3D cell culture offers tremendous promise to revolutionize drug discovery, disease modeling, and regenerative medicine by providing a platform that better recapitulates human biology than conventional 2D cell culture, leading to more accurate predictions of drug efficacy and toxicity and improved understanding of disease mechanisms. However, there are challenges that need to be addressed to fully realize this potential, including the development of standardized protocols and techniques for reproducible and scalable 3D cell culture systems. Additionally, there is a need for innovations in imaging and analysis methods to effectively visualize and characterize the complex cellular interactions and tissue structures within these 3D models. Overcoming these challenges will be crucial to harnessing the full power of 3D cell culture and translating it into meaningful applications in the biomedical field. Corning Life Sciences has been deeply engaged in the 3D cell culture space since its inception, providing critical tools to address and overcome pain points and enable further innovation. In this Q&A, Corning Life Sciences’ Elizabeth Abraham, Ph.D., and Catherine Siler, Ph.D., discuss the evolution of the field and its ongoing challenges, as well as the contributions Corning has and continues to make in its development, with Pharma’s Almanac Editor in Chief David Alvaro, Ph.D.

David Alvaro (DA): While some of this may be a bit obvious, can you explain some of the advantages that 3D cell culture has over more traditional 2D cell culture?

Elizabeth Abraham (EA): Since 2D cell culture has been the primary approach for many years, we have very standardized protocols for 2D cell culture. 2D cell culture is typically much cheaper and simpler, can be automated, and is compatible with high-throughput techniques.

However, 3D cell culture is far superior in providing a more physiological or in vivo–like response. 3D cell culture has been explored to some extent for about 20–25 years, but it became a much bigger part of the conversation in the last 10+ years, since researchers began to be able to demonstrate its similarity to animal model responses.

The 3D approach has been used for drug resistance, drug development, and drug efficacy studies, because the sensitivity of drugs is very similar to what is seen in vivo. It enables the investigation of cell–cell and cell–extracellular matrix (ECM) interactions. It also facilitates co-culturing, making it a heterogeneous system, just like our bodies are, and unlike 2D cell culture, which is typically more homogeneous.

Today, these benefits are widely recognized, and people are seeking ways to move things forward and really take advantage of the possibilities that 3D cell culture presents.

DA: Building off of that, can you discuss the adoption curve — who embraced 3D cell culture early and who was more hesitant, and any key milestones along the way?

Catherine Siler (CS): Beginning 10–15 years ago, we saw a consistent increase every year in the number of publications that referenced 3D cell culture, a trend that has continued to today. Going back to that inflection point, one of the primary drivers of adoption was the lack of success in drug trials at that time.

Cell culture is central to the screening process for new therapeutic compounds. Naturally, it's quite frustrating when years of human effort and funding are invested in trials that ultimately don't go anywhere. There was a realization that the testing methods that led up to trials needed to be reconsidered — they needed to become more physiologically relevant, and 3D cell culture presented an ideal tool to achieve that.

Last year, the U.S. FDA decided to make animal models optional rather than mandatory in the path to clinical trials, which will likely catapult 3D cell culture to the forefront.

As 3D cell culture techniques were evolving, everyone was essentially designing their own testing methods, and there was no real standard and accessible way to embrace these assays. To increase the reach of 3D cell culture and enable much wider adoption, it became critical for manufacturers to develop more standardized technologies.

DA: Besides the fundamental need for more standardization of methods, what other significant challenges needed to be overcome to optimize 3D cell culture, and what challenges remain?

EA: That turning point a decade or so back reflected a shift in the conversation from whether 3D cell culture was possible to how it can be optimized. Since then, we have overcome many challenges, including figuring out how to mimic the complex structure of tissues and organs.

This has been a tough ask because there are many intricacies to an organ or a tissue, but the advent of technologies like organoids, which resemble mini-organs, and advances in bioprinting and tissue engineering enabled great strides in mimicking organs more closely.

Once you succeed in creating these complex structures, new challenges arise, like how to maintain cell viability over extended periods of time. Organoids and spheroids are ball-like structures, which are challenging to feed using more standardized tools, but researchers have worked out how to control the size of the 3D structure to minimize the necrotic core and have improved culturing techniques, such as perfusion systems, that have also helped to increase cell viability.

Another major challenge involved how to control the microenvironment. Researchers found that you need specific ECMs that support 3D cell culture, and Corning stepped up to provide many of those extracellular matrices.

With recent advances in microfluidics, we have found a way to balance pH, gases, and waste removal to continue to optimize these 3D systems.

Imaging is another area where we've seen significant advances. 3D structures are quite dense and compact, so figuring out how to look inside them presented another challenge. Advances in confocal imaging helped us get deeper into these 3D structures and tissues, but some challenges remain around standardization, validation, and guidance.

How do we scale these cultures while keeping the costs low? 3D is more expensive than 2D, and we have to keep that in balance as well going forward.

DA: Can you discuss some of the contributions that Corning Life Sciences has made to the progression of 3D cell culture and what you think differentiates not only your solutions but your engagement with the field and your point of view?

CS: For over 30 years — going back to the dawn of 3D cell culture — Corning Life Sciences has offered products that allowed researchers to grow cells in a more 3D-like environment: for example, our Transwell^® polycarbonate membrane cell culture inserts and other permeable supports, as well as vessels with our ultra-low-attachment surface. Over the past decade, we've focused on providing technologies to make 3D cell culture more consistent and more accessible.

We talked about the challenges associated with keeping the size of your 3D body consistent. We offer spheroid microplates that provide that consistency in every single 3D aggregate with a really familiar footprint. Scientists are very used to working with microplates, and that ease-of-use factor is important.

We have also taken scaling up 3D work into account to try to offer a broader portfolio. We want to allow people to grow more of these 3D structures while still offering the consistency they are used to with 2D culture.

I think that all speaks to our culture of innovation, which is one of our core values. Our R&D teams are always working on the next best technology to solve whatever customers’ problems are, and that really motivates the technical teams as well. At the end of the day, it's our job to solve our customers’ problems, and the nice thing is that with the breadth of products that we offer and the experience that our team has, we can most often do exactly that.

DA: Was there a particular inflection point that led Corning to focus more in the 3D cell culture space, or was it simply a natural, iterative progression from what you had already been doing?

EA: It’s important that researchers remain current with scientific advancements, and we are in the business of providing tools to help them. We listen to our customers intently and follow what the customer community needs. We always pay close attention to the pain points they are facing, and when we start to hear a particular rumble get a little louder, that typically ends up being translated into a product to resolve that pain point through a very disciplined process of product innovation.

We build tools to help those customers to standardize, and the more standardized tools that we provide, the more acceptance we see across the research community.

DA: Are there things that these researchers could be doing themselves to contribute to that standardization?

EA: Collectively, we continue to push for more optimized protocols, consistency, and reproducibility. For example, cancer spheroids are used as a tool in drug discovery and screening. Initially, our customers were using the Corning flat- or round-bottom ultra-low-attachment vessels to generate 3D structures, which is great, but when they would generate spheroids, they would not be uniform. That drove us to continue to innovate our spheroid platform and design plates that encourage the formation of one spheroid per well, which then provided consistency for drug screening.

After that, we heard that in some instances our customers need more signal intensity and thus need multiple spheroids. A few years later, we launched the Elplasia^® platform, which allows multiple spheroids per well, providing both the consistency and the increased signal intensity that our customers were looking for.

DA: How do you see 3D cell culture technology synergizing with some of the other technological advancements — automation, high-throughput screening, AI — that are currently transforming the industry?

CS: As we already mentioned, the FDA has relaxed their animal testing guideline, which is really going to put the spotlight on 3D cell culture. To realize that promise, 3D cell culture is going to have to increase throughput and be compatible with automation. Fortunately, several of our existing technologies — like the spheroid and Elplasia plates — are already compatible with automated plate readers and liquid handlers and fit into that type of workflow. For work that is more focused on ECMs like collagen or Matrigel^® matrix, we have taken steps there to introduce a really user-friendly solution, the Matribot^® bioprinter, that allows users to dispense those matrices in a more automated manner.

However, I think that there will always be room to make things better, faster, and higher throughput, so the coming challenge will be to keep doing that while maintaining physiological relevance, keeping cells happy and functional but in a very complex growth environment.

I think the next question for systems that require an ECM for optimal function is how to consistently miniaturize them to provide a robust path toward high-throughput work.

DA: What very recent developments — either at Corning Life Sciences or elsewhere — do you see really setting the stage for the further evolution of 3D cell culture?

EA: I think the most exciting trend in 3D cell culture revolves around organoids. The importance of organoids in answering drug discovery questions has grown tremendously since 2009, which was when they were first mentioned in the literature by Hans Clevers’ team at HUB Organoids.

Organoids are 3D structures generated from stem cells that self-assemble into mini-organs and recapitulate more structures found within an organ. They are the cornerstones for precision medicine, which is also called personalized medicine. Organoids are generated from biopsies and used to grow specific mini-organs that are used to test various drugs in vitro before selecting a drug that works for that person's cancer. How cool is that?

Corning provides many ECMs, including the gold-standard ECM for organoids, which is our Matrigel matrix. In fact, we have a qualified version specifically for growing organoids, Matrigel matrix for organoid culture. The company is also responding to requests from our customers to better automate organoid growth. Bioprinting is one solution, and Corning has introduced the Corning Matribot Bioprinter, which works with many of our ECMs. Organ-on-chip ecm

technology, meanwhile, allows incorporation of many different organ systems on the same platform to study drug toxicity.

DA: When customers are evaluating the potential value of cell culture systems and platforms, what key factors do they consider, and how does Corning help them with those decisions?

CS: At the core of any cell culture process is the cell or tissue type involved because that dictates everything else in terms of what is needed for successful growth. Will a matrix like collagen or Matrigel matrix be required? Can the cells grow in a scaffold-free environment? Would they do well with a co-cultured cell type, and if so, what type of interactions would be best? If the goal is a 3D aggregate of cells, what size is needed? One of the biggest questions involves the scale of the experiment. Some customers might need a few plates, while others might need dozens to hundreds of plates or flasks.

This set of questions forms a pretty typical framework for a conversation between the field applications team members and customers pursuing new 3D cell culture projects. Corning has team members all across the globe, and there are also extensive resources on our website that provide good starting information, including eBooks and webinars with not only Corning experts but participants from industry and academia.

DA: Can you tell me a little bit more about how you see Corning’s role in advancing the use of 3D cell culture in drug discovery and development?

EA: Corning is a tool provider and are focused on helping researchers by providing state-of-the-art tools that enable them to be successful. We are very tuned into what is happening in research labs and listen closely to learn about pain points so that we can identify product innovations to alleviate those problems.

For example, we have delivered many tools for accelerating precision medicine from the 3D model standpoint and ECMs for organoid growth to Matrigel and collagen-based matrices. We also provide Transwell permeable supports used as scaffolds for 3D cell culture. Our spheroid and Elplasia^® platforms, meanwhile, are examples of scaffold-free options.

Recently, Corning added reagents for better imaging of 3D structures and equipment to support the construction of 3D structures, such as the previously mentioned Corning Matribot^® Bioprinter, as well as cell and organoid counters.

Going forward, we will continue to stay closely connected with our customers so we can provide needed new tools in a timely manner.

DA: Are there any products you have in development for which you can provide some initial information?

EA: One example is a technology for researchers working with spheroids. Corning leveraged microcavity technology to provide 12,000 spheroids in a footprint similar to that of a T-75 flask, called the Elplasia 12K Flask. Users now want access to each spheroid. In response, we will soon be introducing an open-well plate concept that can hold 12,000 spheroids and allow access to them. That product will be launched later in 2023.

Corning has also developed a new platform of synthetic hydrogels for customers that needs more defined platforms than are possible with Matrigel^® matrix. The first Corning Synthegel™ 3D Matrix Kits — which launched recently  — are suitable for growing IPSCs in a 3D format. They allow cells to be grown, passaged, and differentiated in the same 3D structure.

DA: Are there any specific case studies or success stories that you can highlight?

CS: The best example demonstrates the power of organoid models and involves patient tissue samples embedded in Matrigel matrix; Elizabeth mentioned this previously. From one patient, thousands of little mini-organs are generated that have enormous power for predictive medicine. This high-throughput approach can be used to test different combinations of chemotherapeutic agents and regimens to help identify treatment solutions with the greatest likelihood to have a beneficial therapeutic effect. There is a lot of work ongoing in this area to support pancreatic cancer patients, who generally have poor prognoses.

Some researchers are taking this approach to a more population-wide level by creating biobanks of organoids. Population-wide analyses are then possible to determine which types of drugs might be more effective across different patient populations.

Other examples include drugs used to treat cystic fibrosis that were tested on intestinal organoids produced using a Corning ECM. Our permeable supports have also been used to make skin used for wound healing applications in burn victims and diabetes patients. Also of note is the use of Elplasia for bulk production of cellular aggregates for CRISPR gene-editing assays.

DA: To what extent can 3D cell cultures help increase understanding of interactions between tumor cells and the tumor microenvironment (TME)?

EA: Mutual interactions between tumor cells and the non-cellular components that constitute the tumor microenvironment can promote tumor development and progression. The ECM, meanwhile, controls most aspects of tumorigenesis, such as the epithelial-to-mesenchymal transition (EMT), and is very relevant to migration, invasion, tumor formation, vascularization, and apoptosis. Therefore, interaction between the TME and the tumor in the presence of an ECM involves a 3D dialogue that is key to cancer progression.

Corning has long understood the importance of the ECM. Matrigel matrix is itself a reconstituted basement membrane extracted from a mouse tumor rich in ECM proteins. Once this material is isolated, it is approximately 60% laminant, 30% collagen 4, and 8% entactin. The latter is a bridging molecule between the laminin and collagen that contributes to the structural organization of the ECM. Corning Matrigel matrix also contains many growth factors involved in tumor signaling that play roles in enabling the TME to regulate tumor progression.

We also supply many other ECMs, including collagen, laminin, and fibronectin, all of which are part of the TME. These ECMs are not only used to grow cancer cells but also to study cancer cell migration throughout the body. Corning also offers Transwell^® permeable supports, membranes that serve as physical barriers, to help researchers investigate how cancer cells invade tissues.

These tools can be mixed and matched as well. ECMs can be used on the Transwell, for instance, to study their influence on cancer cell movement.

DA: As 3D cell culture tools are further elaborated and optimized, what impact do you think they will have on the use of animal models?

CS: I think there will still be certain applications for which animal models will be the optimum approach, but in an abbreviated fashion. I believe those animal studies might come later in the discovery process and be really complementary to a lot of in vitro work performed early on. As a result, fewer animal models overall will be used for earlier-stage assessments like efficacy and toxicity, with most limited to larger, multi-system settings.

DA: What impact do you think replacing early-stage animal models with 3D cell culture will have on timelines and cost?

CS: Working with animal models is very expensive and creates challenges to maintaining timelines. Animals don’t always do what you need them to do. Switching from animal models to 3D cell culture has real potential to reduce development timeline variation and thereby also reduce cost. There are also, of course, ethical considerations that don’t carry any price tag but are nonetheless important.

DA: Can you tell me about current regulatory considerations and what might be expected from a regulatory standpoint going forward?

EA: The regulatory conversation is ongoing. As Cat mentioned, the FDA Modernization Act will force the scientific community to more carefully consider appropriate regulatory oversight. There are three basic areas that will be useful. Information must be provided about the general application and the specific context of use within that application, particularly with respect to the validity of the model and its ability to provide data that can be used to answer key drug development questions. Systems must also be reliable and perform reproducibly from one experiment to another, one batch to another, and one site to another.

Beyond that, it is important to ensure there are positive and negative controls, define what those controls should look like, determine what endpoints should be measured, and demonstrate that these 3D cell culture models are predictive of relevant animal models and translatable to humans. Finally, it is necessary to establish that the assays used provide results that are reflective of the tissue physiology and that what should and should not be compared are clearly outlined.

Essentially, animal models will still be used as positive controls, and 3D cell culture models will need to be shown to provide data that can be correlated to results obtained using those positive controls. Ideally, 3D cell culture models based on human systems will actually provide improved performance compared with traditional cross-species animal models. Sensitivity and specificity must be considered, as well as cut-off values. 

DA: Can you tell me a little bit more about the scaling challenges for 3D cell culture? 

EA: 3D cell cultures have a far more complex architecture than 2D cultures. Scaling 3D cell culture therefore requires specialized bioreactors and specialized and, in some cases, defined scaffolds to maintain that complexity and structure in a reproducible and consistent manner. Indeed, scaling up to industrial levels while maintaining quality and reproducibility can pose a significant challenge. There is also a need for specialized media and protocols to support 3D cell culture at industrial scale.

Of course, there are also regulatory considerations around safety and quality control. Proof needs to be obtained that 3D cell cultures produced and manipulated at industrial levels are safe for use in humans. The scientific, biotech, and pharma communities are still working to gather that evidence. Overall, the industry is moving forward in this area at a slow but steady pace.

DA: Are there any particular peripheral technologies you would like to see introduced to enable Corning to further advance its 3D cell culture solutions?

CS: Corning has its own R&D team, and innovation is a big part of our culture. There are always internal projects fueled by listening to customers about their evolving needs. We are also open to working with partners that can bring unique technologies to the table that will help Corning solve those customer problems. The focus is always on addressing unmet needs of our customers, and we pursue all possible pathways to achieving that goal.

We have current partnerships with HUB Organoids, which was responsible for the genesis of organoid technology, IT collaborations such as for our organoid counting software, and collaborations with customers to promote the research they are doing using Corning tools.

We also hold both virtual and in-person 3D summits for the 3D cell culture community. The most recent one was held in the fall of 2022 in New York City and brought together close to 200 researchers over the course of two days. Speakers included not only Corning experts but customers sharing best practices around 3D cell culture using all different types of models.

DA: The 3D cell culture field has rapidly evolved over the last decade. What changes do you anticipate that the next 10–15 years will bring?

CS: There are a variety of things that industry partners and academic researchers are going to be working on, some of which we're already tackling at Corning. Those include the synthetic scaffolds Elizabeth mentioned earlier, which provide more defined and tunable environments. Miniaturization, meanwhile, will be leveraged to increase throughput. Challenges around vascularization and keeping these structures alive will be important areas of focus. Another important development target will be increasing the scale of 3D cell cultures, which has been a focus in traditional cell culture and bioprocessing.