A Hypoxia-Reoxygenation Injury Model in Self-Assembling Human Cardioids

Our research focuses on finding new ways to help the heart regenerate after injury. For this, we generate self-assembling cardiac organoids and perform hypoxia-reoxygenation studies. This protocol can be applied to study injury response, therapeutic interventions to promote regeneration, and can be used for patient-specific disease modeling.

To begin, aspirate the D1 cardiac mesoderm specification media and add enough cell detachment solution to entirely cover the human-induced pluripotent stem cell-derived cardiac mesoderm cells. Then place the plate in an incubator at 37 degrees Celsius for five minutes. Using a pipette, add an equal volume of D2 cardiac mesoderm media to each well and gently pipette up and down to release the adherent cells, transfer the cell suspension to a 15 milliliter conical tube.

Using a centrifuge, pellet the cells at 300G for three minutes at room temperature. Resuspend the pellet in one milliliter of D2 cardiac mesoderm specification media containing five micromolar IWP-2 and 50 micrograms per milliliter ascorbic acid in RB minus. Mix 10 microliters of trypan blue with 10 microliters of the cell suspension in a microtube.

Count the cells using the preferred counting method, then dilute the cells to a concentration of 100, 000 cells per milliliter in D2 cardiac mesoderm specification media supplemented with two micromolar thiazovivin. Transfer the cell suspension to an appropriately sized reagent reservoir. Then, using a multichannel pipette, dispense 100 microliters into each well of an ultra-low attachment 96-well plate to achieve a final concentration of 10, 000 cells per well.

Spin the plate at 500G for five minutes at room temperature to induce human cardiac organoid formation in suspension culture. Observe the formation of a large, well-circumscribed cell pellet in each well. If the pellet is not circumscribed, repeat centrifugation.

After 24 hours, prepare 10 milliliters of D3 media containing five micromolar IWP-2 and 50 micrograms per milliliter ascorbic acid in RB minus per plate. Using a multichannel pipette, add 100 microliters of D3 media to each well without removing any existing media. The next day, remove 100 microliters of D3 media from each well.

To minimize aspiration risk, tilt the plate to a 45-degree angle and hold a multichannel pipette at a 90-degree angle to the plate sidewall. Place the pipette firmly on the well wall and slowly aspirate. After removing the media, replace it with 100 microliters of D4 media containing five micromolar IWP-2 in RB minus.

Using a 1, 000 microliter pipette with the tip cut off, transfer the human cardiac organoids to a 1.7 milliliter microcentrifuge tube. Allow the organoids to sink to the bottom of the tube and gently remove the excess media. Add phosphate-buffered saline to wash the organoids once.

Wait a few seconds for them to sink, and gently remove the excess PBS. Next, add 4%paraformaldehyde to fix the organoids and place the tube at room temperature for 30 minutes to allow fixation. Then transfer the desired number of fixed organoids to a 1.7 milliliter microcentrifuge tube.

Remove the fixative solution and wash the organoids with PBS containing 0.1%Tween 20. Prepare the primary antibody solution by diluting the antibody to the desired concentration in staining solution. Using a pipette, add 50 to 100 microliters of the primary antibody solution to each microcentrifuge tube, ensuring the organoids are fully covered.

Following incubation and subsequent washes, incubate the sample with secondary antibody diluted in staining solution for 24 hours at four degrees Celsius on a shaking platform. After incubation, perform washes as previously described. Next, using a cut pipette tip, transfer the organoids onto a positively charged glass slide.

Gently remove excess PBS with Tween 20 and add a few drops of mounting media to cover the organoids. Carefully place a coverslip over the organoids to flatten them. Then proceed to perform fluorescence microscopy.

After incubating the fixed organoids overnight in 30%sucrose at four degrees Celsius, transfer them to a plastic mold and add a small drop of optimal cutting temperature compound to the mold, just enough to cover the organoids. Using fine tweezers or a pipette tip, gently reposition the organoids in the compound so they are not clumped and are away from the edges of the mold. Position the organoids as close to the bottom of the mold as possible to maximize the number present in a single cryosection.

Place the mold into a Styrofoam cooler containing a layer of dry ice to freeze the initial layer of optimal cutting temperature compound. Wait a few minutes for the compound to harden and then move the mold to the benchtop. Add additional optimal cutting temperature compound until the mold is filled.

Allow the first layer to fully melt before returning the mold to the dry ice to complete freezing. Once frozen, store the molds at minus 80 degrees Celsius or proceed to cryosectioning to obtain six micrometer sections. Using a pipette, transfer five milliliters of hypoxia media per 96-well human cardiac organoid plate into a cell culture flask.

Place the flask inside a hypoxia chamber and flush the chamber with anoxic gas composed of 5%carbon dioxide and 95%nitrogen for three minutes. Place the chamber in an incubator at 37 degrees Celsius for at least one hour before initiating hypoxia. Using a multichannel pipette, remove approximately 150 microliters of media from all wells of the human cardiac organoid plate.

Then, using a 200 microliter pipette, carefully remove the remaining media from each individual well without disturbing the organoids. Add 50 microliters of hypoxia media to each well. Place the human cardiac organoid plate into the hypoxia chamber.

And flush the chamber with anoxic gas composed of 5%carbon dioxide and 95%nitrogen for three minutes. Place the hypoxia chamber in an incubator at 37 degrees Celsius for six hours. To initiate reoxygenation, remove the human cardiac organoids from the hypoxia chamber.

Using a multichannel pipette, add 150 microliters of pre-warmed RB+media to each well. Finally, place the plate in an incubator at 37 degrees Celsius and allow at least three hours of reoxygenation before performing downstream assays. After six hours of hypoxia and 24 hours of reperfusion, extracellular lactate dehydrogenase activity was significantly increased in injured human cardiac organoids compared to uninjured controls.

The hypoxia-treated cardiac organoids exhibited TUNEL-positive cardiomyocyte nuclei, and the percentage of TUNEL-positive cardiomyocyte nuclei was significantly higher in injured organoids compared to uninjured controls. A time-dependent increase in scar formation was observed with increasing duration of hypoxia prior to reoxygenation. At 24 hours post-reperfusion, 69%of uninjured human cardiac organoids were beating, whereas none of the injured organoids were beating.

Uninjured human cardiac organoids exhibited synchronized calcium transients across five regions of interest, with peaks occurring at the same time and with similar waveforms. Injured human cardiac organoids did not exhibit coordinated contractions and instead displayed irregular spontaneous calcium transients across regions of interest. This protocol allows researchers to mimic myocardial infarction in iPSC-derived human cardiac organoids.

The greatest technical challenge in ensuring reproducible injury is the complete removal of existing media. Our future studies aim at identifying factors that can promote tissue regeneration in human cardiac organoid models.