Organoids, a type of 3D cell culture configuration, hold great promise for basic research and therapeutic applications. Recent technological advances have made organoids even more suited for use as tissue model systems—speeding their future use in drug discovery and personalized medicine. This article focuses on advances in 3D and organoid cell culture, and their potential future applications.
Imaging organoids
Imaging systems are vital for any cell culture system, and organoids are no exception. Agilent BioTek instruments include automated microscopes for organoid imaging, such as the Cytation Cell Imaging Multi-Mode Readers and Lionheart Automated Microscopes. In addition to multiple processing options, their Gen5 software captures organoid images in the x-, y-, and z-directions, and montages them with z-stacking to assess 3D structure and the distribution of cell types, markers, and expression.
“The analysis may consist of an estimated total cell count based on the maximum projection, changes in intensity for different markers, ratios of two fluorescent markers to each other, or defining cell death modality and quantifying cell death with various treatments,” says Peter Banks, scientific director of BioTek Instruments (part of Agilent). “In other models, the main interest is in the change in size and morphology over time with a low magnification capture of the entire organoid in a single plane or a limited number of planes.”
While the eventual replacement of animal models with organoids is attractive, multiple wrinkles remain to be smoothed out. Data from organoids can be difficult to reproduce because labs create and maintain them differently. Organoids also lack vasculature, which limits their application to biological questions. “The future is bright, however, for automation and standardization of methods to improve reproducibility of workflows, and [for] additional technologies such as microfluidic platforms to provide flow conditions to even better mimic in vivo behavior,” adds Banks.
Making models more physiological
Microfluidics and other innovations are helping to make organoids more closely resemble organs. CN Bio’s PhysioMimix™ OOC Microphysiological System (also known as Organ-on-a-Chip) is designed as an intermediary between preclinical non-human testing and human clinical trials to provide a way to culture predictive organoid models of healthy and disease tissue. “The main advantage that our MPS solution offers scientists working with organoids is the ability to combine organoid cultures with other cell and tissue types to make more complex and translational in vitro models mimic dynamic drug and hormone exposure in an organotypic, fully immersed, microphysiological flow-based environment,” says Tudor Petreus, senior scientist at CN Bio. “Our MPS achieves this through the incorporation of microfluidics tuned to mimic organ-specific blood flow that provides cultures with valuable nutrients, oxygen, and waste removal.”
MPS’s flexibility allows researchers to choose the best culture format for their experiment, which can be influenced by cell types and available materials. It is compatible with 3D matrices, bespoke scaffolds, and Transwell inserts, “the latter of which offers customers significant advantages over the use of 3D matrices for the development of more complex in vitro multi-organoid models interconnected via microfluidics,” reports Petreus.
Indeed, the application often drives the choice of organoid format. Petreus notes that spheroids from immortalized cell lines are often used for tumor biology, while organoids from primary human cells or iPSC-derived cells are often used for models of brain and lung tissues. “Our liver disorder and toxicity-focused customers, however, mainly work with co-cultures of primary liver cells in a 3D set-up using scaffolds to promote the aggregation of organotypic microtissues with in vivo like pathophysiology, which delivers greater metabolic horsepower and clinical marker output versus other less complex models,” he says.
Petreus believes that the next big advance in in vitro models will be interconnectivity. Already, CN Bio’s MPS allows researchers to link individual tissue models, such as a brain organoid and a tumor spheroid, together in a microfluic environment. He says that CN Bio plans to offer technology for controllable flow rates to facilitate the interaction of different organoids: “By linking multiple ‘vascularized’ organoids together, our understanding of inter-organ cross-talk will be greatly advanced, bringing us ever closer to a physiologically relevant body-on-a-chip model.”
More tools specifically for organoids
In addition to Matrigel Matrix, an extracellular matrix used extensively in cell culture, Corning offers their new Matrigel Matrix for organoid culture, which has been optimized for organoid growth and differentiation. Other tools include Corning Matrigel matrix-3D plates for drug discovery assays using organoids; these were recently used in drug screening with airway organoids containing the SARS-CoV-2 receptor. Corning also offers spheroid plates, Transwell permeable supports, ultra low attachment plates, and a 3D Clear Tissue Clearing Reagent for organoid workflows.
Promega also offers a wide range of assays to assess cell health—a key tool in creating and maintaining healthy organoids. Among the most popular tools, according to Terry Riss, Promega’s senior product manager in cell health, are assays that measure live, dead, and apoptotic cells in 3D cultures. Multiplexing is also available to collect additional information from each sample well. According to Riss, “Promega’s customers are utilizing extremely sensitive assays to measure metabolites—such as glucose, lactate, glutamine, glutamate—present in microliter volumes of culture medium to monitor health of organoid cultures.”
Riss believes that organoids derived from stem cells will become more prominent as physiologically relevant and predictive model systems, compared to 2D cultures or less well-defined 3D cultures. “The use of organoids to model specific disease states will expand in the near future because they can provide fit-for-purpose models that display molecular markers and properties more closely representing the in vivo biology,” he adds.
With so many tools available, researchers are continuing to advance disease applications. “More complex, vascularized multi-organoid systems will continue to be developed to advance precision and regenerative medicine closer toward transplantable organs,” says Hilary Sherman, senior applications scientist at Corning Life Sciences. “Protocols and models will continue to be optimized to generate data and improve clinical [predictiveness] of organoid models in pharmacological and toxicity testing, which could potentially mitigate the need for animal models during drug development.”