Enhancing the Scalable Manufacturing of Human Mesenchymal Stromal Cells in Dynamic Culture Using Bioactive Heparin/Collagen Layer-by-Layer Coated Microcarriers.

Expansion of human mesenchymal stromal cells (hMSCs) remains a bottleneck during manufacturing, where high cell yields must be achieved without compromising quality. Microcarriers provide elevated surface areas but often rely on synthetic materials. Here we use heparin/collagen Layer-by-Layer (LbL) coatings on microcarriers to enhance dynamic hMSC culture. HMSCs were cultured on LbL-coated and untreated microcarriers in up to 500 mL flasks, and were assessed over time, up to 18 days across three seeding densities (2600-20,000 cells/cm2), while yield was assessed at the end of culture. Metabolic analysis suggests no induction of a generalized stress response, increased glycolytic activity, and a shift toward a more active secretory state for cells grown on coated surfaces, as measured by noninvasive assays (Luminex multiplex and Raman spectroscopy). At medium and high densities, the cost (18.6%) and yield (60% and 30%, respectively) increases (coated: untreated) were moderately favorable. However, at the lower density, the cost increase remained similar, despite a yield increase of 110%. Together, the findings of this study demonstrate that our LbL surface coatings enhance hMSC functional performance and scalability of microcarrier-based culture, especially at lower initial cell densities, representing a promising approach for scalable cell manufacturing with the potential for GMP adaptation.