Deep-supercooling preservation of stem cell spheroids for chondral defect repairment

Versatile mesenchymal stem cells (MSCs) play an important role in tissue engineering and regenerative medicine. MSCs in 3D spheroid have shown higher secretion and differentiation functions than suspended counterparts, and, thus, in vitro cryopreservation of MSC spheroids is an indispensable technology to bridge the spatiotemporal gaps between spheroid generation and application. Traditional cryopreservation methods are inapplicable for spheroid due to severe thermal stress, toxic cryoprotectants, and ice formation. Here, we constructed and preserved human MSC (hMSC) spheroids via deep supercooling (DSC). Spheroids were DSC preserved at -12°C without ice formation for 7 days, with higher cell viability, energy level, and chondrogenic differentiation capacity than suspended hMSCs. hMSCs embedded in spheroids have close cell-cell interactions via N-cadherin to activate the AKT-cytochrome c-caspase anti-apoptotic cascade during DSC preservation. Finally, preserved hMSC spheroids were capable of chondrogenic differentiation and can be co-delivered with collagen to treat rat cartilage defects in vivo.