with our results using FucMSCs, previous work evaluated the effects of cryopreservation on the differentiation potential of MSCs after thawing, concluding that, although some results are controversial, cryopreservation does not alter MSC osteogenic, adipogenic or chondrogenic differentiation potential (data not shown) ( 56 , 62 – 65 ). In addition, previous studies from our group showed that fucosylated murine and human MSCs are able to efficiently differentiate into adipocyte, osteoblast and chondroblast cell lineages ( 20 , 21 ).…”

Fetal Kidney Grafts and Organoids from Microminiature Pigs: Establishing a Protocol for Production and Long-Term Cryopreservation

“…In this study, we confirmed that porcine MNs can be preserved for 2 years using proper storage procedures. Cell cryopreservation studies have shown that canine and human adipose tissues, platelets, pancreatic cells [8][9][10][11][12], and even small organs, such as rat kidneys, can be cryopreserved [13]. Although intracellular ice crystal formation presents a challenge in the cryopreservation process, it can be averted by adequate immersion in a suitable freezing solution and replacement of water content.…”

Enhanced anti-tumor efficacy with multi-transgene armed mesenchymal stem cells for treating peritoneal carcinomatosis

“…We developed a highly efficient cationic polymer-based transfection method to engineer MSCs to express a therapeutic transgene—cytosine deaminase uracil phosphoribosyl-transferase fused to a green fluorescent protein (GFP) reporter. These engineered cells showed strong anti-cancer potency in in-vitro, in subcutaneous laboratory models [ 20 , 26 ] and in companion animals with naturally occurring cancers [ 27 ]. The non-toxic prodrug (5-flucytosine, 5FC) is converted by CD into 5-flurouracil (5FU) that disrupts the nucleotide biosynthesis, leading to apoptosis [ 28 , 29 ].…”