Human mesenchymal stromal cells (hMSCs) are a promising source for regenerative cell therapy. However, hMSC clinical use has been stymied by product variability across hMSC donors and manufacturing practices resulting in inconsistent clinical outcomes. The inability to predict hMSC clinical efficacy, or potency, is a major limitation for market penetration. Standard metrics of hMSC potency employ hMSCs and third‐party immune cell co‐cultures, however, these assays face translational challenges due to third‐party donor variability and lack of scalability. While surrogate markers of hMSC potency have been suggested, none have yet had translational success. To address this, a high‐throughput, scalable, low‐cost, on‐chip microfluidic potency assay is presented with improved functional predictive power and recapitulation of in vivo secretory responses compared to traditional approaches. Comparison of hMSC secretory responses to functional hMSC‐medicated immune cell suppression demonstrates shortcomings of current surrogate potency markers and identifies on‐chip microfluidic potency markers with improved functional predictive power compared to traditional planar methods. Furthermore, hMSC secretory performance achieved in the on‐chip microfluidic system has improved similarity compared to an in vivo model. The results underscore the shortcomings of current culture practices and present a novel system with improved functional predictive power and hMSC physiological responses.
Mesenchymal stem cells (MSCs) secrete a diversity of factors with broad therapeutic potential, yet current culture methods limit potency outcomes. In this study, we used topographical cues on polystyrene films to investigate their impact on the secretory profile and potency of bone marrow-derived MSCs (hBM-MSCs). hBM-MSCs from four donors were cultured on topographic substrates depicting defined roughness, curvature, grooves and various levels of wettability.
The topographical PS-based array was developed using razor printing, polishing and plasma treatment methods. hBM-MSCs from four donors were purchased from RoosterBio and used in co-culture with peripheral blood mononuclear cells (PBMCs) from Cell Applications Inc. in an immunopotency assay to measure immunosuppressive capacity. Cells were cultured on low serum (2%) for 24–48 h prior to analysis. Image-based analysis was used for cell quantification and morphology assessment. Metabolic activity of BM-hMSCs was measured as the mitochondrial oxygen consumption rate using an extracellular flux analyzer. Conditioned media samples of BM-hMSCs were used to quantify secreted factors, and the data were analyzed using R statistics. Enriched bioprocesses were identify using the Gene Ontology tool
Cell viability was not significantly affected in the topographical substrates, and cell elongation was enhanced at least twofold in microgrooves and surfaces with a low contact angle. Increased cell elongation correlated with a metabolic shift from oxidative phosphorylation to a glycolytic state which is indicative of a high-energy state. Differential protein expression and gene ontology analyses identified bioprocesses enriched across donors associated with immune modulation and tissue regeneration. The growth of peripheral blood mononuclear cells (PBMCs) was suppressed in hBM-MSCs co-cultures, confirming enhanced immunosuppressive potency. YAP/TAZ levels were found to be reduced on these topographies confirming a mechanosensing effect on cells and suggesting a potential role in the immunomodulatory function of hMSCs.
This work demonstrates the potential of topographical cues as a culture strategy to improve the secretory capacity and enrich for an immunomodulatory phenotype in hBM-MSCs.
- NSF-PAR ID:
- 10443868
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Stem Cell Research & Therapy
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 1757-6512
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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