Increasing Desiccation Tolerance of Mammalian Cells Using Late Embryogenesis Proteins

Introduction: The objective of this study is to use a chemical engineering based biomimetic strategy to use late embryogenesis abundant proteins for increasing desiccation tolerance in mammalian cells for lyopreservation. Such a technique can lead to a novel ambient temperature storage strategy for cellular material to support tissue-engineering applications. Late embryogenesis abundant (LEA) proteins are associated with desiccation tolerance in anhydrobiotic organisms. We have identified and sequenced two LEA genes (Afrlea2 and Afrlea3m) in the embryo of desiccation tolerant brine shrimps (Artemia franciscana). AfrLEA3m was shown to be localized in mitochondria while AfrLEA2 is cytoplasmic. In this study, human hepatocellular carcinoma (HepG2) cells with AfrLEA2 and AfrLEA3m was used to evaluate the possibility of improved survivorship during drying. The technique of spin-drying was used to rapidly and uniformly desiccates cells to a moisture content of 0.2 gH2O/gdw at 22⁰C. We report the significant improvement of desiccation tolerance of HepG2 cells using both AfrLEA2 and AfrLEA3m proteins. Desiccated cells showed significantly higher survival after drying and rehydration.

Materials and Methods: Human hepatocellular carcinoma (HepG2) cells were stably transfected to express AfrLEA2 and AfrLEA3m using a tetracycline-inducible expression system. Trehalose was loaded into these cells using a TRET1 trehalose transporter isolated from anhydrobiotic larvae of Polypedilum vanderplanki. Cells attached to glass coverslips were spin dried at 1000 rpm for 60 sec in a trehalose buffer at room temperature. Following spin-drying, the samples were immediately rehydrated and the membrane integrity of the cells following desiccation was assessed using a Syto-13/Ethidium bromide fluorescence assay. Desiccated cells were cultured under normal tissue culture conditions following rehydration for 7 days to evaluate long-term growth characteristics.

Results and Discussion: Control HepG2 cells (without LEA protein or intracellular trehalose) exhibited 0 % membrane integrity (n = 9).  HepG2-TRET1 cells preloaded with trehalose showed 44.5 ± 22.2 % integrity (mean ± SD, n = 3); HepG2-AfrLEA2 cells without trehalose, 57.2 ± 13.0 % (n = 9) (Fig 1); HepG2-AfrLEA2 cells preloaded with trehalose, 98.3 ± 2.2 % (n = 9); HepG2-AfrLEA3 cells without trehalose, 93.6 ± 4.6 % (n = 9); and HepG2-AfrLEA3 preloaded with trehalose, 97.7 ± 3.8 % (n = 9).  Growth studies across subsequent days after rehydration revealed higher proliferation for cells containing LEA protein than those without. Between AfrLEA2 (cytoplasmic) and AfrLEA3m (mitochondrial) proteins, AfrLEA3m was able to provide better protection against desiccation stresses as evidenced by superior proliferation rates. Growth studies also demonstrate that the LEA proteins act synergistically with trehalose in providing protection against desiccation stresses.

Conclusion: This study outlines a biomimetic approach of using LEA proteins to substantially increase the desiccation tolerance of mammalian cells. LEA proteins alone and along with the use of a lyoprotectant can help the development of a desiccation technique for mammalian cell storage at ambient temperatures.