Abstract 10: Effect of Human Fetal Hemoglobin on Reactive Oxygen Species in DMSO Cryopreserved Cord Blood Progenitors Post Thaw

Abstract Introduction Cord blood processing and cryopreservation have emerged as pivotal techniques in regenerative medicine, facilitating the extraction and long-term preservation of stem cells for therapeutic applications. Maximizing cell viability and count post-rejuvenation is paramount for the success of cryopreserved cell-based therapies. However, challenges persist in achieving optimal rejuvenation of cryopreserved cells, necessitating innovative approaches. Objectives In this study, we investigated the potential of Human Fetal Hemoglobin (HFH) supplementation to enhance the rejuvenation of cryopreserved cells post thaw. Utilizing the CCBR’s double centrifugation technique, cord blood samples were processed to extract stem cells and leukocytes. Subsequently, samples were cryopreserved after the addition of DMSO and divided into experimental groups supplemented with HFH and control groups. Flow cytometry was employed to assess cell viability and counts, focusing on leukocytes (LC) and CD34-marked stem cells. Methods The Moxiflow cytometer was utilized for cell counting per the Coulter principle. We determined viability using propidium iodide staining. Biolegend anti-human CD34 antibody clone 581 was used for CD34 count. Results Preliminary findings suggest a notable increase in cell viability and count in HFH-supplemented samples compared to controls. In one sample, we observed 3.08x10^6 cells/mL CD34 (Viability 99.8%), 8.03x10^6 cells/mL LC count (Viability 100%) in HFH-supplemented samples, compared to 1.61x10^4 cells/mL CD34 (Viability 100%), 3.68x10^5 cells/mL LC count (Viability 100%) in controls. Similar results were observed in an additional sample. These results indicate the potential of HFH supplementation in augmenting the rejuvenation of cryopreserved cells, thus improving the efficacy of cell-based therapies. Discussion We hypothesize that HFH exerts its effects by mitigating oxygen radical-induced cell damage, thereby facilitating glucose uptake for enhanced glycolysis and ATP production. We believe the robust glycolytic activity triggers the activation of proliferating pathways in the mitochondria, generating additional ATP. Concurrently, reactive oxygen radicals produced during this process are potentially quenched by the presence of HFH, ensuring cellular integrity and function. These preliminary findings underscore the significance of HFH supplementation as a promising strategy to enhance the effectiveness of cryopreserved cell-based therapies. Further investigation into the underlying mechanisms and clinical validation of this approach are warranted to fully exploit its therapeutic potential and translate it into clinical practice. Moxiflow Results