John P. Wikswo

Virtual electrodes in cardiac tissue: a common mechanism for anodal and cathodal stimulation.

Traditional cable analyses cannot explain complex patterns of excitation in cardiac tissue with unipolar, extracellular anodal, or cathodal stimuli. Epifluorescence imaging of the transmembrane potential during and after stimulation of both refractory and excitable tissue shows distinctive regions of simultaneous depolarization and hyperpolarization during stimulation that act as virtual cathodes and anodes. The results confirm bidomain model predictions that the onset (make) of a stimulus induces propagation from the virtual cathode, whereas stimulus termination (break) induces it from the virtual anode. In make stimulation, the virtual anode can delay activation of the underlying tissue, whereas in break stimulation this occurs under the virtual cathode. Thus make and break stimulations in cardiac tissue have a common mechanism that is the result of differences in the electrical anisotropy of the intracellular and extracellular spaces and provides clear proof of the validity of the bidomain model.

Current injection into a two-dimensional anisotropic bidomain

A two-dimensional sheet of anisotropic cardiac tissue is represented with the bidomain model, and the finite element method is used to solve the bidomain equations. When the anisotropy ratios of the intracellular and extracellular spaces are not equal, the injection of current into the tissue induces a transmembrane potential that has a complicated spatial dependence, including adjacent regions of depolarized and hyperpolarized tissue. This behavior may have important implications for the electrical stimulation of cardiac tissue and for defibrillation.

Microfluidic Single-Cell Array Cytometry for the Analysis of Tumor Apoptosis

Limitations imposed by conventional analytical technologies for cell biology, such as flow cytometry or microplate imaging, are often prohibitive for the kinetic analysis of single-cell responses to therapeutic compounds. In this paper, we describe the application of a microfluidic array to the real-time screening of anticancer drugs against arrays of single cells. The microfluidic platform comprises an array of micromechanical traps, designed to passively corral individual nonadherent cells. This platform, fabricated in the biologically compatible elastomer poly(dimethylsiloxane), PDMS, enables hydrodynamic trapping of cells in low shear stress zones, enabling time-lapse studies of nonadherent hematopoietic cells. Results indicate that these live-cell, microfluidic microarrays can be readily applied to kinetic analysis of investigational anticancer agents in hematopoietic cancer cells, providing new opportunities for automated microarray cytometry and higher-throughput screening. We also demonstrate the ability to quantify on-chip the anticancer drug induced apoptosis. Specifically, we show that with small numbers of trapped cells (approximately 300) under careful serial observation we can achieve results with only slightly greater statistical spread than can be obtained with single-pass flow cytometer measurements of 15,000-30,000 cells.

SQUID magnetometers for biomagnetism and nondestructive testing: important questions and initial answers

For two decades, academic and industrial researchers worldwide have used SQUID magnetometers to measure magnetic signals from the heart, brain, lungs, liver, nerves, skeletal muscle, stomach, intestines, eyes, and other organs, and have invested heavily in developing and promoting this technology. While there are as yet few accepted clinical applications of SQUIDs, various trends are encouraging. The introduction of SQUIDs to the nondestructive testing (NDT) of aircraft and other structural systems and materials is following a similar course: most of the effort is directed towards instrumentation development and demonstrations in simple systems, and instruments suitable for specific commercial applications are just now being prototyped. To assess the potential of either technology, it is useful to ask critical questions: why are we doing this, what have we learned so far, how easy is it, what does it cost, how might we best utilize advances in digital SQUIDs and high-temperature superconductivity, and what can competing technologies provide? Answers to such questions can help identify those specific technological niches for which SQUIDs are uniquely suited, and guide the optimization of SQUID systems that are targeted for particular NDT or biomagnetic measurements.<<ETX>>

Scaling and systems biology for integrating multiple organs-on-a-chip.

Coupled systems of in vitro microfabricated organs-on-a-chip containing small populations of human cells are being developed to address the formidable pharmacological and physiological gaps between monolayer cell cultures, animal models, and humans that severely limit the speed and efficiency of drug development. These gaps present challenges not only in tissue and microfluidic engineering, but also in systems biology: how does one model, test, and learn about the communication and control of biological systems with individual organs-on-chips that are one-thousandth or one-millionth of the size of adult organs, or even smaller, i.e., organs for a milliHuman (mHu) or microHuman (μHu)? Allometric scaling that describes inter-species variation of organ size and properties provides some guidance, but given the desire to utilize these systems to extend and validate human pharmacokinetic and pharmacodynamic (PK/PD) models in support of drug discovery and development, it is more appropriate to scale each organ functionally to ensure that it makes the suitable physiological contribution to the coupled system. The desire to recapitulate the complex organ-organ interactions that result from factors in the blood and lymph places a severe constraint on the total circulating fluid (~5 mL for a mHu and ~5 μL for a μHu) and hence on the pumps, valves, and analytical instruments required to maintain and study these systems. Scaling arguments also provide guidance on the design of a universal cell-culture medium, typically without red blood cells. This review presents several examples of scaling arguments and discusses steps that should ensure the success of this endeavour.

SQUIDs for nondestructive evaluation

We attempt a comprehensive review of all published research in nondestructive evaluation (NDE) performed with the superconducting quantum interference device (SQUID) magnetometer since the first work was reported in the mid-1980s. The SQUID is the most sensitive detector of magnetic flux known. The energy sensitivity of the SQUID may make it the most sensitive detector of any kind. The research on SQUIDs for NDE is based on the promise of that sensitivity and on the various other desirable properties developed for SQUID instrumentation in biomagnetism and other fields. The sensitivity of SQUID instruments down to very low frequencies allows them to function as eddy-current sensors with unparalleled depth resolution, and to image the static magnetization of paramagnetic materials and the flow of near-dc corrosion currents. The wide dynamic range of the SQUID makes it possible to image defects in steel structures and to measure the magnetomechanical behaviour of ferromagnetic materials with high sensitivity. In the last decade SQUID instrumentation designed specifically for NDE has appeared and improved the spatial resolution of most work to roughly 1 mm, with promise of another order of magnitude improvement within the next five years. Algorithms for flaw detection and image deconvolution have begun to flourish. With many talented, industrious people in the field, the future of SQUID NDE looks bright, provided the crucial first niche can be found.

Microfluidic single cell arrays to interrogate signalling dynamics of individual, patient-derived hematopoietic stem cells

Stem cells hold great promise as a means of treating otherwise incurable, degenerative diseases due to their ability both to self-renew and differentiate. However, stem cell damage can also play a role in the disease with the formation of solid tumors and leukaemias such as chronic myeloid leukaemia (CML), a hematopoietic stem cell (HSC) disorder. Despite recent medical advances, CML remains incurable by drug therapy. Understanding the mechanisms which govern chemoresistance of individual stem cell leukaemias may therefore require analysis at the single cell level. This task is not trivial using current technologies given that isolating HSCs is difficult, expensive, and inefficient due to low cell yield from patients. In addition, hematopoietic cells are largely non-adherent and thus difficult to study over time using conventional cell culture techniques. Hence, there is a need for new microfluidic platforms that allow the functional interrogation of hundreds of non-adherent single cells in parallel. We demonstrate the ability to perform assays, normally performed on the macroscopic scale, within the microfluidic platform using minimal reagents and low numbers of primary cells. We investigated normal and CML stem cell responses to the tyrosine kinase inhibitor, dasatinib, a drug approved for the treatment of CML. Dynamic, on-chip three-color cell viability assays revealed that differences in the responses of normal and CML stem/progenitor cells to dasatinib were observed even in the early phases of exposure, during which time normal cells exhibit a significantly elevated cell death rate, as compared to both controls and CML cells. Further studies show that dasatinib does, however, markedly reduce CML stem/progenitor cell migration in situ.

Quatrefoil reentry in myocardium: an optical imaging study of the induction mechanism.

Quatrefoil Reentry in Myocardium. Introduction: The “critical point hypothesis” for induction of ventricular fibrillation has previously been extended to infer the coexistence of four critical points, and hence four simultaneous spiral reentries or a quatrefoil reentry, resulting from only one premature stimulus delivered to the same location as the pacing stimulus. An optical imaging technique was used to explore its existence and to study the induction mechanism of this peculiar reentry pattern.

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel.

Deciphering the signaling pathways that govern stimulation of naïve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of naïve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.

Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor.

The blood-brain barrier (BBB) is a critical structure that serves as the gatekeeper between the central nervous system and the rest of the body. It is the responsibility of the BBB to facilitate the entry of required nutrients into the brain and to exclude potentially harmful compounds; however, this complex structure has remained difficult to model faithfully in vitro. Accurate in vitro models are necessary for understanding how the BBB forms and functions, as well as for evaluating drug and toxin penetration across the barrier. Many previous models have failed to support all the cell types involved in the BBB formation and/or lacked the flow-created shear forces needed for mature tight junction formation. To address these issues and to help establish a more faithful in vitro model of the BBB, we have designed and fabricated a microfluidic device that is comprised of both a vascular chamber and a brain chamber separated by a porous membrane. This design allows for cell-to-cell communication between endothelial cells, astrocytes, and pericytes and independent perfusion of both compartments separated by the membrane. This NeuroVascular Unit (NVU) represents approximately one-millionth of the human brain, and hence, has sufficient cell mass to support a breadth of analytical measurements. The NVU has been validated with both fluorescein isothiocyanate (FITC)-dextran diffusion and transendothelial electrical resistance. The NVU has enabled in vitro modeling of the BBB using all human cell types and sampling effluent from both sides of the barrier.