Using Protein-Functionalized Microchannels for Stem Cell Separation

Abstract

Current hematopoietic stem cell (HSC) separation methods utilize immunomagnetic techniques where antibodies are conjugated to paramagnetic or iron-dextran particles so that the cells can be precipitated using a magnetic field. Antibodies are usually targeted against CD34 antigen, a known HSC marker that is lost as the cell matures into a terminally differentiated blood cell. While this method is able to produce stem cell purities greater than 80% in most cases, cell yield is usually low (<50%) and there is evidence to suggest that not all HSCs express CD34 and hence, cannot be selected using these methods. Our long-term aim is to move away from immunologic isolation methods and instead focus on general HSC behavior in the presence of specific proteins. Selectins are a group of adhesion molecules that have been shown to selectively retard HSC rolling in comparison to more mature blood cells so that, under the right conditions, HSC isolation from whole blood should be possible. Since this functional method is independent of specific HSC surface marker, it would enable isolation of all HSC sub-populations as well as improve the overall yield. We have used two geometries for HSC isolation using selectins. The first assembly was a commercially available parallel plate flow chamber with rectangular cross-section. This proved to be inefficient for cell separation due to the emergence of dead zones within the chamber and excessive cell accumulation along the tubing and fittings that comprised the inlets and outlets. We have evaluated this chamber and two redesigns using finite elements software. Ultimately we decided on a simpler approach — to replace the flow chamber with a capillary tube, which reduced unwanted cell accumulation due to gravity or dead zones while increasing the effective area for separation, and hence allowing for better HSC isolation.

P-Selectin–Coated Microtube for Enrichment of CD34+ Hematopoietic Stem and Progenitor Cells from Human Bone Marrow

“…Ϫ leukocytes on selectin surfaces indicated a possibility that this phenomenon could be exploited for the selective capture of HSPCs from blood flow by simply implanting selectin-coated stents into circulation (14,17 ). Supporting this hypothesis are the observations that selectins are implicated in the homing of HSPCs to bone marrow (12)(13)(14) and that sP ligand, PSGL-1, is expressed on HSPCs (15 ).…”

“…Selectins have been implicated in the homing of HSPCs to the bone marrow (12)(13)(14). HSPCs express at least 1 P-selectin (sP) ligand, sP glycoprotein ligand-1 (PSGL-1/CD162), and another selectin ligand, HCELL (15,16 ) Primitive CD34 ϩ HSPCs have been demonstrated to exhibit stronger rolling adhesion on selectins than mature CD34 -MNCs (14,17 ). We have exploited this differential rolling characteristic of CD34 ϩ HSPCs on selectins to capture and enrich them in flow.…”