Multiplex Functional Testing of Bioreactor-Upscaled First Trimester Human Umbilical Cord Perivascular Cells (FTM HUCPVC) and Bone Marrow-Derived Mesencymal Stem Cells (BMSC) using the chemstress® fingerprinting assay, reveals hidden differences between cell therapy candidates

Abstract

Background & Aim FTM HUCPVC are a rich source of young MSC, with angiogenic and immunomodulatory properties superseding older MSC. We developed the xeno-free, bioreactor-based protocol to produce FTM HUCPVC for clinical translation. The cell therapy field requires reliable, robust, functional benchmark tests for cell therapy candidates, as often only viable yield and immunophenotype are production success criteria. Methods, Results & Conclusion FTM HUCPVC, optimized and upscaled in a xeno-free (HPL) bioreactor system (Terumo) were compared to clinically applicable BMSC (RoosterBio), using the ChemStress® assay from Valitacell. ChemStress® is an information-rich functional profiling tool based on the cell response to an array of bioprocess-relevant chemical stressors that generates a profile of cell health, stability and phenotype. Chemstress® assay targets glucose metabolism, ER and Golgi function, programmed cell death, kinase activity and ROS production. We hypothesized that Chemstress® fingerprinting can reveal key differences between cell therapy products, as a benchmark test. FTM HUCPVC showed higher resistance to chemical stressors targeting glucose metabolism and oxidative energy production: CoCl2 (hypoxia), 2dg (glucose deprivation), NaOthV (ATP depletion) compared to BSMC. Chemical stressors targeting the secretory compartment: BrefA, BSO, had a more severe impact on viability and proliferation of BMSC compared to FTM HUCPVC. When comparing FTM HUCPVC and BMSC to their further expanded counterparts (4-5 additional PD), BMSC showed an altered stress response in the ChemStress® assay with increased PD. No such difference was measured in FTM HUCPVC, suggesting a less PD-dependent proliferative potential in a stressor environment. Angiogenic factor array on the media of FTM HUCPVC and BMSC from chemical stressor wells corresponding to ischemic conditions: 2dG, CoCl2 and oxidative MS showed, that FTM HUCPVC increased angiogenic factor secretion (Angp2, DPPIV, ET-1) in the ischemia-like environment. No such effect was observed in BMSC cultures. Fluorescence microscopy revealed evident differences in cell density and morphology between stressor culture groups in both FTM HUCPVC and BSMC ChemStress® cultures. These observations matched the viability/proliferation fingerprints. Our results suggest that the Chemstress® assay can be applicable as a a multiplex, quantitative, quality control (QC) measure for cell therapy candidates, both for bioreactor protocol optimization and cell type comparison.