A novel method for enriching CD34+ hematopoietic cells from adult bone marrow using immobilized selectins

Hematopoietic stem cell transplantation (HSCT) is used to treat both malignant and non-malignant diseases and enrichment of the hematopoietic stem and progenitor cells (HSPCs) has the potential to reduce the likelihood of graft vs. host disease or relapse, potentially fatal complications associated with the therapy. Current commercial HSPC isolation technologies rely solely on the CD34 surface marker and while they have proven to be invaluable, HSCT that utilize very pure samples of CD34+ HSPCs is associated with higher incidences of failure and longer time for engraftment. Since HSPCs must interact with selectins to successfully home to the marrow, a functional technique to isolate HSPCs based on their interaction with immobilized selectins might improve HSCT by simultaneously purifying the HSPCs and selecting only those cells with the highest probability of homing to the marrow.;To assess the behavior of adult bone marrow (ABM) cells on immobilized selectins, purified CD34+ and CD34- ABM cells were perfused over immobilized P-, E- and L-selectin-IgG at physiologic wall shear stresses. CD34+ ABM cells generally exhibited lower rolling velocities and higher retention than CD34- ABM cells on all three selectins. For the experimental conditions used, it is theoretically possible to increase the purity of CD34+ ABM cells starting from 1-5% to between 5.2%-36.1%. Additionally, hybrid surfaces of immobilized antibody and selectin were used to create differences in rolling velocities between CD34+ KG1a cells and CD34- HL60 cells, acute myeloid leukemia cell lines with no inherent differences in rolling velocity on P-selectin-IgG alone. The relationship between yield and purity is reciprocal, but optimal cell enrichment should be achievable within 10 minutes, much faster than most current commercially available systems.;Devices that might enrich CD34+ ABM cells by capitalizing on the differential rolling velocity remain an unfeasible option due to geometric and manufacturing constraints. Laboratory-scale testing of the efficiency of P-selectin-mediated CD34+ ABM cell enrichment exploited the differential cell retention using a cylindrical flow chamber and various wall shear stresses and perfusion times. Short perfusion times and low wall shear stresses consistently resulted in the highest CD34+ ABM cell yield, and the enrichment procedure could be completed in a short time (≤ 15 minutes), however a threshold for the enrichment was observed, generally ∼ 3-fold. Although CD34+ ABM cells enriched using Dynabeads immunomagnetic cell enrichment technique had higher purities and fold enrichment, the yield was lower than P-selectin-mediated enrichment depending on the flow conditions used.;Gravitational sedimentation was used to recruit cells to the surface to ensure maximum cell accumulation and interaction, but this reduced the efficiency of the device by at least 50% since only the lower surfaces were used.;Experimental study and computational fluid dynamics (CFD) was used to investigate how three tubing geometries (straight, helical and pinched), two P-selectin-IgG bulk concentrations (0.5 or 2.5 mug/mL), and two inlet volumetric flow rates (1.123 or 6.741 mL/min) influenced yield and efficiency of recruitment of CD34+ KG1a cells. CFD results predicted that the pinched tube should consistently recruit more cells than the straight or helix, but experimental results were lower than expected, and likely due to the high wall shear stresses generated within this geometry. Similar experiments were performed using micro-pillars in a rectangular flow channel, to simulate expansions and contractions in the pinched tubes without causing the high increases in wall shear stresses. There was high cell recruitment and robust fluid recirculation between pillars, which was subsequently confirmed by CFD.;Selectin-mediated hematopoietic cell enrichment has the potential to improve HSCT by selecting the cells that are most likely to complete the homing and engraftment process. Further characterization of the enriched cells as well as in vivo testing to prove their repopulating capabilities are necessary next steps in developing a useable product.