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Whole cell assays for compound detection and drug screening have become an increasingly attractive approach that achieves a functional response without the traditional high costs of animal studies. Cardio-active pharmaceutical screening is particularly wellsuited to cell-based approaches, as myocytes have the ability to generate their own electrical signature ñ the cardiac action potential. However, several subtleties complicate the design of systems relying on spontaneous action potentials: first, spontaneous activity is highly dependent upon environmental factors such as temperature and pH; second, compounds may inhibit spontaneity while the tissue remains excitable; third, some tissues spontaneously depolarize at very slow rates, making assays slow and signal averaging for SNR improvement time-consuming; and lastly, not all cardiac cell preparations continually exhibit spontaneous activity. Electrical stimulation affords the investigator a means by which to elicit propagated action potentials that might otherwise be absent, at a controlled rate. Furthermore, stimulation itself confers certain advantages which are absent in spontaneous cultures, including the ability to control the beat rate of the culture precisely, and with it rate-dependent action potential duration changes, the ability to assess excitability via stimulation threshold and do so at any relevant rate, and the ability to assess the refractory period. While traditional techniques using manual or computer-controlled open-loop stimulators can begin to address these questions, threshold determination is slow, accuracy is limited, transient or dynamic effects are missed, and pacing itself may generate undesired and unobserved physiological responses. In addition, many traditional stimulation protocols measure threshold, typically at the outset, and add a safety factor, assuming modulation of threshold remains within those limits, while drugs that drastically alter excitability may break this assumption.

This dissertation presents an electrical stimulation system that utilizes a closed-loop algorithm to solve many of these problems. The system achieves closed-loop stimulation of clonal HL-1 cardiomyocytes cultured on planar microelectrode arrays, enabling culturing, stimulation, and analysis over a period of many days. Recording of electrical field potentials evoked by stimulation through electrodes on the same substrate is made possible by stimulus artifact extraction algorithms, which reveal and allow detection of action potentials which could otherwise be undetectable. A novel closed-loop algorithm that is tolerant of the high quantization and low data rates inherent in this cell-based system was designed to enable feedback using the efficacy of stimulation, although feedback of any quantifiable physiological parameter is possible. This feedback enables real-time and long-term monitoring of the stimulation threshold that achieves complete or partial capture of the cell culture and permits real-time monitoring of this threshold during exposure to potential pharmaceuticals, toxins, or other changes in environmental conditions. In addition, since stimulus efficacy is a property emerging from highly nonlinear physiological processes, real-time tracking of this property may lead to considerable increases in screening assay sensitivity for compounds affecting appropriate targets. The hybrid hardware/ software stimulation system, along with temperature control circuitry and a custom fluidic perfusion system, comprise a desktop hardware suite that is easily transportable and flexible, enabling convenient cell-level analysis in other laboratories and with other cardiomyocyte cell types. Furthermore, the architecture permits scaling of the hardware to achieve medium-throughput screening of potential cardio-active pharmaceuticals. The system is characterized using various physiological stimuli, including temperature variation, ion channel block, electrolyte disturbance, and ‚-receptor stimulation and blockade. The device extends the capabilities of electrical stimulation as a mode of electrophysiological analysis and this dissertation introduces the tool as a drug screening technique.

 

Whittington, R. H., ìReal-time control of electrical stimulation: a novel tool for compound screening and electrophysiologic analysis,î Ph.D. Thesis, Stanford University, CA, 2006.