The research aims to advance in vitro cardiac microphysiological models by integrating material-based photostimulation methods. It focuses on overcoming limitations in longevity and stimulation invasiveness, providing reliable tools for studying cardiac physiology, improving drug screening processes, and fostering applications in disease modeling. The recent advancements in biostimulation leverage, like for precise non-invasive cellular control.
Optogenetics enables genetic modification to regulate cellular activity, while emerging material-based phototransducers offer non-genetic alternatives. This innovation provide a significant breakthrough in studying and modulating neurons, cardiomyocytes, and skeletal muscle cells across diverse applications. Current experimental challenges include achieving consistent light delivery to deep tissues, optimizing the biocompatibility and stability of phototransducers, and improving the precision of light-based stimulation techniques.
Additionally, integrating these methods with complex biological systems while maintaining cellular viability and functionality remains a key hurdle. This protocol addresses the research gap of the need for non-invasive precise stimulation techniques in cardiac microphysiological models. By using material-based phototransducers, like Ziapin2, this approach overcomes the limitations of traditional electrical stimulation and optogenetics, offering enhanced temporal and spacial contour while preserving tissue viability and function.
My findings will advance research by introducing a non-invasive, material-based light stimulation technique that offers greater precision and control over cellular activity in cardiac tissues. This approach eliminates the need for genetic modifications, providing a versatile tool for modeling heart function, studying disease mechanisms, and developing more effective therapeutic strategies. To begin, adhere two layers of laboratory tape, one white and one blue, to a one millimeter thick clear, scratch and ultraviolet-resistant acrylic sheet.
Cut the chip pattern on the tape according to the intended design, then, using a carbon dioxide laser engraver, cut the acrylic sheet into circles. Remove the two layers of tape inside the innermost line using tweezers. Soak the chips in pure bleach for 30 minutes to one hour to remove thick lines and dark spots from cutting, leaving a sharp line, and rinse the chips in a beaker with running deionized water overnight or for at least three hours.
Sonicate the chips for 10 minutes in the polydimethylsiloxane, or PDMS stamps, with line groove features for 30 minutes in clean 70%ethanol. Transfer the chips and stamps to a clean area under a hood and let them dry under airflow for approximately one to two hours. Next, sonicate the freshly prepared gelatin for 15 minutes, then return it to the 65 degree Celsius water bath until ready for use.
Place the microbial transglutaminase, or MTG tube, in a desiccator with the cap slightly loosened and slowly turn on the vacuum to remove the bubbles. After degassing, return the MTG tube to the 37 degree Celsius water bath. Then, cover a grid sheet with clean Parafilm and place the chips on the grid.
Keep the PDMS stamp nearby for use. Add five milliliters of MTG to five milliliters of the gelatin solution, pipetting carefully to avoid bubbles. Now, quickly aliquot approximately 0.5 milliliters of the gelatin mixture onto each chip, ensuring the mixture covers the chip area.
Place the line-patterned PDMS stamp on top and apply a 200 gram weight to ensure the gelatin is patterned parallel to the tissue's longitudinal axis. Once all chips are molded, cover them with a glass jar to avoid environmental disturbance and allow them to cross-link overnight. Transfer the chip and PDMS stamp sandwiched to a new P150 dish filled with PBS to hydrate the gelatin for 30 minutes to one hour to facilitate the separation of the PDMS stamp from the chip.
After removing any excess unmold gelatin around the chip, transfer the clean chip to a new P150 dish filled with PBS. Store the PDMS stamps in 70%ethanol. To sterilize the chips, soak them in ethanol for 10 minutes under the hood.
Transfer the chips to PBS, soak them for 10 minutes, followed by a three times PBS wash. For the coating solutions, mix 20 micrograms per milliliter fibronectin with a one to 100 dilution of Geltrex in culturing media without supplement. Coat the chips with this solution for two hours in an incubator at 37 degrees Celsius and 5%carbon dioxide.
Thaw and seed human-induced pluripotent stem cell-derived cardiomyocytes in RPMI medium containing 10 micromolar Y-27632. Replace the medium with RPMI devoid of Y-27632 after 24 hours. Three days after cell seeding, use tweezers to carefully remove the white tape from the chips.
Prepare the optical mapping apparatus consisting of a modified tandem lens microscope equipped with a high-speed camera and a 200 milliwatt mercury lamp as the excitation light source. Place a dichroic mirror in front of the designated calcium imaging camera. For optical pacing, apply optical point stimulation at one end of the tissue using an LED light source to stimulate Ziapin2, the phototransducer.
Pace the tissues at a frequency of 0.5, or one hertz, via a temporally regulated optical fiber positioned one millimeter away from the tissue. Incubate the sample with two micromolar X-Rhod-1 added to the culturing medium for 30 minutes at 37 degrees Celsius. After washing the chips with fresh culture medium, transfer them to phenol red-free RPMI 1640 medium, supplemented with B-27 minus insulin and HEPES.
Place the tissue chips in a temperature-controlled dish set to physiological temperature and start recordings at a frame rate of 2.5 frames per second. Calcium wave propagation was successfully visualized, with a clear spatial and temporal resolution during photostimulation at either 0.5 or one hertz frequencies, with conduction velocities calculated as approximately 4.5 centimeters per second, consistent with physiological values. Calcium transient parameters, including amplitude, rise time, maximum decay, slope, and decay time, were quantitatively similar between light stimulation and electrical stimulation, confirming comparable functional responses.
