Dielectrophoretic enrichment of live chemo-resistant circulating-like pancreatic cancer cells from media of drug-treated adherent cultures of solid tumors.

Due to low numbers of circulating tumor cells (CTCs) in liquid biopsies, there is much interest in enrichment of alternative circulating-like mesenchymal cancer cell subpopulations from in vitro tumor cultures for utilization within molecular profiling and drug screening. Viable cancer cells that are released into the media of drug-treated adherent cancer cell cultures exhibit anoikis resistance or anchorage-independent survival away from their extracellular matrix with nutrient sources and waste sinks, which serves as a pre-requisite for metastasis. The enrichment of these cell subpopulations from tumor cultures can potentially serve as an in vitro source of circulating-like cancer cells with greater potential for scale-up in comparison with CTCs. However, these live circulating-like cancer cell subpopulations exhibit size overlaps with necrotic and apoptotic cells in the culture media, which makes it challenging to selectively enrich them, while maintaining them in their suspended state. We present optimization of a flowthrough high frequency (1 MHz) positive dielectrophoresis (pDEP) device with sequential 3D field non-uniformities that enables enrichment of the live chemo-resistant circulating cancer cell subpopulation from an in vitro culture of metastatic patient-derived pancreatic tumor cells. Central to this strategy is the utilization of single-cell impedance cytometry with gates set by supervised machine learning, to optimize the frequency for pDEP, so that live circulating cells are selected based on multiple biophysical metrics, including membrane physiology, cytoplasmic conductivity and cell size, which is not possible using deterministic lateral displacement that is solely based on cell size. Using typical drug-treated samples with low levels of live circulating cells (<3%), we present pDEP enrichment of the target subpopulation to ∼44% levels within 20 minutes, while rejecting >90% of dead cells. This strategy of utilizing single-cell impedance cytometry to guide the optimization of dielectrophoresis has implications for other complex biological samples.

Microfluidic Blood Separation: Key Technologies and Critical Figures of Merit.

Blood is a complex sample comprised mostly of plasma, red blood cells (RBCs), and other cells whose concentrations correlate to physiological or pathological health conditions. There are also many blood-circulating biomarkers, such as circulating tumor cells (CTCs) and various pathogens, that can be used as measurands to diagnose certain diseases. Microfluidic devices are attractive analytical tools for separating blood components in point-of-care (POC) applications. These platforms have the potential advantage of, among other features, being compact and portable. These features can eventually be exploited in clinics and rapid tests performed in households and low-income scenarios. Microfluidic systems have the added benefit of only needing small volumes of blood drawn from patients (from nanoliters to milliliters) while integrating (within the devices) the steps required before detecting analytes. Hence, these systems will reduce the associated costs of purifying blood components of interest (e.g., specific groups of cells or blood biomarkers) for studying and quantifying collected blood fractions. The microfluidic blood separation field has grown since the 2000s, and important advances have been reported in the last few years. Nonetheless, real POC microfluidic blood separation platforms are still elusive. A widespread consensus on what key figures of merit should be reported to assess the quality and yield of these platforms has not been achieved. Knowing what parameters should be reported for microfluidic blood separations will help achieve that consensus and establish a clear road map to promote further commercialization of these devices and attain real POC applications. This review provides an overview of the separation techniques currently used to separate blood components for higher throughput separations (number of cells or particles per minute). We present a summary of the critical parameters that should be considered when designing such devices and the figures of merit that should be explicitly reported when presenting a device's separation capabilities. Ultimately, reporting the relevant figures of merit will benefit this growing community and help pave the road toward commercialization of these microfluidic systems.

Multichannel impedance cytometry downstream of cell separation by deterministic lateral displacement to quantify macrophage enrichment in heterogeneous samples.

The integration of on-chip biophysical cytometry downstream of microfluidic enrichment for inline monitoring of phenotypic and separation metrics at single-cell sensitivity can allow for active control of separation and its application to versatile sample sets. We present integration of impedance cytometry downstream of cell separation by deterministic lateral displacement (DLD) for enrichment of activated macrophages from a heterogeneous sample, without the problems of biased sample loss and sample dilution caused by off-chip analysis. This required designs to match cell/particle flow rates from DLD separation into the confined single-cell impedance cytometry stage, the balancing of flow resistances across the separation array width to maintain unidirectionality, and the utilization of co-flowing beads as calibrated internal standards for inline assessment of DLD separation and for impedance data normalization. Using a heterogeneous sample with un-activated and activated macrophages, wherein macrophage polarization during activation causes cell size enlargement, on-chip impedance cytometry is used to validate DLD enrichment of the activated subpopulation at the displaced outlet, based on the multiparametric characteristics of cell size distribution and impedance phase metrics. This hybrid platform can monitor separation of specific subpopulations from cellular samples with wide size distributions, for active operational control and enhanced sample versatility.

Biophysical quantification of reorganization dynamics of human pancreatic islets during co-culture with adipose-derived stem cells.

Islet transplantation is a potential therapy for type 1 diabetes, but it is expensive due to limited pancreas donor numbers and the variability in islet quality. The latter is often addressed by co-culture of harvested islets with stem cells to promote in vitro remodeling of their basement membrane and enable expression of angiogenic factors for enhancing vascularization. However, given the heterogeneity in islet size, shape and function, there is a need for metrics to assess the reorganization dynamics of single islets over the co-culture period. Based on shape-evolution of individual multi-cell aggregates formed during co-culture of human islets with adipose derived stem cells and the pressures required for their bypass through microfluidic constrictions, we present size-normalized biomechanical metrics for monitoring the reorganization. Aggregates below a threshold size exhibit faster reorganization, as evident from rise in their biomechanical opacity and tightening of their size distribution, but this size threshold increases over culture time to include a greater proportion of the aggregates. Such biomechanical metrics can quantify the subpopulation of reorganized aggregates by distinguishing them versus those with incomplete reorganization, over various timepoints during the co-culture.

On-chip microfluidic buffer swap of biological samples in-line with downstream dielectrophoresis.

Microfluidic cell enrichment by dielectrophoresis, based on biophysical and electrophysiology phenotypes, requires that cells be resuspended from their physiological media into a lower conductivity buffer for enhancing force fields and enabling the dielectric contrast needed for separation. To ensure that sensitive cells are not subject to centrifugation for resuspension and spend minimal time outside of their culture media, we present an on-chip microfluidic strategy for swapping cells into media tailored for dielectrophoresis. This strategy transfers cells from physiological media into a 100-fold lower conductivity media by using tangential flows of low media conductivity at 200-fold higher flow rate versus sample flow to promote ion diffusion over the length of a straight channel architecture that maintains laminarity of the flow-focused sample and minimizes cell dispersion across streamlines. Serpentine channels are used downstream from the flow-focusing region to modulate hydrodynamic resistance of the central sample outlet versus flanking outlets that remove excess buffer, so that cell streamlines are collected in the exchanged buffer with minimal dilution in cell numbers and at flow rates that support dielectrophoresis. We envision integration of this on-chip sample preparation platform prior to or post-dielectrophoresis, in-line with on-chip monitoring of the outlet sample for metrics of media conductivity, cell velocity, cell viability, cell position, and collected cell numbers, so that the cell flow rate and streamlines can be tailored for enabling dielectrophoretic separations from heterogeneous samples.

Self-aligned sequential lateral field non-uniformities over channel depth for high throughput dielectrophoretic cell deflection.

Dielectrophoresis (DEP) enables the separation of cells based on subtle subcellular phenotypic differences by controlling the frequency of the applied field. However, current electrode-based geometries extend over a limited depth of the sample channel, thereby reducing the throughput of the manipulated sample (sub-µL min-1 flow rates and <105 cells per mL). We present a flow through device with self-aligned sequential field non-uniformities extending laterally across the sample channel width (100 µm) that are created by metal patterned over the entire depth (50 µm) of the sample channel sidewall using a single lithography step. This enables single-cell streamlines to undergo progressive DEP deflection with minimal dependence on the cell starting position, its orientation versus the field and intercellular interactions. Phenotype-specific cell separation is validated (>µL min-1 flow and >106 cells per mL) using heterogeneous samples of healthy and glutaraldehyde-fixed red blood cells, with single-cell impedance cytometry showing that the DEP collected fractions are intact and exhibit electrical opacity differences consistent with their capacitance-based DEP crossover frequency. This geometry can address the vision of an "all electric" selective cell isolation and cytometry system for quantifying phenotypic heterogeneity of cellular systems.

High-throughput dynamical analysis of dielectrophoretic frequency dispersion of single cells based on deflected flow streamlines.

Phenotypic quantification of cells based on their plasma membrane capacitance and cytoplasmic conductivity, as determined by their dielectrophoretic frequency dispersion, is often used as a marker for their biological function. However, due to the prevalence of phenotypic heterogeneity in many biological systems of interest, there is a need for methods capable of determining the dielectrophoretic dispersion of single cells at high throughput and without the need for sample dilution. We present a microfluidic device methodology wherein localized constrictions in the microchannel are used to enhance the field delivered by adjoining planar electrodes, so that the dielectrophoresis level and direction on flow-focused cells can be determined on each traversing cell in a high-throughput manner based on their deflected flow streamlines. Using a sample of human red blood cells diluted to 2.25 × 108 cells/mL, the dielectrophoretic translation of single cells traversing at a flow rate of 1.68 µL/min is measured at a throughput of 1.1 × 105 cells/min, to distinguish positive versus negative dielectrophoresis and determine their crossover frequency in media of differing conductivity for validation of the computed membrane capacitance to that from prior methods. We envision application of this dynamic dielectrophoresis (Dy-DEP) method towards high-throughput measurement of the dielectric dispersion of single cells to stratify phenotypic heterogeneity of a particular sample based on their DEP crossover frequency, without the need for significant sample dilution. Grapical abstract.