Affinity Flow Fractionation for label-free cell sorting

Abstract

Capture and isolation of flowing cells from body fluids such as peripheral blood, bone marrow or pleural effusion has enormous implications in diagnosis, disease monitoring, and drug testing. However, in many situations the conventional methods of cell sorting are of limited use due to complex sample preparation steps, high costs, or low sensitivity. Drawing inspiration from nature, a novel platform technology for cell separation known as Affinity Flow Fractionation (AFF) was developed. AFF relies on interaction of cells with asymmetric patterns of weak adhesive molecules allowing for continuous sorting of cells with high purity without irreversible capture of cells. Cells are sorted in a single step, which is a significant advance over conventional immunocapture methods, especially for point-of-care and point-of-use applications. In this work, first, the interaction of cells under shear flow with asymmetric patterns of weak adhesive molecules was studied systematically to highlight the underlying mechanism of AFF at a phenomenological level. Next, an optimized separation device was fabricated and its performance was characterized using model cell lines. A detailed predictive mathematical model, which accounts for the major transport processes involved in cell separation by AFF, was developed and the results validated using experiments. Finally, AFF was applied for rapid isolation of neutrophils from blood, which is important for several applications where conventional procedures involve multiple steps and time-intense manual skills. It was demonstrated that asymmetric patterns of Pselectin, a weak adhesive molecule involved in cell trafficking, can directly draw neutrophils out of a continuously flowing stream of blood, with high purity (92%). As cells exhibiting non-specific adhesion are not drawn out of the flowing stream, an ultrahigh 400,000-fold enrichment of leukocytes over erythrocytes is achieved. Moreover, the sorted neutrophils remain viable, unaltered, and functionally intact. The lack of background erythrocytes enabled direct enumeration of neutrophils by a downstream detector, which could distinguish the activation state of neutrophils in blood. This method is compatible with capillary microfluidics and may find use in isolation of neutrophils for diagnosis of sepsis, genetic analysis, HLA typing, assessment of chemoreadiness, and other applications. Weak molecular interactions govern a large number of important physiological processes such as stem cell homing, inflammation, immune modulation and cancer metastasis. Yet, currently there are no effective technologies that can separate cells based on weak interactions alone. We believe, AFF would fulfill this un-met need in the area of cell sorting and enabling new discoveries. Keywords: Microfluidics, Cell sorting, cell rolling, selectin, blood, point-of-care, neutrophils.