High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

This protocol is significant because it enables researchers to mature commercial or in-house-made hiPSC-CMs to an adult-like phenotype that significantly improves the predictive value of hiPSC-CMs in cardiotoxicity screening. The main advantage of this protocol is that it delivers mature hiPSC-CMs in a high throughput format for optical mapping of calcium or voltage change. This protocol may be used to investigate disease mechanisms or to screen drugs for cardiotoxicity.

Demonstrating the procedure will be Jeffrey Creech, a research associate from my laboratory. Begin by washing the maturation-inducing extracellular matrix, or MECM, plates two times with 200 microliters of Hank's balanced salt solution, or HBSS, 1 hour prior to cardiomyocyte plating. Keep the wells hydrated.

Transfer the cardiomyocyte tubes from the liquid nitrogen tank to dry ice, and release the pressure by slightly opening the tube caps. Next, reseal the tube caps and thaw them in the water bath for 4 minutes. After the thawing of the cells, spray the tubes with 70%ethanol before opening them.

Transfer the cells into 15-milliliter conical tubes with a 1-milliliter pipette. Add 1 milliliter of plating medium to the cryovial and transfer the wash into the 15-milliliter conical tube. Then, slowly drip 1 milliliter of plating medium and agitate the tube.

Repeat this until the transfer of 8 milliliters of plating medium. Centrifuge the 15-milliliter tube at 300 x g for 5 minutes and resuspend the pellet in 1 milliliter of the plating medium. Remove an aliquot, and perform live cell counting with a hemocytometer before adding a plating medium to obtain 7.5 x 10 to the 5th cells per milliliter.

Dispense 100 microliters of the cell suspension per well of an MECM-coated 96-well plate using a multichannel pipette, and incubate the cells for 2 days. Next, replace the medium with 200 microliters of maintenance medium, and maintain the plates for 7 days, with a change of medium on day 5. On day 7, perform EP assay, as described in the text.

Ensure to change the medium every other day when opting for extending the cell culture. To purify hiPSC-CM via magnetic-activated cell sorting, or MACS, aspirate the cell culture medium and wash the cells with 1 milliliter of HBSS-Then, dissociate the cells by adding 1 milliliter of 0.25%trypsin-ethylenediaminetetraacetic, or EDTA, and incubating the cells for 10 minutes. Then, inactivate the trypsin/EDTA by resending and singularizing the cells with 2 milliliters of plating medium.

From the six wells, collect the cells into a 50-milliliter conical tube using a 70-micrometer strainer. After washing the strainer with 3 milliliters of plating medium, count the cells. After counting, centrifuge and wash the cell pellet with 2 milliliters of ice-cold MACS separation buffer before pelleting and resending the cells in 80 microliters of cold MACS separation buffer per 5 x 10 to the 6th cells.

Add 20 microliters of cold non-cardiomyocyte depletion cocktail and mix the suspension before incubating on ice for 10 minutes. After washing the cells with 4 milliliters of cold MACS separation buffer at 300 x g for 5 minutes, resuspend the pellet in 80 microliters of cold MACS separation buffer. Add 20 microliters of cold anti-biotin microbeads per 5 x 10 to the 6th cells, and gently mix the cell suspension before incubating on ice for 10 minutes.

While the samples are incubating, place the positive depletion columns fitted with the 30 micrometer pre-separation filters on the MACS separator, and place the labeled 15-milliliter collection tubes below the columns. Next, prime each column with 3 milliliters of cold MACS separation buffer. Mix the antibody-treated cell suspension with 2 milliliters of MACS separation buffer and add it to the column.

Add 2 milliliters of MACS separation buffer to each column and collect 12 milliliters of flowthrough cardiomyocyte suspension. Centrifuge the cardiomyocytes and discard the supernatant before resuspending the cardiomyocytes in a 1-milliliter plating medium. To determine the concentration, count the cells and adjust the volume to the desired seeding density before plating the purified cardiomyocyte cells on the MECM 96-well plates.

Aspirate and replace the cardiomyocyte maintenance medium with 100 microliters of voltage-sensitive dyes, or VSD, or calcium-sensitive fluorophores, or CSF, per well of a 96-well plate. After 30 minutes of incubation, replace the dyes with an assay medium or HBSS. Equilibrate the cells at 37 degrees Celsius before acquiring baseline data optical mapping using a high-throughput optical mapping device.

To treat the cells with drugs for acute exposure testing or map the cells chronically exposed to drugs of interest, dilute the drugs in dimethyl sulfoxide, or DMSO, and store them as stock solutions at 20 degrees Celsius before diluting them in HBSS to the desired concentrations. For cardiotoxicity testing in 96-well plates, add four doses of a compound with at least eight wells per dose. Use doses ranging from below to above the clinically-effective therapeutic plasma concentration, including the effective dose.

Similar to the baseline electrophysiology measurements before drug application, capture electrophysiology recordings at least 30 minutes after drug treatment for chronic studies. After baseline recordings, apply at least four doses of each medication to mature hiPSC-CMs monolayers expressing GCaMP6m, with at least eight wells per dose. Equilibrate the medications on the cells for 30 minutes, and warm the cells to 37 degrees Celsius before and during the data acquisition.

Following baseline recordings of an entire plate, add 500 nanomolar isoproterenol to every well to enable robust drug response data and quantify the effects of isoproterenol on monolayer beat rate, contraction amplitude, and calcium transient duration. To acquire optical mapping data, open the front drawer and position the plate on the plate heater. After opening the acquisition software and determining the file saving location in the software, select 10 to 30 seconds duration and frame rate of acquisition for higher temporal resolution, and click Start Acquisition.

Open the analysis software, and in the Import Filter tab, select either Browse for a single file or Tile Multiple to reconstruct a plate. Select the files and enter a number of rows and columns and select Automatic. Next, select Update Pixel Size, enter distance per pixel, and use the Well Wizard to determine the wells'location in the image before clicking Process Save and moving to the next tab.

Open the ROIs, or Regions of Interest tab, and choose to draw the ROIs manually, automatically, or not use ROIs at all, which will be considered for the analysis of the entire plate. After selecting the region in the well, click the Process Save button to move to the next step. Check the Hide Wells and Only Show Filtered boxes to visualize the ROIs.

Open the Analysis tab, select each well or ROI to confirm the accuracy of automatic beat detection. Add or remove beats from the traces by selecting an individual beat and pressing Delete on the keyboard. Once done, click on Save.

Next, click the Time-Space Plot button in the Analysis tab and add the line to determine the time-space plot to visualize data for each well or the ROI. After selecting a horizontal or vertical line, click on Generate Plot. After ensuring the accurate beat detection, proceed to the Export tab and select File Format, enter Beat Statistic option before clicking Export.

Once exported, open data files and run the chosen statistical analysis routine. Phase contrast imaging showed that the hiPSC-CMs plated on the MECM matured and became structurally distinct from the same batch of hiPSC-CMs replated on the mouse ECM. Mature cells became rod-shaped, while immature cells retained a circular shape.

Cardiomyocytes stained with alpha-actinin antibodies showed the typical shape of the cells cultured on each ECM condition. Consistent with the TnI staining, the alpha-actinin staining indicated that the hiPSC-CMs matured on the MECM promoted a rod-shaped mature phenotype and induced greater sarcomere organization. Mitochondrial content and activity were distinct between cells cultured on the mouse ECM and the MECM.

The electrophysiological data for action potentials recorded using VSDs and the optical mapping system showed that the atrial-specific cells have a significantly faster spontaneous beat rate and shorter action potential duration, or APD80, than the ventricular-specific cells. Whole-plate heat maps for parameters such as APD80 revealed the reproducibility of a given parameter within a plate. Typical action potentials of mature hiPSC-CM monolayers indicated the action potential morphology of isolated and tested adult cardiomyocytes.

A typical action potential spontaneous rhythm was also displayed. In response to isoproterenol, activation of the beta-1-adrenergic receptors caused positive chronotropy, positive inotropy, and positive lusitropy. HiPSC-CM response to human Ether-a-go-go-related gene, or hERG, channel blocker, including E-4031, domperidone, vandetanib, and sotalol, was studied using GCaMP6m calcium fluorescence to monitor rhythm.

The study showed the detection of early after-depolarizations caused by the E-4031 hERG channel blockade. Work fast with the cells in suspension and avoid shear stress by pipetting cells gently. Additionally, perform media change carefully to avoid damaging the hiPS syncytia.

Following this procedure, cells are amenable for protein, RNA, DNA, and lipids collection for analysis with investigators'favorite technique. The technique empowers researchers to investigate diseases and test for cardiotoxicity of new drugs using adult-like cardiomyocytes.