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Abstract Understanding mesenchymal stromal cells (MSCs) growth mechanisms in response to surface chemistries is essential to optimize culture methods for high‐quality and robust cell yields in cell manufacturing applications. Heparin (HEP) and collagen 1 (COL) substrates have been reported to enhance cell adhesion, growth, viability, and secretory potential in MSCs. However, the biomolecular mechanisms underlying the benefits of combined HEP/COL substrates are unknown. This work used HEP/COL bilayered surfaces to investigate the role of integrin‐HEP interactions in the advantages of MSC culture. The layer‐by‐layer approach (LbL) was used to create HEP/COL bilayers, which were made up of stacks of 8 and 9 layers that combined HEP and COL in an alternate arrangement. Surface spectroscopic investigations and laser scanning microscopy evaluations verified the biochemical fingerprint of each component and a total stacked bilayer thickness of roughly 150 nm. Cell growth and apoptosis in response to IC₅₀and IC₇₅levels of BTT‐3033 and Cilengitide, α2β1 and αvβ3 integrin inhibitors respectively, were evaluated on HEP/COL coated surfaces using two bone marrow‐derived MSC donors. While integrin activity did not affect cell growth rates, it significantly affected cell adhesion and apoptosis on HEP/COL surfaces. HEP‐ending HEP/COL surfaces significantly increased FAK‐ERK½ phosphorylation and endogenous cell COL deposition compared to COL, COL‐ending HEP/COL and uncoated surfaces. BTT‐3033 but not Cilengitide treatment markedly affected FAK‐ERK½ activity levels on HEP‐ending HEP/COL surfaces supporting a major role for α2β1 activity. BTT‐3033 treatment on HEP‐ending bilayers reduced MSC‐mediated macrophage inhibitory activity and altered the cytokine profile of co‐cultures. Overall, this study supports a novel role for HEP in regulating the survival and potency of MSCs via enhancing the α2β1‐FAK‐ERK½ signaling mechanism. 

Abstract BackgroundMesenchymal 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. MethodsThe 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 toolenrichGOfrom theclusterprofiler.One-way and two-way ANOVAs were carried out to identify significant changes between the conditions. Results were deemed statistically significant for combinedP < 0.05 for at least three independent experiments. ResultsCell 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. ConclusionsThis 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.