Development of a miniaturized stimulation device for electrical stimulation of cells
© Xiong et al. 2015
Received: 23 May 2015
Accepted: 9 August 2015
Published: 4 September 2015
Directing cell behaviour using controllable, on-demand non-biochemical methods, such as electrical stimulation is an attractive area of research. While there exists much potential in exploring different modes of electrical stimulation and investigating a wider range of cellular phenomena that can arise from electrical stimulation, progress in this field has been slow. The reasons for this are that the stimulation techniques and customized setups utilized in past studies have not been standardized, and that current approaches to study such phenomena rely on low throughput platforms with restricted variability of waveform outputs.
Here, we first demonstrated how a variety of cellular responses can be elicited using different modes of DC and square waveform stimulation. Intracellular calcium levels were found to be elevated in the neuroblast cell line SH-SY5Y during stimulation with 5 V square waves and, stimulation with 150 mV/mm DC fields and 1.5 mA DC current resulted in polarization of protein kinase Akt in keratinocytes and elongation of endothelial cells, respectively. Next, a miniaturized stimulation device was developed with an integrated cell chamber array to output multiple discrete stimulation channels. A frequency dividing circuit implemented on the device provides a robust system to systematically study the effects of multiple output frequencies from a single input channel.
We have shown the feasibility of directing cellular responses using various stimulation waveforms, and developed a modular stimulation device that allows for the investigation of multiple stimulation parameters, which previously had to be conducted with different discrete equipment or output channels. Such a device can potentially spur the development of other high throughput platforms for thorough investigation of electrical stimulation parameters on cellular responses.
The study and manipulation of cell behaviour are currently predominantly conducted with biologics or chemical factors such as protein growth factors, blocking antibodies or small molecular entities that bind to specific protein moieties in the cell. However, these biochemical agents may be difficult to synthesize and could be limited by quality and costs. In addition, the pharmacological effects of biochemical agents depend on multiple factors, which include their pharmacokinetics, interactions with other molecules in the body, clearance rates from the body and toxicity . On the other hand, physical stimuli such as electrical currents provide a more controllable form of on-demand stimuli for the cells by altering the cell membrane potential and subsequent ion flux across the membrane [2, 3]. The physiological effects of electrical stimuli have been shown to be due to the redistribution of cell surface receptors and cytoskeleton reorganization in response to the applied electrical fields .
Specifically, electrical stimulation with direct current (DC) signals of 100–400 mV/mm have been shown to affect intracellular calcium dynamics,  epithelial cell proliferation,  cell migration, [7, 8] and stem cell differentiation . Also, Zhao et al. demonstrated cathodal (−) electrotaxis for keratinocytes upon the application of a 150 mV/mm DC stimulation . Similar electrotactic responses have also been observed for neural cells, adipose-derived stem cells and osteoblasts [10–12]. While DC signals have gained the attention of biomedical scientists and engineers in recent years, other waveforms such as alternating current (AC) or square waves have not been studied as extensively in non-excitable cell types. Although FDA-approved functional electrical stimulation devices (FES) that have been configured to deliver AC or square waveforms are already in clinical use for neuromuscular stimulation and wound healing [13–15], investigations into the effects of AC signals on physiological processes have yet to be elucidated.
In fact, emerging evidence of the potential of AC signals are just beginning to emerge; for instance in mesenchymal stem cell differentiation into osteoblasts . Unlike continuous DC stimulation, square waveforms minimise the deleterious irreversible non-faradaic reactions at the tissue/electrode interface . Charge-balanced biphasic stimuli are also an attractive option for electrical manipulation of cellular behaviour as they theoretically leave no residual charges at the site. Currently, the published studies on square and sinusoidal waveform stimulation use a wide range of waveforms that vary in stimulation mode (voltage or current mode), frequency and amplitude [16, 18–20]. Frequency-dependent neuronal responses such as elongation and neurite outgrowth are well-known . The proliferation of MC3T3-E1 bone cells have been known to occur at electrical fields of 60 Hz , and at 10 kHz, sinusoidal electrical fields promoted mesenchymal stem cell differentiation . In another study, rectangular pulses of 1 kHz were shown to induce cathodal neurite growth and branching . As abovementioned, the deployment of different stimulation frequencies to probe physiological responses vary greatly, and only a few studies have been conducted to investigate them systematically.
As such, this current study describes the use of a commercially-available stimulation board to systematically investigate physiological responses of DC and square waveforms on the intracellular localization of migration pathway-associated factor, cellular morphology and calcium signalling. Subsequently, a circuit board stimulation device integrated with a cell chamber array was designed to allow investigation into the effects of varying waveforms simultaneously on cell responses. The circuit design implements a ‘divide-by-2’ frequency divider to receive an input voltage waveform with frequency f0 from a waveform generator or a programmable microcontroller and give output pulses with f0/2, f0/4 and f0/8. Output pins next to each biocompatible chamber in the array will connect platinum wires to provide the stimuli. Using the stimulation device, the impedance frequency response of an ITO-coated glass was characterized. The miniature stimulation device is proposed to be used as a platform to study the effects of stimulation frequencies on cell behaviour and serves as a platform to develop a high throughput stimulation device with programmable output waveforms in future. In addition, a lower height profile and improved portability over existing common designs was targeted.
Results and discussion
Calcium signalling in SH-SY5Y cells using pulsed voltages
DC stimulation of PH-Akt-GFP overexpressing keratinocytes
DC stimulation of endothelial cells
Challenges of current cell stimulation techniques
From the cell stimulation experiments, we have demonstrated the physiological responses, such as increased Ca2+ concentration, localisation of Akt and cell elongation, in various cell types in response to electrical stimulation. We have employed a commercially-available stimulation electrode board (C-Dish™) in combination with other discrete electrical equipment. However the C-Dish™ stimulation board was designed to be used in conjunction with the C-Pace EP Cell Stimulator (IonOptix, USA), with pulsed waveforms ranging from 1–100 Hz. Although the stimulation board fits multi-well culture plates, only a single waveform could be used for them simultaneously, as all electrodes will receive signals from a single channel on the stimulator. Current cell stimulators on the market meant for biological use are limited to DC and pulsed voltages (monophasic and biphasic), and do not have kHz frequency outputs. This is consistent with that fact that most cell stimulation experiments today only require DC or low frequency waveforms [17, 23–26]. The exploration of high frequency waveforms and their physiological effects have only been explored in setups similar to ours that utilize a discrete waveform generator [16, 25]. The disadvantage of using a waveform generator is that they are designed for use with electronic circuits with fixed resistances/impedances. Cellular studies frequently require voltage- or current-controlled outputs that can compensate for changes in biological responses as the culture medium undergoes slight changes in volume or when the intrinsic impedance of cells change as a result of physiological responses.
In addition, as with most customized setups used currently, the stimulation chambers have a large profile, and may occupy a large volume inside the cell culture incubators. Conducting long-term higher throughput studies require a number of discrete components with many wires running into and out of the incubators. In order to circumvent these challenges, a miniature stimulation device with on-board integrated circuits (ICs) and a biocompatible cell array was proposed. Such a device should also allow the output of non-identical waveforms into each cell chamber through the functions of on-board processing ICs.
Frequency divider circuit design and simulations
Cell chamber array fabrication and integration into miniature stimulation device
Miniature stimulation device performance
DC frequency response of ITO-coated glass used with stimulation device
As various studies, including ours, have shown that cellular behaviour can be regulated by material surface chemistry [35–38], one area of interest will be to study the influence of chemical functional groups on materials and if any demonstrated effects are in synergism with electrical stimuli. Some cell stimulation studies have been conducted by applying electrical potentials on cells seeded directly on redox-active materials such as polypyrrole  and indium tin oxide (ITO)  to investigate the effects of surface charge on biological behaviour e.g. cell adhesion and cell spreading. In light of these studies, one criteria that was incorporated into the design of the stimulation device was to enable users to use a conductive or electroactive substrate seeded with cells as the working electrode. Fitting the platinum wire under the wire holder and pushing the wire holder down into the cell chamber ensured that the platinum wire could be interfaced tightly with substrate (Fig. 7c).
The aim of this study was to demonstrate the use of various modes of electrical stimulation in guiding cellular behaviour, and subsequently develop a miniaturized cell stimulation platform to enable research on physiologically-relevant stimulation signals. In parallel with what is known in the literature, cell calcium signaling, polarization of Akt, and cell elongation were demonstrated with discrete components. The stimulation device for performing frequency division of input AC signals to a cell array was designed to enable higher throughput where multiple stimulation frequencies can be applied simultaneously in the same study. In order to perform substrate-based stimulation, the electronic properties of the substrate have to be understood. In this study, the impedance frequency response of conductive ITO-coated glass was characterized using the stimulation device. The miniature stimulation device can be implemented for further cellular studies on cell-material interaction and synergism with electrical stimulation. This modular design consisting of the circuit board layout and 3D-printed cell chambers has the potential to address systematically the effects of different stimulation parameters on cell behaviour. More complex signal-processing ICs can also be implemented on this platform in future.
SH-SY5Y neuroblast cells (ATCC® CRL-2266™) were purchased from ATCC (Manassas, VA), while human umbilical vein endothelial cells (HUVECs) and EndoGRO-LS complete medium were purchased from Merck Millipore (Billerica, MA). Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) and X-Rhod-1 AM calcium-binding dye were ordered from Life Technologies (Carlsbad, CA). FuGene®6 transfection reagent was purchased from Promega Corporation (Madison, WI).
Pulsed voltage stimulation of SH-SY5Y cells
DC stimulation of endothelial cells
HUVECs were cultured in EndoGRO-LS complete medium. 1.0 × 106 cells were seeded in each well of the 6-well plate. A DC power supply was connected to the C-Dish™ electrode board to generate the required DC current. A 100-Ohm resistor was connected in series with the waveform generator to determine the DC current though 1 mL of cell culture medium (calculated from the voltage drop across the resistor and Ohm’s law, R = V/I). A current of 1.5 mA was set for the stimulation through tuning of the DC voltage. The stimulation was carried out for 12 h in a CO2 incubator at 37 °C. The morphology of the HUVECs were observed under a light microscope (AxioVert, Carl Zeiss, Germany) and phase contrast images were taken using the camera attachment. The elongation of HUVEC cells was quantitated by measuring the long and short axis of 10 cells from each field before stimulation and after 12 h stimulation, and expressed as a ratio of long/short axis.
DC stimulation of PH-Akt-GFP transfected keratinocytes
The human keratinocyte cell line HaCaT was cultured in DMEM with 10 % FBS and maintained at less than 50 % confluency. 5 × 104 cells were then seeded on a glass coverslip and allowed to reach 50 % confluency. A pCMV vector with the PH-Akt-green fluorescence protein (GFP) construct was transfected into HaCaT cells using FuGene®6 transfection reagent following the manufacturer’s protocol. 24 h post-transfection, the glass coverslips were transferred to a 6-well plate and cells were checked for green fluorescence protein (GFP) expression under the fluorescence microscope. The C-dish™ electrode board connected to a DC power supply was used to provide a constant voltage of 150 mV/mm across each well. A single cell expressing PH-Akt was focused on and images were taken using the GFP filter set (Ex: 470/40, Em: 525/50) every 15 min for 1 h to monitor the redistribution of PH-Akt-GFP within the cell. The total signal intensity of each cell, as well as a region of interest (ROI) was marked around the anodal end of the cell, and measured in ImageJ. The localization of PH-Akt-GFP at the anodal was then determined from the proportion of signal intensity in the ROI/the total signal intensity in the whole cell.
This research was supported by funding from the Ministry of Education (Singapore) Academic Research Fund (AcRF Tier 1: RG52/13 2013-T1-002-227). G.M.X. acknowledges sponsorship from the Economic Development Board of Singapore Industrial Postgraduate Programme (EDB-IPP). The authors would like to thank Dr. Tan Nguan Soon, Andrew (School of Biological Science, NTU) for providing the PH-Akt-GFP plasmids and HaCaT cell lines, and Dr. Hu Xiao (School of Materials Science and Engineering, NTU) for the ITO-coated glass used in the study.
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