A high-throughput microfluidic device based on controlled incremental filtration to enable centrifugation-free, low extracorporeal volume leukapheresis

Abstract

Leukapheresis, the extracorporeal separation of white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), is a life-saving procedure used for treating patients with cancer and other conditions, and as the initial step in the manufacturing of cellular and gene-based therapies. Well-tolerated by adults, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of standard centrifugation-based machines represents a particularly large fraction of these patients’ total blood volume. Here we describe a novel high-throughput microfluidic device (with a void volume of 0.4 mL) based on controlled incremental filtration (CIF) technology that could replace centrifugation for performing leukapheresis. The CIF device was tested extensively using whole blood from healthy volunteers at multiple hematocrits (5–30%) and flow rates (10–30 mL/min). In the flow-through regime, the CIF device separated WBCs with > 85% efficiency and 10–15% loss of RBCs and PLTs while processing whole blood diluted with saline to 10% hematocrit at a flow rate of 10 mL/min. In the recirculation regime, the CIF device demonstrated a similar level of separation performance, virtually depleting WBCs in the recirculating blood (~ 98% reduction) by the end of a 3.5-hour simulated leukapheresis procedure. Importantly, the device operated without clogging or decline in separation performance, with minimal activation of WBCs and PLTs and no measurable damage to RBCs. Compared to the typical parameters of centrifugation-based leukapheresis, the CIF device had a void volume at least 100-fold smaller, removed WBCs about twice as fast, and lost ~ 2–3-fold fewer PLTs, while operating at a flow rate compatible with the current practice. The hematocrit and flow rate at which the CIF device operated were significantly higher than previously published for other microfluidic cell separation methods. Finally, this study is the first to demonstrate a highly efficient separation of cells from recirculating blood using a microfluidic device. Overall, these findings suggest the feasibility of using high-throughput microfluidic cell separation technology to ultimately enable centrifugation-free, low-ECV leukapheresis. Such a capability would be particularly useful in young children, a vulnerable group of patients who are currently underserved.

Red blood cell rosetting enables size-based separation of specific lymphocyte subsets from blood in a microfluidic device

“…However, the recovery plateau was still below the theoretical maximum (∼95% for a device with FR = 20) likely because of the natural tendency of blood cells to build up near the posts of the device as described in our previous studies. 12,15,18 The overlap of values for devices with FR = 12 and 20 allowed us to confirm the effect of the increasing FR on recovery: for a given , CD3 + T cell recovery was higher for a CIF device with larger FR. For example, for a CIF device with = 1.70 × 10 −3 the recovery was 82.0 ± 1.6% at FR of 12 and 88.2 ± 3.9% at FR of 20.…”

“…As described in our previous work, is a reference value of f gap for a given central channel width ( = 130 μm for all devices discussed herein), the combination of which is known to produce a size cut-off that is well-suited to the intended application of the CIF device. 12,14,18 For a given , the higher the value of , the larger the size cut-off of the device. 12,14,18…”

“…12,14,18 For a given , the higher the value of , the larger the size cut-off of the device. 12,14,18…”

“…Although in this study we used one standalone CIF device to demonstrate the feasibility of the RESIZE method, CIF technology is inherently scalable and large-volume separations could be performed using many CIF devices multiplexed in parallel to achieve higher throughput (flow rates as high as 10–50 mL min −1 were demonstrated in our previous studies 12,18 ). Such a capability would be particularly useful for isolating cells of interest ( e.g.…”

Rapid, label‐free enrichment of lymphocytes in a closed system using a flow‐through microfluidic device

“…In our own previous studies, a syringe pump (or a hydrostatic pressure gradient created by gravity, or pressure applied to the input bag pneumatically) was used to drive the sample through the device because of its requirement for stable, oscillation-free flow. 13,34,42,44 Similarly, other groups have been utilizing various syringe pump-based approaches to minimize the pressure/flow oscillations which inevitably degrade the performance of most if not all microfluidic cell separation devices. 32 However, common syringe pumping methods are not strictly compatible with the closed-system processing required in the clinical setting.…”