Michael D. Vahey

Microfluidic arrays for logarithmically perfused embryonic stem cell culture

We present a microfluidic device for culturing adherent cells over a logarithmic range of flow rates. The device sets flow rates through four separate cell-culture chambers using syringe-driven flow and a network of fluidic resistances. The design is easy to fabricate with no on-chip valves and is scalable both in the number of culture chambers as well as in the range of applied flow rates. Using particle velocimetry, we have characterized the flow-rate range. We have also demonstrated an extension of the design that combines the logarithmic flow-rate functionality with a logarithmic concentration gradient across the array. Using fluorescence measurements we have verified that a logarithmic concentration gradient was established in the extended device. Compared with static cell culture, both devices enable greater control over the soluble microenvironment by controlling the transport of molecules to and away from the cells. This approach is particularly relevant for cell types such as embryonic stem cells (ESCs) which are especially sensitive to the microenvironment. We have demonstrated for the first time culture of murine ESCs (mESCs) in continuous, logarithmically scaled perfusion for 4 days, with flow rates varying >300x across the array. Cells grown in the slowest flow rate did not proliferate, while colonies grown in higher flow rates exhibited healthy round morphology. We have also demonstrated logarithmically scaled continuous perfusion culture of 3T3 fibroblasts for 3 days, with proliferation at all flow rates except the slowest rate.

High-Throughput Cell and Particle Characterization Using Isodielectric Separation

Separations can be broadly categorized as preparative, where the objective is to extract purified quantities of a sample from a complex mixture, or analytic, where the goal is to determine and quantify the contents of the original mixture. Here we demonstrate the application of a new microfluidic separation method, isodielectric separation (IDS), to a range of analytic separations involving cells and particles spanning several orders of magnitude in volume and electrical conductivity. In IDS, cells are dielectrophoretically concentrated to the region along an electrical conductivity gradient where their polarizability vanishes; by measuring this position--the isodielectric point (IDP)--as operating conditions such as the frequency and voltage of the applied electric field are varied, we are able to sort cells or particles with distinct IDPs while simultaneously characterizing their electrical properties. We apply this technique to measure the electrical properties of polystyrene microspheres, viable and nonviable cells of the budding yeast Saccharomyces cerevisiae , and murine pro B cells, including how these electrical properties vary with the electrical conductivity of the surrounding solvent.

Microfluidic genome-wide profiling of intrinsic electrical properties in Saccharomyces cerevisiae.

Methods to analyze the intrinsic physical properties of cells - for example, size, density, rigidity, or electrical properties - are an active area of interest in the microfluidics community. Although the physical properties of cells are determined at a fundamental level by gene expression, the relationship between the two remains exceptionally complex and poorly characterized, limiting the adoption of intrinsic separation technologies. To improve our current understanding of how a cells genotype maps to a measurable physical characteristic and quantitatively investigate the potential of using these characteristics as biomarkers, we have developed a novel screen that combines microfluidic cell sorting with high-throughput sequencing and the haploid yeast deletion library to identify genes whose functions modulate one such characteristic - intrinsic electrical properties. Using this screen, we are able to establish a high-content electrical profile of the haploid yeast gene deletion strains. We find that individual genetic deletions can appreciably alter the electrical properties of cells, affecting ~10% of the 4432 gene deletion strains screened. Additionally, we find that gene deletions affecting electrical properties in specific ways (i.e. increasing or decreasing effective conductivity at higher or lower electric field frequencies) are strongly associated with an enriched subset of fundamental biological processes that can be traced to specific pathways and complexes. The screening approach demonstrated here and the attendant results are immediately applicable to the intrinsic separations community.

Emergent behavior in particle-laden microfluidic systems informs strategies for improving cell and particle separations.

Colloidal particles placed in an energy landscape interact with each other, giving rise to complex dynamic behavior that affects the ability to process and manipulate suspensions of these particles. Propagating across scales ranging from the local behavior of 10s of particles to non-local behavior encompassing >10(6) particles, these particle interactions are pervasive and challenging to describe quantitatively, especially in the confined environments typical of microfluidic devices. To better understand the effects of particle interactions in this context, we have performed experiments and simulations involving a simple microfluidic device in which hydrodynamic and electrostatic forces are leveraged to concentrate and separate particle mixtures. These investigations reveal the mechanisms underlying the dynamic patterns formed by micron-scale particles as they impinge on a dielectrophoretic force barrier: their tendency to aggregate and recirculate under constant operating conditions, and to reorganize when the operating conditions are changed. The emergent behaviors of these ensembles of interacting particles exhibit features of dynamical frustration and cooperativity that suggest non-intuitive strategies for concentrating and sorting suspensions. Finally, we present a simple analytic model based on hydrodynamic coupling that captures important features of strongly interacting particle suspensions.

Isodielectric separation and analysis of cells.

Measuring the electrical properties of a cell provides a fast and accessible means of identifying or characterizing cells whose biological state differs from the population as a whole. This chapter describes a microfluidic method for characterizing the electrical properties of cells based upon their convergence to equilibrium in an electrical conductivity gradient. The method, called isodielectric separation, uses the dielectrophoretic force induced on polarizable objects in spatially nonuniform electric fields to deflect cells to the point in the conductivity gradient where their polarization charge vanishes. This equilibrium position encodes the cells electrical properties and can be used to identify cells that are electrically distinct from a background population, to determine the extent of this difference, and to physically isolate them for further study.

Handheld 2-channel impedimetric cell counting system with embedded real-time processing

Lab-on-a-chip systems have been attracting a growing attention for the perspective of miniaturization and portability of bio-chemical assays. Here we present a the design and characterization of a miniaturized, USB-powered, self-contained, 2-channel instrument for impedance sensing, suitable for label-free tracking and real-time detection of cells flowing in microfluidic channels. This original circuit features a signal generator based on a direct digital synthesizer, a transimpedance amplifier, an integrated square-wave lock-in coupled to a Σ▵ ADC converter, and a digital processing platform. Real-time automatic peak detection on two channels is implemented in a FPGA. System functionality has been tested with an electronic resistance modulator to simulate 1% impedance variation produced by cells, reaching a time resolution of 50μs (enabling a count rate of 2000 events/s) with an applied voltage as low as 200mV. Biological experiments have been carried out counting yeast cells. Statistical analysis of events is in agreement with the expected amplitude and time distributions. 2-channel yeast counting has been performed with concomitant dielectrophoretic cell separation, showing that this novel and ultra compact sensing system, thanks to the selectivity of the lock-in detector, is compatible with other AC electrical fields applied to the device.