Erythrocytes (ERYs) play an important role in regulating blood flow via a pathway involving ERY-derived adenosine triphosphate (ATP) and endothelium-derived nitric oxide (NO). Impaired ATP release from ERYs of diabetic patients is potentially responsible for diabetic complications, thereby therapies involving stimulation of ERY-derived ATP release might limit such complications. This work explores 3D-printing to fabricate a novel microfluidic device to mimic the physiology of ERYs. 3D-printing enables fabrication of the device following a standard 96-well plate geometry for efficient and high throughput readout with a plate reader. This 3D-printed rugged device was reusable after simple rinsing, which enables the detection of a batch of samples on the same device during a long-term experiment. This 3D-printed fluidic device facilitated the investigation of the efficacy of C-peptide on stimulating ERY-derived ATP. Without albumin, C-peptide and Zn2+ cannot increase ERY-derived ATP, suggesting the indispensable role of albumin in the process. The glutamic acid at the 27 position of C-peptide participated in the binding to albumin. Collectively, the ensemble of albumin, C-peptide, and Zn2+ enhances ERY-derived ATP, which may reduce diabetes complications.To make the results more physiologically conclusive, an Organs-on-a-Chip platform that combined pancreatic β-cells, ERYs and endothelial cells as a blood barrier mimic was developed. The secretion profiles of the β-cells on the device simulate the physiological secreting process well. Subsequent cell-cell communication investigations showed that β-cell secretions do not affect the endothelial cells but increase ATP release from ERYs, which in turn, exerts a downstream effect on endothelial cells by stimulating NO production. Currently approved hyperglycemic ERY storage solutions impair ATP release from ERYs. This work continues to investigate the reversibility of ATP release from stored ERYs and shows that 15 days of storage is a turning point, after which the ATP release is no longer reversible. This result is consistent with the clinical reports that blood stored longer than 2 weeks is more likely to result in transfusion complications. The mechanism by which hyperglycemia impairs ATP release was also explored by studying ERY deformability using a 3D-printed demand-based cell filter, finding that hyperglycemia permanently alters the deformability of ERYs after 5 days of storage. A 3D-printed intravenous device was developed to mimic a transfusion process in vitro. Addition of β-cells on the platform showed that hyperglycemia-stored ERYs failed to respond to the endocrine cells.Summarily, 3D-printing yields reusable, robust and reproducible microfluidic devices, and demand-based devices. The ensemble of albumin, C-peptide and Zn2+ can be a potential therapy for diabetes complications, and the current ERY storage protocol adversely alter the physiology of stored ERYs. A normoglycemic alternative may avoid this problem.
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