Multicentre comparison of biological and functional properties of mesenchymal stromal cells from different sources cultivated using a harmonised manufacturing workflow

Background Mesenchymal stromal cells (MSCs), commonly sourced from adipose tissue, bone marrow and umbilical cord, have been widely used in many medical conditions due to their therapeutic potential. Yet, the still limited understanding of the underlying mechanisms of action hampers clinical translation. Clinical potency can vary considerably depending on tissue source, donor attributes, but importantly, also culture conditions. Lack of standard procedures hinders inter-study comparability and delays the progression of the field. The aim of this study was A-to assess the impact on MSC characteristics when different laboratories performed analysis on the same MSC material using harmonised culture conditions and B-to understand source-specific differences. Methods Three independent institutions performed a head-to-head comparison of human-derived adipose (A-), bone marrow (BM-), and umbilical cord (UC-) MSCs using harmonised culture conditions. In each centre, cells from one specific tissue source were isolated and later distributed across the network to assess their biological properties, including cell expansion, immune phenotype, and tri-lineage differentiation (part A). To assess tissue specific function, angiogenic and immunomodulatory properties and the in vivo biodistribution were compared in one expert lab (part B). Results By implementing a harmonised manufacturing workflow, we obtained largely reproducible results across three independent laboratories in part A of our study. Unique growth patterns and differentiation potential were observed for each tissue source, with similar trends observed between centres. Immune phenotyping verified expression of typical MSC surface markers and absence of contaminating surface markers. Depending on the established protocols in the different laboratories, quantitative data varied slightly. Functional experiments in part B concluded that conditioned media from BM-MSCs significantly enhanced tubulogenesis and endothelial migration in vitro . In contrast, immunomodulatory studies reported superior immunosuppressive abilities for A-MSCs. Biodistribution studies in healthy mice showed lung entrapment after administration of all three types of MSCs, with a significantly faster clearance of BM-MSCs. Conclusion These results show the heterogeneous behaviour and regenerative properties of MSCs as a reflection of intrinsic tissue-origin properties while providing evidence that the use of standardised culture procedures can reduce but not eliminate inter-lab and operator differences. Highlights In this study, we have: ### Competing Interest Statement The authors have declared no competing interest. * A : Adipose BLI : Bioluminescence Imaging BM : Bone Marrow CM : conditioned Media CPD : Cumulative Population Doublings DEAE-dextran : Diethylaminoethyl-Dextran DMSO : Dimethyl Sulfoxide DPBS : Dulbecco’s phosphate-buffered saline EGM : Endothelial Growth Medium EU : European Union FACSs : Fluorescence Activated Cell Sorting FBS : Foetal Bovine Serum HEK : Human Embryonic Kidney cells HLA-DR : Human Leukocyte Antigen – DR Isotype HUVEC : Human Umbilical Cord Endothelial Vein Cells IDO : Indoleamine 2,3-dioxagenase IFN- : γ Interferon gamma IGFBP : Insulin-like Growth Factor Binding Protein IL : Interleukin ISCT : International Society for Cell and Gene Therapy ITN : Innovative Training Network IV : Intravenously LV : Lentiviral Vector MCP-1 : Monocyte Chemoattractant Protein 1 MEM- : α Minimum Essential Medium Alpha MFI : Mean Fluorescence Intensity MHC : Major Histocompatibility Complex MOI : Multiplicity of Infection MSC : Mesenchymal Stromal Cell PBMC : Peripheral Blood Mononuclear Cells PD : Population Doublings PDT : Population Doubling Time PHA : Phytohemagglutinin-L ROI : Region of Interest RPMI : Roswell Park Memorial Institute Medium SC : Subcutaneous SD : Standard Deviation UC : Umbilical Cord VEGF : Vascular Endothelial Growth Factor