University of Veterinary Medicine Vienna No simple way to averaging out: Pooled mesenchymal stromal cells do not reflect average donor characteristics Mesenchymal stromal cells (MSCs) are promising candidates for numerous regenerative therapies. Still, clinical translation is complicated by the heterogeneity of MSCs and related shortcomings in preclinical research. Pooling MSCs from multiple donors is increasingly being advocated as an effective way to mitigate donor variability. However, it remains unclear whether the range of individual cell characteristics is equally reflected in pooled cultures, or if pooling rather leads to a homogenized cell population dominated by the fittest donor, which would lead to skewed results. This study investigates whether MSC pools are functionally representative for their respective donor MSCs and whether dominant donors emerge over time. MSCs from nine human donors were categorized into low-, middle-, and high-fitness groups. Individual MSCs were then pooled according to their fitness groups, complemented by a mixed-fitness pool. Functional assays for proliferation, metabolic activity, differentiation, migration and senescence were performed to evaluate the pools versus the individual MSCs. Donor representation within pools was tracked using fluorescence microscopy and qPCR. The high-fitness pool, as well as its individual donor MSCs, displayed the most rapid proliferation and highest metabolic activity. However, while for proliferation, the pool data aligned well with the individual donor data, all other assays revealed discrepancies between the pooled cultures and individual donor cells. Interestingly, particularly the mixed fitness pool showed inferior metabolic activity and differentiation potential in comparison with the respective individual donor MSCs. Cell tracking showed that over one passage, even pools composed of donors with similar cell fitness became dominated by the donor with the highest cellular fitness. The discrepancy between pooled and individual donor data emphasizes the importance of biological replicates to capture donor variation and ensure that MSC research reflects natural diversity. en TEXT journal article Wissenschaftlicher Artikel Articolo scientifico Documento PDF Articolo scientifico Text Wissenschaftlicher Artikel Text journal article Mesenchymal Stromal Cells Human MSCs Adipose-derived Donor Variability Heterogeneity Cellular Fitness Cell Pooling Functional Characterization Cell Tracking 2025 2025 Dea Kukaj Sabine Niebert Christoph Biehl Ursula Reichart Christiane Schueler Janina Burk Elsevier