(489s) Hematopoietic Stem Cell Enrichment with Microfluidic Stem Cells

Stem cells have been a focus of research due to their great promise to develop new treatments for human diseases such as Parkinson's disease, diabetes, and cancers. Motivated by this promise, enormous efforts have been made in fields ranging from identification and enrichment of stem cells, to understanding their underlying biology. Hematopoietic stem cells (HSCs) are adult stem cells found in bone marrow that give rise to all the blood cell types. Significant progress has been achieved in identification and enrichment of HSCs in recent years. For example, CD34 and other lineage markers have been discovered that identify HSCs in blood and techniques such as fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS), have been developed to enrich HSCs for blood transplantation. However, current enrichment techniques have limitations such as low purity, low throughput, and poor reproducibility in yield. Here we propose an immunoaffinity based microchip with specific antibody coated on its surface to enrich HSCs with high performance. Same as FACS and MACS, we use CD34, a heavily glycosylated trans-membrane protein that is expressed on HSCs but lost upon their differentiation to mature blood cells, as the bio-marker for HSCs. The specificity of the interaction between anti-CD34 antibody on the surface of the microchip and CD34 on HSC ensures high purity of the enrichment. The large surface area-to-volume ratio of microfluidic device and the versatility of microfluidics for scaling out to large surface area, such as integrating microposts, help achieve high throughput. In addition to the high purity and throughput, our microchip technique significantly reduces the pre- and post- processing steps required for FACS and MACS to minimize the losses of cell viability and ?stemness?. In this microchip technique, since cells are captured in a dynamic flow. Thus flow rate and shear force profile are the key factors for the overall performance. Optimum shear forces for capturing and releasing target cells were firstly studied in a parallel shear force chamber. The purity and throughput of the enrichment was tested in a model system, where a mixture of two phenotypically pure cell lines at same total cell count (108 cells/ml) and same concentration (1%) of CD34+ cells as bone marrow mobilized human peripheral blood was prepared and the enrichment of fluorescent labeled CD34+ cells were evaluated. Results showed very good enrichment performance in this model system. We successfully achieved 99% of purity even at low shear force (0.5 dynes/cm2). The estimated throughput of the current two-dimensional design was 106 cells/s, and the number would be largely increased after integrating three dimensional microposts. Furthermore, our preliminary results with mobilized human peripheral blood were promising as well, though the complexity of raw blood sample raised some problems such as lower affinity to the surface due to lower expression of CD34 on HSCs, a higher degree of non-specific binding from the varied receptors on all different blood cell types, and a high variation of sample quality due to the different disease condition of individual patient. But we are on the way of solving these problems and we already achieved a 20% purity of CD34+ cells after enrichment from a sample with 1% CD34+ cells originally. We are also testing the differentiation capacity of the enriched CD34+ cells with colony forming assay to verify the ?stemness? of the enriched population.