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.

The authors declare that they have no conflict of interest.

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.

<table><tbody><tr><td>&lt;strong&gt;Electrical stimulator&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Operational amplifier</td><td>Motorola</td><td>LF-353N</td><td>----&lt;br/&gt; Quantity: 1</td></tr><tr><td>Resistors</td><td>----</td><td>----</td><td>22 k&amp;Omega;&lt;br/&gt; Quantity: 1</td></tr><tr><td>Resistors</td><td>----</td><td>----</td><td>10 k&amp;Omega;&lt;br/&gt; Quantity: 3</td></tr><tr><td>Resistors</td><td>----</td><td>----</td><td>2.6 k&amp;Omega;&lt;br/&gt; Quantity: 2</td></tr><tr><td>Resistors</td><td>----</td><td>----</td><td>2.2 k&amp;Omega;&lt;br/&gt; Quantity: 1</td></tr><tr><td>Resistors</td><td>----</td><td>----</td><td>1 k&amp;Omega;&lt;br/&gt; Quantity: 1</td></tr><tr><td>Resistors</td><td>----</td><td>----</td><td>220 &amp;Omega;&lt;br/&gt; Quantity: 2</td></tr><tr><td>Resistors</td><td>----</td><td>----</td><td>22 &amp;Omega;&lt;br/&gt; Quantity: 5</td></tr><tr><td>Resistors</td><td>----</td><td>----</td><td>10 &amp;Omega;&lt;br/&gt; Quantity: 1</td></tr><tr><td>Resistors</td><td>----</td><td>----</td><td>6.8 &amp;Omega;&lt;br/&gt; Quantity: 1</td></tr><tr><td>Resistors</td><td>----</td><td>----</td><td>3.3 &amp;Omega;&lt;br/&gt; Quantity: 2</td></tr><tr><td>Polyester capacitors</td><td>----</td><td>----</td><td>1 nF&lt;br/&gt; Quantity: 2</td></tr><tr><td>Polyester capacitors</td><td>----</td><td>----</td><td>100 nF&lt;br/&gt; Quantity: 1</td></tr><tr><td>VHF Band Amplifier Transistor JFET</td><td>Toshiba</td><td>2SK161</td><td>----&lt;br/&gt; Quantity: 1</td></tr><tr><td>Power transistor BJT NPN</td><td>Mospec</td><td>TIP 31C</td><td>----&lt;br/&gt; Quantity: 1</td></tr><tr><td>Zener diode</td><td>Microsemi</td><td>1N4148</td><td>----&lt;br/&gt; Quantity: 1</td></tr><tr><td>Switch</td><td>Toogle Switch</td><td>SPDT - T13</td><td>----&lt;br/&gt; Quantity: 3</td></tr><tr><td>Toroidal ferrite core</td><td>Caracol</td><td>----</td><td>T*22*14*8&lt;br/&gt; Quantity: 1</td></tr><tr><td>Cooper wire</td><td>Greenshine</td><td>----</td><td>AWG &amp;ndash; 24&lt;br/&gt; Quantity: 1</td></tr><tr><td>Relimate header with female housing</td><td>ADAFRUIT</td><td>----</td><td>8 pin connectors&lt;br/&gt; Quantity: 1</td></tr><tr><td>Relimate header with female housing</td><td>ADAFRUIT</td><td>----</td><td>2 pin connectors&lt;br/&gt; Quantity: 1</td></tr><tr><td>Female plug terminal connector</td><td>JIALUN</td><td>----</td><td>4mm Lantern Plugs (Plug + Socket) 15 A&lt;br/&gt; Quantity: 1</td></tr><tr><td>Aluminum Heat Sink</td><td>AWIND</td><td>----</td><td>For TIP 31C transistor&lt;br/&gt; Quantity: 1</td></tr><tr><td>Led</td><td>CHANZON</td><td>----</td><td>5 mm red&lt;br/&gt; Quantity: 1</td></tr><tr><td>Integrated circuit socket connector</td><td>Te Electronics Co., Ltd.</td><td>----</td><td>Double row 8-pin DIP&lt;br/&gt; Quantity: 1</td></tr><tr><td>3 pin connectors set</td><td>STAR</td><td>----</td><td>JST PH 2.0&lt;br/&gt; Quantity: 3</td></tr><tr><td>2 pin screw connectors</td><td>STAR</td><td>----</td><td>For PCB&lt;br/&gt; Quantity: 1</td></tr><tr><td>3 pin screw connectors</td><td>STAR</td><td>----</td><td>For PCB&lt;br/&gt; Quantity: 1</td></tr><tr><td>Banana connector test lead</td><td>JIALUN</td><td>----</td><td>P1041 - 4 mm - 15 A&lt;br/&gt; Quantity: 7</td></tr><tr><td>Bullet connectors to banana plug charge lead</td><td>JIALUN</td><td>----</td><td>4 mm male-male/female-female adapters - 15 A&lt;br/&gt; Quantity: 1</td></tr><tr><td>Case</td><td>----</td><td>----</td><td>ABS&lt;br/&gt; Quantity: 1</td></tr><tr><td>Electrodes</td><td>----</td><td>----</td><td>Stainless &amp;ndash; steel&lt;br/&gt; Quantity: 2</td></tr><tr><td>Electrode support</td><td>----</td><td>----</td><td>Teflon&lt;br/&gt; Quantity: 2</td></tr><tr><td>Printed circuit board</td><td></td><td></td><td>Quantity: 1</td></tr><tr><td>&lt;strong&gt;Magnetic stimulator&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cooper wire</td><td>Greenshine</td><td>----</td><td>AWG &amp;ndash; 18&lt;br/&gt; Quantity: 1</td></tr><tr><td>AC power plugs</td><td>----</td><td>----</td><td>120 V AC &amp;ndash; 60 Hz&lt;br/&gt; Quantity: 1</td></tr><tr><td>Banana female connector test lead</td><td>JIALUN</td><td>----</td><td>1Set Dual Injection - 4 mm &amp;ndash; 15 A&lt;br/&gt; Quantity: 2</td></tr><tr><td>Banana male connector test lead</td><td>JIALUN</td><td>----</td><td>1Set Dual Injection - 4 mm 15 A&lt;br/&gt; Quantity: 1</td></tr><tr><td>Cell culture well plate support</td><td>----</td><td>----</td><td>PMMA&lt;br/&gt; Quantity: 1</td></tr><tr><td>Fuse</td><td>Bussmann</td><td>2A</td><td>----&lt;br/&gt; Quantity: 1</td></tr><tr><td>Transformer</td><td>----</td><td>----</td><td>1A &amp;ndash; 6 V AC&lt;br/&gt; Quantity: 1</td></tr><tr><td>Tube</td><td>----</td><td>----</td><td>PVC&lt;br/&gt; Quantity: 1</td></tr><tr><td>Variable rheostat</td><td>MCP</td><td>BXS150</td><td>10 &amp;Omega;&lt;br/&gt; Quantity: 1</td></tr><tr><td>&lt;strong&gt;General equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Digital dual source&amp;nbsp;</td><td>PeakTech</td><td>DG 1022Z</td><td>2 x 0 - 30 V / 0 - 5 A CC / 5 V / 3 A fijo&lt;br/&gt; Quantity: 1</td></tr><tr><td>Digital Oscilloscope</td><td>Rigol</td><td>DS1104Z Plus</td><td>100 MHz, bandwidth, 4 channels&lt;br/&gt; Quantity: 1</td></tr><tr><td>Digital multimeter</td><td>Fluke</td><td>F179</td><td>Voltage CC &amp;ndash; CA (1000 V). Current CC &amp;ndash; CA 10 A. Frequency 100 kHz&lt;br/&gt; Quantity: 1</td></tr></tbody></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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5.0K Views.  Universidad Nacional de Colombia. Biofysische stimulus is gebruikt om de cel- en moleculaire dynamiek in verschillende weefsels te stimuleren. Bepaalde studies hebben de impact van elektrische en magnetische velden in verschillende soorten cellen geëvalueerd, zoals chondrocyten, osteoblasten en fibroblasten, weefselimplantaten en steigers. Hoewel verschillende stimulerende apparaten zijn ontwikkeld onder de specifieke kenmerken om biologische weefsels te stimuleren, is het noodzakelijk om elektrische en magnetische apparaten...

Video: Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Medicine

Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. It has been extensively used in various clinical settings. Based upon recent discoveries related to the systemic effects of voluntary breathing and intrinsic physiological interactions among systems during voluntary breathing, a new EStim protocol, Breathing-controlled Electrical Stimulation (BreEStim), has been developed to augment the effects of electrical stimulation. In BreEStim, a single-pulse electrical stimulus is triggered and delivered to the target area when the airflow rate of an isolated voluntary inspiration reaches the threshold. BreEStim integrates intrinsic physiological interactions that are activated during voluntary breathing and has demonstrated excellent clinical efficacy. Two representative applications of BreEStim are reported with detailed protocols: management of post-stroke finger flexor spasticity and neuropathic pain in spinal cord injury.

Breathing-controlled Electrical Stimulation BreEStim for Management of Neuropathic Pain and Spasticity

The purpose is to present a new method, breathing-control electrical stimulation (BreEStim) for management of neuropathic pain and spasticity.

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. ...

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

Neuroscience

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

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