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

Current bioreactor research often focuses on hematopoietic stem cells rather than mature red blood cells (RBCs). Mature RBCs lack nuclei but still have intricate metabolic and signaling pathways. Prior research has shown that RBCs undergo metabolic and structural changes during storage. No prior work had resolved how to dynamically study RBCs under controlled conditions. Traditional storage methods like closed bags limit access to extracellular data. This gap motivated the development of a new bioreactor design. The need for a system that minimizes shear stress and allows continuous sampling was clear. Existing methods do not support real-time monitoring of RBC metabolism and signaling.

The aim of this work was to create a small-scale perfusion bioreactor for RBC studies. The system needed to mimic storage conditions in transfusion medicine. Researchers wanted to evaluate RBC behavior under aerobic and anaerobic environments. The design had to allow for easy sampling of extracellular medium. The goal was to measure glucose consumption and lactate production over time. The system also needed to track hemolysis and microvesiculation during storage. The researchers aimed to compare traditional closed systems with the new fluidized bed design. This approach could help understand RBC aging and signaling under various conditions.

The bioreactor design used a fluidized bed with a 7.6 mL volume. It contained 3·10^9 RBCs perfused at 8.5 μL/min. The system mimicked RBC storage in saline-adenine-glucose-mannitol (SAGM) solution. Two bioreactors were used in parallel for aerobic and anaerobic conditions. RBCs were perfused with modified SAGM over 14 days at room temperature. Extracellular medium was sampled regularly for glucose and lactate analysis. Hemolysis and microvesiculation were measured as indicators of RBC aging. Metabolomics was used to assess changes in glycolysis and purine pathways.

Hemolysis and microvesiculation increased during storage but were lower under anaerobic conditions. Glucose levels decreased over time, while lactate levels increased as expected. Extracellular glucose consumption was higher under aerobic conditions. Metabolomics showed depletion of glycolysis and pentose phosphate pathway metabolites. Purine metabolite end-products accumulated during the 14-day period. The fluidized bed design allowed continuous sampling without agitation. The system minimized shear stress and separated extracellular medium from RBCs. This setup enabled the measurement of both on- and off-line parameters in real time.

The fluidized bed bioreactor provides a novel method for dynamic RBC studies. It allows real-time monitoring of extracellular parameters without requiring agitation. The design reduces shear stress and enables continuous sampling. The system supports the study of RBC aging under aerobic and anaerobic conditions. The results suggest that anaerobic conditions may reduce hemolysis and microvesiculation. The bioreactor could help investigate RBC behavior under various physiological or pathological conditions. It may also be useful for studying RBC responses to different stimuli. The authors propose that this system could improve understanding of RBC metabolism and signaling.