A device for separated and reversible co-culture of cardiomyocytes.

A novel technique is introduced for patterning and controllably merging two cultures of adherent cells on a microelectrode array (MEA) by separation with a removable physical barrier. The device was first demonstrated by separating two cardiomyocyte populations, which upon merging synchronized electrical activity. Next, two applications of this co-culture device are presented that demonstrate its flexibility as well as outline different metrics to analyze co-cultures. In a differential assay, the device contained two distinct cell cultures of neonatal wild-type and beta-adrenergic receptor (beta-AR) knockout cardiomyocytes and simultaneously exposed them with the beta-AR agonist isoproterenol. The beat rate and action potential amplitude from each cell type displayed different characteristic responses in both unmerged and merged states. This technique can be used to study the role of beta-receptor signaling and how the corresponding cellular response can be modulated by neighboring cells. In the second application, action potential propagation between modeled host and graft cell cultures was shown through the analysis of conduction velocity across the MEA. A co-culture of murine cardiomyocytes (host) and murine skeletal myoblasts (graft) demonstrated functional integration at the boundary, as shown by the progression of synchronous electrical activity propagating from the host into the graft cell populations. However, conduction velocity significantly decreased as the depolarization waves reached the graft region due to a mismatch of inherent cell properties that influence conduction.

Modeling conduction in host-graft interactions between stem cell grafts and cardiomyocytes.

Cell therapy has recently made great strides towards aiding heart failure. However, while transplanted cells may electromechanically integrate into host tissue, there may not be a uniform propagation of a depolarization wave between the heterogeneous tissue boundaries. A model using microelectrode array technology that maps the electrical interactions between host and graft tissues in co-culture is presented and sheds light on the effects of having a mismatch of conduction properties at the boundary. Skeletal myoblasts co-cultured with cardiomyocytes demonstrated that conduction velocity significantly decreases at the boundary despite electromechanical coupling. In an attempt to improve the uniformity of conduction with host cells, differentiating human embryonic stem cells (hESC) were used in co-culture. Over the course of four to seven days, synchronous electrical activity was observed at the hESC boundary, implying differentiation and integration. Activity did not extend far past the boundary, and conduction velocity was significantly greater than that of the host tissue, implying the need for other external measures to properly match the conduction properties between host and graft tissue.

A discrete-time control algorithm applied to closed-loop pacing of HL-1 cardiomyocytes.

Electrical stimulation represents a useful tool for electrophysiologic investigation of electrically excitable cells such as cardiomyocytes. The stimulation threshold and electrophysiologic response to precisely timed pulses yields valuable information regarding physiologic processes. However, determining these parameters accurately, while simultaneously resolving time-dependent or transient effects has been difficult or impossible with previous methods. This paper presents a discrete-time algorithmic controller used for closed-loop electrical stimulation of HL-1 clonal cardiomyocytes cultured on, and stimulated using, a planar microelectrode array. We introduce the temporal error-controlled algorithm (TECA), that is well-suited to control using capture fraction, a low data rate, highly quantized feedback parameter describing stimulation efficacy. HL-1 cardiomyocytes were electrically stimulated and resulting parameters were used to develop a representative model of partial capture, enabling extensive analysis of the algorithm. The performance of this approach is compared via computer simulation to a previously introduced conditional convergence algorithm to quantify its performance and relative advantages. Operation of the TECA is demonstrated by tracking the real-time biological response of stimulation threshold to a rapid increase in extracellular potassium concentration in four independent cell cultures. This work enables the use of stimulation threshold as a real-time, continuously monitored parameter with considerable utility in cardiac pharmacology, electrophysiology, and cell-based biosensing.

Temporal resolution of stimulation threshold: a tool for electrophysiologic analysis.

Electrical stimulation of cardiac cultures with closed-loop control permits the determination of threshold in real time. The temporal response of stimulation threshold and underlying cell membrane excitability is valuable information for understanding the complex electrophysiologic processes within cardiac cells and can aid in understanding the mechanisms and effects of pharmaceuticals or other stimuli. This work presents the temporal response of stimulation threshold measured using HL-1 cardiac myocytes when exposed to changes in temperature and extracellular potassium concentration. These changes mimic systemic alteration of excitability and conditions that can result from ischemia in the heart. The results demonstrate the efficacy of stimulation threshold as a physiologic indicator and illustrate transient effects with both fast and slow time constants that can be resolved using a system that determines stimulation threshold in real time.

A closed-loop electrical stimulation system for cardiac cell cultures.

An integrated electrical stimulation and recording system was designed for closed-loop control and analysis of cardiac cultures on planar microelectrode arrays. Stimulated action potentials from HL-1 clonal myocyte cultures were digitized, stimulation artifacts were removed using nulling and filtering methods, and analysis was performed to determine stimulation efficacy in real time. Results of this analysis were used to determine future stimulation waveform parameters such as polarity, amplitude, pulse duration, and rate or pattern. Algorithms were designed utilizing real-time analysis and control to maintain a desired electrophysiological response of the culture, such as an arbitrary capture fraction value. This paper presents the hardware and software design of the stimulus pulse circuitry, artifact extraction, analysis, and control components of the system. Applications of this technology include the study of cardiac cell physiology, improving the speed and accuracy of traditional open-loop stimulation protocols, pharmacological screening, and improving the performance of biosensors based on sensing electrical activity in cardiac cultures.

Sensitivity of cell-based biosensors to environmental variables.

Electrically active living cells cultured on extracellular electrode arrays are utilized to detect biologically active agents. Because cells are highly sensitive to environmental conditions, environmental fluctuations can elicit cellular responses that contribute to the noise in a cell-based biosensor system. Therefore, the characterization and control of environmental factors such as temperature, pH, and osmolarity is critical in such a system. The cell-based biosensor platform described here utilizes the measurement of action potentials from cardiac cells cultured on electrode arrays. A recirculating fluid flow system is presented for use in dose-response experiments that regulates temperature within +/-0.2 degrees C, pH to within +/-0.05 units, and allows no significant change in osmolarity. Using this system, the relationship between the sensor output parameters and environmental variation was quantified. Under typical experimental conditions, beat rate varied approximately 10% per degree change in temperature or per 0.1 unit change in pH. Similar relationships were measured for action potential amplitude, duration, and conduction velocity. For the specific flow system used in this work, the measured environmental sensitivity resulted in an overall beat rate variation of +/-4.7% and an overall amplitude variation of +/-3.3%. The magnitude of the noise due to environmental sensitivity has a large impact on the detection capability of the cell-based system. The significant responses to temperature, pH, and osmolarity have important implications for the use of living cells in detection systems and should be considered in the design and evaluation of such systems.