UC Riverside

Cryopreservation is an essential tool for the storage of biological material while retaining their biological processes; however, reduced cell and tissue viability persists due to ice crystal formation and cryoprotectant agent (CPA) toxicity. Further, cryopreservation of larger tissues or 3D cultures presents additional challenges due to temperature and CPA gradients. Incorporation of tardigrade stress resistance genes is an untested mechanism to enhance cell viability during cryopreservation. Here, we show the expression of tardigrade stress resistance genes (Dsup, MAHS, and RvLEAM) into U87 cells to improve resistance to cryopreservation related stresses. The study reveals that cells expressing Dsup and MAHS genes had a greater survival rate than those expressing RvLEAM when exposed to chronic and acute DMSO. Furthermore, MAHS provided increased cell viability during cryopreservation. Our results demonstrate that tardigrade stress resistance genes improve cell viability under CPA and cryogenic stressors in 2D and 3D cell cultures. These findings offer valuable insights into using tardigrade gene expression to boost cryopreservation viability and reduce the use of CPA.