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

Summary

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.

Abstract

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.

Introduction

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, neuroimmunity^2,3,4, 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 cytosol^9,10. 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 organelles^13,15. 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.

Protocol

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

1. Isolate primary cardiomyocytes (CMs) using a commercial kit following manufacturer’s guidelines.
2. Prepare complete DMEM for primary cell isolation supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine.
3. To isolate myofibroblast (MFs) from the MFs-CMs suspension, pre-plate the isolated cells on a tissue culture dish (cells isolated from 2 hearts from step 1.1. per 100 mm dish) for 1 h. As CMs need a fibronectin or collagen treated surface to adhere, only MFs will attach to tissue culture surface.
4. Aspirate the enriched CMs cell suspension. These CMs can be used for hetero-cellular coupling experiments as described in section 3. Rinse the MFs with DMEM to eliminate any remaining CMs from the MFs dish.
5. Add fresh culture medium to MFs and allow them to proliferate until they are ~80% confluent (2-4 days). Change the medium every other day.
6. When the cells are ready (80% confluent), prepare SiNWs for the hybridization step below (step 1.7).
   NOTE: Many types of SiNWs can be used for this. In this experiment, SiNWs that were grown by chemical vapor deposition (CVD) with a core-shell p-i-n junction configuration were used as described previously¹⁶. During the CVD growth, the SiNWs are grown on a silicon wafer substrate, and are eventually kept as silicon chips covered with SiNWs.
7. Cut a 3 mm x 3 mm chip from a wafer with CVD grown SiNWs using a diamond scribe. Use sharp forceps to handle the wafer and minimize the surface area that is touched with the forceps, as this may break the SiNWs.
8. Sterilize the chip by rinsing it with 70% ethanol and allowing the ethanol to dry for 30 min under UV light in a biosafety laminar flow hood.
9. Transfer the chip into a sterile microcentrifuge tube and rinse excess ethanol using complete culture media.
10. Add 1 mL of culture media and sonicate the chip in sonication bath for 1-10 min. The media should turn cloudy as SiNWs are released into the media.
    NOTE: Sonication time and power should be optimized for different sonicators or different cells, as shorter durations and lower powers will yield longer SiNWs.
11. Add the SiNWs suspension into 5 mL of culture media and seed it onto the 100 mm tissue culture dish with MFs. Allow the SiNWs to internalize for 4 h and rinse the excess SiNWs off 5x with media. Allow partially internalized SiNWs to complete the internalization by allowing them to sit for another 1 h before use.
    NOTE: Different cell types may need different SiNWs concentration and/or internalization times.
12. Prepare collagen coating solution by diluting collagen stock solution (3 mg/mL) with sterile 20 mM acetic acid at a ratio of 1:50. Add 0.5 mL coating solution to a 35 mm glass bottom dish and allow it to sit for 1 h in 37 °C. Remove the solution and rinse dish with sterile PBS.
13. Harvest the cell-SiNWs hybrids by treating the cells with 3 mL of trypsin for 2 min at 37 °C. Add 10 mL of culture media and rinse the hybrids vigorously by pipetting. Centrifuge the cells gently at 200 x g for 5 min to avoid damaging the cells with the internalized SiNWs. Remove excess media, suspend cells with 1 mL media and seed them onto the collagen treated glass bottom dish.
    NOTE: The hybrids can be seeded alone, to investigate intracellular or intercellular coupling or with CMs to investigate hetero-cellular coupling in vitro.
14. Perform a final verification of SiNW internalization by labeling cells’ cytosol (calcein-AM, 4 µM) and membrane (membrane marker, 2 µM) for 30 min at 37 °C, and imaging the cell using confocal microscopy. As SiNWs are highly reflective, reflected light can be used instead of fluorescence to visualize them.

2. Preparation of cells for intra- and intercellular investigations

1. Prepare collagen coating solution as in 1.12
2. For intracellular electrical stimulation, culture hybrids with low seeding densities. Use a standard hemocytometer to count cells from section 1.11. and seed 50,000 cells on a 35 mm glass bottom dish in culture media. For intercellular investigations, use higher cell densities (500,000 cells per dish). For intercellular coupling between CMs and MFs, co-culture the hybrids with freshly isolated CMs. 
3. Allow cells to attach overnight before performing optical stimulation experiments. For intercellular investigations, allow 48 h at 37 °C for the cells to express intercellular gap junctions before the experiment is conducted.
4. Prepare calcium sensitive dye stock solution (may be kept in -20 °C) by adding 50 µL of DMSO to 50 µg Fluo-4 AM. Prepare staining solution by diluting 1 µL of dye in 1 mL of DMEM.
5. Aspirate the culture medium from cells and add 1 mL of staining solution. Allow dye to internalize into cells for 20-30 min at 37 °C. Aspirate the dye and rinse twice with sterile PBS.
6. Finally, add 1 mL of pre-warmed phenol-red free DMEM Media, and allow the intracellular Fluo-4 to undergo de-esterification for 30 min in 37 °C.
7. Transfer cells to microscope for imaging and stimulation.

3. Optical imaging and stimulation

1. Pre-heat a humidified microincubator to 37 °C and bubble air-CO₂ mixture (95:5).
2. Use a microscope with a collimated laser line coupled into the light path for calcium imaging and optical stimulation.
   NOTE: A scanning confocal microscope is the most straightforward option due to its point stimulation capabilities. However, this procedure can be done using any standard florescence microscope by coupling a collimated laser beam into the infinity space of the light path using a beam splitter. Any microscope objective is designed so that a collimated laser passed through it will be focused to a diffraction limited laser spot at the focal plane. The laser wavelength should be close to the excitation light, so it will be reflected by the dichroic mirror and passed by the excitation filter.
3. Visualize the SiNWs and determine the stimulation site using brightfield microscopy, transmitted light, or reflective light. Then, reconfigure the light path to fluorescence mode, while maintaining the stimulation point at the predefined location of the SiNW.
4. Validate the optimal stimulation power and pulse length for each SiNW size and cell type, to minimize photothermal damage to stimulated cells. For a typical stimulation protocol, perform a 2-10 s baseline recording of the intracellular calcium activity. Then, apply a single laser pulse of 1-10 mW power and 1-10 ms duration (corresponding to 30-300 kW/mm²) to stimulate the SiNW, and record the resulting calcium wave for another 2-10 s.
   NOTE: It is essential to optimize the optical power and pulse length for each SiNW size and cells. This is necessary to minimize photothermal damage to the stimulated cells.
5. Transfer the recorded movies of the optical stimulation, if necessary, for further analysis.

4. Video processing

1. Visualize changes in Fluo-4 fluorescence using the “dF over F” macro¹⁷ available for ImageJ¹⁸, which calculates the change in fluorescence counts for each pixel, and normalizes by the average value of the resting baseline. Convert the output (floating point format) to 8 bits for further processing.
2. Process the dF/F movie further using the “Remove outliers” selective median filter available in ImageJ. Define parameters to remove pixels where their value is more than 10 above the median value in a 2-pixel radius and replace that pixel value with the median.
3. Calculate the optical flow in each frame via the Lucas-Kanade algorithm, as implemented in the Matlab Computer Vision toolbox. The output of this function was a vector field containing the x- and y-components of the optical flow at each point in each image (code is available in Supplementary File 1).
   NOTE: The mean optical flow within a cell, <ν>, corresponds to the development of calcium flux within a cell. The differential of the optical flow, Δ<ν> provides the time point at which calcium signaling was activated, by identifying where the signal was maximized. Additionally, the magnitude of Δ<ν> at its maximum is correlated to the rate at which the calcium wave front progresses through the cell.
4. Calculate the intercellular speed of calcium transmission according to the following equation.
   v_Ca²⁺ = r_ij / ( t_max,j − t_max,i ),
   where t_max,j and t_max,i are the times of activation for cells j and i, respectively, and r_ij is the distance between the centroids of the cells.

Results

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 µm long into car...

Discussion

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...

Materials

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