Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes

• DOI: 10.1371/journal.pone.0209201
• Publication Date: 2018-12-21T15:19:31Z
• Authors: Stephen C. Rogers; Laura B. Dosier; Timothy J. McMahon; Hongmei Zhu; David Timm; Hengtao Zhang; Joseph Herbert; Jacqueline Atallah; Gregory M. Palmer; Asa Cook; Melanie Ernst; Jaya Prakash; Mark Terng; Parhom Towfighi; Reid Doctor; Ahmed Said; Matthew S. Joens; James A. J. Fitzp...; Gabi Hanna; Xue Lin; Julie A. Reisz; Travis Nemkov; Angelo D’Alessandro; Allan Doctor
• Keywords: Erythrocyte fragility

Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter sample analysis, we developed a research-specific RBC cryopreservation protocol assessed its impact on data fidelity key biochemical physiological assays. RBCs drawn from healthy volunteers were aliquotted immediate analysis or following glycerol-based cryopreservation, thawing, deglycerolization. phenotype was by (1) scanning electron microscopy (SEM) imaging standard morphometric indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge superoxide thermal source; SOTS-1), as well in vivo quantification (following human mouse xenotransfusion) of (9) oxygenation content mapping flow dynamics (velocity adhesion). Our revised glycerolization (40% v/v final) resulted >98.5% recovery freezing (-80°C) thawing (37°C), no difference compared the reported method w/v final). Full deglycerolization (>99.9% glycerol removal) 40% final samples total cumulative lysis ~8%, ~12–15% method. The post cryopreservation/deglycerolization indistinguishable that fresh regard physical parameters (morphology, volume, density), adhesivity, O2 vasoregulation, metabolomics, dynamics. These results indicate does not significantly (compared cells).