Three-dimensional cultures are playing increasingly prominent roles in life science research, with a multitude of methods developed over the past 15 years.
3D aggregate cultures are a subset of 3D cultures in which cells clump together in suspension, growing as aggregates instead of as individual cells. In this article, we will examine the historical and current contributions of 3D cultures to research and provide expert advice on selecting a 3D model for your lab.
Three-dimensional cultures have a long history in biology, dating back as early as 1902. In 1910, anatomist Ross Harrison developed a technique for cultivating frog neurons in drops of partially coagulated serum or lymph suspended from an inverted slide. Researchers have since developed the hanging drop method into a versatile technique for producing 3D aggregate cultures.
In the 1970s and 1980s, neuroscientists used animal brain sections to develop the first organotypic cultures, which are thick slices of organs cultured in media for further study. Researchers have used organotypic cultures to gain insight into reproductive biology, brain development, neurodegenerative disorders, and other areas of study.
3D experiments on tumor cells grown in aggregate cultures called spheroids began in the 1970s with the work of Robert Sutherland and colleagues. Not long after, in the mid-1980s, Matrigel was introduced—a solubilized basement membrane preparation derived from the Engelbreth-Holm-Swarm murine tumor and rich in extracellular matrix (ECM) proteins. After a 1989 study reported that Matrigel could support the development of functional 3D mammary gland organoid cultures, Matrigel became a central tool for laboratories working in 3D, and it remains so today.
Researchers developed several additional 3D culture techniques in the 1990s and 2000s. Since the early 2010s, they have seen an exponential increase in both the number and impact of 3D culture studies.
The principal three-dimensional in vitro biological models include spheroids, organoids, and tissue constructs.
Researchers primarily use 3D spheroid models in cancer biology, but they also apply these models in infectious disease and stem cell research. Drug discovery workflows can also benefit from the adoption of 3D spheroid models. In work reported in Drug Metabolism and Disposition, a research team used hepatocyte spheroids grown in Corning® Elplasia® 96-well plates to predict how the human liver will process drug candidates. The researchers state that this method offers advantages over existing in vitro methods when evaluating certain types of compounds.
Helpful tools for culturing healthy spheroids include Corning® spheroid microplates and Corning® Elplasia® plates.
Compared with spheroids, which typically consist of one cell type that grows as a simple, spherical aggregate, 3D organoid culture models include multiple cell types that are arranged into complex and diverse structures. Researchers use organoid models in precision medicine and across various biological fields. Scientists in the laboratory of Benjamin Freedman at the University of Washington are using kidney organoids derived from induced pluripotent stem cells (iPSCs) in the search for therapies for polycystic kidney disease (PKD), an inherited disorder. According to research published in Stem Cell Reports, the team used iPSCs carrying genetic mutations found in individuals with PKD to develop a more realistic organoid model of the disease and identify and test a promising PKD drug candidate.
Tools for organoid cultures include pre-coated Corning® Matrigel® matrix-3D plates and a variety of natural and synthetic hydrogels.
Tissue models are sophisticated 3D constructs of organs such as the skin, lungs, or gut, designed with permeable membranes that establish controlled microenvironments or maintain separation between distinct cell populations. Corning® Transwell® inserts are permeable membrane inserts that help researchers create various tissue models by supporting cell polarization, co-cultures, cell migration, and other cellular processes.
For example, researchers in Nicholas Zachos’ laboratory are using a tissue model that combines 2D and 3D methods to explore the interactions between gut epithelial cells and immune cells. By using Corning® Transwell® inserts to co-culture a monolayer of gut cells with immune cells, researchers in the laboratory create enteroids and colonoids, which are tissue models of the small and large intestine, respectively. The team uses these tissue models to study gut immune mechanisms and explore how viruses like COVID-19 interact with the gut, as published in Cellular and Molecular Gastroenterology and Hepatology.
We spoke with experts at Corning to learn how laboratories should choose a 3D model. Here's what they recommend:
Whitney Cary Wilson, Field Application Scientist at Corning Life Sciences, recommends that scientists begin by defining the research question they aim to answer and the cell type they are working with. To aid in selecting the right 3D cell model, scientists should also consider the starting material, such as a primary culture from a donor or a stem cell line.
"That's one of the first decision points that I see my customers go through that'll be dependent upon whether they have stem cell expertise in their lab, and if they have access to patient tissue to generate those organoids," Wilson said. You may also consider which 3D models other labs have used to ask similar questions.
Catherine Siler, Field Application Scientist Manager at Corning Life Sciences, recommends considering the method's throughput and scale.
"I always encourage my users to think about what scale this ultimately needs to be at so we can help them start on the right foot," Siler said. She also noted that it is possible to start with simpler, smaller-scale methods as a proof-of-concept and then move to more advanced models.
Mikael Garcia, Field Application Scientist for Corning EMEA, states that labs should consider the end goal of their 3D work (e.g., research or clinical use) and assess whether the chosen method can effectively support that goal.
"If it's a model where they are testing drugs, in terms of quality, it's not the same as if they want to go to a patient. So, the question of where we're going to go at the end is very important because, according to that, the strategy at the very beginning will be very different," Garcia said.
When asked about advice for early-career scientists, Garcia emphasizes the importance of researching the 3D models most relevant to their research question. It can also be helpful to learn how other research groups have successfully utilized those models.
Wilson agreed and added, "Read the publications that are already out there and take the advice of people who know what they're doing because it's not such an easy plug-and-play the way that 2D culture can be."
Maintaining tight control of the process is also crucial, helping to ensure consistency of 3D cultures.
"Broadly speaking, it's typically easier to get consistency in processes with 2D cultures,” Wilson states. “With 3D cultures, particularly organoid differentiation, it can be challenging to achieve reproducibility from experiment to experiment, or even within the same experiment when growing multiple organoids. Developing very robust techniques at the beginning is really important.”
Wilson also encourages researchers to monitor and address phenotypic signs that 3D aggregate cultures are unhealthy, such as shadowing or signs of necrosis in the center of the structures.
Due to the diversity of 3D models and their varying levels of complexity, partnering with an expert can be extremely helpful. Advice and assistance from Corning's experts can help you find and optimize the best tools and models for your work.
Explore Corning products designed specifically for 3D cell culture models, 3D tissue models, and more.
