Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

The overall goal of this technique is to generate three dimensional engineered heart tissue that can be used as an in vitro contraction analysis platform for drug screening and disease modeling. This method can help answer key questions in the cardiac field about disease modeling, safety pharmacology and the effects of gene variants, or drugs on contractile force. The main advantage of this technique is that contractile force can be measured in engineered heart tissues from different species under a sterile conditions, for extended periods of time.

Scientists new to this method could struggle with lack of cellular quality. Or lack of a in the medium. It is important to optimize self preparation and avoid cycles of the Demonstrating this procedure will be Anika Benzin and Umber Saleem.

Before beginning the procedure, add 1.6 milliliters of warm two percent agarose into the first aid wells of a 24 well plate. And immediately place polytetrafluoroethylene in spacers into the agarose. After about 10 minutes at room temperature, the agarose will become opaque, signifying its solidification.

Remove the spacers and place PDMS racks into the castings so that pairs of rack posts reach into each agarose mold. When all of the racks have been placed, we suspend the cardiomyocytes in medium at a minimum concentration of 1.5 times 10 to the seven cells per milliliter and freshly prepared reconstitution mix with fibrinogen 10 to 15 times with the serological pipette until the fibrinogen clot is dissolved. Check the reconstitution mix in the pipette for homogeneity.

Then for each engineered heart tissue, or EHT, mix 100 microliters of reconstitution mix with three microliters of thrombin in a 200 microliter tube. And immediately aliquot 100 microliters of this EHT thrombin mixture into the agarose slots. Use a new filter tip for each EHT.

Once all of the slots have been filled with reconstitution mix, close the lid of the cell culture dish and incubate the preparations for two hours at 37 degrees Celsius and seven percent carbon dioxide. At the end of the incubation, place the plate in a sterile hood and cover each well with 200 to 500 microliters of medium. Shake the plate gently and return the EHT's to the incubator.

After 10 minutes, carefully transfer the racks into a new 24 well plate containing 1.5 milliliters of EHT medium per well and place the plate in a cell culture incubator under the appropriate culture conditions. Feeding the EHT's every Monday, Wednesday and Friday, by transferring the PDMS racks to a second plate with fresh medium. Seven to 10 days after casting, the EHT's should demonstrate spontaneous contractions deflecting the posts of the PDMS racks.

To analyze the EHT's contractions, on the day of the experiment, turn on the heating device in the EHT analysis instrument two hours before the measurement. Immediately before the analysis, turn on the gas and electronic supplies of the axis system. For a baseline recording, place the EHT plate into the EHT analysis instrument and start the contraction analysis software according to the manufacturers instructions.

Using the software manual instructions to start a new run, click the protocol tab to enter the relevant study information. Open the set up tab and choose the wells for analysis. Due to time constraints, this is demonstrated only for two EHT's in this video.

Then click the camera view tab and start the camera live mode with automatic figure detection mode. Adjust the camera position with the xyz coordinates for each selected well in the 24 well dish, taking care that each EHT is in the center of the window and in focus. Make sure that the blue crosses are in the top and bottom ends of the EHT's and moving to match the contractions of the EHT's.

And press the position okay button to save the set up positions of each well. Now click the parameters tab to define the criteria by which the software identifies a contraction peak, marking the peaks with green squares. Save all entered settings by pressing the use parameters on all wells button.

Then click the automatic tab and the start button to record the baseline EHT contractions and to calculate the spontaneous baseline contractility for each EHT. The live contraction analysis will be displayed in the real time tab. Make sure that the blue crosses are moving only vertically to match the contractions of the EHT's.

To analyze the effects of a specific drug of interest on the contractions, first transfer a 24 well plate filled with protein free thyroid solution under a sterile hood and place carbon electrodes into the wells. Transfer the EHT's in their PDMS racks on top of the electrodes and return the EHT's to the incubator. After about a half an hour, place the EHT's into the the interlock of the EHT analysis instrument and record the spontaneous baseline contractility for 20 seconds per EHT.

Next, connect the carbon electrodes to pacing cable and begin electrically stimulating the EHT's. Record the paced baseline for 10 seconds per EHT, taking care that all of the EHT's are properly paced. The pacing signal is now marked by blue vertical lines, included in the contraction graft prior to every peak.

After the baseline recording, transfer the electrodes and the PDMS racks to a new 24 well plate containing the first drug concentration and immediately return the EHT's to the interlock of the EHT analysis instrument for a 20 minute incubation. Reconnect the pacing cables, adjust the figure recognition and record the EHT contractions in response to the first drug concentration. After all of the drug concentrations have been tested, save all of the resulting PDF recordings and check each recording for figure recognition and the correct positioning of the contraction peak squares and artifacts.

For offline analysis, click select run for the respective run from the runs database and in the parameter tab, change the parameter for the contraction analysis. Then in the offline tab, choose offline analysis to reanalyze the videos. To adjust the figure recognition and to record a new video file.

Adjust the figure recognition in the real time tab and select sample review. The EHT will now be analyzed according to the new positions. The analysis of each EHT is based on figure recognition at the top and the bottom ends of the tissue.

The filmed EHT contractions are then analyzed automatically by the EHT analysis instrument software algorithm and the peaks are determined according to the relevant predefined criteria. The long term measurement of EHT contraction analysis demonstrates that there are no apparent time dependent changes in the contraction force, beating frequency, T1 contraction or T2 relaxation times for up to 20 hours. Indicating a high level of stability for the constructs.

Indeed, the results for each signal EHT are reproduce able with a low variability. Exposure to a hERG channel blocker however, results in a significant prolongation of the T2 relaxation time, beginning at the concentration. And an irregular beating pattern at higher concentrations.

Once mastered, 24 EHT's can be generated within 30 minutes if the preparation is performed properly. After it's development, this technique paved the way for researchers in the field of disease modeling to explore the effect of gene variants in vitro. The versatility of this model, allows it to be combined with human induced stem cells or the genome editing technology.

Following this procedure, additional methods like histology or RNA and protein analysis can be used to answer additional questions about gene transcription and expression. After watching this video, you should have a good idea on how to cast and analyze EHT's. If you have any questions regarding this procedure, please don't hesitate and contact us directly.