Rapid cell fractionation of whole immune organs using a novel microfluidic device

Abstract Separation and isolation of specific cell populations from solid immune organs are essential steps in immunological research. While antibody-labeling based cell sorting techniques, such as FACS, offer the possibility of isolating rare cells from a large background population, the cost and lengthy preparation time associated with these approaches remain burdensome for many researchers. Here, we report a rapid partitioning method that allows the separation of immune cells based on their size and density using a novel, spiral designed, microfluidic device (MFD) (Hou et al., 2016) Representing less than 0.5% of the total thymic cells, thymic epithelial cells (TECs) are critical for the generation of diverse T cell populations, essential for acquired immunity. Due to their rarity, separating TECs from the large pool of thymocytes is challenging. Using a MFD, we achieved substantial enrichment of TECs (up to 20X), high cell recovery (>2.5X of the traditional density gradient method) and excellent cell viability (>90%) within 7 minutes/thymus. High-purity TEC populations were obtained through further depletion of CD45+ thymic stromal cells using MACS. Thymus organoids reconstructed with TECs can support the development of both CD4+ and CD8+ T cells in vitro, proving the function of the isolated TECs. In addition, MFD could also be utilized to separate immune cell subtypes from spleen and lymph nodes. Notably, significant enrichment of CDllc+ MHCII+ dendritic cells (~15X) was achieved without RBC lysis and antibody labeling. In conclusion, our data suggest that the MFD partitioning method is efficient and cost-effective for rapid size-based separation of immune cells from primary and secondary immune organs for downstream applications.