Cell-Based Assays: How to Decide Between 2D and 3D

Although traditional two-dimensional (2D) cell cultures offer many advantages for scientific research, they are now rivaled by three-dimensional (3D) alternatives. This article looks at the pros and cons of 3D cultures and shares guidance for when to use them.

Advantages and limitations of 2D cell cultures

For more than a century, 2D cell-based assays have been used for researching biological processes. According to Carolina Lucchesi, Ph.D., Principal Scientist of the Microphysiological System (MPS) Program at ATCC, such methods have stood the test of time due to their ease of set up and analysis, cost-effectiveness, and capacity to support large-scale screening for testing cellular responses to drug candidates. Yet these advantages come at a price—namely, a lack of physiological relevance. “Cells in the human body exist in a 3D matrix with complex interactions and signaling,” says Lucchesi. “The flat substrate in 2D cultures fails to replicate these natural conditions, potentially leading to results that may not fully translate to in vivo scenarios. Additionally, the limited cell-cell interactions in a 2D environment can oversimplify the understanding of biological phenomena.”

Reasons for persevering with 2D

While the recognized constraints of 2D cell-based assays are driving a shift toward the use of 3D models, 2D cultures are still more common. “One reason for this is that many 2D cell-based assays are part of an established or validated Standard Operating Procedure (SOP) that would require significant resources to validate using a 3D model system,” reports Terry Riss, Scientific Ambassador, Cell Health, at Promega Corporation. “Another is that, depending on the scientific question being asked, 2D cell-based assays may be the best or even the only option for many experimental designs,” adds Joseph Boyd, Ph.D., Research Scientist in the Cell Biology group at MilliporeSigma (the life science business of Merck KGaA, Darmstadt, Germany in the U.S. and Canada). “Ultimately, both 2D and 3D cell-based assays have value for scientific research and can complement one another to enable a deeper insight and range of exploration.”

Types of 3D cultures

During the past few decades, many different types of 3D cultures have been developed. “Spheroids represent one of the most commonly used 3D cell culture models,” says Lucchesi. “They are formed by the aggregation of cells, either suspended in a biomatrix or self-assembled, and mimic the natural intercellular contacts found in tissues to allow for cell-cell signaling and the formation of nutrient and oxygen gradients.” Spheroids derived from established tumor cell lines or primary tumor cells (sometimes called tumoroids) are used extensively in cancer research due to their ability to mirror tumor heterogeneity.

Organoids are more complex than spheroids, typically consisting of multiple cell types. “Organoids are derived from stem cells or primary tissues and more closely resemble in vivo structures and physiology than spheroids,” explains Boyd. “They are usually cultured using a 3D scaffold, either a synthetic hydrogel or an animal-component-derived extracellular matrix, although some organoids can be grown as monolayers on membrane inserts. For example, gastrointestinal organoids cultured on MilliCell^® hanging inserts represent a useful modality for studying topics such as barrier integrity, permeability, and transporter function.”

Other types of 3D cultures include microphysiological systems (MPS) such as organ-on-a-chip and body-on-a-chip. “Microphysiological systems often contain multiple organoids derived from different tissue or organ types, which are connected using some kind of microfluidic device to create fluid flow,” says Riss. Importantly, MPS enable researchers to model the complex interactions between different cell types within an organ, or among organs, including responses to physiological cues, drugs, and toxins.

3D cell cultures can also be bioreactor-based, whereby factors such as oxygen levels, nutrient supply, and mechanical forces are tightly controlled to allow for studying cell behaviors and tissue development under dynamic conditions. In addition, bioprinting is used to precisely position cells and biomaterials in a 3D space, thus generating multi-layered structures, and has broad utility for studying tissue development, disease progression, and drug responses.

Advantages of 3D cultures

As well as providing more physiologically relevant data than 2D cell-based assays, 3D models can help researchers to follow the ‘3Rs’ principle of replacing, reducing, and refining the use of animals for research. “With the passing of the FDA Modernization Act 2.0, there is keen interest in finding better in vitro models that can be used instead of animal testing,” explains Hilary Sherman, Senior Applications Scientist at Corning Life Sciences. “I expect that 3D models will start to play a much larger regulatory role in the near future.” It is also possible using patient-derived 3D models in drug screening and toxicity assessments to better predict side effects and reveal patient-to-patient variability. Boyd highlights the value of patient-derived organoid biobanks for these types of studies, including MilliporeSigma’s recently launched 3dGRO™ colorectal cancer and pancreatic ductal adenocarcinoma biobanks, which comprise 20 lines each from individual patients, representing a range of patient variation and oncogene mutational status.

Challenges for using 3D cultures

Despite the obvious benefits, working with 3D cultures presents several challenges. “Since the cells are not attached to microplates, more optimization can be required for liquid handling in order to avoid disturbing growing cultures,” cautions Sherman. “Additionally, 3D structures can be more difficult to image and assay compared to 2D models due to light and reagent penetration.” Lucchesi also notes that the costs associated with 3D cell culture are generally higher than that of 2D cell culture, limiting its accessibility for some researchers.

“Another major challenge, especially with the generation of organoid models, is the greater variability among replicate samples,” comments Riss. “Cryopreservation of 3D models is an active area of research that will help to address this. Reproducibility can also be improved by ensuring that assays have been fully validated for use with 3D models. Researchers can either do this themselves or purchase validated assays from vendors such as Promega.”

Deciding between 2D and 3D

So, how do you decide whether a 2D or 3D cell-based assay is right for you? “First, determine what you want to know at the end of the experiment,” advises Riss. “In many cases, it is possible to design and utilize a less expensive ‘fit-for-purpose’ assay, perhaps 2D, that will serve to achieve your experimental goals.” Sherman echoes this, noting that sometimes simpler is best provided you can get the information you need.

Next, if you plan to go down the 3D route, take some time to identify which type of culture best meets your research requirements. “For some questions, a hybrid ‘2.5D’ approach, in which cells are grown on a flat surface like a membrane insert but develop some structural characteristics of organoids such as specialized secretory regions, offers several advantages of 3D while largely retaining 2D culture technique,” comments Boyd.

Finally, remember to consider available resources and budget restraints. “While 2D assays offer simplicity and affordability, 3D assays provide a more physiologically relevant environment, but at a greater cost and decreased throughput,” summarizes Lucchesi. “Where possible, combining 2D and 3D approaches may yield more comprehensive and representative results for your project.”