Abstract
Genomic and epigenomic studies require the precise transfer of microliter volumes among different types of tubes in order to purify DNA, RNA, or protein from biological samples and subsequently perform analyses of DNA methylation, RNA expression, and chromatin modifications on a genome-wide scale. Epigenomic and transcriptional analyses of human blood cells, for example, require separation of purified cell types to avoid confounding contributions of altered cellular proportions, and long-term preservation of these cells requires their isolation and transfer into appropriate freezing media. There are currently no protocols for these cellular isolation procedures on the International Space Station (ISS). Currently human blood samples are either frozen as mixed cell populations (within the CPT collection tubes) with poor yield of viable cells required for cell-type isolations, or returned under ambient conditions, which requires timing with Soyuz missions. Here we evaluate the feasibility of translating terrestrial cell purification techniques to the ISS. Our evaluations were performed in microgravity conditions during parabolic atmospheric flight. The pipetting of open liquids in microgravity was evaluated using analog-blood fluids and several types of pipette hardware. The best-performing pipettors were used to evaluate the pipetting steps required for peripheral blood mononuclear cell (PBMC) isolation following terrestrial density-gradient centrifugation. Evaluation of actual blood products was performed for both the overlay of diluted blood, and the transfer of isolated PBMCs. We also validated magnetic purification of cells. We found that positive-displacement pipettors avoided air bubbles, and the tips allowed the strong surface tension of water, glycerol, and blood to maintain a patent meniscus and withstand robust pipetting in microgravity. These procedures will greatly increase the breadth of research that can be performed on board the ISS, and allow improvised experimentation by astronauts on extraterrestrial missions.
Highlights
The ability to collect biosamples onboard the international Space Station (ISS) is extremely limited
We evaluated the fluid handling steps required for density gradient centrifugation (DGC), including the loading of Ficoll, the overlay of blood, and the removal and preparation for cryopreservation of isolated cells, in microgravity conditions
Common perception is that handling of open liquids would be difficult in microgravity, we found that all pipetting steps related to density gradient centrifugation were performed in microgravity using analog fluids as well as human blood products (Figure 1)
Summary
The ability to collect biosamples onboard the international Space Station (ISS) is extremely limited. There would be a science benefit for ISS to expand the frequency of flight sampling and to isolate purified cells from blood or tissue while onboard the ISS, including peripheral blood mononuclear cells (PBMCs) or positively isolated cell populations (e.g. CD4+, CD8+, or CD19+ cells) This ability would facilitate a host of cellular and -omics analyses for which segregation of distinct cell populations has become increasingly important. It is generally perceived that techniques for isolating cells, including density gradient centrifugation (DGC) with Ficoll or magnetic separation of cells, are gravity-dependent This perception is based on the assumption that open liquids would be difficult to manipulate in microgravity, or that sensitive density-dependent steps such as overlaying of fluids or cellular band removal would be compromised without gravity. The overlay pipetting of actual diluted blood and the removal of isolated PBMCs was accomplished
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