Perfusion bioreactor-based cryopreservation of 3D human mesenchymal stromal cell tissue grafts

The generation of an off-the-shelf in vitro engineered living tissue graft will likely require cryopreservation. However, the efficient addition and removal of cryoprotective agents (CPA) to cells throughout the volume of a three-dimensional (3D) tissue graft remains a significant challenge. In this work, we assessed whether a perfusion bioreactor-based method could be used to improve the viability of cryopreserved mesenchymal stromal cell- (MSC) based tissue constructs as compared to using conventional diffusion-based methods. The bioreactor was first used to saturate 3D constructs with CPA under perfused flow. Following cryopreservation, the bioreactor was then also used for the efficient removal of the CPA from the 3D tissues. We demonstrate that addition and removal of CPA under perfused flow significantly increased the viability of MSC within cryopreserved 3D tissue constructs as compared to conventional diffusion-based methods.