Screening and identification of cryopreservative agents for human cellular biotechnology experiments in microgravity.

The field of cryobiology originated in 1949, when Polge, et al., described the cryoprotective property of glycerol in the freezing of spermatozoa (1). Lovelock and Bishop reported the cryoprotective ability of dimethyl sulfoxide (DMSO) on red blood cells in 1959 (2). Fortytwo years later, DMSO was used to preserve the first cryogenically-stored cells launched onboard STS-105 for use on the first human cellular biotechnology payload, Cellular Biotechnology Operations Support System (CBOSS-01), on the International Space Station (ISS). DMSO is the most common cryoprotective agent used in the laboratory. While DMSO is easily eliminated in ground-based experiments, its removal in flight-based experiments is more difficult due to microgravity, hardware limitations, and on-orbit constraints. Because of the deleterious effects of DMSO on cells at noncryogenic temperatures, there was concern regarding the difficulties of removing the DMSO after thawing the cells in microgravity. Our primary goal was to find an alternate cryoprotectant to DMSO to be used for future CBOSS Exploration Cell Science investigations. While systematically screening for potential permeating and non-permeating cryoprotectants, we used a human colorectal carcinoma cell line, MIP-101 (3) that has flown on several biotechnology payloads (4), including CBOSS01. We utilized data from immediate post-thaw viability, culture recovery and clonogenic assay to determine a candidate alternate cryoprotectant to DMSO. Once alternates were identified, we explored the use of a binary system consisting of permeating and non-permeating cryoprotectants (5) at concentrations suboptimal in single cryoprotectant systems in an effort to maximize cryoprotective effect at lower concentrations. MIP-101 cells were resuspended in cryopreservatives using RPMI culture media as a carrier solution. Afterwards, they were frozen according to either a twostep procedure involving initial cooling at -1C/min overnight in a Nalgene Cryocooler at 4C followed by storage in liquid nitrogen (LN2) vapor, or by storing cells directly in the LN2 vapor phase at -10C/min. The frozen cells were thawed by immersion and agitation in a 37C water bath. A sample was taken immediately and assayed by Guava Viacount to determine the post thaw viability and cell density. Ability to preserve cellular function after cryopreservation was assessed by the recovery of viable cells in shortand long-term cell culture experiments. Culture Recovery: Cells were plated in 6-well plates and cultured. After 96 hours, cells were harvested and viability was determined by Guava Viacount. In the assay to determine cell growth kinetics, cells were harvested at 12, 24, 36, and 48 hours. Clonogenic Assay: Cells were seeded in 35-mm tissue culture plates and cultured for 10-12 days. Individual colonies were counted after staining with crystal violet. Binary Cryoprotectant: Suboptimal levels of ethylene glycol and raffinose were combined to cryopreserve cells.