Heparan Sulfate-Collagen Surface Multilayers Support Serum-Free Microcarrier Culture of Mesenchymal Stem Cells

The increasing cost of high-volume cultures and dependence on serum and growth factor supplementation limit the affordability of mesenchymal stromal cell (MSC) therapies. This has spurred interest in developing strategies that support adherent cell expansion while reducing raw material costs. Culture surfaces coated with sulfated glycosaminoglycans (GAGs), specifically heparan sulfate (HS), are an alternative to prolong growth factor retention in cell cultures. Unlike heparin, recombinant HS (rHS) offers strong binding affinity for multiple growth factors and extracellular matrix components, such as collagen I, without undesirable anticoagulant effects or xenobiotic health risks. The potential of rHS as a factor reservoir in MSC cultures remains underexplored. This study investigated the impact of rHS on the growth and anti-inflammatory properties of undifferentiated bone marrow MSCs in both planar and microcarrier-based cultures. It was hypothesized that rHS would enable MSC growth with minimal growth factor supplementation in a sulfation level-dependent manner. Cell culture surfaces were assembled via the layer-by-layer (LbL) method, combining alternating collagen I (COL) and rHS. These bilayers support cell adhesion and enable the incorporation of distinct sulfation levels on the culture surface. Examination of pro-mitogenic FGF and immunostimulatory IFN-γ release dynamics confirmed prolonged availability and sulfate level dependencies. Sulfated surfaces supported cell growth in low serum (2% FBS) and serum-free (SF) media at levels equivalent to standard culture conditions. Cell growth on rHS-coated surfaces in SF was comparable to that on heparin-coated surfaces and commercial surface-coated microcarriers in low serum. These growth benefits were observed in both planar and microcarrier (μCs) cultures. Additionally, rHS surfaces reduced β-galactosidase expression relative to uncoated surfaces, delaying cell senescence. Multivariate analysis of cytokines in conditioned media indicated that rHS-containing surfaces enhanced cytokine levels relative to uncoated surfaces during IFN-γ stimulation and correlated with decreased pro-inflammatory macrophage activity. Overall, utilizing highly sulfated rHS with COL reduces the need for exogenous growth factors and effectively supports MSC growth and anti-inflammatory potency on planar and microcarrier surfaces under minimal factor supplementation.