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PLoS One. 2018 Dec 21;13(12):e0209201. doi: 10.1371/journal.pone.0209201. eCollection 2018.

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

Author information

1
Department of Pediatrics, Divisions of Critical Care Medicine, Washington University in Saint Louis, School of Medicine, Saint Louis, MO, United States of America.
2
Department of Biochemistry & Molecular Biophysics, Washington University in Saint Louis, School of Medicine, Saint Louis, MO, United States of America.
3
Department of Pediatrics, Duke University School of Medicine, Durham, NC, United States of America.
4
Department Medicine, Duke University School of Medicine, Durham, NC, United States of America.
5
Departments of Medicine, Durham VA Medical Center, Durham, NC, United States of America.
6
Department of Radiation Oncology, Duke Univ. School of Medicine, Durham, NC, United States of America.
7
Washington University Center for Cellular Imaging, Washington University in Saint Louis, School of Medicine, Saint Louis, MO, United States of America.
8
Departments of Neuroscience and Cell Biology & Physiology, Washington University in Saint Louis, School of Medicine, Saint Louis, MO, United States of America.
9
Department of Biochemistry, University of Colorado Denver-Aurora, CO, United States of America.

Abstract

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

PMID:
30576340
PMCID:
PMC6303082
DOI:
10.1371/journal.pone.0209201
[Indexed for MEDLINE]
Free PMC Article

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