An Improved Culture Protocol of Murine Mesenchymal Stromal Cells From Compact Bone

Mesenchymal stromal/stem cells (MSCs) are promising candidates for cell therapy and regenerative medicine. However, murine bone marrow-derived MSCs (mBMSCs) are much more difficult to isolate and purify because of their high heterogeneity and very limited numbers within bone morrow. To this end, this protocol was developed to establish an appropriate method for isolation and expansion of mBMSCs for preclinical research. Cells were dissociated and isolated mainly from compact bones via an enzyme digestion method to dissociate sufficient cell pools for further expansion ex vivo. Adherent cells collected were spindle-shaped with colony-forming units, and expressed a series of surface markers, including Oct4, CD29, and Sca-1 (stem cell antigen-1). Additionally, mBMSCs possessed potent capacity to differentiate into osteoblasts, adipocytes, and chondrocytes under appropriate culture conditions. Therefore, this study provides a standardized and reliable method for quick expansion of murine BMSCs. © 2026 Wiley Periodicals LLC. Basic Protocol: Enzymatic digestion method.