Purification of cell subpopulations via immiscible filtration assisted by surface tension (IFAST)

Abstract

The selective isolation of a sub-population of cells from a larger, mixed population is a critical preparatory process to many biomedical assays. Here, we present a new cell isolation platform with a unique set of advantages over existing devices. Our technology, termed Immiscible Filtration Assisted by Surface Tension, exploits physical phenomena associated with the microscale to establish fluidic barriers composed of immiscible liquids. By attaching magnetically-responsive particles to a target cell population via immunocapture, we can selectively transport this population across the immiscible barrier and into a separate aqueous solution. The high interfacial energy associated with the immiscible phase / aqueous phase boundaries prevents unwanted cells or other contaminants from inadvertently crossing the immiscible phase. We have demonstrated, using fluorescent particles, stromal cells, and whole blood as “background”, that we can successfully isolate ~70% of a target breast cancer cell population with an average purity of >80%. Increased purity was obtained by coupling two immiscible barriers in series, a modification that only slightly increases operational complexity. Furthermore, several samples can be processed in parallel batches in a near-instantaneous manner without the requirement of any washing, which can cause dilution (negative selection) or significant uncontrolled loss (positive selection) of target cells. Finally, cells were observed to remain viable and proliferative following traverse through the immiscible phase, indicating that this process is suitable for a variety of downstream assays, including those requiring intact living cells.

Integrated Analysis of Multiple Biomarkers from Circulating Tumor Cells Enabled by Exclusion-Based Analyte Isolation

“…One of the most significant hurdles is analyte loss that occurs during standard cell staining or nucleic acid extraction when isolating rare cell populations (12–14). Most systems are designed for a single endpoint while other multiplexed analyses are add-ons that increase sample loss and impair throughput through transfer of samples or analytes.…”

“…ESP utilizes microscale constrictions to stabilize the positioning of immiscible fluids side-by-side, creating an immiscible barrier between two aqueous fluids. By pulling paramagnetic particles (PMPs) bound to specific analytes of interest through these immiscible barriers, we isolate the analytes from a general sample without dilution, splitting or perturbation (13, 14, 19–23). The VERSA (Versatile Exclusion-Based Rare Sample Analysis) was designed to use ESP technology for CTC isolation and multi-endpoint analyses (13, 14, 19–22, 24–26) thus achieving modularity without compromising each individual assay.…”

Microfluidic sample preparation for diagnostic cytopathology

“…As with traditional centrifugation – but with much smaller volumes – this approach allows for separation of plasma from whole blood and the subsequent mixing of the plasma with lyophilized reagents for detection 79 . A recent innovation that works most effectively with microscale volumes separates cells specifically bound to magnetic micro beads by a surface antigen by, instead of moving fluid, passing the bead-bound cells themselves through an immiscible phase into a second wash volume 80 .…”

Liquid carry-over in an injection moulded all-polymer chip system for immiscible phase magnetic bead-based solid-phase extraction

“…This is done by transporting the target-MB cluster through an immiscible phase, such as oil [4][5][6], wax [4,7,8], or air [8], often using an external permanent magnet. Such systems have been demonstrated for a number of applications including enzyme linked immunosorbent assays [6,9,10], nucleic acid extraction [4,7,8,[11][12][13][14], protein extraction [8], and cell extraction [15,16].…”