Extracellular Matrix: The Secret Sauce of 3D Cell Culture

With 3D cell culture making strong inroads into preclinical studies and basic research, the focus of vendors and investigators alike has turned from discovery to optimization. What kinds of organoids, spheroids, or nondescript 3D cultures work best? What should their cellular composition be? How do we keep them alive and functioning for the duration of long experiments? And, more comprehensively, How do we industrialize the process for creating 3D cultures to make them more accessible, robust, and reproducible?

What we're talking about here is industrialization, which will require re-examination of the basics of 3D culture itself, including, but not limited to, the extracellular matrix (ECM), the framework on which many of the more advanced 3D cultures are built. ECM supplies the physical structure in which cells live, and in/around which they receive signals from neighboring cells.

Three roles

ECM plays three major roles in cell culture. It provides a surface for cellular attachment, a barrier through which cells invade, and a source of biological relevance.

"ECM is especially important when cell polarity is relevant to the model," says Hilary Sherman, Senior Applications Scientist at Corning Life Sciences. Examples include organoids, mammospheres, or other cyst-like structures in which cells are oriented toward either the inside or the outside. "Cells in vitro use the ECM to orient themselves similarly to how they would in the body." If orientation is unimportant, as is true for some 3D cancer models or suspension cultures, there may be little or no value to using an ECM. "The question to ask is what model is being mimicked and would an ECM be biologically relevant?"

The in vivo ECM is generally tissue-specific (e.g. lungs vs. skin vs. bone) and heterogenous. It typically consists of water, proteins and polysaccharides. Commercial biologically derived ECMs, for example Matrigel™, are typically a complex, heterogeneous, solubilized basement membrane preparation. More simply constituted ECMs, derived from collagen, laminin, or fibronectin, are more chemically and structurally defined.

Because ECM plays such an important role in so many fundamental cell processes, ECM selection depends primarily on the cell type, culture geometry (e.g. 3D vs. 2D), the anticipated role of the replicated in vivo environment, and whether the experiment is looking at healthy or diseased cells.

"Researchers need to take into consideration whether the ECM would provide the required architecture and biochemical cues to support adhesion and cell behavior and whether, based on the application, these characteristics are best provided by a biologically derived or synthetically produced ECM," Sherman tells Biocompare.

With 3D cell culture-based disease models hitting their stride just in time for the pandemic, references to Corning's ubiquitous ECM have found their way into nearly 750 publications since January, 2020. During this time Matrigel-based organoids have been used to characterize the responses of model human intestinal and airway tissue to coronavirus and influenza virus. This, is in addition to standard applications like modeling cancer, evaluating new drugs, and the creation of cardiomyocytes using  "mattress" technology with Matrigel matrix, which uses a bed of undiluted Matrigel to create stem cell-derived 3D cultures.

Not all ECMs are equal

Composed of a secret sauce of structural and signaling components, ECM is the dynamic biological meshwork where cells reside in nature, and which scientists strive to replicate, in microplates, with 3D cell cultures. Along with chemical signaling and nearby cells, ECM is the physical or mechanical component of the cellular microenvironment, or niche.

When designing 3D cultures—organoids, spheroids, organ-chips, etc.—scientists tend to look for an off-the-shelf answer, much as they do with cell culture media. But in nature ECM composition varies significantly throughout organisms, from tissue to tissue and organ to organ. "Its structure and biochemical composition are highly specific to organs and disease states," says Tanya Yankelevich, Director of Product Management, Xylyx Bio.

Many commonly used ECM substrates are synthetic, composed of a single biochemical component. "These products are too simple to provide the myriad biochemical factors that make up the native cellular environment, and thus support only limited cell growth and function," Yankelevich tells Biocompare. "While basement membrane extracts derived from tumor cells certainly accelerate cell growth, they may also lead to dysregulation of normal cellular function, which is not conducive to studying natural cell behavior."

These limitations on in vitro 3D cell culture explain, at least to some degree, the lack of sufficiently predictive models in pharmaceutical drug development. Xylyx Bio claims its tissue-specific ECM substrates recreate native cell environments more faithfully by providing the full milieu of ECM components—proteins, proteoglycans, glycoproteins, growth factors, and others—normally found in specific tissues.

"These ECM substrates support cell attachment, viability, and phenotypic maintenance for organoids and spheroids used in drug discovery, toxicology assays, bioprinting, organ-on-a-chip, cancer research, tissue engineering, regenerative medicine, and more," Yankelevich says.

"In cancer research, tissue-specific bone, liver, and lung ECMs provide the right composition and mechanics to model colonization of tumor cells in common secondary sites. ECM substrates that are directly derived from diseased tissue re-create the disease microenvironment, which significantly improves in vitro disease modeling and drug development in a setting that is more disease-relevant,” she adds.

Because 3D cultures represent a huge step from conventional 2D cell cultures, scientists should be prepared for all-around greater complexity, from the assembly of organoids or spheroids to their ongoing culture to the multiple of data they can expect compared with 2D cultures. Traveling the last mile of industrialization for 3D cultures demands special attention to the basics, among which ECM ranks prominently.