iPSC: SELECTION OF O-VE IPSC CLONES FOR HIGH-DENSITY RED BLOOD CELL PRODUCTION IN A SCALABLE PERFUSION BIOREACTOR SYSTEM

ABSTRACT

Background & Aim: Blood is one of the most vital resources in modern medicine. Blood transfusions have become an essential and often lifesaving procedure for accidents, during surgery, for patients with chronic disorders such as anemia, sickle cell disease, cancer, and myriad other circumstances. However, despite the rapidly growing world population, the availability of healthy blood donors is declining with aging populations. Furthermore, natural and man- made calamities often produce sudden and concentrated shocks in demand, which strains global supply chains. The COVID-19 pandemic has demonstrated this issue on a global scale by reducing the number of blood drives and donations, resulting in 39% of blood centers in the United States being left with only one- to two-day supplies, and a 50% drop of blood units collected in countries such as Zambia. Additionally, storage limitations of 42 days for donor blood limits stock availability during peak demand. Large-scale generation of universal red blood cells (RBCs) from O-ve human induced pluripotent stem cells (hiPSCs) offers the potential to alleviate blood shortages and provide a secure year-round supply. Mature iPSC-derived RBCs and reticulocytes could also find important applications in research in malaria and COVID-19 studies. (Figure Presented) Fig. 1 ( 700). Methods, Results & Conclusion: In this study, we have reprogrammed hiPSC from CD34+ O-ve cells and demonstrated the smallscale generation of high-density cultures of erythroblasts in a stirred perfusion bioreactor system. Twenty O-ve iPSC lines were derived, screened, and characterized for their ability to differentiate towards the erythroid lineage, showing high expression of mesoderm (KDR+, 64.9%), hematopoietic (CD34+/CD45+, 68.4%;CD34+/CD43+, 84.9%), and erythroid markers (CD235a+, 83,5%), and were able to undergo enucleation in vitro. Using the best clones, we were able to achieve erythroblast peak cell density of 34.7 million cells/mL with 92.2% viability in an Applikon perfusion bioreactor using an ultrasound system (Sonosep) to concentrate cells while removing waste media. This resulted in a cumulative-fold expansion of over 1,500 after 29 days of culture. Cells carried O2 effectively as demonstrated by hemoglobin dissociation curves. The perfusion culture platform paves the way for controlled high-density bioreactor culture for the generation of RBCs.