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Cryopreservation did not adversely affect the migratory potential of MSC overexpressing CD::UPRT::GFP. (A) Unmodified MSCs, freshly modified or thawed MSC overexpressing CD::UPRT::GFP (500,000 cells each group) were stained with anti-human CXCR4 antibody conjugated to a PE fluorophore (Cat. 306,506, Biolegend) at 1:10 dilution. Staining of cells were analyzed by flow cytometry. Percentage of expression was measured using FACS. The population of CXCR4 positive MSCs was gated relative to their isotype controls. (B) Four hundred thousand A549 or RPMI 2650 cells were seeded on the bottom chamber of a trans-well (Corning) plate in DMEM supplemented with 10% FBS. Twenty-four hours later, the media was replaced with serum free DMEM before the adding of 150,000 MSC overexpressing CD::UPRT::GFP (unmodified, freshly modified or cryopreserved modified) in the 8 µM matrigel coated cell inserts. The inserts were transferred into the wells containing the cancer cells. Twenty-four hours later, cells on the flip side of the inserts were stained with Hoechst 33,342 and imaged using a microscope. A total of three frames were imaged and counted. The graph represents mean ± SD of migratory cells per frame (n = 3). MSC overexpressing CD::UPRT::GFP without cancer cells (blank) served as negative control. Significant differences in cell count per frame between unmodified and modified MSCs were calculated using two-tailed Student’s t test. **P < 0.005
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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 suc...
Citations
... 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]. ...
Background
Mesenchymal stem cells (MSCs) have garnered significant interest for their tumor-tropic property, making them potential therapeutic delivery vehicles for cancer treatment. We have previously shown the significant anti-tumour activity in mice preclinical models and companion animals with naturally occurring cancers using non-virally engineered MSCs with a therapeutic transgene encoding cytosine deaminase and uracil phosphoribosyl transferase (CDUPRT) and green fluorescent protein (GFP). Clinical studies have shown improved response rate with combinatorial treatment of 5-fluorouracil and Interferon-beta (IFNb) in peritoneal carcinomatosis (PC). However, high systemic toxicities have limited the clinical use of such a regime.
Methods
In this study, we evaluated the feasibility of intraperitoneal administration of non-virally engineered MSCs to co-deliver CDUPRT/5-Flucytosine prodrug system and IFNb to potentially enhance the cGAS-STING signalling axis. Here, MSCs were engineered to express CDUPRT or CDUPRT-IFNb. Expression of CDUPRT and IFNb was confirmed by flow cytometry and ELISA, respectively. The anti-cancer efficacy of the engineered MSCs was evaluated in both in vitro and in vivo model. ES2, HT-29 and Colo-205 were cocultured with engineered MSCs at various ratio. The cell viability with or without 5-flucytosine was measured with MTS assay. To further compare the anti-cancer efficacy of the engineered MSCs, peritoneal carcinomatosis mouse model was established by intraperitoneal injection of luciferase expressing ES2 stable cells. The tumour burden was measured through bioluminescence tracking.
Results
Firstly, there was no changes in phenotypes of MSCs despite high expression of the transgene encoding CDUPRT and IFNb (CDUPRT-IFNb). Transwell migration assays and in-vivo tracking suggested the co-expression of multiple transgenes did not impact migratory capability of the MSCs. The superiority of CDUPRT-IFNb over CDUPRT expressing MSCs was demonstrated in ES2, HT-29 and Colo-205 in-vitro. Similar observations were observed in an intraperitoneal ES2 ovarian cancer xenograft model. The growth of tumor mass was inhibited by ~ 90% and 46% in the mice treated with MSCs expressing CDUPRT-IFNb or CDUPRT, respectively.
Conclusions
Taken together, these results established the effectiveness of MSCs co-expressing CDUPRT and IFNb in controlling and targeting PC growth. This study lay the foundation for the development of clinical trial using multigene-armed MSCs for PC.
... 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. ...
Fetal organs and organoids are important tools for studying organ development. Recently, porcine organs have garnered attention as potential organs for xenotransplantation because of their high degree of similarity to human organs. However, to meet the prompt demand for porcine fetal organs by patients and researchers, effective methods for producing, retrieving, and cryopreserving pig fetuses are indispensable. Therefore, in this study, to collect fetuses for kidney extraction, we employed cesarean sections to preserve the survival and fertility of the mother pig and a method for storing fetal kidneys by long-term cryopreservation. Subsequently, we evaluated the utility of these two methods. We confirmed that the kidneys of pig fetuses retrieved by cesarean section that were cryopreserved for an extended period could resume renal growth when grafted into mice and were capable of forming renal organoids. These results demonstrate the usefulness of long-term cryopreserved fetal pig organs and strongly suggest the effectiveness of our comprehensive system of pig fetus retrieval and fetal organ preservation, thereby highlighting its potential as an accelerator of xenotransplantation research and clinical innovation.
... 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)(63)(64)(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). ...
Mesenchymal stem/stromal cells (MSCs) are being increasingly used in cell-based therapies due to their broad anti-inflammatory and immunomodulatory properties. Intravascularly-administered MSCs do not efficiently migrate to sites of inflammation/immunopathology, but this shortfall has been overcome by cell surface enzymatic fucosylation to engender expression of the potent E-selectin ligand HCELL. In applications of cell-based therapies, cryopreservation enables stability in both storage and transport of the produced cells from the manufacturing facility to the point of care. However, it has been reported that cryopreservation and thawing dampens their immunomodulatory/anti-inflammatory activity even after a reactivation/reconditioning step. To address this issue, we employed a variety of methods to cryopreserve and thaw fucosylated human MSCs derived from either bone marrow or adipose tissue sources. We then evaluated their immunosuppressive properties, cell viability, morphology, proliferation kinetics, immunophenotype, senescence, and osteogenic and adipogenic differentiation. Our studies provide new insights into the immunobiology of cryopreserved and thawed MSCs and offer a readily applicable approach to optimize the use of fucosylated human allogeneic MSCs as immunomodulatory/anti-inflammatory therapeutics.
Adult mesenchymal stromal/stem cells (MSCs) are a standard component of de novo tissue generation to treat and study injury, disease, and degeneration. Canine patients constitute a major component of veterinary practice, and dogs share numerous pathologic conditions with humans. The relative abundance of adipose-derived stromal/stem cells (ASCs) in various canine adipose tissue depots is well described. Refined isolation, characterization, and differentiation techniques contribute to the collective knowledge of ASC phenotypes and subpopulations for specific tissue targets. Continued efforts to advance the knowledge of canine ASC behavior in vivo are critical to harnessing the full potential of primary cell isolates. This chapter contains a description of techniques to isolate, characterize, and differentiate canine ASCs.
Multipotent mesenchymal stromal/stem cell (MSC) spheroids generated in three-dimensional culture are of considerable interest as a novel therapeutic tool for regenerative medicine. However, the lack of reliable methods for storing MSC spheroids represents a significant roadblock to their successful use in the clinic. An ideal storage medium for MSC spheroids should function as both a vehicle for delivery and a cryoprotectant during storage of spheroids for use at a later time. In this study, we compared the outcomes after subjecting MSC spheroids to a freeze/thaw cycle in three Good Manufacturing Practices-grade cryopreservation media, CryoStor10 (CS10), Stem-Cellbanker (SCB), and Recovery Cell Culture Freezing Media (RFM) or conventional freezing medium (CM) (CM, Dulbecco's modified Eagle's medium containing 20% fetal bovine serum and 10% dimethyl sulfoxide) as a control for 2 months. The endpoints tested were viability, morphology, and expression of stem cell markers and other relevant genes. The results of LIVE/DEAD™ assays and annexin V/propidium iodide staining suggested that viability was relatively higher after one freeze/thaw cycle in CS10 or SCB than after freeze/thaw in CM or RFM. Furthermore, the relative "stemness" and expression of MSC markers were similar with or without freeze/thaw in CS10. Scanning electron microscopy also indicated that the surface morphology of MSC spheroids was well preserved after cryopreservation in CS10. Thus, even though it was tested for a short-term period, we suggest that CS10, which has been approved for clinical use by the U.S. Food and Drug Association, is a promising cryopreservation medium that would facilitate the development of MSC spheroids for future clinical use.
