Exploring Engineered Desiccation Tolerance in Mammalian Cells

Abstract

Engineered desiccation tolerance in mammalian cells has historically been an area of great interest but also significant challenges. One emerging research avenue has explored the potential of imparting the functionality of proteins expressed by tardigrades, a model anhydrobiotic organism, to mammalian cells. However, progress has been slowed due to limited understanding of the working mechanisms of protectant molecules, as well as a lack of standardization in experimental desiccation protocols. This study aims to design a more standardized and reproducible desiccation protocol and analyze the impact that protectant molecules have on 3T3 fibroblasts and HeLa cells. We explore three main desiccation protectants: trehalose and the cytoplasmic (CAHS) and secreted (SAHS) abundant heat soluble tardigrade disordered proteins (TPDs). The functionality of these molecules is assessed with membrane integrity and metabolic activity viability assays, as well as with optical and electron microscope imaging. In addition, other methodologies adjacent to desiccation, including osmotic pressure, cold storage, and lyophilization are explored. For TDPs, we find that dried SAHS protein exhibits a porous structure with concentration dependent fibril formation. In addition, we identify a unique phase separation behavior in RVCAHS, which differs from the uniform cytoplasmic distribution and vitrification seen in other CAHS proteins. Despite promising signs of forming membraneless organelles and promoting structural integrity, all CAHS proteins studied exhibit significant cytotoxicity.