Lyophilization of Human Pluripotent Stem Cells for Long-Term Storage Based On Intracellular Loading of Trehalose By Cell Cenetrating Ceptides

Human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) hold great potential for cell therapy and regenerative medicine. Efficient preservation of hPSCs without affecting their survival and functions is essential for cell-based clinical applications. There is growing interest in lyophilization of cells since stabilization of mammalian cells in desiccated state can significantly simplify the storage and transportation and provide enormous benefit for clinical applications. At present, freeze-drying method has been successfully applied in the preservation of diverse kind of cells, including sperm and platelet. However, the possibility of long-term preservation of hPSCs using this method has not yet been studied. Due to a higher glass transition temperature and dihydrate crystalline structure, trehalose can provide a better stability to the system than other carbohydrate. However, the major challenge in using trehalose for freeze-drying of cells is to overcome the impermeability of the plasma membranes to introduce them at high intracellular concentrations. In this study, a novel cell penetrating peptide (CPP) was designed to deliver trehslose intracellularly based on the molecular simulations. A sequence rich in basic amino acid combined with a sequence having the ability of coupling with trehalose through non-covalent bonding was developed. We evaluated the cytotoxicity of the designed CPP to hPSCs and further examined the efficiency of the designed CPP entering into hPSCs. The capacity of intracellular trehalose loading using the designed CPP was also determined. The results indicated that CPPs has high loading rate of trehalose and no significant toxic effects. In addition, freeze-dried hPSCs loaded with trehalose after rehydration showed significantly higher cell viability than the control. The establishment of freeze-drying method of hPSCs loaded with trehalose based on CPPs may prove a powerful tool for long-term and stable storage of hPSCs for various clinical and research applications.