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.
