Cryopreserved Spontaneous Spheroids from Compact Bone-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

Spontaneously formed spheroids from mouse compact bone-derived mesenchymal stromal cells (CB-MSCs) possess enhanced stemness and superior plasticity. In this study, the effect of cryopreservation on viability, stemness, and osteogenic differentiation capability of spontaneous CB-MSC spheroids were investigated. CB-MSCs were isolated from mouse femur and tibia. Spheroids were cryopreserved with various concentrations of dimethyl sulfoxide (DMSO). After thawing, the number of living and dead cells was measured. The expression levels of stem cell markers and osteogenic marker genes were analyzed. The cryopreserved and noncryopreserved spheroids were transplanted in mice with a beta-tricalcium phosphate as a scaffold to evaluate the in vivo bone-forming capability. The percentage of living cells was highest when 5% DMSO was used as a cryoprotectant, confirmed by the number of dead cells. The expression of stem cell marker genes and osteogenic differentiation capability were maintained after cryopreservation with 5% DMSO. The cryopreserved spontaneous CB-MSC spheroids showed remarkable new bone formation in vivo, identical to that of the noncryopreserved spheroids even without osteogenic induction. The cryopreserved spontaneous CB-MSC spheroids retained stemness and osteogenic differentiation capability and highlight the utility of spontaneous CB-MSC spheroids as ready-to-use tissue-engineered products for bone tissue engineering.