Name: silicon nanowires and optical stimulation for investigations of intra- and intercellular electrical coupling
Uploaded: 2021-01-28T14:00:00
Description: Watch this Scientific Journal Video about Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling at JoVE.com

This work is supported by the Air Force Office of Scientific Research (AFOSR FA9550-18-1-0503).

To ensure compliance with ethical standards, all animal procedures related to isolating cardiomyocytes from rodent hearts were first approved by the University of Chicago Institutional Animal Care and Use Committee (IACUC). Additionally, all animal experiments were conducted in complete accordance with guidance from the University of Chicago IACUC.
1. Preparation of cell-SiNWs hybrids
<ol>
	<li>Isolate primary cardiomyocytes (CMs) using a commercial kit following manufacturer&#8217;s guidelines....

<ol>
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We have demonstrated here a simple way to perform intracellular electrical stimulation of cells. In this demonstration, we used MFs that were prehybridized with SiNWs, then co-cultured with CMs. In general, most proliferating cells have the tendency to internalize SiNWs, which allows the use of this methodology with many other cell types. Moreover, while we demonstrated the intracellular stimulation of cells, the same principles can be used to perform extracellular stimulation of cells. This can be done by blocking endos...

All biological organisms use electricity, in the form of ions, to regulate the cellular behavior. Cell membranes contain various types of specific ion channels allowing the passive and active transport of ions. These ions govern the functions of excitable cells, such as neuronal activity and skeletal and cardiac muscle contractility. However, bioelectricity also plays an important role in non-excitable cells, governing many cellular functions such as cell proliferation1, neuroimmunity2,3,4, and stem cell differentiation5.
In recent decades, the field of bioelectricity has drawn an increasing level of interest, which has contributed to the development of numerous technologies for bioelectronic interfaces. Microelectrode patch pipettes are the gold standard of intracellular recording and stimulation6. In this methodology, a glass pipette is pulled under specific conditions to form a sharp edge with a pore size of few microns. This pipette is filled with a buffer and the pipette allows direct contact of the buffer with the intracellular volume. This results in a bioelectric interface that yields extremely high signal to noise ratios, precise control over cellular electrical activity, and extremely high temporal resolution. Although this methodology is an extremely powerful tool, which was recently downscaled to a nano-pipette configuration7, it is associated with several important technical limitations. The cytosol dilution effect8, as well as mechanical vibrations, limits its utility to short term interrogations, and it requires expensive specialized equipment and a high level of technical skill. Moreover, its bulkiness limits the number of cells that can be recorded or stimulated simultaneously, and due to its invasiveness, it cannot be reconfigured throughout an experiment. To overcome these limitations, microelectrode arrays were developed, but the size of the electrodes limits the spatial resolution as well as intracellular access. Nanoelectrode arrays allow intracellular recording and stimulation but require abrasive electroporation to access the cytosol9,10. In addition, all these methodologies are substrate bound and are thus limited to in vitro cell cultures, or to external superficial cells, with no access to cells that are inside a 3-dimensional (3D) tissue.
Optogenetics11 is widely used to address these 3D and in vivo limitations. However, optogenetic methods are based on the perturbations of light-activated plasma membrane ion channels that are distributed at the plasma membrane, limiting the 3D spatial resolution12 and intracellular capabilities.
We have recently shown that silicon nanowires (SiNWs) can be used to perform intracellular bioelectric interrogation with submicron spatial resolution with different non-excitable cells, namely cardiac myofibroblasts and oligodendrocytes13. Moreover, we used these SiNWs to perform ex-vivo cell specific interrogation within a 3D cardiac tissue, to investigate how cardiac cells electrically couple in vivo14. A major advantage of this methodology is its simplicity; it does not require any genetic modification or bulky instrumentation. Many cells will spontaneously internalize photo-responsive SiNWs with no need for sonication or electroporation15. In addition, they will spontaneously escape the endosomal encapsulation and form a seamless integration with the cytosol and intracellular organelles13,15. These cell-SiNWs composites, termed cell-silicon hybrids, possess the dynamic, soft and versatile nature of the original cell, as well as the optoelectric capabilities of the SiNWs. After hybridization, the cell-SiNW hybrid can be harvested using standard tissue culture techniques and used for various applications such as intracellular bioelectric stimulation; studying intercellular bioelectric coupling in vitro; and for in vivo cell specific interrogation. As an effective stimulation requires co-localization of high optical power densities and SiNWs, one can achieve high spatial resolution both in 2D and 3D. In this protocol we describe in detail the methodology, as well as how the results can be analyzed. The focus is placed on the intra- and intercellular investigation in vitro, but the in vivo implementation of this methodology can be directly utilized for many other biological scenarios.

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			<td height="21" style="height:21px;width:216px;">35 mm Glass bottom dishes</td>
			<td style="width:121px;">Cellvis</td>
			<td style="width:161px;">D35-10-0-N</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3i Marianas Spinning Disk Confocal</td>
			<td style="width:121px;">3i</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcein-AM</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">C1430</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellMask Orange Plasma membrane Stain</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">C10045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagen I, rat tail</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">A1048301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deluxe Diamond Scribing Pen</td>
			<td style="width:121px;">Ted Pella</td>
			<td style="width:161px;">54468</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM, high glucose, pyruvate, no glutamine</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">10313039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO, Anhydrous</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">D12345</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Falcon Standard Tissue Culture Dishes</td>
			<td style="width:121px;">Falcon</td>
			<td style="width:161px;">08-772E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum, certified, heat inactivated,</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">10082147</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fibronectin Human Protein, Plasma</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">33016015</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fisherbrand 112xx Series Advanced Ultrasonic Cleaner</td>
			<td style="width:121px;">Fisher Scientific</td>
			<td style="width:161px;">FB11201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-4, AM, cell permeant</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">F14201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FluoroBrite DMEM Media</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">A1896701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Glutamine (200 mM)</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">25030081</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">OKO full environmental control chamber (constant temperature, humidity and CO2)</td>
			<td style="width:121px;">OKO</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS, pH 7.4</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">10010023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pierce Primary Cardiomyocyte Isolation Kit</td>
			<td style="width:121px;">Thermo Scientific</td>
			<td style="width:161px;">88281</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td style="width:121px;">Gibco</td>
			<td style="width:161px;">25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The ability of this methodology to allow direct access to the intracellular cytosol depends on the spontaneous internalization of the SiNW into the cells. Although SiNWs will undergo spontaneous internalization into many cell types15, some cells, such as cardiomyocytes and neurons, will need the SiNWs to be treated to allow their internalization19. In this protocol we describe the internalization process of p-i-n SiNWs with 200-300 nm diameter and ~1-3 &#181;m long into car...

Watch this Scientific Journal Video about Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling at JoVE.com

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

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 ...

This technique allows intracellular bioelectrical interrogation with extremely high spatial resolution and cell specificity using only standard optical microscopy. This method facilitates stimulation and electrical interrogation of cells and specific locations within the cells and can be performed in vitro as well as in 3D ex vivo tissue preparations. This technique enables the bio-electronic investigation of many cellular arenas such as cardiac or brain cells, as well as the electrical communication between all our non-excitable cells.To isolate myofibroblasts from a myofibroblast cardiomyocyte suspension, pre-plate the isolated cells on a 100 millimeter tissue culture dish for one hour, as cardiomyocytes need a fiber necked and treated surface to adhere. At the end of the incubation, collect the enriched cardiomyocyte containing supernatant for downstream co-culture. Rinse the myofibroblasts with DMEM to eliminate any remaining cardiomyocytes and feed the cells with fresh culture medium before an additional two to four days of incubation.When the cells reach 80%confluency, use a diamond scribe to cut a three by three millimeter chip from a wafer with chemical vapor deposition-grown silicon nanowires. And use sharp forceps to rinse the chips in 70%ethanol. After rinsing, air-dry the chips for 30 minutes under ultraviolet light in a bio-safety laminar flow hood, before transferring the chips to a sterile micro centrifuge tube.Use a complete cell culture medium rinse to remove any remaining ethanol, before sonicating the chips in one milliliter of fresh culture medium in a sonication bath for one to 10 minutes. The supernatant should turn cloudy as the nano wires are released. Mix the Silicon nanowire suspension into five milliliters of fresh culture medium and seed the nano wire solution onto the dish of myofibroblasts.After four hours in the cell culture incubator, rinse the dish five times with fresh culture medium to remove any uninternalized nanowires and continue to incubate the cells for another hour to allow any partially internalized nanowires to be completely incorporated. Next add 500 microliters of freshly prepared collagen coating solution to a 35 millimeter glass bottom dish. After a one hour incubation at 37 degrees Celsius, rinse the dish with sterile PBS and harvest the Silicon nanowire myofibroblast hybrids, with three milliliters of trypsin, for two minutes at 37 degrees Celsius.When the cells have detached, stop the reaction with 10 milliliters of culture medium, rinse vigorously by pipetting and do not centrifuge over 200 G to avoid damaging the cells. Then re-suspend the hybrid cell pellets in one milliliter of fresh medium, and seed the cells on the collagen coated glass bottom dish. To verify the nano wire internalization, label the cells with fluorescent cytosol and membrane dyes, according to standard protocols, and use a confocal microscope to image the cells.As silicon nanowires are highly reflective, reflected light can be used instead of fluorescence for their visualization. To set up a myofibroblast silicon nanowire hybrid cardiomyocyte co-culture, seed the appropriate number of each cell type onto a collagen coated glass bottom dish. And culture the cells at 37 degrees Celsius for 48 hours, to allow the cells to form intercellular gap junctions.On the day of the experiment, replace the culture supernatant with one milliliter of culture medium supplemented with freshly prepared calcium sensitive dye, for a 20 to 30 minute incubation at 37 degrees Celsius. At the end of the incubation, wash the plate two times with sterile PBS, before treating the cells with one milliliter of pre-warmed phenol red free DMEM. Then allow the intracellular calcium dye to undergo de-esterification for 30 minutes at 37 degrees Celsius, before acquiring baseline images.Before optical imaging and stimulation, preheat a humidified micro-incubator on a microscope with a collimated laser line coupled into the light path, for calcium imaging and optical stimulation to 37 degrees Celsius and a 5%bubble era carbon dioxide mixture. When the microscope is ready, place the myofibroblast hybrid cardiomyocyte co-culture into the micro incubator and visualize the nanowires by bright field microscopy to locate an appropriate stimulation site. When a site has been identified, reconfigure the light path to fluorescence mode, while maintaining the stimulation point at the pre-defined location of the nano wire, and validate the optimal stimulation power and pulse length, for each silicon nanowire size and cell type.Acquire a two to ten second recording of the baseline intracellular calcium activity, before stimulating the nano wire with a single laser pulse of one to 10 melowatts of power and a one to 10 millisecond duration, recording the resulting calcium wave for another two to 10 seconds. At the end of the experiment transfer the recorded movies of the optical stimulation to the appropriate software program, for additional analysis. Using standard phase contrast optics, confluence cells are easily viewed by light microscopy.Silicon nanowires, however, are barely visible, making their locations impossible to define by this method. Super imposition of light and dark field images, however, allows visualization of the Perry nuclear arrangement of the light reflective silicon nanowires. Confocal microscopy can be used to visualize fluorescent marker stained cytosol and plasma membranes, making the intracellular location of the silicon nanowires more evident.Using standard microscopy, a bright field image can be obtained to identify the location of the silicon nanowire to be stimulated. A short baseline video of the spontaneously beating cardiomyocytes and the resting myofibroblasts can then be acquired. After stimulation, the calcium propagation within the co-culture can be recorded.The time at which the different cells become excited can be determined by the point at which the change in average optical flow reaches its maximum. The optical flow can then be calculated to aid in the identification of the time of activation within each cell region. The intracellular velocities can be determined for each cell using Khaimah graphs, with the slope of the line representing the inverse of the intracellular propagation speed.For example, here, a summary of the different inter and intracellular velocities measured for the cell interactions within a single co-culture can be observed. This method can be utilized for in vivo studies by injecting the cell Silicon hybrids directly into the tissue and using 3D ex vivo preparations to study the in vivo electrical company.

Research

JoVE Journal

Bioengineering

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the  extracellular matrix of animal tissue to provide structural scaffolding and  mechanical strength. These native ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...