Cryopreserved spontaneous spheroids from compact bone-derived MSCs for bone tissue engineering.

Spontaneously formed spheroids from mouse compact bone-derived cells (CBDCs) possess enhanced stemness and superior plasticity. Here, the effect of cryopreservation on viability, stemness, and osteogenic differentiation capability of spontaneous CBDCs spheroids were investigated. CBDCs 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 were measured. The expressions of stem cell markers and osteogenic marker genes were analyzed. The cryopreserved and non-cryopreserved spheroids were transplanted in mice with a β-TCP 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 CBDCs spheroids showed remarkable new bone formation in vivo, identical to that of the non-cryopreserved spheroids even without osteogenic induction. The cryopreserved spontaneous CBDCs spheroids retained stemness and osteogenic differentiation capability and highlight the utility of spontaneous CBDCs spheroids as ready-to-use tissue-engineered products for bone tissue engineering.