Macromolecular crowding in animal component-free, xeno-free and foetal bovine serum media for human bone marrow mesenchymal stromal cell expansion and differentiation

Abstract

Background: Cell culture media containing undefined animal-derived components and prolonged in vitro culture periods in the absence of native extracellular matrix result in phenotypic drift of human bone marrow stromal cells (hBMSCs).Methods: Herein, we assessed whether animal component-free (ACF) or xeno-free (XF) media formulations maintain hBMSC phenotypic characteristics more effectively than foetal bovine serum (FBS)-based media. In addition, we assessed whether tissue-specific extracellular matrix, induced via macromolecular crowding (MMC) during expansion and/or differentiation, can more tightly control hBMSC fate.Results: Cells expanded in animal component-free media showed overall the highest phenotype maintenance, as judged by cluster of differentiation expression analysis. Contrary to FBS media, ACF and XF media increased cellularity over time in culture, as measured by total DNA concentration. While MMC with Ficoll™ increased collagen deposition of cells in FBS media, FBS media induced significantly lower collagen synthesis and/or deposition than the ACF and XF media. Cells expanded in FBS media showed higher adipogenic differentiation than ACF and XF media, which was augmented by MMC with Ficoll™ during expansion. Similarly, Ficoll™ crowding also increased chondrogenic differentiation. Of note, donor-to-donor variability was observed for collagen type I deposition and trilineage differentiation capacity of hBMSCs.Conclusion: Collectively, our data indicate that appropriate screening of donors, media and supplements, in this case MMC agent, should be conducted for the development of clinically relevant hBMSC medicines.

Gums as Macromolecular Crowding Agents in Human Skin Fibroblast Cultures

“…Despite the notable advances in MMC technology in cell culture context, there is no widely accepted MMC agent, and there are remarkable differences in the effectiveness of those that have been used to date. For example, although polysucrose [24][25][26][27], hyaluronic acid [28], dextran sulphate [29][30][31][32], polyvinylpyrrolidone [33], polysodium-4-styrene sulfonate [34], and cocktails thereof (e.g., dextran sulphate and polysucrose [35]) have shown potential as MMC agents, none has come close to the efficacy and efficiency of carrageenan (CR), largely attributed to its negative charge and polydispersity [36,37]. Considering though the rather questionable direct or indirect association of CR with colitis [38][39][40] that may restrict its use in biomedicine, despite its regulatory history, it is imperative to identify alternative MMC agents with a clear regulatory clearance pathway.…”