Late embryogenesis abundant protein 3-modified human umbilical cord mesenchymal stem cell sheet exhibits antifreeze...

Cell sheet technology (CST) is used clinically to repair various epithelial tissue defects in human, but long-term preservation of cell sheet remains a challenge. Late embryogenesis abundant proteins (LEAs) can improve cell survival in extreme environments such as low temperature and drought dehydration, but whether they can be used in human cell sheets to improve cryopreservation has not been reported. In this study, we applied the tetracycline-controlled (Tet-On) system to enable the AfrLEA3m (LEA3, a LEA from Artemia franciscana) proteins "express on demand" to efficiently adapt to the freezing environment and avoid the additional nutrient consumption of sustained expression on cells. We firstly confirmed the expression and degradation of LEA3 by western blotting and immunofluorescence in human umbilical cord mesenchymal stem cells (UCMSCs), and then the UCMSCs expressing LEA3 were cultured into cell sheets. The Tet-On system was activated to express LEA3 before freezing the cell sheets, and then the cell sheets were stored in liquid nitrogen after programmed cryopreservation. Finally, the recovered LEA3 protein-modified cell sheets were used to repair mouse skin defects. The results showed that LEA3-modified cell sheets had a higher cell survival rate and performed better in skin repair. In conclusion, our finding demonstrated that the LEA3-modified human UCMSCs sheet exhibits antifreeze properties, which provides a technological basis for the establishment of a cell sheet storage bank.