Cryopreservation method for spheroids and fabrication of scaffold-free tubular constructs

Cryopreservation is a method used for preserving living cells by cooling them to very low temperatures. Although cryopreservation methods for oocytes and embryos have been developed for use in reproductive medicine, there are no established methods yet for preserving cell aggregates (spheroids) in regenerative medicine. We have developed a bio-three-dimensional (3D) printer that can fabricate scaffold-free 3D constructs by loading spheroids onto a needle array. We fabricated several constructs such as blood vessels, liver, diaphragm, and a conduit for nerves by using this method. These constructs have the potential to be applied in patients. However, the process of fabricating tissue constructs (harvesting cells, expanding cells, making spheroids using cultured cells, printing constructs, and maturing constructs) is time-consuming. Therefore, cryopreservation methods for spheroids or constructs should be developed to increase the efficiency of this method for clinical use. Here, we developed a method for cryopreserving spheroids, which were then used to fabricate constructs. Fibroblast cell-based spheroids were cryopreserved in phosphate-buffered saline or cryopreservation solution at -80°C for 1 week. After thawing, spheroids in cryopreservation solution began to fuse on day 1. Cryopreserved spheroids were printed onto a needle array to fabricate a scaffold-free tubular construct using a bio-3D printer. After 7 days, the printed spheroids fused and formed scaffold-free constructs. We confirmed the viability of cells in the cryopreserved spheroids and fabricated tubular constructs. Our results indicate that spheroids can be cryopreserved and used to prepare scaffold-free constructs for clinical use.