Small-Scale Perfusion Bioreactor of Red Blood Cells for Dynamic Studies of Cellular Pathways: Proof-of-Concept

Abstract

To date, the development of bioreactors for the study of red blood cells (RBCs, daily transfused in the case of disease or hemorrhage) has focused on hematopoietic stem cells. Despite the fact that mature RBCs are enucleated and do not expand, they possess complex cellular and metabolic pathways, as well as post-translation modification signaling and gas-exchange regulation. In order to dynamically study the behavior of RBCs and their signaling pathways under various conditions, a small-scale perfusion bioreactor has been developed. The most advanced design developed here consists of a fluidized bed of 7.6 mL containing 3·109 cells and perfused at 8.5 μL/min. Mimicking RBC storage conditions in transfusion medicine, as a proof-of-concept, we investigated the ex vivo aging of RBCs under both aerobic and anaerobic conditions. Hence, RBCs stored in saline-adenine-glucose-mannitol (SAGM) were injected in parallel into two bioreactors and perfused with a modified SAGM solution over 14 days at room temperature under air or argon. The formation of a fluidized bed enabled easy sampling of the extracellular medium over the storage period used for the quantitation of glucose consumption and lactate production. Hemolysis and microvesiculation increased during aging and were reduced under anaerobic (argon) conditions, which is consistent with previously reported findings. Glucose and lactate levels showed expected trends, i.e., decreased and increased during the 2-week period, respectively; whereas extracellular glucose consumption was higher under aerobic conditions. Metabolomics showed depletion of glycolsis and pentose phosphate pathway metabolites, and an accumulation of purine metabolite end-products. This novel approach, which takes advantage of a fluidized bed of cells in comparison to traditional closed bags or tubes, does not require agitation and limit shear stress, and constantly segragates extracellular medium from RBCs. It thus gives access to several difficult-to-obtain on- and off-line parameters in the extracellular medium. This dynamic bioreactor system does not only allow us to probe the behavior of RBCs under different storage conditions, but it also could be a powerful tool to study physiological or pathological RBCs exposed to various conditions and stimuli.

Restoration of Physiological Levels of Uric Acid and Ascorbic Acid Reroutes the Metabolism of Stored Red Blood Cells

“…These approaches were not able to improve the storage. The reduction of oxygen has been proposed by several groups [38][39][40][41]. In general, ATP levels were better preserved during the storage and haemolysis was reduced.…”

Oxygen in Red Blood Cell Concentrates: Influence of Donors’ Characteristics and Blood Processing

“…With the TB bags system, blood components are first separated and only the RBCs are filtrated and stored for up to 42 days in SAGM additive solution at 4 • C. In this process, WB units were rested at room temperature for 14.1 ± 3.6 h before RBCs were separated from the plasma and the buffy coat (used for fabrication of platelet concentrates). With TT bags system, blood components are extracted by centrifugation after WB filtration and RBCs resuspended in PAGGSM additive solution usually within 12.0 ± 1.9 h to be stored up to 49 days at 4 • C. TT plasma can be used for transfusion, and in such case, male donors are preferentially selected in order to limit the risk of adverse effect associated with HLA antibodies (that may occur during pregnancy) (Pandey and Vyas, 2012). It explains why proportion of TT RCCs donated by men was five times higher than those donated by women.…”

“…Since RCCs are known to get oxygenated during hypothermic storage ( Yoshida et al, 2017 ) and knowing that storage lesions are driven by oxidative stress ( D’Alessandro et al, 2015 ; Bardyn et al, 2018 ), storage of high sO 2 -level units might accelerate the storage lesions. Indeed, it is known that the cold storage under anaerobic or reduced O 2 content reduces the lesions to RBCs ( Yoshida et al, 2007 ; Burns et al, 2016 ; Prudent et al, 2016 ). Of interest, it was reported that the hemolysis rates are lower in RCCs from females ( Jordan et al, 2016 ).…”

Cysteine redox proteomics of the hemoglobin‐depleted cytosolic fraction of stored red blood cells

“…Knowing that tissue oxygenation depends on RBC deformability, the study of early events in this cascade is of particular interest to control and reduce the storage lesion. Identification of target proteins such as catalase, peroxiredoxin‐1 and DJ‐1 merit a closer investigation of their oxidation forms, as well as provide valuable oxidation markers to test different conditions to alleviate oxidation with prolonged storage.…”