Name: A Hypoxia-Reoxygenation Injury Model in Self-Assembling Human Cardioids (Video) | JoVE Journal
Uploaded: 2026-03-17T12:35:00.000+00:00
Duration: 10 min 41 s
Description: Learn to model hypoxia-reoxygenation injury in self-assembling human cardiac organoids derived from iPSCs. This protocol enables reproducible ischemia-reperfusion studies for testing regenerative therapies.

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This work is supported by R01 HL157277 (RK), AHA 24PRE1186062 (LP), F30 HL175908-01 (LP), and the Duke University Medical Scientist Training program T32 5T32GM145449-03 (LP).

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</ol>

Presented here is a step-by-step protocol for generating and injuring self-assembling hCOs with H/R. Ischemic heart diseases are characterized by pathophysiological remodeling, including cardiomyocyte death, fibrotic remodeling, electrical dysfunction, and functional decline. These perturbations are observed in the hCO model system, including increased TUNEL+ CMs, an increase in LDH release, an increase in Vimentin+ tissue area, and alterations in calcium handling leading to loss of functional synch...

Ischemic heart disease is a leading cause of morbidity and mortality worldwide, affecting at least 20.5 million people in the United States alone1,2,3. Without adequate perfusion, prolonged hypoxia results in cardiomyocyte (CM) death and myocardial dysfunction. Myocardial ischemia is primarily treated with revascularization via percutaneous intervention, thrombolytic therapy, or coronary artery bypass grafting. However, revascularization is a double-edged sword and can cause additional injury via the release of reactive oxygen species4,5. While reperfusion strategies have been immensely successful, approaches to limit CM loss during ischemic insults or after reperfusion have been more elusive. Though many attempts to salvage infarcted tissue prove effective in animal models of ischemia-reperfusion, these same therapies have largely been unable to achieve significant benefit in human trials, raising a need for human models to evaluate CM-sparing or replenishing therapeutics6,7,8,9,10,11,12.
Human platforms used to model cardiac injury include primary tissues and human induced pluripotent stem cell (hiPSC)-derived systems13,14. Because primary tissues are constrained by sample availability, hiPSC systems have emerged as the dominant human platform for cardiac disease modeling, with the distinct advantages of being genetically tractable and scalable15,16,17,18,19. Traditional 2D hiPSC-derived cardiomyocyte (hiPSC-CM) cultures are limited by the immaturity of the resulting CMs, the absence of complex tissue architecture, and an inability to reproduce the full cellular diversity found in the human heart15,20. To address these shortcomings, engineered 3D heart tissues (EHTs) have become increasingly adopted20,21,22,23,24,25,26. EHT approaches include mixing pre-differentiated cardiac cells into spheroids24 or seeding differentiated cardiac cells into scaffolds or molds21,22,27. Although EHTs can better model heterotypic cellular interactions and have relatively more maturity than 2D cultures, the requirements for custom scaffolds or bioreactors to create and maintain EHTs currently limit their scalability21,27,28.
Self-assembling cardiac organoids (hCOs), or cardioids, are a new and powerful in vitro human model system20,25,26,29. hCOs have several advantages. First, they can include multiple cell types, including endothelial cells, fibroblasts, and epicardial cells, without the need for multiple specialized differentiation protocols26,29,30. Second, most of their differentiation occurs in 3D suspension culture, obviating the requirements for specialized molds or tissue matrix25,26,29,30. Finally, hCOs have a stereotypical spherical morphology with a central cavity, enabling morphological analyses. Thus, hCOs have scalability similar to 2D culture systems while retaining some of the tissue complexity of EHTs. hCOs have been used for drug toxicity testing, patient-specific disease modeling, and to model aspects of cardiac development20,26,30,31,32,33,34,35,36,37,38,39. While promising, hCOs have several limitations compared to EHTs, including an inability to directly measure force output. Further, there is no native immune cell population in the described hCO models, though it is possible to populate hCOs with macrophages. Nevertheless, hCOs are an emerging and rapidly evolving human model system20,26,30,31,32,33,34,35,36,37,38,39.
To date, modeling cardiac injury with hCOs has been limited. A cryoinjury model has been described, but injuries are highly variable, and the need to individually injure hCOs limits scalability29. This manuscript describes a user-friendly protocol for generating and injuring self-assembling hCOs with hypoxia-reoxygenation (H/R) injury. Compared to other protocols for generating hCOs, this protocol uses a simplified differentiation medium and incorporates the 2D-3D mesoderm differentiation strategy described by Hofbauer et al.29. The protocol for H/R injury can be performed any time after hCOs begin beating, which typically occurs around differentiation day 8 (D8) to D11. H/R duration can be titrated between 6 h and 24 h to achieve the desired level of injury, ranging from minimal to near complete CM loss. Additionally, hCOs stereotypically develop a collagenous core with Vimentin+ cells after H/R injury that also scales in severity with H/R duration. This high-throughput, scalable hCO model of H/R injury potentially addresses the need for human models to develop and test new therapeutic avenues for promoting CM recovery or regeneration after injury.

<table><tbody><tr><td>Accutase solution</td><td>Sigma-Aldrich</td><td>A6964-500ML</td><td></td></tr><tr><td>Anti-Cardiac Troponin T antibody</td><td>Abcam</td><td>EPR20266</td><td></td></tr><tr><td>B-27 Supplement (50X), serum free</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>B-27 Supplement, minus insulin</td><td>Gibco</td><td>A1895602</td><td></td></tr><tr><td>BioTek Synergy Neo2 Plate Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>Cal-520, AM</td><td>AAT Bioquest</td><td>21131</td><td></td></tr><tr><td>CardioCount V1</td><td>Karra Lab</td><td>PMID: 38984052</td><td></td></tr><tr><td>Cell line (&lt;em&gt;Homo sapiens&lt;/em&gt;, male)- DU11 iPSC Line</td><td>Duke University Stem Cell Core</td><td></td><td></td></tr><tr><td>Charged Microscope Slides, White Glass</td><td>VWR</td><td>89500-496</td><td></td></tr><tr><td>CHIR 99021</td><td>Tocris</td><td>4423</td><td></td></tr><tr><td>CloneR</td><td>STEMCELL Technologies</td><td>5888</td><td></td></tr><tr><td>Corning 96-Well Clear Ultra Low Attachment Microplates</td><td>Fisher</td><td>7007</td><td></td></tr><tr><td>Disposable Pipetting Reservoirs</td><td>VWR</td><td>89094-678</td><td></td></tr><tr><td>DMEM/F-12, HEPES</td><td>Gibco</td><td>11330057</td><td></td></tr><tr><td>EVOS Fluorescent Microscope</td><td>EVOS</td><td></td><td></td></tr><tr><td>Geltrex Reduced-Growth Factor Basement-Membrane Matrix, LDEV-free, stem-cell qualified</td><td>Gibco</td><td>A1413302</td><td></td></tr><tr><td>Human Activin A Recombinant Protein</td><td>PEPROTECH INC/Life Technologies</td><td>120-14E-50UG</td><td></td></tr><tr><td>IWP-2</td><td>STEMCELL Technologies</td><td>72124</td><td></td></tr><tr><td>L-Ascorbic Acid (Crystalline/Certified ACS)</td><td>Fisher Scientific</td><td>A61-100</td><td></td></tr><tr><td>Mouse anti-Vimentin</td><td>DSHB</td><td>AMF-17b</td><td></td></tr><tr><td>mTeSR Plus</td><td>STEMCELL Technologies</td><td>100-0276</td><td></td></tr><tr><td>Multiwell Cell Culture Plates &amp;ndash; 12 well</td><td>VWR</td><td>10062-894</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td></td></tr><tr><td>ReLeSR</td><td>STEMCELL Technologies</td><td>100-0483</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Gibco</td><td>11875119</td><td></td></tr><tr><td>RPMI 1640 Medium, no phenol red</td><td>Gibco</td><td>11835-030</td><td></td></tr><tr><td>Thiazovivin</td><td>STEMCELL Technologies</td><td>72254</td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>SAKURA FINETEK USA INC (VWR)</td><td>25608-930</td><td></td></tr><tr><td>Zeiss Spinning Disk Confocal Microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

a hypoxia-reoxygenation injury model in self-assembling human cardioids

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

Without rapid revascularization, ischemic heart disease leads to irreversible loss of cardiomyocytes, scar formation, and decreased cardiac function. The ability to model hypoxia-reoxygenation injuries and evaluate potential therapeutic interventions in human tissues is an unmet need. Human cardiac organoids (hCOs) are an emerging model system, but beginning work with hCOs can be challenging due to their small size, requirements for suspension culture, and variability in differentiation efficiency. This protocol offers a user-friendly guide for generating and maintaining human iPSC-derived self-assembling hCOs; methods to process and histologically analyze hCOs; and an approach to injure hCOs with hypoxia-reoxygenation that mimics the fibrosis and apoptosis seen with human ischemia/reperfusion. This protocol provides guidance for using hCOs as a complement to animal models of ischemia-reperfusion injury and could be used to identify and test factors that may boost human myocardial recovery.&#160;

Following the above protocol should yield well-formed hCOs that begin beating between differentiation days 7-12 (D7-12). As hCOs mature past day 25, it is possible for spontaneous contractions to no longer be visible without electrical pacing. H/R injury can be performed at any time after hCOs begin beating - we typically perform injury on 15-day-old (D15) hCOs or later.
Confirmation of Injury 
Like human myocardial infarction, H/R injury of hCOs results in cell death. Clin...

Self-assembling human cardiac organoids (hCOs) are an emerging system for modeling human cardiac development and disease. Here, a methodology for generating and injuring hCOs with hypoxia-reoxygenation and details of expected histological and functional outcomes after injury are provided.

Without rapid revascularization, ischemic heart disease leads to irreversible loss of cardiomyocytes, scar formation, and decreased cardiac function. The ...

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.

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Research

JoVE Journal

Medicine

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

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