TY - JOUR
T1 - Evaluation of techniques for performing cellular isolation and preservation during microgravity conditions
AU - Rizzardi, Lindsay F.
AU - Kunz, Hawley
AU - Rubins, Kathleen
AU - Chouker, Alexander
AU - Quiriarte, Heather
AU - Sams, Clarence
AU - Crucian, Brian E.
AU - Feinberg, Andrew P.
N1 - Funding Information:
We thank Jerry Myers and John McQuillen (NASA Glenn Research Center) for advice and critical reading of the manuscript. We also thank Terry Lee, Kerry McMannis, D. Del Rosso and the pilots and other crew members from NASA’s Reduced Gravity Office for coordinating our use of the C-9 aircraft for parabolic flights. We thank Dr. Richard Simpson, Dr. Cherie Oubre, Mr. Andrew Beitman and Mr. Todd Nelman for participating as evaluators in the parabolic flight activity. We thank NASA photographers/videographers James Blair and Regan Geeseman for documenting our work. Additionally, we thank Veronica Seyl for use of the Education Office’s glove box for these experiments as well as Marisa Covington and everyone at Johnson Space Center for their contributions to the success of this work. This work was supported by funding from NASA (NNX14AH28G awarded to A.P.F. and a Division Innovation award from the NASA Biomedical Research and Environmental Sciences Division to B.E.C).
Publisher Copyright:
© 2016 Macmillan Publishers Limited.
PY - 2016/1/7
Y1 - 2016/1/7
N2 - 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.
AB - 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.
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U2 - 10.1038/npjmgrav.2016.25
DO - 10.1038/npjmgrav.2016.25
M3 - Article
AN - SCOPUS:85038919276
SN - 2373-8065
VL - 2
JO - npj Microgravity
JF - npj Microgravity
M1 - 16025
ER -