Comparative Characterization of Placental Mesenchymal Stromal Cell-Derived Extracellular Vesicles From 3D Hollow Fiber Bioreactor and 2D Tissue Flask Culture

Introduction: : Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are emerging as promising acellular therapeutics due to their distinct immunomodulatory and regenerative potential. Placenta-derived MSCs (PMSCs) offer a non-invasive, ethically accessible, and potent source for clinical-scale EV production. However, scalable, reproducible, and GMP-compatible manufacturing platforms remain a key bottleneck to clinical translation. Both MSCs and MSC-EVs have been investigated in clinical trials for their therapeutic potential across various disease areas including joint conditions and neurological diseases. However, many of these trials fail to achieve safety or efficacy due to poorly characterized and poorly understand mechanisms of action in a disease-relevant context. A major cause of this disconnect between early stage research promise and lackluster clinical trial results lies in MSC culture conditions for EV production. Clinical trial study designs often incorporate bioreactor cultures to ensure efficient EV production without characterizing the differences between 2D and 3D culture systems during translational scale-up. To address this gap in knowledge, we performed a head-to-head comparison of EVs produced by PMSCs in 2D flask culture and 3D bioreactor culture. By doing so, the physicochemical and functional differences of both PMSCs and PMSC-derived EVs were comprehensively characterized in the context of translational scale-up for clinical trial testing.

Materials and

Methods: : Human, Good Manufacturing Practice (GMP) grade PMSCs were seeded into either 2D tissue culture-treated flasks or a 3D hollow fiber bioreactor (FiberCell Duet; FiberCell Systems). Four PMSC donor lines were tested to account for biological variation. PMSCs were cultured for 7 days in 2D conditions and 30 days in 3D conditions, and PMSC-derived EVs were isolated from conditioned media by tangential flow filtration. PMSC characterization included trilineage differentiation, karyotyping, MSC phenotyping by flow cytometry and potency testing via an established neuroprotection assay. EVs isolated from both culture conditions were physicochemically characterized by nanoparticle tracking analysis for size and concentration, cryogenic transmission electron microscopy for morphology, and super resolution microscopy for EV marker validation. Additionally, EV composition was assessed via quantitative proteomic analysis to observe differences arising from culture conditions. Lastly, EV therapeutic potency was evaluated in an established neuroprotection assay and lymphocyte proliferation assay.

Results, Conclusions, and Discussions:: PMSCs exhibited comparable growth dynamics across the 30-day culture period in the hollow fiber bioreactor, with similar glucose consumption rates, lactate levels in growth media, and doubling times. PMSCs grown in the bioreactor maintained typical MSC phenotypic markers (positive for CD90, CD105, CD73; negative for CD34, CD45), trilineage differentiation potential, and were exhibited a normal karyotype. PMSCs also maintained their neuroprotective capacity with >1.2 fold change compared to the untreated control. PMSC-derived EVs exhibited a consistent and normal size, morphology, and tetraspanin expression from both 2D and 3D cultures. 3D-cultured EVs were observed to have decreased heterogeneity in surface protein expression. 3D-cultured EVs were isolated at ~40x yield compared to donor-matched 2D-cultured EVs per passage. Following proteomic analysis, 3D-cultured EVs isolated from each week throughout the 30-day continuous culture possessed distinct protein profiles compared to one another and the 2D-cultured EVs. Several pathways including ganglioside catabolism and neuron cell homeostasis were significantly enriched in 3D-cultured EVs. 3D-cultured EVs also exhibited increased neuroprotective potential compared to 2D-cultured EVs.

These results demonstrate through comprehensive characterization maintained EV size and morphology across culture conditions, altered protein expression profiles per EV and between entire EV samples, and increased therapeutic potential in 3D-cultured EVs. 3D-cultured EVs were thereby successfully produced in a high quality, high throughput, and scalable manner using the hollow fiber bioreactor and tangential flow filtration systems without apparent deficiencies in composition or functionality. However, proteomics results revealed large differences in EV proteomes across culture conditions and timepoints, suggesting underlying differences EV functionality that require additional studies to elucidate. Altogether, the potential mechanistic differences and functional differences were reported in this study, establishing a foundational knowledge base for future translational applications.

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