Key Considerations When Adopting 3D Cell Culture Models

As a result of the FDA Modernization Act 2.0, researchers are looking at alternatives to animal testing, especially for early drug discovery. Many are turning toward 3D cellular models, hoping they can deliver on their promise to replicate the physiological environment. 3D cell models have evolved significantly in terms of offering more cellular heterogeneity, predictability, tunability, ease of maintenance, and longer shelf-life, which has led to their increased use in diverse applications. However, the question whether these 3D models truly represent what happens in vivo and in disease continues to be raised. To minimize this ambiguity, it is imperative that researchers ask all the right questions before designing and developing 3D models for their studies. Biocompare recently hosted a Bench Tips webinar where a panel of senior post-doctoral fellows discussed some of those key questions and how their research has been impacted.

What are the benefits of using 3D versus 2D cell models?

“One benefit of 3D cultures is that they better model what you would see in vivo, and they maintain some of the tissue and cellular heterogeneity that you would see,” explained Mackenzie Callaway, Ph.D., Postdoctoral Associate in the Cold Spring Harbor Cancer Center. She uses 3D models to study how pregnancy and associated hormones influence breast cancer composition, development, and metastasis since they recapitulate the in vivo tissue features and composition.

Similarly, Cristina Antich Acedo, Ph.D., Postdoctoral Fellow in the National Center for Advancing Translational Sciences at National Institutes of Health, uses human 3D models for high-throughput testing of drug candidates and for studying disease pathologies to help identify potential therapeutic targets for new treatments. Her studies with 3D cardiac model systems for testing drug toxicity have shown that 3D systems can better replicate what is seen in patients, when compared to data obtained using 2D systems.

3D cell cultures are also very tunable to suit specific applications. Callaway recalled that when studying pancreatic cancer, she was able to model the hollow luminal structures of pancreatic ducts by creating Matrigel droplets. “Since the model is tunable you can change the extracellular matrix (ECM) inside of the droplets and generate hollow spheroids to mimic the luminal structures,” she noted.

However, there is a lot of variability in 3D cells since they use tissues derived from different animal models or patients. Therefore, each model must be well characterized and validated to make sure it represents what is being studied. It also takes time and expertise to culture, grow, and maintain the 3D cells for cellular integrity and functional response. The time for culturing the cells and running the experiments then must closely align with the biological endpoints being looked at. All these factors must be considered before deciding if 3D models truly outweigh the use of 2D systems. 2D cultures, on the other hand, are often an efficient, economical, fast, and easy way to get some preliminary information on what is happening in the cellular environment.

How do you decide which type of 3D model to use?

There are many ways to culture cells in 3D. Callaway encouraged people to think about their hypothesis, the biological question, and access to the tissue source, either mouse models, stem cells, human tissues, or immortalized cell lines. Based on the application, the 3D cells can be in multicellular microfluidic devices to mimic vasculature and fluid flow. 3D cell matrices are useful when the architecture of the cells needs to be varied by changing the composition of the ECM. 3D tissue slices can be grown and cut to observe image dynamics happening in endogenous tissues. Hollow or solid 3D spheroids and 3D organoids can be generated for a number of applications.

The 3D models range from simple 3D organoids to complex organ-on-a-chip systems to try and reproduce the physiological complexity of the human body. “Bioengineering approaches that balance complexity and scalability are the best option for functional studies and for disease modeling,” Acedo explained. She added that there are four different components that are crucial to developing these tissue systems. The first is the type of cell that is used to generate the model. The second is the biomaterial used, which can be natural or synthetic polymers, and the kind of organization needed. Depending on the tissue the support can be soft, like hydrogels, or more rigid, like scaffolds. The third component is the regulatory signal needed to induce proper differentiation. That includes chemical stimulation like growth factors, cytokines, or hormones added to the media, as well as physical regulators such as mechanical forces or atmospheric conditions. Finally, there is the choice of the biofabrication tool. There are self-assembled systems, molding devices, microfluidic, or 3D bioprinting systems. “We decided to use bioprinting technology, since it allows us to biofabricate tissues in a high-throughput automated manner with high resolution and reproducibility,” Acedo noted.

How to ensure that the organoids develop and grow to mimic what happens in vivo?

“It is very important that we define the different culture methods and make sure that we're providing cells with the appropriate matrix and media conditions that they need,” said Callaway. “Different organoids require different chemical factors to maintain their differentiation status.” She recommended starting with established protocols because it's much easier to tune it to fit your application than trying to establish something from the ground up. Acedo mentioned that when she was developing a 3D placental model for her studies, she had to monitor the media components, concentrations, and regulatory signals to induce proper fetal vasculature. She had to pay attention not only to the cell type but also the volume.

Being realistic about the timeline for the experiment is important to make sure that physiological conditions are replicated. “Some of these organoid cultures can take up to a month or more to differentiate before you can use them in experiments,” said Callaway. With many 3D systems, the stability decreases over time. Planning for how to maintain the structural support, how to passage the cells, and thinking about how the cell populations might change over time and impact results, is absolutely necessary.

Simon Schafer, Ph.D., is working to develop 3D brain models to study the onset of neurogenesis by mimicking an environment that replicates colonization of the brain with precursors of microglial cells. He recently finished his post-doctoral work at the Salk Institute of Biological Studies and joined the Technical University of Munich (TUM) as Assistant Professor for advanced organoid technologies. He too finds that time is an important technical consideration when generating 3D models. “Microglia, like other cell types, undergo a very extensive developmental phase and so how long can you keep these models and how long is it physiological to maintain them in an organized structure is one aspect that we looked into,” Schafer explained.

In addition, making sure that the 3D models represent the organ or disease that is being studied is very important. Callaway mentioned that the organoids she developed using mammary glands had to maintain a lot of the heterogeneity with adipocytes, epithelia, immune cells, and fibroblasts to really understand what was happening in these tissues in response to different conditions. “Our lab put a lot of time into validating that this is a good system. We showed, using single-cell RNA sequencing and other techniques, that both the epigenetic profile and the gene expression from these organoids mimics what is happening in mice and humans during pregnancy.”

At the same time, it is also important to consider what cell types are missing from the model and how that may be impacting the tissue development and the effects being looked at. “With our mammary gland organoids, we saw a decrease in immune cells within the first week of culture. So, to interrogate any cellular interactions you have to make sure that these cell types are present in the model,” added Callaway.

For more details on the ideas and insights shared by the expert panel during the webinar, view the on-demand version here.