Three-dimensional (3D) organoid models are increasingly being used to investigate disease mechanisms, drug responses and cellular function. By reproducing important aspects of tissue architecture and biology, organoids can provide a more physiologically relevant model than conventional two-dimensional (2D) cell cultures.
But moving from 2D cultures to 3D models creates an important analytical challenge:
How can researchers accurately measure mitochondrial function and cellular energy metabolism in organoids?
Accurate organoid metabolic analysis can help researchers understand mitochondrial function, cellular energy metabolism and responses to experimental treatments. Researchers therefore need analytical approaches capable of measuring these processes while accommodating the complex three-dimensional structure of organoids.
Why measure metabolism in organoids?
Cellular metabolism is closely linked to cell function. Changes in mitochondrial respiration, glycolysis and ATP production can reveal how cells respond to disease, environmental conditions and experimental treatments.
Traditional 2D cell cultures remain valuable research tools, but cells grown in a monolayer do not fully reproduce the architecture, cellular interactions and microenvironment found within tissues.
Organoids offer a more physiologically relevant 3D environment and are increasingly being investigated across cancer research, cardiovascular disease, neuroscience, developmental biology, toxicology and drug discovery.
Patient-derived organoids (PDOs) are particularly interesting for translational research because they enable researchers to investigate biological and treatment responses using models derived from patient tissue.
As organoid models become more sophisticated, researchers need methods that provide meaningful functional information about what is happening inside them.
The challenge of organoid metabolic analysis
Organoids are commonly grown within extracellular matrix-containing hydrogels that support their three-dimensional structure and development. This creates additional considerations when performing metabolic assays that were traditionally designed around 2D cell cultures.
An effective organoid metabolic analysis workflow needs to accommodate both the 3D architecture and surrounding matrix while enabling researchers to quantify mitochondrial respiration and glycolytic activity.
In other words, researchers need a way to measure metabolism in organoids while maintaining the physiological advantages that made them choose a 3D model in the first place.
Measuring organoid metabolism with Agilent Seahorse XF Flex
The Agilent Seahorse XF Flex platform provides a workflow designed to interrogate cellular energy metabolism in organoid cultures.
The workflow combines the Seahorse XF Flex Analyser with a specialised XF Flex organoid microplate that enables organoids to be cultured within an extracellular matrix-containing scaffold prior to metabolic analysis.
Organoids can be maintained in the microplate for several days and exposed to experimental treatments during the culture period. Researchers can then investigate how different compounds or experimental conditions influence cellular energy metabolism.
Using the Seahorse XF Flex workflow, researchers can measure metabolic parameters including:
- Oxygen consumption rate (OCR) to assess mitochondrial respiration
- Proton efflux rate (PER) or extracellular acidification rate (ECAR) to investigate glycolytic activity
- ATP production rates
- Changes in metabolic function following experimental treatments
Together, these measurements provide a functional picture of how an organoid's energy metabolism responds under different experimental conditions.
Why does metabolic analysis in 3D matter? The metformin example
Metformin provides an example of how 3D organoid metabolic analysis can be used to investigate drug responses.
In an Agilent study, HCT116-H2B-GFP colorectal cancer cell-derived organoids were cultured in Matrigel and exposed to increasing concentrations of metformin before analysis using the Seahorse XF Mito Stress Test.
Metformin produced a concentration-dependent inhibition of mitochondrial respiration in the cancer organoids.
By measuring oxygen consumption rate (OCR), researchers could evaluate changes in basal respiration, ATP-production-coupled respiration, maximal respiration and spare respiratory capacity.
This demonstrates the value of looking beyond organoid morphology or viability alone. Measuring cellular metabolism provides additional functional information about how a 3D model responds to a drug or experimental treatment.
Towards more physiologically relevant research
The increasing use of organoids also aligns with the broader development of New Approach Methodologies (NAMs) and human-relevant experimental models aimed at improving the translational relevance of preclinical research and reducing reliance on animal models where appropriate.
As organoid technologies continue to evolve, the ability to characterise mitochondrial function and cellular metabolism will become increasingly valuable across disease modelling, drug discovery and toxicology.
The Agilent Seahorse XF Flex organoid workflow provides researchers with a practical approach to organoid metabolic analysis, helping them investigate mitochondrial respiration, glycolysis and cellular energy metabolism in physiologically relevant 3D models.
Want to explore metabolic analysis in your organoid research?
Vito Technologies can help researchers determine how Agilent Seahorse XF technology can be incorporated into existing 3D cell culture and organoid workflows.
Contact our team to discuss your organoid model, research objectives and metabolic analysis requirements.
