Cardiac Organoids

Cardiovascular disorders, including cardiovascular disease (CVD) and congenital heart defects (CHD), are the leading cause of death worldwide, accounting for an estimated 17.9 million deaths annually. Despite this, the development of organoids for cardiovascular disease research has lagged behind that for other organs such as the kidney, brain, and intestine, possibly due to the structural complexity of the heart.

To date, various cardiac models, including animal models and spheroid cultures, have been used to study disease mechanisms and support drug discovery. However, many of these models consist of only specific cell types and cannot fully recapitulate the multicellular interactions—including those involving epicardial and endocardial cells—or the structural complexity of the organ in vivo.

To address these challenges, researchers developed 3D cardiac tissue (engineered heart tissue), which is created from a mixture of individually differentiated cardiomyocytes, vascular endothelial cells, fibroblasts, and other cells. 3D cardiac tissue excels at modeling the mechanical and electrophysiological properties of tissue, and numerous disease models have been reported for recapitulating the phenotypes of various heart diseases and for drug discovery research. However, because this approach is based on the artificial assembly of differentiated cells, it cannot readily reproduce the natural processes of early embryonic heart development or the accompanying formation of the endogenous extracellular matrix (ECM).

In contrast, cardiac organoids, a field that has attracted growing interest in recent years, form through the self-organization of stem cells during cardiac differentiation. As a result, their structure more closely resembles that of the actual heart, including naturally formed layers. With current technology, 3D cardiac tissue is built from cells that are already relatively well differentiated, whereas the cells in cardiac organoids often remain at early stages of development. Because cardiac organoids more closely recapitulate in vivo developmental processes, they can be used to model malformations by introducing specific genetic mutations or altering culture conditions. They are therefore expected to become a more effective platform for elucidating the mechanisms of heart development and studying developmental disorders—areas that have been difficult to investigate using conventional models.

Cardiac Organoids vs. 3D Cardiac Tissue

	Cardiac Organoids	3D Cardiac Tissue
Structure	Cell arrangement and interactions closely resemble those in vivo Contain layered structures and a lumen	Organized with fibrin, gels, or similar materials in a form that differs from native tissue
Generation	Formed through spontaneous self-organization	Formed by combining differentiated cells on scaffolds or in molds
Disease Modeling	Mimic developmental processes, making them well suited for modeling embryonic development and developmental abnormalities	Models of various hereditary and non-hereditary diseases have been reported
Variability	High tissue-to-tissue variability	Low tissue-to-tissue variability

We present protocols and recommended reagents for generating cardiac organoids.
Please refer to the references for detailed protocol information. (Tian, Y. et al., 2022 , Voges, HK. et al., 2023 )

Protocol examples 1 (Tian, Y et al., 2022)

	iPSCs	Differentiation of cardiac organoids
Media	hPSC Medium	RPMI
Serum/Additives		B27-insulin				B27-insulin
Cell Dissociation Reagents	Accumax
Small Molecules	Y-27632	CHIR99021		Wnt-C59		CHIR99021
Cytokine		BMP4/ ActivinA
Equipment	96-well plate

*Created based on data from Tian, Y. et al., 2022

Protocol examples 2 (Voges, HK. et al., 2023)

	iPSCs	Differentiation of cardiomyocytes and stromal cells			Cell dissociation	Cardiac organoid culture
Media	hPSC medium	RPMI			MEMα	MEMα	DMEM-glucose
Serum/Additives		B27-insulin/PS/L-Ascorbic Acid/L-Ala-L-Gln		B27/PS/L-Ascorbic Acid /L-Ala-L-Gln	PS/L-Ascorbic Acid/FBS/L-Ala-L-Gln	PS/L-Ascorbic Acid/B27/ L-Ala-L-Gln	PS/L-Ascorbic Acid/B27-insulin/ L-Ala-L-Gln/Aprotinin/D-glucose/Palmitic Acid
								Insulin
Cell Dissociation Reagents					Collagenase I/Trypsin-EDTA
Scaffolding Materials	EHS gel matrix				Collagen I /EHS gel matrix/10×DMEM	PDMS
Small Molecules		CHIR99021	IWP-4
Cytokine		BMP4/ActivinA/FGF2				PDGF-BB/FGF2
Equipment	T25 flask				T25 flask	96-well plate

*Created based on data from Voges, HK. et al., 2023

1. Volmert, B. et al.: Nat Commun., 14 (2023).
2. Drakhlis, L. et al.: Nat Biotechnol., 39, 737 (2021).
3. Lewis-Israeli, YR et al.: Nat Commun., 12, 5142 (2021).
4. Tian,Y. et al.: Front Cell Dev Biol., 10 (2022).
5. Voges, HK. et al.: STAR Protoc., 4 (2023).
6. Tani, H., Tohyama, S.: Organoid research, 99 (2024).