This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.
Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.
EFs and MFs have been shown to modify cell dynamics, stimulating proliferation and increasing synthesis of the main molecules associated with the extracellular matrix of tissues1. These biophysical stimuli can be applied in different ways by using specific settings and devices. Regarding the devices to generate EFs, direct coupling stimulators use electrodes that are in contact with biological samples in vitro or implanted directly into tissues of patients and animals in vivo2; however, there are still limitations and deficiencies that include insufficient biocompatibility by the electrodes in contact, changes in the pH and molecular oxygen levels1. On the contrary, indirect coupling devices generate EFs between two electrodes, which are placed in parallel to biological samples3, allowing a non-invasive alternative technique to stimulate biological samples and avoid direct contact between tissues and electrodes. This type of device can be extrapolated to future clinical applications to perform procedures with minimal invasion to the patient. In relation to devices that generate MFs, inductive coupling stimulators create a time-varying electric current, which flows through a coil that is located around cell cultures4,5. Finally, there are combined devices, which use EFs and static MFs to generate transient electromagnetic fields1. Given that there are different configurations to stimulate biological samples, it is necessary to consider variables such as tension and frequency when biophysical stimuli are applied. Voltage is an important variable, since it influences the behavior of biological tissues; for instance, it has been shown that cell migration, orientation and gene expression depend on the amplitude of applied voltage3,6,7,8,9,10. Frequency plays an important role in biophysical stimulation, as it has been evidenced that these occur naturally in vivo. It has been demonstrated that high and low frequencies have beneficial effects on cells; especially, in cell membrane voltage-gated calcium channels or endoplasmic reticulum, which trigger different signaling-pathways at intracellular level1,7,11.
According to the abovementioned, a device for generating EFs consists of a voltage generator connected to two parallel capacitors12. This device was implemented by Armstrong et al. to stimulate both the proliferative rate and the molecular synthesis of chondrocytes13. An adaptation of this device was performed by Brighton et al. who modified cell culture well-plates by drilling their top and bottom lids. Holes were filled by cover slides, where the bottom glasses were used to culture biological tissues. Electrodes were placed on each cover slide to generate EFs14. This device was used to electrically stimulate chondrocytes, osteoblasts and cartilage explants, showing an increase in cell proliferation14,15,16 and molecular synthesis3,17. The device designed by Hartig et al. consisted of a wave generator and a voltage amplifier, which were connected to parallel capacitors. Electrodes were made of high-quality stainless-steel located in an insulating case. The device was used to stimulate osteoblasts, showing a significant increase in proliferation and protein secretion18. The device used by Kim et al. consisted of a biphasic current stimulator chip, which was built using a manufacturing process of complementary semiconductors of high-voltage metal oxide. A culture well-plate was designed to culture cells over a conductive surface with electrical stimulation. Electrodes were coated in gold over silicon plates19. This device was used to stimulate osteoblasts, showing an increase in the proliferation and the synthesis of the vascular endothelial growth factor19, and stimulating the production of alkaline phosphatase activity, calcium deposition and bone morphogenic proteins20. Similarly, this device was used to stimulate the proliferative rate and expression of vascular endothelial growth factor of human bone marrow mesenchymal stem cells21. The device designed by Nakasuji et al. was composed of a voltage generator connected to platinum plates. Electrodes were built to measure the electric potential at 24 different points. This device was used to stimulate chondrocytes, showing that EFs did not alter cell morphology and increased proliferation and molecular synthesis22. The device used by Au et al. consisted of a glass chamber equipped with two carbon rods connected to a cardiac stimulator with platinum wires. This stimulator was used to stimulate cardiomyocytes and fibroblasts, improving cell elongation and fibroblast alignment23.
Different MF devices have been manufactured based on Helmholtz coils to stimulate several types of biological samples. For instance, Helmholtz coils have been used to stimulate proliferation and molecular synthesis of chondrocytes24,25, enhance proteoglycan synthesis of articular cartilage explants26, improve gene upregulation related to bone formation of osteoblast-like cells27, and increase proliferation and molecular expression of endothelial cells28. Helmholtz coils generate MFs throughout two coils located one in front the other. The coils must be placed with a distance equal to the radius of the coils to ensure a homogeneous MF. The disadvantage of using Helmholtz coils lies in the coil dimensions, because they need to be big enough to generate the required MF intensity. Additionally, the distance between coils must be adequate to ensure a homogeneous distribution of MFs around biological tissues. To avoid issues caused by Helmholtz coils, different studies have been focused on solenoid coils manufacturing. Solenoid coils are based on a tube, which is wound with copper wire to generate MFs. Copper wire inputs can be connected directly to the outlet or a power supply to energize the coil and create MFs in the center of the solenoid. The more turns the coil has, the greater the MF generated. The MF magnitude also depends on the voltage and current applied to energize the coil29. Solenoid coils have been used to stimulate magnetically different kind of cells such as HeLa, HEK293 and MCF730 or mesenchymal stem cells31.
Devices used by different authors have not considered either the adequate size of electrodes or correct length of the coil to homogeneously distribute both EFs and MFs. Furthermore, devices generate fixed voltages and frequencies, limiting their use to stimulate specific biological tissues. For this reason, in this protocol a computational simulation guideline is performed to simulate both capacitive systems and coils to ensure homogeneous distribution of EFs and MFs over biological samples, avoiding the edge effect. Additionally, it is shown that the design of electronic circuits generate voltages and frequency between the electrodes and the coil, creating EFs and MFs that will overcome limitations caused by impedance of cell culture well-plates and air. These modifications will allow the creation of non-invasive and adaptive bioreactors to stimulate any biological tissue.
1. Simulation of EFs and MFs
NOTE: Simulation of EFs and MFs was performed in COMSOL Multiphysics.
2. Design and manufacturing of the electrical and magnetic stimulation devices
Computational simulation
Distributions of EFs and MFs are shown in Figure 3. On the one hand, it was possible to observe the homogeneous distribution of EFs in the capacitive system (Figure 3A). The EF was plotted to observe in detail the magnitude of the field inside the biological sample (Figure 3B). This simulation was useful to parametrize the size of the electrodes and manufacture them to avoid the edge effect. On the other hand, it was possible to observe the homogeneous distribution of MFs generated by the solenoid coil (Figure 3C). The MF was plotted to observe in detail the magnitude of the field inside the coil (Figure 3D). This simulation was important measure the distance where the MF is the same and build the PMMA support. This support ensures a homogeneous distribution of the MF not only in the center of the coil, but also in the biological samples to be stimulated.
Signals generated by electrical and magnetic stimulators
Output signals generated by electrical stimulator are shown in Figure 4. It is relevant to highlight that signals captured by the oscilloscope were directly taken in the electrodes, as whether the measurement is taken directly to the output cables, the voltages will be higher (Figure 4A). This voltage variation is given by the capacitance of electrodes. The output voltage oscillates in a range of ± 5V at 60 kHz; for instance, the output signals were 54.9 Vp-p (Figure 4B), 113 Vp-p (Figure 4C), 153 Vp-p (Figure 4D) and 204 Vp-p (Figure 4E) for 50, 100, 150 and 200 Vp-p, respectively.
The output signal generated by the magnetic stimulator is shown in Figure 5. The signal captured by the oscilloscope were directly taken in the output cables of the coil (Figure 5A). The output voltage oscillates in range of ± 15V p-p at 60 Hz (Figure 5B).
Figure 1. Electrical stimulation device. A) Circuit that generates tensions of 50, 100, 150 and 200 Vp-p at 60 kHz sine wave-form. B) Printed circuit board within the case. C) Electrodes inside the incubator. Please click here to view a larger version of this figure.
Figure 2. Magnetic stimulation device. A) Schematic representation of the magnetic stimulator device and the PMMA support. B) Circuit to generate the MFs. Please click here to view a larger version of this figure.
Figure 3. Computational simulation of EFs and MFs. A) Distribution of EFs inside and outside the capacitive system. B) Distribution of EFs within the hydrogel, the region of interest is indicated in a red detail. C) Distribution of MFs inside and outside the coil. D) Distribution of MFs in the center of the coil, the region of interest is indicated in a red detail. Please click here to view a larger version of this figure.
Figure 4. Sinusoidal signal generated by electrical stimulator. A) Signal verification generated by the electrical stimulator. B) Signal at 50 Vp-p. C) Signal at 100 Vp-p. D) Signal at 150 Vp-p. E) Signal at 200 Vp-p. All measurements oscillate in a range of ± 5V at 60 kHz. Please click here to view a larger version of this figure.
Figure 5. Sinusoidal signal generated by the magnetic stimulator. A) Signal verification generated by the magnetic stimulator. B) Signal at 15 Vp-p at 60 Hz. Please click here to view a larger version of this figure.
System | Components | Width (mm) | Height (mm) |
Electrical system | Air | 100 | 100 |
Electrodes | 50 | 5 | |
Well-plate | 7 | 20 | |
Hydrogel | 3.5 | 3.5 | |
Culture media | 6 | 8 | |
Magnetic system | Air | 500 | 600 |
Coil | 2 | 250 |
Table 1. Dimension of geometries that compose electric and magnetic systems.
System | Components | Relative Permittivity (ε) | Conductivity (σ) |
Electrical system | Air | 1 | 0 |
Electrodes | 1 | 1.73913 [MS/m] | |
Well-plate | 3.5 | 6.2E-9 [S/m] | |
Hydrogel | 8.03E3 | 7.10E-2 [S/m] | |
Culture media | 2.67E4 | 7.20E-2 [S/m] | |
Magnetic system | Coil | 1 | 5.998E7[S/m] |
Table 2. Dielectric properties of elements that compose electric and magnetic systems.
Treatments used to heal different pathologies that affect human tissues are pharmacological therapies32 or surgical interventions33, which seek to relieve pain locally or replace affected tissues with explants or transplants. Recently, autologous cell therapy has been proposed as an alternative therapy to treat injured tissues, where cells are isolated from patient and expanded, through in vitro techniques, to be implanted at the site of the injury34. Given that autologous cell therapy has demonstrated to have direct influence over tissue recovery, different strategies has been developed to increase the effectiveness of this technique. For instance, biophysical stimuli have been used as a non-invasive alternative therapyto stimulate several types of biological samples, modulating cell functionality by improving cell proliferation and molecular synthesis35,36. Among the most used biophysical stimuli, electrostimulation and magnetotherapy have been widely applied to stimulate cells, tissue explants and scaffolds. It has been evidenced that electrostimulation reduces pain and increases healing processes of several tissues37. Regarding the magnetotherapy, it has been described that this stimulus improves integration of implants with host tissues, accelerates healing processes, relieves pain locally and increases scar strength8,38.
Considering the mentioned above, the combination of biomaterials, cell culture and external biophysical stimuli such as EFs and MFs, at in vitro level, has been introduced in tissue engineering as an alternative therapeutic technique to heal injured tissues8,39. However, finding a bioreactor that helps to stimulate different tissues, whether healthy or affected by traumatic pathologies, is a challenge. In this context, the present protocol aims to develop both electrical and magnetic stimulators. Currently, there are two possible schemes for applying EFs. The first method consists of generating EFs through direct coupling systems, which are used to evaluate cell migration and orientation40,41,42. However, there are limitations such as alterations in biocompatibility of the cell culture medium by electrodes in contact, possible changes in pH and molecular oxygen levels1. Additionally, direct coupled stimulation cannot amplify high-frequency signals. The output tends to vary with time, generating supply voltage changes. It has little temperature stability, due to this its operating points change and at low frequencies the capacitor fails and acts as an open circuit43. Considering these limitations, the second method was implemented, where external parallel electrodes were used. This indirect coupling system method has evidenced an increase in cell proliferation and molecular synthesis3,7,17,22,44,45; however, the devices developed by different authors have not considered the size of electrodes to distribute homogeneously EFs. For instance, devices generate fixed voltages and frequencies, limiting their use in stimulating specific cells and tissues. Accordingly, in this study the size of the electrodes was modelled to ensure a homogeneous distribution of EFs over biological tissues. In addition, a circuit was designed to generate a frequency and high voltages between electrodes, creating different EFs that overcome the limitations caused by the impedance of cell culture well-plates and air.
Solenoid coils are versatile devices that can be used to stimulate biological samples within the incubator, allowing that atmospheric conditions remain stable without affecting physiological features of biological samples. This advantage elucidates that solenoid coils are feasible alternatives more than Helmholtz coils, as these need to be bigger in size, preventing stimulation inside incubators46. Stimulation of biological samples outside the incubator can lead in several issues such as cell culture contamination, cell stress, pH changes of culture media, among others. Given that different stimulator devices have been developed to stimulate several cell types and tissues24,25,26,27, it is relevant to build devices where MF intensities can be varied to stimulate a wide range of biological samples29,30. Accordingly, in this protocol the magnetic stimulator is connected to a rheostat, which can vary the current that flows through the solenoid by modifying their resistance and current, parameters that are directly related to the generation of MFs. Another important feature to consider at the moment of building magnetic devices is the distribution of MFs. Here, a computational simulation was used to simulate the MF distribution inside the solenoid coil. This simulation allowed to calculate the number of turns of the copper wire and the length of the coil to generate homogeneous MFs in the middle of the coil. The computational simulation is a useful tool to calculate the number of biological samples to be stimulated, ensuring that all samples receive the same field strength47.
The biophysical stimulators developed in this protocol have some limitations. First, the electronic circuit designed for electrical stimulator generates four output voltages at a specific frequency. Although the circuit overcome the limitation of generating high voltages between electrodes1, it could be improved to generate variable voltages and frequencies. The circuit can be modified to generate different frequencies just calculating either resistors or capacitors using equation (1); however, it is possible to use variable resistors to vary manually the resistor value. Similarly, a variable resistor may be use in the amplification stage of the circuit to vary the output voltage. Second, the electronic circuit of the electrical stimulator generates sinusoidal signals. It would be useful to generate different kind of signals such as square, triangular, trapezoidal and ramp, as these types of signals could be used to stimulate a wide range of cells and biological samples48,49. To generate different type of signals, the operational amplifier can be replaced by a monolithic function generator, which can produce high quality waveforms of high-stability and accuracy with low amplitude, and the amplification stage can be replaced by a non-inverting operational amplifier or a stage with NPN transistors. Third, even though the magnetic stimulator generates small MF magnitudes, it has been evidenced that these intensities have direct impact over dynamics of biological samples24,28,30,38; however, the magnetic device could be improved to generate variable MFs and frequencies to stimulate a wide range of biological tissues29.
Overall, this protocol is a useful tool which provides a technological contribution to the scientific community that works on biophysical stimulation of biological tissues. These devices will allow researchers to use EFs and MFs to stimulate the function of healthy biological tissues or those altered by a particular pathology.Considering this in further in vivo studies, different parameters and variables such as electrodes size, number of turns of the coil, stimuli strength and stimulation times would be determined to homogeneously distribute both EFs and MFs in animals such as pigs, calves, guinea pigs or rabbits. Additionally, bioreactors designed in this protocol can be extrapolated to clinical settings to improve regenerative techniques such as autologous cell implantation. Here, bioreactors can play an important role by stimulating biological samples, at the in vitro level, to improve the cellular and molecular featuresof cells, tissues and scaffolds before being implanted in the patient.
The authors have nothing to disclose.
The authors thank the financial support provided by "Fondo Nacional de Financiamiento para la Ciencia, la Tecnología, y la Innovación -Fondo Francisco José de Caldas- Minciencias" and Universidad Nacional de Colombia through the grant No. 80740-290-2020 and the support received by Valteam Tech – Research and Innovation for providing the equipment and technical support in the edition of the video.
Electrical stimulator | |||
Operational amplifier | Motorola | LF-353N | —- Quantity: 1 |
Resistors | —- | —- | 22 kΩ Quantity: 1 |
Resistors | —- | —- | 10 kΩ Quantity: 3 |
Resistors | —- | —- | 2.6 kΩ Quantity: 2 |
Resistors | —- | —- | 2.2 kΩ Quantity: 1 |
Resistors | —- | —- | 1 kΩ Quantity: 1 |
Resistors | —- | —- | 220 Ω Quantity: 2 |
Resistors | —- | —- | 22 Ω Quantity: 5 |
Resistors | —- | —- | 10 Ω Quantity: 1 |
Resistors | —- | —- | 6.8 Ω Quantity: 1 |
Resistors | —- | —- | 3.3 Ω Quantity: 2 |
Polyester capacitors | —- | —- | 1 nF Quantity: 2 |
Polyester capacitors | —- | —- | 100 nF Quantity: 1 |
VHF Band Amplifier Transistor JFET | Toshiba | 2SK161 | —- Quantity: 1 |
Power transistor BJT NPN | Mospec | TIP 31C | —- Quantity: 1 |
Zener diode | Microsemi | 1N4148 | —- Quantity: 1 |
Switch | Toogle Switch | SPDT – T13 | —- Quantity: 3 |
Toroidal ferrite core | Caracol | —- | T*22*14*8 Quantity: 1 |
Cooper wire | Greenshine | —- | AWG – 24 Quantity: 1 |
Relimate header with female housing | ADAFRUIT | —- | 8 pin connectors Quantity: 1 |
Relimate header with female housing | ADAFRUIT | —- | 2 pin connectors Quantity: 1 |
Female plug terminal connector | JIALUN | —- | 4mm Lantern Plugs (Plug + Socket) 15 A Quantity: 1 |
Aluminum Heat Sink | AWIND | —- | For TIP 31C transistor Quantity: 1 |
Led | CHANZON | —- | 5 mm red Quantity: 1 |
Integrated circuit socket connector | Te Electronics Co., Ltd. | —- | Double row 8-pin DIP Quantity: 1 |
3 pin connectors set | STAR | —- | JST PH 2.0 Quantity: 3 |
2 pin screw connectors | STAR | —- | For PCB Quantity: 1 |
3 pin screw connectors | STAR | —- | For PCB Quantity: 1 |
Banana connector test lead | JIALUN | —- | P1041 – 4 mm – 15 A Quantity: 7 |
Bullet connectors to banana plug charge lead | JIALUN | —- | 4 mm male-male/female-female adapters – 15 A Quantity: 1 |
Case | —- | —- | ABS Quantity: 1 |
Electrodes | —- | —- | Stainless – steel Quantity: 2 |
Electrode support | —- | —- | Teflon Quantity: 2 |
Printed circuit board | Quantity: 1 | ||
Magnetic stimulator | |||
Cooper wire | Greenshine | —- | AWG – 18 Quantity: 1 |
AC power plugs | —- | —- | 120 V AC – 60 Hz Quantity: 1 |
Banana female connector test lead | JIALUN | —- | 1Set Dual Injection – 4 mm – 15 A Quantity: 2 |
Banana male connector test lead | JIALUN | —- | 1Set Dual Injection – 4 mm 15 A Quantity: 1 |
Cell culture well plate support | —- | —- | PMMA Quantity: 1 |
Fuse | Bussmann | 2A | —- Quantity: 1 |
Transformer | —- | —- | 1A – 6 V AC Quantity: 1 |
Tube | —- | —- | PVC Quantity: 1 |
Variable rheostat | MCP | BXS150 | 10 Ω Quantity: 1 |
General equipment | |||
Digital dual source | PeakTech | DG 1022Z | 2 x 0 – 30 V / 0 – 5 A CC / 5 V / 3 A fijo Quantity: 1 |
Digital Oscilloscope | Rigol | DS1104Z Plus | 100 MHz, bandwidth, 4 channels Quantity: 1 |
Digital multimeter | Fluke | F179 | Voltage CC – CA (1000 V). Current CC – CA 10 A. Frequency 100 kHz Quantity: 1 |