The authors thank the financial support provided by &#34;Fondo Nacional de Financiamiento para la Ciencia, la Tecnolog&#237;a, y la Innovaci&#243;n -Fondo Francisco Jos&#233; de Caldas- Minciencias&#34; 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.

1. Simulation of EFs and MFs
NOTE: Simulation of EFs and MFs was performed in COMSOL Multiphysics.
<ol>
	<li>Select an axisymmetric 2D configuration to represent both domains electric and magnetic.</li>
	<li>In the physic configuration, select either the Electric Current interface to compute EFs in parallel electrodes or the Magnetic Field interface to compute MFs around coils.</li>
	<li>In the study configuration, select Frequency Domain to...

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...

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.&#160;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.

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</table>

electric and magnetic field devices for stimulation of biological tissues

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.

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 e...

Watch this Scientific Journal Video about Electric and Magnetic Field Devices for Stimulation of Biological Tissues at JoVE.com

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

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, ...

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