Choosing Extracellular Matrix for Cultured Cells

The extracellular matrix (ECM) is a complex mixture surrounding live cells, composed of fibrous proteins that provide structure and physical support, and other types of molecules that function in signaling and cell health. Common components include collagen, fibronectin, laminin, glycoproteins, and proteoglycans. In vivo, each tissue type has its own distinct ECM recipe, which may be incompletely understood, yet approximating it helps cultured cells to thrive. “It is well established that the architecture of the ECM plays a significant role in cell responses, therefore, only a combination of various tissue-specific ECM components can provide the essential biochemical and biomechanical structure that can promote physiologic cell growth and function,” says Evelyn Aranda, Director of Applications at Xylyx Bio. This article discusses factors to consider when choosing ECM components for cultured cells.

Identify your goal for the cells

You should consider your experimental goal or desired outcome, because the ECM you choose will influence your cells toward (or away from) it. “If culturing stem cells, do you want them to maintain pluripotency or differentiate?” says Bowman Bagley, CEO of Advanced BioMatrix. “Do you want cancer cells to migrate, or are you trying to limit migration?” Different ECM choices can play a significant role in such plans.

Once you’ve identified a goal for your cells, check the literature for relevant protocols that can be a starting point for your own process of trial and error. “The main components of ECM used in culture are proteins of the natural ECM, such as laminin, fibronectin, elastin, gelatin and many others, combined together or with synthetic polymers such as poly-D-lysine and poly-L-lysine,” says Joseph Boyd, Scientist for Cellular Assays R&D at MilliporeSigma, the U.S. and Canada Life Science Business of Merck KGaA. “It’s hard to determine the right ECM for specific cell types or specific applications, but there are a lot of data in the literature for all cell types.”

General selection guidelines

Cells are more likely to thrive in an environment that more closely resembles their tissue of origin. “We observe significant differences in cell growth and function when culturing cells in 2D without ECM or in collagen I, compared to ECMs that we extract from native tissues,” says Aranda. Choosing ECM proteins that normally exist in your cells’ milieu is an important first step. Advanced BioMatrix offers an array slide containing 36 combinations of ECM proteins, so researchers can seed cells and then evaluate where they attach best.

Besides tissue specificity, some other general guidelines can be helpful when beginning a new project. Matrisome material (such as Matrigel^® matrix), which contains a mixture of different proteins and factors extracted from tissues, is compatible with a broad range of cell types in 2D and 3D (though concentrations will need to be optimized, and you might need to add specific purified proteins to tailor the ECM to your cells). “For stem cells and progenitor cells, we can think about Matrigel matrix, fibronectins, collagens, gelatins, and laminins,” says Sherwin (Xiaoyu) Zhu, Scientific Support Specialist at Corning Life Sciences. “For hepatocytes, we usually recommend Matrigel matrix, collagens, and laminins.” Another possibility is ECM mimics, or synthetic ECM components. These are helpful in defining the ECM composition in some types of research —for example, stem cell-related therapeutic research, especially if manufactured in a cGMP-compliant facility (such as the Corning Synthemax substrate).

ECM considerations in 3D

Cells cultured in 3D are especially sensitive to ECM composition, requiring more components than their 2D counterparts. “This mixture and variety of ECM components is critical for 3D cultures, as matrices consisting of only one or two ECM components, or synthetic matrices, fail to support growth of most 3D cultures,” says Nurit Becker, Human Protein Biology R&D Head at MilliporeSigma.

Two main types of 3D cell cultures (organoid and spheroid) differ greatly in their uses of ECM components. Organoid cultures grow on scaffolding made of ECM components of an optimized stiffness and concentration. “For a dome culture, researchers can start from 50% Matrigel matrix concentration, and can increase to 70% in some cases,” says Zhu. “If a sandwich strategy is used, cells can be embedded in 25–40% Matrigel matrix and overlaid by 2–5% Matrigel matrix or other ECMs.”

For spheroids, the culturing method is designed to encourage cells to aggregate together into a spheroid, suspended in the medium without attaching to a plate. This is facilitated by coating the culture vessel with a hydrogel such as Corning Ultra-Low Attachment surface. “In some cases, hepatocyte culture for example, we can use the sandwich method,” says Zhu. “Cells form spheroids when grown in between two ECM layers, such as collagen and Matrigel matrix.”

Selecting ECM components can influence both the strength and stiffness of hydrogels in which 3D cultures are grown. The strength of a hydrogel is determined by the ECM protein type (e.g., enzyme- vs acid-extracted collagen) and concentration. “The higher concentration of protein leads to stronger gels, but [these have] lower porosity, which can affect cellular migration,” says Bagley. “Again, the right product depends on the desired cellular outcome.”

The stiffness of hydrogels is also influenced by ECM selection. “It is important to optimize the stiffness of the microenvironment according to each tissue type; we have observed significant differences in cell growth and function when reconstituting ECM-based hydrogels at various concentrations,” says Aranda. Other tools such as methacrylated forms of ECM components can help to tailor a hydrogel for your cells. “Methacrylated ECMs can be solubilized at various concentrations and photocrosslinked to fully customize and tune the hydrogel to fit your application,” says Bagley. Becker also adds that greater crosslinking can be achieved by raising the concentration of entactin in the ECM mixture. “This increases the tensile strength of the ECM and can help with some cultures that proliferate better with a more robust structural support,” he says.

The stiffness of the substrate beneath the ECM is also important. “Tissue culture plastic is gigapascals hard, while brain tissue is closer to 0.2 kPa (very soft) and heart tissue is between 5–30 kPa, for example,” says Bagley. “Culturing neurons or cardiomyocytes on plastic or glass doesn’t provide the cells with a truly native-like environment.” Advanced BioMatrix’s CytoSoft^® plates have a PDMS layer of a specific stiffness (0.2–64 kPa). “You coat the PDMS with the desired ECM, and suddenly your cells are exposed to the correct protein and substrate stiffness—getting you that much closer to an in vivo environment,” he says.

Common problems, and solutions

When researchers start optimizing ECM components, common problems can include death or abnormal growth of cells, failure of cells to attach, and differences between 2D and 3D cultures in terms of growth rates and phenotypes. “Many of these issues may be dependent on the particular cell type and can be resolved by incorporating cell-specific media, growth factors that are required for optimal cell growth, and optimizing the concentration or stiffness of the ECMs based on the quantity of cells that are being cultured,” says Aranda.

Whichever ECM materials you select, take the time to read the accompanying information, as it may spare you trouble later. “ECMs are sensitive to temperature and pH, and sometimes the product turns into precipitates, gelation, or insoluble,” explains Zhu. “This might be caused by improper temperature or pH, so researchers need to pay attention to the product’s user guidelines.”

Bagley warns not to begin with a complex mixture of ECM components. “Researchers love to overcomplicate,” he adds. “I always start at the basics and build, one step at a time.”