We demonstrate countless method for all optical control and observation of triggered cellular activity in iPSC derived cardiomyocytes for high-throughput drug screening and toxicity test. Enable multi parametric quantification of the phenotypic patterns in time and space, allow observing the effect of drugs over hours or days. This method can be applied to different type of cells, such as, neuros, or beta cells, for functional screening and toxicity test.
Demonstrating the procedure will be Wan-Chi Su, a bio-imaging engineer from my laboratory. Prepare the multi-well plate before iPSC cardiomyocytes are removed from cold storage for thawing. Coat each well of the 96 well micro plate with 100 microliters of 0.02%gelatin solution with 50 micrograms per milliliter of fibronectin to cover all surfaces thoroughly.
Transfer the iPSC cardiomyocytes from the liquid nitrogen storage tank to a 37 degrees Celsius water bath. Take the vial to a class two bio-safety cabinet, and gently transfer the contents to a new 15 milliliter conical tube containing nine milliliters of room temperature medium. Centrifuge the cells at 200 times G for three minutes at room temperature.
Discard the supernatant by pipette, and gently re-suspend the cells in one milliliter of medium with 10 micromolar ROCK inhibitor Y27632. Plate cells encoated 96 well plates at the density of 100, 000 to 150, 000 cells per square centimeter, as per manufacturer's instructions. After 24 hours, ensure that the cells attached to the surface of the plate are in contact with other cells.
After plating the cells for 72 hours thaw the GECIs viral kits on ice. Prepare a double strength viral kit stock solution with the maintenance medium. Prepare the cells for infection by adjusting the medium volume to 100 microliters per well.
Add 100 microliters of the premixed viral solution to wells, and incubate for eight to 16 hours at 37 degrees Celsius. After incubation, replace with 200 microliters pre-warmed medium. Visualize the GECIs expression 24 hours post transduction using an imaging system.
Maintain cells in the humidified incubator before functional assays are performed. Switch on all the devices of the high content imaging system. Verify that the chamber temperature reaches 37 degrees Celsius with 5%carbon dioxide supplementation.
Open the imaging software, and choose the plate setting for a 96 well plate. Place an un-lidded 96 well plate into the chamber, and load the live cell sealing ring on top. Choose the 20X, 60X, and 20X water immersion objective lens, according to the spatial resolution required.
Adjust the focus to take clear images before experimental acquisition. Select three to five regions randomly per well for calcium activity recording. Choose appropriate filters for different indicators.
Set the appropriate light emitting diode power for each imaging channel used. Set the camera exposure time to a maximum of 40 milliseconds or 25 hertz, and a recording duration of 30 seconds for stream acquisition to observe calcium transients, reported by MNG-GECO and K-GECO probes expressed in cardiomyocytes. Increase the LED power if the signal to noise ratio is less than two at higher frame rates.
For optogenetic stimulation, optimize the power and frequency of blue light at one hertz, and start acquisition. Re-suspend the E4031 Dofetilide, and Verapamil in DMSO, and dilute them with Tyrode's buffer. Arrange compounds to the corresponding target well.
Add 100 microliters of the double strength compound via the auto micro-fluidic system into the wells, and start acquisition after the desired incubation period. Isolate the signal area from the background by automatically detecting the pulses feature, over time resolution, with the software. Use the calcium peak analysis software to analyze the beating frequency, peak signal, calcium transient duration 50%calcium transient duration 90%rise time, and decay time.
The observation of spontaneous calcium oscillation in NMG-GECO expressed by iPSC derived cardiomyocytes with or without drug treatments is demonstrated in the video. Kinetic traces obtained using MNG-GECO showed that small molecular ion channel inhibitors Verapamil, Dofetilide, and E4031 affected calcium transients as expected. To evaluate the dose response of E4031 under standardized, paced conditions channelrhodepsin-2, and K-GECO expressing iPSC CMs were optically controlled using one hertz and 470 nanometer pulses of light, and observed using the red K-GECO signal.
Progressive reductions of peak amplitude, and increase in the decay time of the calcium transients, occur with increasing concentrations of E4031. A dose dependent effect of E4031 was most apparent for the reductions in peak amplitude. Clear calcium transients remain visible a month after viral transduction, or without the additional light used for optical pacing.
The graph depicts a drug that appears to increase beat rate, regularize contraction interval, and increase the transient calcium amplitude, in the LVNC iPSC CM mode. The variability of beat frequency, and the subsequent impact on calcium transient duration, also change as iPSC CM are maintained in culture. Several GECIs, including, ER LAR-GECO, MTG-Sepia, and NIR-GECO, have been developed for expression singly, or in combination in cell models for real time calcium activity measurement arising in the endoplasmic reticulum, sarcoplasmic reticulum, mitochondria, and cytosol, respectively.
The GECIs can be combined in the iPSC CM model to study different intracellular calcium stores. K-GECO expressing cardiomyocytes showed consistent beating behavior over time. However, Fluo-4 loading impacted both, the beat frequency, and CTD in this model, suggesting Fluo-4, itself, might influence the results of such experiments.
We deliver a simple way to study phenotypic profiles from control and patient derived cells, while iPSC intermediate may shed light on drug discovery in various disease.
