Cryopreservation of vascularizable tissue with temperature-controlled-cryoprinting

Abstract

Advancements in regenerative medicine have made it possible to fabricate complex, engineered tissues which closely mimic in vivo tissue. As with in vivo tissue, vascularization is crucial for supplying cells in the engineered tissue with nutrients. However, cryopreserving engineered tissues remains challenging due to their large 3D volume. Without effective cryopreservation techniques, it is difficult to use vascularized tissues at scale for drug development or to create banks for patient transplantation. Previously, our group developed Temperature-Controlled-Cryoprinting as a novel technology for simultaneously fabricating and cryopreserving 3D bioprinted tissue. During Temperature-Controlled-Cryoprinting, a cell-laden bioink is printed and frozen layer-by-layer under optimal cooling rates. In this study, we demonstrate that this approach can be used to cryopreserve the cell types which are most sensitive to cryopreservation: primary cells and stem cells. Human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) were encapsulated in a collagen bioink and cryoprinted. The tissues were stored at −80 °C, and then thawed at 37 °C. After thawing, the HUVECs and hMSCs naturally self-assembled into hollow capillaries, creating vascularized tissue. Analysis with Fiji found that vascular network formation was not impeded by cryopreservation and resembled that of a non-cryopreserved tissue. The ability to cryopreserve vascularizable tissue is an important advance, as it allows these tissues to become a shelf-stable product that can be shipped or stored long-term.