Description

Generating reproducible human cardiac organoids from pluripotent stem cells can be challenging. Variability in embryoid body formation, precisely timed medium changes, and inefficient progression through early cardiac lineage stages may lead to inconsistent organoid morphology or delayed functional development. Researchers therefore need a standardized cardiac organoid differentiation workflow that coordinates each induction stage while remaining practical for routine laboratory use.

XR Cardiac Organoid Differentiation Kit is a chemically defined, staged culture system designed to direct human pluripotent stem cells, including induced pluripotent stem cells, toward multi-lineage cardiac organoids. The protocol begins with single-cell aggregation in a low-attachment U-bottom plate and proceeds through sequential treatment with Cardiac Organoid Differentiation Media A, B, C, and D.

Each medium is assigned to a defined phase of the differentiation workflow, supporting progression from embryoid body formation through mesendoderm induction, cardiac lineage specification, and cardiac organoid maintenance. Under the documented culture conditions, rhythmic spontaneous beating may be observed by approximately Day 8–10, providing a visible functional readout of cardiac differentiation.

This cardiac organoid differentiation kit supports the generation of up to 48 organoids and provides a structured platform for researchers studying human heart development, cardiomyocyte differentiation, three-dimensional cardiac models, and related cardiac research applications.

Specifications

Product Name	XR Cardiac Organoid Differentiation Kit
Product Type	Human pluripotent stem cell cardiac organoid differentiation medium kit
Starting Cell Type	Human pluripotent stem cells, including human iPSCs
Culture Format	Embryoid body-based three-dimensional differentiation
Recommended Plate Format	96-well low-attachment U-bottom plate
Differentiation System	Sequential four-medium induction workflow
Medium A	10 mL
Medium B	40 mL
Medium C	20 mL
Medium D	60 mL
Recommended Volume	200 μL per well during the documented differentiation workflow
Kit Capacity	Supports differentiation of up to 48 cardiac organoids
Functional Observation	Rhythmic spontaneous beating may be observed at approximately Day 8–10 under the documented conditions
Grade	For Research Use Only

Kit Components

Component	Volume	Workflow Stage
Cardiac Organoid Differentiation Medium A	10 mL	Initial cardiac differentiation induction after embryoid body formation
Cardiac Organoid Differentiation Medium B	40 mL	Continued lineage induction during the early differentiation phase
Cardiac Organoid Differentiation Medium C	20 mL	Subsequent cardiac organoid differentiation stage
Cardiac Organoid Differentiation Medium D	60 mL	Cardiac organoid maintenance and maturation phase

Features

Staged cardiac organoid differentiation system for human pluripotent stem cells and human iPSCs
Chemically defined workflow designed to reduce dependence on undefined induction components
Four sequential cardiac organoid media corresponding to distinct differentiation stages
Embryoid body-based protocol compatible with 96-well low-attachment U-bottom plates
Supports generation of multi-lineage three-dimensional cardiac organoids
Documented workflow supports up to 48 cardiac organoids per kit
Rhythmic spontaneous beating may become visible at approximately Day 8–10
Suitable for standardized human cardiac organoid culture and differentiation studies

Cardiac Organoid Differentiation Workflow

The protocol uses a timed, sequential medium-change strategy. The starting iPSC culture should be in good condition and reach approximately 80% confluence before differentiation begins. Individual cell lines may require routine upstream optimization to achieve consistent aggregation and differentiation performance.

Preparation: iPSC Culture

Begin the differentiation experiment when the iPSC culture reaches approximately 80% confluence.

EB Formation: 24 Hours Before Day 0

Dissociate iPSCs into single cells and culture them in a 96-well low-attachment U-bottom plate for 24 hours to generate embryoid bodies.

Day 0: Differentiation Medium A

After embryoid body formation, define this point as Day 0. Replace the culture medium with Cardiac Organoid Differentiation Medium A at 200 μL per well and culture for 36–40 hours.

Day 1.5: Differentiation Medium B

Replace Medium A with Cardiac Organoid Differentiation Medium B. Continue culture for three consecutive days, replacing the medium daily.

Day 5.5: Differentiation Medium C

Replace Medium B with Cardiac Organoid Differentiation Medium C. Perform an additional Medium C change on Day 6.5.

Day 7.5 and Later: Differentiation Medium D

Replace Medium C with Cardiac Organoid Differentiation Medium D. Continue changing Medium D every other day until the cardiac organoids reach the desired stage.

Functional observation: Under the documented conditions, cardiac organoids may show rhythmic spontaneous beating when examined by microscopy at approximately Day 8–10.

Human iPSC cardiac organoid differentiation timeline showing embryoid body formation and staged cardiac induction.

Performance Data

Organoid Formation During Staged Differentiation

The documented morphology images show the progression from an iPSC culture to a compact embryoid body and a gradually enlarging three-dimensional organoid during the staged differentiation process. Images collected at Day 0, Day 1.5, Day 5.5, and Day 7.5 illustrate changes in aggregate size and organization as the culture advances through the cardiac induction workflow.

Consistent initial aggregate formation is an important starting point because differences in embryoid body size, shape, or cell number can influence downstream cardiac organoid differentiation. Researchers should use healthy pluripotent stem cell cultures and maintain consistent dissociation, seeding, and medium-change practices.

Cardiac Marker Expression

Immunofluorescence images supplied with the product documentation show cardiac-associated staining for α-actinin and cardiac troponin T. α-Actinin is commonly used to visualize sarcomeric organization in differentiated cardiomyocytes, while cardiac troponin T is a widely used cardiac lineage marker.

Alpha-actinin and cardiac troponin T immunofluorescence staining in human iPSC-derived cardiac organoids.

Applications

XR Cardiac Organoid Differentiation Kit provides a three-dimensional human cardiac differentiation platform for laboratories that need greater structural and biological complexity than conventional two-dimensional cardiomyocyte cultures. The staged system can be incorporated into cardiac research workflows that require EB-based organoid formation, cardiac lineage induction, and observation of spontaneous contractile activity.

Human Cardiac Development Research

Human cardiac organoids can provide an in vitro system for examining cellular organization and phenotypic changes during early cardiac differentiation. The timed transition from embryoid body formation to cardiac lineage induction makes the kit suitable for research focused on human heart development and developmental biology.

iPSC Cardiac Differentiation Studies

The kit supports directed differentiation of human iPSCs through multiple cardiac induction stages. It may be used to investigate how cell-line characteristics, starting culture quality, or experimental treatments influence cardiomyocyte differentiation and cardiac organoid development.

Three-Dimensional Cardiac Models

Unlike a conventional two-dimensional cardiomyocyte monolayer, an iPSC cardiac organoid provides a three-dimensional aggregate format. This format can support research involving tissue-level organization, cell-to-cell interactions, cardiac marker expression, and spontaneous beating behavior.

Cardiac Disease Modeling

When combined with appropriately characterized patient-derived or genetically modified iPSC lines, cardiac organoid culture may support research into disease-associated cardiac phenotypes. Experimental suitability should be validated for each cell line and disease model.

Drug Screening and Cardiotoxicity Research

Beating cardiac organoids may be incorporated into exploratory drug-screening or cardiotoxicity-testing workflows that assess morphology, viability, cardiac marker expression, or changes in contractile behavior. The kit itself does not include analytical assays, imaging systems, or compound-response validation tools.

Cardiac Regenerative Medicine Research

The system may support basic research into cardiac lineage specification, cardiomyocyte development, tissue organization, and other questions relevant to cardiac regenerative biology. The product is intended exclusively for research use and is not intended for clinical or therapeutic applications.

Human iPSC cardiac organoid differentiation
Human heart organoid culture
Embryoid body-based cardiac differentiation
Cardiomyocyte differentiation research
Cardiac progenitor and cardiac lineage studies
Human heart development research
Three-dimensional cardiac model development
Cardiac disease modeling using selected iPSC lines
Exploratory drug screening and cardiotoxicity studies
Cardiac marker expression and organoid morphology analysis

Why Choose a Staged Cardiac Organoid Differentiation System?

Cardiac organoid differentiation is highly dependent on timing. Applying the same culture conditions throughout the entire process may not provide the changing signals required as pluripotent cells progress through early lineage specification and cardiac organoid development.

XR Cardiac Organoid Differentiation Kit separates the workflow into four sequential medium stages. This format provides researchers with a defined schedule for initiating differentiation, continuing early cardiac induction, supporting subsequent organoid development, and maintaining the resulting cardiac organoids.

The 96-well embryoid body format also supports parallel processing of multiple organoids. By standardizing the plate format, medium volume, and timing of medium changes, laboratories can establish a more structured cardiac organoid culture workflow while retaining the ability to optimize cell-line-specific parameters.

Important Considerations

Use healthy, undifferentiated human pluripotent stem cell cultures before beginning the protocol.
Begin differentiation at approximately 80% iPSC confluence, as stated in the documented workflow.
Generate uniform embryoid bodies by maintaining consistent single-cell preparation and seeding conditions.
Follow the specified medium sequence and timing because each medium corresponds to a different differentiation stage.
Use 200 μL per well during the documented 96-well differentiation workflow.
Monitor aggregate morphology and spontaneous beating by microscopy during cardiac organoid development.
Optimize starting cell number, dissociation conditions, and upstream iPSC culture practices for each cell line when necessary.

FAQ

What is the XR Cardiac Organoid Differentiation Kit used for?

XR Cardiac Organoid Differentiation Kit is used to direct human pluripotent stem cells, including human iPSCs, through an embryoid body-based workflow toward three-dimensional cardiac organoids. The kit contains four sequential differentiation media for cardiac induction and organoid maintenance.

What is the difference between a cardiac organoid and a two-dimensional cardiomyocyte culture?

A cardiac organoid is a three-dimensional cellular aggregate that can provide greater structural organization and cell-to-cell interaction than a conventional two-dimensional cardiomyocyte monolayer. This kit is specifically designed for EB-based cardiac organoid differentiation rather than only flat cardiomyocyte culture.

Can this kit be used for human iPSC cardiac organoid differentiation?

Yes. The documented protocol begins with human iPSCs and directs them through embryoid body formation followed by sequential cardiac differentiation using Media A, B, C, and D.

How are embryoid bodies generated for cardiac organoid culture?

Human iPSCs are dissociated into single cells and cultured for 24 hours in a 96-well low-attachment U-bottom plate. The resulting embryoid bodies are designated as Day 0 before Cardiac Organoid Differentiation Medium A is added.

How many cardiac organoids can be generated with one kit?

The supplied medium volumes support differentiation of up to 48 cardiac organoids when the documented 96-well workflow and recommended medium volumes are followed.

When do iPSC-derived cardiac organoids begin to beat?

Under the documented culture conditions, rhythmic spontaneous beating may be observed by microscopy at approximately Day 8–10. The exact timing may vary with the iPSC line, starting culture quality, aggregate uniformity, and laboratory conditions.

What is the cardiac organoid differentiation timeline?

Embryoid bodies are prepared for 24 hours before Day 0. Medium A is used from Day 0 for 36–40 hours, Medium B begins at Day 1.5 with daily changes for three days, Medium C begins at Day 5.5 and is replaced again on Day 6.5, and Medium D begins at Day 7.5 with every-other-day medium changes.

What cardiac markers are shown in the product data?

The product documentation includes immunofluorescence images for α-actinin and cardiac troponin T. These markers are commonly used in cardiac differentiation research to assess cardiomyocyte-associated structural and lineage characteristics.

Is this a chemically defined cardiac differentiation system?

Yes. The product is described as a chemically defined cardiac organoid-directed differentiation system consisting of four staged culture media.

Can this cardiac organoid kit be used for disease modeling?

The resulting iPSC cardiac organoids may be incorporated into research workflows involving cardiac disease modeling when suitable patient-derived, control, or genetically modified iPSC lines are used. Researchers should validate the organoid phenotype and experimental endpoints for each model.

Can beating cardiac organoids be used for drug screening or cardiotoxicity testing?

Cardiac organoids may be incorporated into exploratory drug-screening and cardiotoxicity research workflows that measure viability, morphology, cardiac marker expression, or contractile behavior. Additional validated assays and analytical equipment are required because they are not included with the differentiation kit.

Does the kit include a low-attachment plate or cell dissociation reagent?

The documented kit contents list Cardiac Organoid Differentiation Media A, B, C, and D. A 96-well low-attachment U-bottom plate, cell dissociation reagent, iPSC culture reagents, extracellular matrices, and analytical assays are not listed as kit components.

Is XR Cardiac Organoid Differentiation Kit intended for clinical use?

No. This cardiac organoid differentiation kit is intended for research use only and is not intended for diagnostic, therapeutic, transplantation, or clinical applications.

For Research Use Only. Not intended for diagnostic, therapeutic, or clinical use.

XR iPSC-Derived Cardiac Organoid Differentiation Kit