3D Cell Culture: A Definition

Most cells in living tissue grow in a three-dimensional architecture, where they communicate and interact with each other and their surroundings. Animal cells are embedded in the extracellular matrix (ECM), which is composed of proteoglycans and fibrous proteins (mainly collagen, elastin, and fibronectin). This complex, dynamic, and tissue-specific 3D structure provides physical scaffolding for the cells and initiates cues that influence cell differentiation and behavior.

When cultured in a traditional, two-dimensional in vitro environment, the cells are attached to a flat surface (e.g., a monolayer in a standard cell culture dish) and can only grow and migrate on the substrate. In a 3D in vitro setup, the cells are grown in suspension on a non-adhesive surface, or they can be embedded in or on a 3D matrix (e.g., Matrigel^® or Collagen I) that mimics the ECM and allows them to grow in all three directions.

In many cases, a 3D environment reflects the in vivo situation more accurately. This should be considered when analyzing cell behavior, differentiation, response to drug treatment, and gene expression. It is therefore not surprising that many cell culture approaches have been adapted to a 3D setting. This includes drug screenings that use spheroids and organoids, which are indispensable nowadays as tumor models.

3D culture of an IL-22-treated mouse small intestine organoid grown in Matrigel^® drops using an ibidi µ-Slide 8 Well. Cell image by Naveen Parmar, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.

Although 3D cell culture-based experiments already offer a more in vivo-like approach when compared to 2D culture, multiple advancements to obtain even more realistic culture conditions have recently been established. This includes the dynamic flow culture of 3D cells, the simultaneous co-cultivation of multiple cell types in the same dish, and 3D printer-based bioprinting.

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3D cell culture applications in comparison to the traditional 2D monolayer.

Common 3D Cell Models

Scaffold-based 3D cell culture models provide a structural framework that mimics the extracellular matrix, supporting the growth and organization of cells in three dimensions. Unlike scaffold-free systems (which are self-forming aggregates without artificial supports, including spheroids and organoids), these models incorporate biocompatible materials—such as hydrogels, synthetic polymers, or natural fibers—that facilitate cell attachment, proliferation, and differentiation. This environment not only closely resembles in vivo conditions but also allows for the inclusion of multiple cell types, which can interact and develop complex tissue structures. Such models are crucial for studying cell interactions, tissue development, and disease progression, and are increasingly used in tissue engineering and regenerative medicine. Scaffold-based models enhance the physiological relevance of the studies, thereby improving the predictive accuracy of drug responses and biological behaviors.

Scaffold-based 3D culture of single cells in a matrix that mimics the physiologic environment.

Spheroids, clusters of cells forming under three-dimensional, non-adherent culture environments, are primarily composed of fully differentiated cells due to their usual lack of stem cells. This composition generally precludes them from self-renewal and further differentiation. However, tumor cell spheroids are an exception. Owing to the inherent unlimited proliferative capacity of cancer cells, these spheroids can divide and renew themselves, making them an invaluable tool for studying tumor cell behavior, especially in large-scale drug screenings. The creation of spheroids often employs techniques like the hanging drop method, which is a scaffold-free suspension approach. Spheroids offer important insights into cellular behaviors in a more in vivo-like environment, enhancing the relevance of findings in biomedical research.

Spheroids are cell aggregates, which are often generated from cancer cells.

Organoids are cultured "mini organs" and can be generated from pluripotent stem cells (PSCs). When these cells are cultivated in a three-dimensional scaffold (e.g., Matrigel^® or collagen), they differentiate into organ-specific cell types, undergoing important steps of organogenesis. Cultivation methods for organoids are becoming more and more sophisticated, making these 3D cell models increasingly accurate representations of real organs.

The first generation of intestinal organoids, created from an Lgr5⁺ stem cell by Sato et al., initiated many protocols for organoid generation from different organs, such as intestine, liver, brain, prostate, kidney, pancreas, lung, and thyroid. Importantly, organoids can be edited using technologies such as CRISPR, making them a powerful tool for studies about organogenesis, personalized medicine, and drug screening.

Several procedures for the generation and cultivation of 3D cells were established. While the generation of spheroids usually follows highly standardized protocols, the cultivation of organoids often requires a more careful revision of existing procedures.

Organoids are cultured miniature versions of organs, which are derived from stem cells.

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Spheroid formation of NIH-3T3 cells (murine embryo fibroblasts) on 200 µm adhesion spots using ibidi µ-Patterning. Spheroid generation was documented for 64 hours. Phase contrast live cell imaging, 4x objective lens.