Abstract 19 CD34+ Cell Isolation from Cord Blood: A Novel Option for Faster, More Consistent Outcomes

Abstract

Abstract Introduction Immunomagnetic isolation of cells has been long established but still suffers from drawbacks when used for hemopoietic stem cells (HSCs) in cord blood. [Kekarainen et al., 2006] Limitations with HSC isolation from cord blood include steep learning curves, time consuming manual labor, and inconsistent outcomes. [de Wynter et al., 1999; Perdomo-Arciniegas and Vernot, 2011] FerroBioTM has developed a semi-automated system for CD34+ cell isolation from unmodified cord blood (Fig 1A) as a means to provide fast, effective, and consistent isolations. Objectives The purpose of this work is to explore process improvements for the FerroBioTM system. Additionally, this work aims to characterize the resulting cell isolates. Methods The cell isolation protocol was evaluated by flow cytometry for cell counts, viability, and purity (total n=195). Protocol modification included incubation time at various steps, magnetic bead dosing, temperatures, and volumes. The ‘percent of cells captured’ was calculated as the difference between starting CD34 and non-captured CD34 cells. Cryopreservation tolerance was evaluated by freeze-thawing cells in a dimethyl sulfoxide-based media and measuring viability via hemocytometry (n=3). Sterility testing on cell isolates was performed via BacT/ALERT iNST & iAST (n=2). Results Incubation time did not have a significant effect on the efficiency of bead-cell binding (Fig 1B). As the FerroBioTM protocol progressed, final cell purity and viability increased and became more consistent (Fig 1C, D). Additionally, over the same phases of process development, final cell recovery increased 4-fold, from 6.5%±5.9% to 28.1%±9.7%. Post-cryopreservation viability was 78.8%±3.3%. Sterility tests were negative for anaerobic and aerobic bacteria. Discussion By establishing that bead-cell incubation duration did not significantly influence outcomes, the incubation time was considerably reduced, bringing the total processing time to ~3hrs. That said, if an overnight incubation is preferable for an operator’s workflow, the data suggests the process could be flexible to meet such needs. Iterations of the process have also resulted in significant improvements to final cell recovery while maintaining high purity and viability. Additional work is being done to further improve recovery. The initial investigation of sterility and cryopreservation was encouraging, and assays for cell functionality are underway. Figure 1. (A) The FerroBioTM hardware, shown with a cartridge in which cell isolation occurs. (B) The proportion of cells that successfully bound magnetic beads as a function of bead-cell incubation time. Final cell (C) purity and (D) viability, shown at different phases of chronological process development. Statistics: GraphPad Prism 9, 1-way ANOVA followed by a Tukey’s HSD. * p < 0.05, **** p < 0.0001. Each data point corresponds to one CBU.