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A MULTI-PARAMETER, FEEDB ACK-BASED ELECTRICAL STIMULATION SYSTEM FOR CARDIOMYOCYTE CULTURES

R.H. Whittington, K.H. Gilchrist, L. Giovangrandi, G.T.A. Kovacs Department of Electrical Engineering, CIS-202X, Stanford Universiv, Stanford, CA, 94305-4075 USA

ABSTRACT Cardiac myocytes are cultured to confluency on a 36-ele-

ment planar microelectrode array. Spontaneously-active car- diomyocyle cultures are electrically stimulated, eliciting action potentials (APs), and the resulting data is amplified, filtered, and stimulation artifacts are removed during data acquisition. Stimulation circuitry allows real-time control of stimulation waveform amplitude, duration, and rate,via 1 0 hit digital-to-analog converters (DACs). By extracting arti- facts and processing AP properties in real time, we incorpo- rate these properties into real-time stimulation algorithms, creating a sensitive feedback-regulated myocyte-based trans- ducer of cell electrophysiological properties. Applications of this technology include toxin detection, automated phar- maceutical screening, and hasic cardiac electrophysiology.

I. BACKGROUND :. Utilizing traditional photolithographic processes to pro- duce planar arrays of microelectrodes has proven to be of immense value to the cardiac and neuroscience communi- ties. There are numerous reports describing the plating of various cell types on planar arrays, including neurons, brain .slices, primary cardiomyocytes, and clonal myocyte lines [l- 61. We present work based on the HL-1 cell line, a clonal murine atrial myocyte allowing continuous culturing in vitro

Cardiac myocytes in culture under proper media and environmental conditions exhibit spontaneous and synchro- nous action potential activity throughout the culture. By analyzing the signals generated by spontaneous cultures using a microelectrode array, we are atile to monitor the con- tinu.ous electrophysiological state of the culture, and its vari- ation in space .and time. The action potential (AP) morphology, beat rate, variation of beatrate, and velocity of signal propagation, among others, are pakameters of interest in this system. Monitoring the parameters of the APs sensed by the electrode m a y during exposure to potential pharma- ceuticals, unknown toxins, or other agents is one important approach to the realization of cell-based biosensor{. Bio- sensing systems with detection and classification capabilities based on the analysis of cell metabolism or electrophysiol- ogy have been and are being developed [8-121.

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II. INTRODUCTION Electrical stimulation of cardiac cultures offers a new

paradigm for investigating the physiological effects of unknown compounds or drugs. By pacing the cardiac cul- ture at a specific rate and energy, one can extract additional

information regarding the electrophysiological properties of the cells and the cell culture. In addition, whereas traditional analysis of spontaneous cultures relies on regular and spon- taneons heating, by electrically pacing the tissue this some- times confounding variable is eliminated. This allows more in-depth investigation of the possible causes of irregularity or extraction of other parameters in the presence of rate- depressing or spontaneity-inhibiting agents.

Electrical stimulation and pacing of cardiac cultures is achieved by injecting a square, hiphasic, current pulse through a planar electrode undemeath the cell culture. The resulting local change in potential modifies the transmem- brane voltage (either hyperpolarizing or depolarizing the cell), which initiates an AP via voltage-gated transmembrane ion channels. This AP then propagates naturally throughout the culture as a result of the gap-junction-coupled cell mem- branes of adjacent cells.

Varying the magnitude and duration of the current pulse injected into the electrode in the positive and negative phases of the signal pennits control of the charge delivered to the culture and electrode. The excitability of. the culture can be revealed by determining the pacing threshold, the current amplitude or duration necessary to elicit an action potential at a given rate. Likewise, by varying the pacing rate, the effects of continuous heating above the natural physiological rate can he determined, providing an indication of the refrac- toriness of the culture (period of inexcitahility after each AP).

Integrating real-time analysis of the paced culture with control of the stimulation parameters and protocol enables more complex investigation of the electrophysiological properties of cardiac cultures, while facilitating more time- 'efficient and accurate determination of threshold and rate parameters. The abilities of this system and its potential for algorithm development bil l be discussed.

IILMETHODS

A. System Hardware

Microelectrode Array and Recording System HL-I ,cardiac myocytes are cultured on microelectrode

arrays fabricated in the Stanford Nanofahrication Facility. The microelectrode arrays are fabricated on glass substrates, with gold routing and platinum electrodes, and have been described by Borkholder [13]. Arrays are mounted in CER- DIP-40 packages with a 2mL polystyrene dish providing a fluid bath for the tissue and easy access for cell culturing, feeding, and cleaning. Each array has 36 circular electrodes,

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each with 22pm diameter arranged in a 6x6 array with 100pm spacing between electrodes. The media bath is grounded by two large reference electrodes of area 6x105 pm2 on opposite sides of the array. Two smaller stimulating electrodes of area 2x105 pmz are situated as shown in Figure 1. The recording electrodes are electroplated with platinum- black to reduce their impedance. Four representative sam- ples yielded impedances at lkHz of 118kQ and phase angle - 49.6' (d = 50.2kQ 7.76O, n=144). The reference and stimu- lation electrodes have impedances at 1kHz of 675Q 1-31.6' (d = 53.44 4.51°, n=7) and 4.78w2 / -41.2' ($ = 148Q 1.03O, n=7), respectively. _. _ _

Figure 1: Microelectrode o r r q chip with mounted ZmL Petri dishfor contain- ment of culncre medin. Cells ore cultured within the cir- cular chamber at chip cen- ter

Imet: Micrograph of elec- tmde array showing 36 electrodes and IWO stimula- tion electmdes. A r r q elec- trodes are plnn'num, spaced IWpn apan, stimulation eleclrdes am Z00x100m.

The CustomIdesigned recording system consists of a 36 Channel amplifier with gain 100OX, 4Hz high-pass cutoff, and selectable eight-pole low-pass cutoff from 500Hz to 6kHz. Further details of the recording system have been described in detail by Gilchrist [lo]. Thin-film heaters, inte- grated temperature control circuitry, and an aluminum enclo- sure maintain temperature at 37OC within OS0C, media pH at 7.4 using 5% COz ambient, and high humidity via a satu- rated sponge inside the enclosure.

Stimulation Circuit The stimulation circuit is designed to generate square,

biphasic, return-to-zero current pulses with independent con- trol of positive and negative pulse amplitudes, positive and negative pulse duration, and signal repetition rate. Typical current amplitudes lie in the l-40pA range, durations range from 0.1-4Oms, and rates typically extend from 0 to 300 beats per minute (bpm). A schematic diagram of the circuit, implemented with discrete components, is shown in Figure 2. Stimulus parameters are programmable via an 8-channel ]@bit DAC with serial digital interface. The serial interface is controlled by custom software discussed below. A volt- agecontrolled oscillator (VCO) sets the rate of pulse genera- tion based on DAC input control voltage. The VCO then mggers the generation of a ramp waveform whose slope can he configured by selection of integrating capacitor to provide the coarse range of durations desired. Specific duration of each phase of the pulse is set by a comparator network that generates pulses corresponding to ramp voltage less then or greater than DAC references. These constant-voltage com-

Figure 2 Schematic ofstimulation circuit allowing independent control of pulse mte and positive and negative amplitude and duration vi0 an 8- channel IO-bit DAC interface. SofMi(lre routines inrefme with the circuit to setparameters in red time, based on the result ofpacing.

parator pulses are then DC-converted to the positive and negative amplitudes desired before being convened to cur- rent pulses via a Howland-configured operational amplifier. The voltage-to-current conversion ratio is determined by choice of feedback resistance, thus setting the coarse range of amplitudes.

B. System Software

Action potential data sensed by the microelectrode array is amplified and filtered by the recording system and is sub- sequently captured by a 16 channel 12-bit ADC, capable of 330ksps (DAS 16/330, Measurement Computing). We uti- lize two of these cards to sample 32 channels at IOksps. Data is subsequently acquired and processed using a custom Matlab GUI'. Matlab routines were integrated in a GUI for the purpose of visualizing 32 channels of action potential data in real time. In addition, real-time extraction routines display beat rate, signal amplitude, slope, and conduction dynamics across the m a y during data acquisition. Further, more complex, post-processing iS done offline in Matlab. For the purposes of stimulation analysis and control, further development of the acquisition software was needed; rou- tines for the serial DAC interface, artifact extraction, and stimulation algorithms were developed for this purpose.

Artifact extraction and data processing Artifact extraction is a key component to the design of

any system utilizing electrical stimulation, either in vitro or in vivo. Many techniques have been developed to address the incompatibility of stimulus voltages relative to signal voltages and the electrical artifacts introduced by stimula- tion. The wide variation in technique is explained by the wide variety of artifacts observed in different environments, and the best solution for a given environment is often empir- ically dictated. Previous methods for artifact prevention or removal include active gain control, amplifier input ground- ing, transient low impedance stimulator design, template subtraction, and filtering [14-18].

Typical action potential amplitudes for HL-I cardiomyo- cytes recorded extracellularly on our arrays range from 200pVp.p to 3mVp.p. while stimulus amplitudes vary from 100 to 8OOmV,,, typically. The relative proportions of these

I . Matlab is a registered trademark of The Mathworks, Inc.

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signals allow threshold detection of artifacts in the presence of paced APs (see Figure 3). Data above the artifact thresh- old, typically 1.5-2mV, are considered saturated and are nulled. Data 3ms prior to the artifact trigger, which includes the artifact rising edge hut not paced AP data, is also nulled. The nulled data is then filtered to remove the remaining slow decay resulting from electrode discharge, and the filter step response is removed by nulling lOms following the artifact. Results of artifact extraction on a typical signal are shown below. Over the normal range of stimulation pulse energies, this method allows reliable recovery of action potential data within lOms of the end of the artifact. For thephysical spac- ing of stimulation to recording electrodes (0.5mm) and range of normal conduction velocities, this window of data loss is acceptable. Latencies from stimulation pulse rising edge to AP are rarely less than 30ms, and this relaxes the constraints on the artifact ,reduction considerably, enabling a simple scheme to obtain high efficacy.

i:/ 4 6 ' . w ID A rao 7k .4 M II) ,m 12p ,a Time (ms) Time@"

Figum 3 Example of oclion polenrial extraction (solid) from mrfacl- contominnred dam (doshedi using nulling,andfZlc~ing. Right figure is close up of lqfrfigure shbving voltage range -1mV 10 1mV Here, on AP with posiiive and ~ g n f i v e peak mplirlules of +I 0.Zm V is errrocred in thepresence of an onifacrwirh nega1ivepeak0mplirde -2.9mV

IV. RESULTS

A. Stimulation of cardiac cultures

Electrical stimulation is accomplished by injecting anodic-first current pulses into an electrode on which cells are directly adhered. Several factors determine efficacy of stimulation; these include confluency over the stimulation electrode area, degree of coupling between cell and stimula- tion electrode, and excitability of the cell membrane. The major dependent variable is cell membrane excitability, which can he a function of the extracellular and intracellular milieu, but also the general physiological state of the cell and culture. Inherent in this state are the complex metabolic and molecular pathways that determine electrical response.

Stimulus amplitude and duration are varied in order to determine the stimulus threshold at a given pacing rate. If threshold is reached, APs appear following each stimulus at a fixed delay determined mainly by the conduction velocity of the culture. Pacing fraction is determined by computing the percentage of stimulus pulses eliciting APs with a fixed latency. Together with the threshold data, this experiment generates a three-dimensional array of threshold and pacing

Pulse amplitude w) Pulse amplitude ($A)

Figure 4: Example oflhreshold deleminnlion before and &r exposure 10 ZoDnM Nifedipine. Pulse omplirude (horizontal oxis) and duration (venicnl axis) are swepr from I-lOpA curmnr omplirude, and l ~ l 0 m s pulse durorion per phose, consecutively, in one-unit sreps. Black squares indicate 1WX pacing, end while 0% ofpulses elicilng response

percentage, shown in Figure 4; dark colors represent increas- ing pacing percentage. Further data may he obtained by varying the pacing rate using a supra-threshold pulse energy. This method provides information on the dynamic ionic equilibrium maintained by ion channels and pumps. Figures 4 and 5 demonstrate these concepts using data extraction before and after exposure to 200nM of Nifedipine, a Ca++- channel blocker.

While normal determination of pacing thresholds or rate would he done by sweeping parameters successively, soft- ware control allows one to determine this protocol with com- plete freedom, varying sweep duration and amplitude or rate randomly or in any prescribed order. By monitoring the results during stimulation, protocols may be adjusted during an experiment to automatically extract particular regions from the stimulation parameter space.

Figure 5 Pacing fraction plotted ogoinrt pocing: rule for pu1.w amplitude 5pA ond durorion per phase 5ms. At 0.50 pacing f m c ~ lion, e v e v orher pulse elic- ils M AI: on average. Plols indicale pacing versus rate

before (01 and @er (+) erposure Io 20hM Nife-

Pacing Rate (bpm) dipinr.

B. Adaptive algorithms and results

The feedback-based stimulation system allows us to per- form more sophisticated analyses and control of the stimula- tion parameters. Stimulation pulse parameters are set, the culture response is recorded and analyzed for a given time period (typically 1 to 3 seconds), and the resulting pacing percentage or AP signal properties are used in the determina- tion of the next set of pacing parameters. There are many ways to apply this control algorithm to myocyte cultures, including adaptive step-size algorithms, and successive approximation of stimulation or culture parameters.

Whereas normal threshold determination relies on step- ping through the parameter space at fixed intervals, the adap- tive step-size algorithm performs a coarse sweep with equal

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steps until the target pacing percentage is achieved, at which point the previous step is repeated, the step size is reduced, and the sweep continues until reaching the target pacing fraction. The process canbe iterated until the desired resolu- tion is achieved. This allows the assignment of step size res- olution to the specific region of interest without the need for performing long sweeps with high step counts. This tech- nique and its iteration allows considerably more accurate determination of thresholds, maximum rate at a given ampli- tude and pacing fraction, or other parameters in an auto- mated fashion with highly reduced sweep time.

Figure 6 Example of adaprive step-size olgo- rithm 01 a single pulse duration of Zms. Upon attainment of 100% poc- ing, I @ coarse step size (square) is reduced to

O . l @ , the mplirude value ir decrementedf" 4pA IO 3pA, ond w e e p resumes using the finer s t e ~ size of 0. I U A fcirclesj

Another feedback-based algorithm is the determination of the minimum amplitude required to achieve pacing at a given percentage. By determining rate-dependency of pac- ing threshold rather than maximum rate, one investigates the temporal ability to generate an AP as a function of the excit- ability of the cell membrane. Converging on the rate at which 50% of pulses elicit responses for a given pulse energy allows accurate determination of maximum rate at a

algorithms possible with such a system, and there'are consid- erable opportunities for further investigation through design .of stimulus and conb-ol protocol.

V. CONCLUSIONS We have demonstrated the hardware and software design

of a system that allows the implementation of real-time algo- rithms to extract electrophysiological information from car- diomyocyte cultures in a manner not previously explored. The flexibility of this approach lies in the ability to dynam- cally determine pacing properties and incorporate cell elec- trical responses into the stimulation feedback and control algorithm. The ability to arbitrarily assign resolution to parameter-space regions of interest offers a tremendous time-saving benefit, allowing the detection of short time- course physiological events and fast screening-type assays. There is immense freedom and hence potential for an anto- mated stimulation system for the investigation of cardiac pharmacology, toxin detection, and fundamental research related to clinical cardiac pacing.

VI. ACKNOWLEDGEMENTS The authors would like to thank those who contributed to

this work, including Chris Storment for fabrication of the electrode mays, Steve Young for circuit development, as well as William Claycomh and Proctor and Gamble for pro- viding the HL-1 cells. Funding for this research was pro- vided by Defense Advanced Research Projects Agency contract N66001-99-C-8642.

VII. REFERENCES given stable reference point representing the inability of the culture to generate consecutive An example of this protocol is shown in Figure 7. At a current ma@itude Of

5pA, the target pacing fraction of 0.5 is determined by suc- cessively decreasing step-size while converging on the target

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2w1.. Figure 7: Plot of stimu- lation rme Y e m u step count (Is rate converges on 50% pacing and duration is s t e p p e d f " Jms to 6ms in Ims steps. Pulre amplitude is main- - toined at 5pA rhmugh-

E X 4 out. Rote convergence ir defined as ' I e p fan- j n g 0'6 bpm' Hori- zontol lines indicate

350 - 543-56.2Nl

E &a

a" .I - -

37, 1982.

w 0 20 40 w en 1m

Step Count (Discrete Time Base) converged

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