As cytosensors and cell-based therapies move from research laboratories to medical use, the need increases to transport cryopreserved cell lines over long distances. The NASA-funded Institute for Cell Mimetic Space Exploration (CMISE) is focused on monitoring and maintaining the health of the astronauts during long interplanetary flights, such as from the Earth to Mars. We are working to develop mammalian cell sentinel cytosensors to monitor the effects of the environment of space, including high dose radiation, and to serve as surrogates for potential risk to the astronauts. Due to energy and payload restrictions in spacecraft, we propose to use single cell culture in a microelectromechanical system (MEMS). The purpose of the current investigations was to determine whether isolated single mammalian cells could be cryopreserved and subsequently recovered in a viable state. We used limiting dilution in 96-well plates and examined individual wells for the presence of single cells. Cryopreservation was then performed with 7.5% dimethylsulfoxide (DMSO) as cryoprotectant, with the temperature reduced gradually (placed on ice for ˜1hr, and stored at -80°C). After 3-5 days, cells were thawed by addition of pre-warmed, 37°C culture medium, appropriate for the specific cells. The plates were cultured at 37°C in 95% air and 5% CO2. We observed single cell viability for lung carcinoma cells to average 52% (range 29-78%). We observed the average viability of single fibroblasts or lymphoblastoid cell lines to be 75% or 33% (range 67-80% or 29-38%), respectively. We conclude that single cells can be cryopreserved and recovered in a viable state, though viability may depend on the specific cell line. We are modifying the protocol to attempt to improve cryoprotection. The ability to cryopreserve and to recover viable single cells will enable MEMS-based sentinel cytosensors and permit us to monitor the potential impact of space on the health of the astronauts.
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