Collagen-heparan sulfate microcarriers support mesenchymal stem cell culture in microgravity and streamline post-flight recovery.

Mesenchymal stem cells (MSCs) hold broad therapeutic potential, yet the absence of validated, scalable adherent culture methods remains a barrier to orbital biomanufacturing. Here, we report the first integrated workflow for MSC culture on pre-seeded microcarriers (µCs) encompassing cryopreservation, ambient thaw, and expansion aboard the International Space Station. We engineered heparan sulfate-collagen (HS/COL) µCs with defined sulfation levels to modulate integrin engagement and factor-release kinetics and benchmarked them against commercial µCs and suspension controls. Our results demonstrate that pre-seeded MSCs remain viable and adherent throughout the spaceflight lifecycle, with ambient workflows enabling parallel evaluation of multiple donors and surface chemistries while minimizing crew intervention. In microgravity, MSC proliferation peaked 3 days earlier than on Earth, with highest cell yields being obtained with low-sulfated HS/COL µCs. Secretome analyses revealed increased pro-collagen and extracellular vesicle release with preserved bioactive microRNA cargo. While HS/COL surfaces buffered microRNA homeostasis, suspension and commercial µC cultures exhibited orbit-specific miRNA signatures associated with cytoskeletal remodeling and immune regulation. Post-flight, MSCs recovered from µCs retained proliferative capacity and multipotency, though microgravity exposure favored adipogenic over chondrogenic differentiation. Collectively, these results establish a scalable culture strategy using pre-seeded µCs for stem cell research and biomanufacturing in space.