Exploring the Red Blood Cell from Erythroid Culture to Deformation Studies

Abstract

Red blood cells (RBCs) are vital for oxygen delivery, enduring extreme mechanical deformation throughout their lifespan. This thesis investigates RBC biology, combining molecular, metabolic, and biophysical approaches to provide new insights into deformability and stress response.

A significant focus was the optimisation of erythroid culture systems, addressing the variability in medium and supplementation strategies, that complicates reproducibility. Ensuring the generation of high-quality erythroid cells from in vitro cultures was foundational to this thesis, as it influences all ensuing studies on cell characteristics. By employing detailed assays, including lipidomics, this work demonstrated that plasma-based cultures require cholesterol supplementation to produce stable, filterable reticulocytes, challenging the common assumption of interchangeability of plasma and serum.
I also investigated the roles of PIEZO1 and GLUT1 in RBC membrane functionality. The PIEZO1 studies contributed to defining the newly discovered Er-blood group, demonstrating that novel mutations associated with this blood group do not impair channel functionality when assessed by automated patch-clamp. Additionally, I found that the absence of GLUT1 does not impede human erythroblast proliferation, differentiation, or enucleation. This is the first report on enucleated human reticulocytes lacking GLUT1. GLUT1-deficient reticulocytes showed no significant changes in membrane composition or deformability but exhibited metabolic shifts, including reduced glucose import, altered antioxidant metabolism, and increased osmotic fragility. Despite these changes, the lack of a developmental phenotype or impaired deformability challenges the presumed essentiality of GLUT1 in RBC biology and aligns with clinical findings that GLUT1 deficiency does not cause anaemia.
To mimic the repeated deformation and recovery cycles RBCs endure, I employed an electrodeformation assay based on dynamic fatigue to enhance understanding of erythroid membrane characteristics, despite reproducibility challenges.

Overall, the work presented here advances our understanding of RBC biology by tackling significant gaps in culture optimisation, PIEZO1 and GLUT1 functionality, and by exploring dynamic deformability of cultured reticulocytes.

Date of Award	13 May 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Ash M Toye (Supervisor) & Jan Frayne (Supervisor)