NonFreezable Preservation of Human Red Blood Cells at −8 °C

Abstract

Red blood cell (RBC) preservation is very important in human health. The RBCs are usually preserved at 4 ± 2 °C without freezing or at a very low temperature (−80 °C or liquid nitrogen) with deep freezing. Herein, non freezable preservation of RBCs at a subzero temperature is reported to prolong the preservation time compared with that at 4 ± 2 °C. By adding glycerol and poly­(ethylene glycol) (PEG) (average number molecular weight 400, PEG-400) into the preservation solution, the freezing point is decreased and the hemolysis is kept low. The cell metabolism of stored RBCs at −8 °C is reduced, and the shelf life of RBCs extends up to at least 70 days. At the end of preservation, the pH decreases a little bit to demonstrate the low metabolic rate of RBCs stored at subzero temperatures. After quick washing, the RBC survival rate is ca. 95%. The adenosine triphosphate, 2,3-diphosphoglycerate, and cell deformation ability of the washed RBCs are maintained at a high level, while the malondialdehyde is relatively low, which verifies the high quality of RBCs stored at this condition.

Comparing two extracellular additives to facilitate extended storage of red blood cells in a supercooled state

“…, the molecular weight of 35,000 g/mol) is the most used subtype in many of these applications, data from one of our pilot studies indicated that PEG400 is preferable for the supercooled preservation of RBCs ( Supplementary Figure S1 ). In fact, Zhu et al (2022) , in one of the few published studies describing supercooled RBC preservation, showed that PEG400 (75 mM) can diminish hemolysis during long-term storage at −8°C in the presence of 7.5%–10% (w/v) of glycerol ( Zhu et al, 2022 ). Our study provides further evidence of the protection imparted by PEG400 in supercooled RBCs, with all tested concentrations (27.5–165 mM) providing an improvement in deformability with an increase in concentration at −4°C ( Figure 2B ).…”

“…Preservation in an unfrozen state at high sub-zero temperatures (i.e., supercooling) down to −15°C has been a recently popularized strategy to avoid ice-induced damage and extend organ storage limits that currently contribute to organ shortages ( Monzen et al, 2005 ; Berendsen et al, 2014 ; de Vries et al, 2019 ). While supercooling has been considered as a strategy to store a variety of different cell types of therapeutic interest, supercooled storage of RBCs has only recently received attention ( Usta et al, 2013 ; Puts et al, 2015 ; Huang et al, 2018 ; Huang et al, 2020 ; Zhu et al, 2022 ). One of the major challenges with supercooling is to ensure that ice formation does not occur in the metastable “supercooled” state that exists between the liquid-solid phase transition; an objective that is exceedingly hard to achieve in large-volume systems like RCCs.…”

Biomolecular Pathways of Cryoinjuries in Low-Temperature Storage for Mammalian Specimens

“…Biopreservation for mammalian specimens is an enabling technology to bridge the spatiotemporal gap between procurement sources and application destinations for living cells, tissues, organs, and biomacromolecules (such as proteins and mRNA) [ 1 , 2 , 3 , 4 ]. Since mammalian specimens can slow down or even suspend their in vitro metabolic activity and decay time at low temperatures, they are usually stored in non-physiological low-temperature conditions to prolong their preservation period [ 5 ].…”