Methods: Murine MSC were characterized by tri-lineage differentiation inducement and flow cytometry analysis. The immune properties of allogeneic MSC at serial passage numbers were tested by co-culturing with spleen mononuclear cells in vitro and utilizing a delayed-type hypersensitivity model in vivo.
Results: The surface molecule profile and successful tri-lineage differentiation verified the identity of murine MSC. By in vitro co-culture testing, the intensity of immunogenicity gradually increased and that of immuno-inhibitory effect gradually decreased with serial passage after P8. Consistent results were observed on the footpad swelling upon the stimulation of allogeneic MSC. Further study found that the immuno-inhibition of MSC at a late passage number was dominated by soluble molecules and was impaired by cell-cell contact once the immunogenicity was obtained. Transwell separation and chamber constraint of MSC would rescue the immuno-inhibitory effect. An extensive in vivo study demonstrated a complex mechanism was involved in these immune activities of MSC.
Conclusion: MSC will lose the immune advantages after prolonged ex-vivo culture, but keep the immuno-inhibitory property for a long period of time in a secretion manner. Therefore, to get better integration, the passage numbers must be considered when allogeneic MSC are used for tissue regeneration. And the separation of allogeneic MSC will help to achieve a longer and better immuno-inhibitory effect for immunological therapy.