Guide to Extracellular Matrices (and How to Avoid Common Problems)

An integral part of mimicking the microenvironment of specific cell types in a tissue culture dish is the use of extracellular matrices (ECM). ECMs are a mixture of proteins, such as collagen, and glycosaminoglycans, a type of negatively charged polysaccharide, secreted by fibroblasts in the body. The ECM lends structural support to cells and subsequently the tissues that make up organs. It may, mistakenly, be thought of only as a passive scaffolding, but the ECM is also a biologically active mixture of molecules. As an example, integrins, transmembrane proteins that sit on the cell surface, link the cellular cytoskeleton to the ECM leading to a plethora of signaling cascades that regulate everything from proliferation and morphology, to adhesion and cell death; this underscores the importance of choosing the appropriate ECM for particular cell types.

When used correctly, ECM gel supports a variety of in vitro assays including neurite outgrowth, cell invasion, and angiogenesis tube formation, according to Karina Durlacher-Betzer, Cell Biology R&D Head at MilliporeSigma, and can be used with many cell types including neurons, tumor cells, and stem cells. However, scientists may run into issues with cell attachment, growth, and differentiation when they use the wrong type of substrate, explains Evelyn Aranda, Director of Applications at Xylyx Bio. “To avoid these problems, researchers should study the characteristics of the cellular microenvironments in the body by identifying the necessary nutrients (cell media and exogenous factors) and optimal biochemical composition and biomechanical conditions (ECMs) that are required to promote successful cell attachment, proliferation, and efficient differentiation.”

ECMs can also improve cellular function, says Hilary Sherman, Senior Scientist at Corning, “[But] ECMs vary widely in their structure and function, so choosing something biologically relevant to the in vivo model being studied, while ensuring it has the ease of use to work for the in vitro application are equally important.”

In this guide, we’ll briefly explore ECM properties scientists should consider when designing experiments, as well as discuss common issues that may arise when working with ECMs.

Do your research before the experiment

In addition to studying the unique environment cells experience in the body, and translating that into the appropriate ECM, as well as media and growth factors, Aranda points out that optimal biochemical composition and biomechanical conditions must also be considered when choosing ECMs; this is required to promote the very things scientists may struggle with—successful cell attachment, proliferation, and efficient differentiation. “For example, in non-cancer related models, incorporating the milieu of ECM components that are derived from native tissues ensure that the source of the microenvironment aligns with the tissue model being developed.”

Xylyx Bio offers primary ECMs from seven major tissue types. “[This] allows the study of physiologically relevant mechanisms in a more native environment,” adds Aranda.

Some cells, such as stem cells or induced pluripotent stem cells, are very delicate and may be more challenging to work with. These cells may require a more stringent protocol and experienced handling, according to Durlacher-Betzer. “In the planning stage of the cell assay, it is recommended to search as much as possible updated specific protocols and acquire products suitable to the cell line to be researched.”

Understand that ECM can influence cells in big ways (and vice versa)

“The cells and the ECM have a two-way reciprocal relationship. Cells produce, secrete, deposit, and remodel ECM to mediate ECM composition and topography, says Durlacher-Betzer. “The ECM, in turn, transmits signals through ECM receptors to influence cell characteristics and activities. Such a feedback mechanism is essential for rapid response of cells to surrounding environmental changes.” She explains that the biochemical properties of the ECM let cells sense and interact with their environment through signal transduction pathways, especially adhesive proteins such as fibronectin, integrin, and non-integrin receptors, as well as growth factors and other signaling molecules.

While the composition of the ECM can activate distinct cellular responses, the physical properties of the matrix, such as its rigidity, density, porosity, and topography also provide physical signals to the cell, continues Durlacher-Betzer. “The mechanical properties are essentially sensed by integrins that connect extracellular ECM to the actin cytoskeleton inside of the cells. Stiff matrices induce integrin clustering, robust focal adhesions, and Rho and MAP kinase activation, leading to increased proliferation, differentiation, and contractility. For example, on soft matrices, mesenchymal stem cells favor a neurogenic path, and on stiff ones they favor an osteogenic path.”

Scientists should consider how the stiffness of the microenvironment affects cell function and behavior in the body in order to create more physiologically relevant models, adds Aranda. “Solubilized ECMs can be utilized as coatings for 2D assays and reconstituted as hydrogels that provide the natural biomechanics and composition of the endogenous 3D environment.”

Know that there is variability between lots

“One of the most common issues in working with ECMs is that they are biologically derived products, which can result in some inherent variability," says Sherman. “Normalizing each experiment for the ECM’s protein concentration can go a long way to help reduce some of the variability.” Sherman says that Corning offers different formulations of products like Matrigel Matrix, such as growth factor reduced (GFR) and human embryonic stem cell (hESC) qualified, which can help to reduce some of the variability.

Durlacher-Betzer recommends trying to reserve as many vials of product from the same batch as needed for the experiment to also cut down on inconsistencies.

For temperature sensitive ECMs pre-chill everything (really)

Temperature sensitive ECMs like Matrigel and collagen can give new users the most trouble. These ECMs will quickly polymerize at even slightly elevated temperatures. But a few simple steps can go a long way to make handling easier.

Prechill everything that comes into contact with the gel, including media, tips, tubes, and plates, advises Sherman. Also, it is crucial to thaw ECM slowly overnight at 2–8° C and to store it on ice during use, says Durlacher-Betzer.

Aliquot the Matrigel into single-use vials. “ECM gel is a very delicate product, repeated thaw and refreeze can affect the quality and efficiency of the product,” says Durlacher-Betzer. She recommends diluting the ECM with cold medium before adding it to the plate and using frequent mechanical disruption, like pipetting up and down, to help protect against polymerization.

Go beyond the ECM ingredient list

“Most researchers look at the components of the ECM, but it is also important to understand its fit for different applications, "says Durlacher-Betzer. She notes, for example, that thinner coats of ECM are better for cell proliferation, while thicker coats are better for 3D culture.

Scientists should also determine whether they need standard ECM, which is typically used for less sensitive cells, or growth factor reduced ECM. MilliporeSigma offers optimization assays to determine the best ECM protein and concentration for specific experiments.

Going forward

ECMs have the potential to take tissue culture to the next level. But only if properly understood and utilized. Aranda points out that ECMs “can be applied to disease modeling, bioprinting, drug development, and organoids among other advanced cell culture techniques.”

On the horizon, synthetic ECMs are being studied and developed. And this would help to remove some of the inherent inconsistency issues that come from animal-derived products. Sherman says, “This is definitely a new area and one with lots of interest. 3D systems can be so complex so any way to reduce variability is a huge help. Corning will be launching a synthetic ECM later this year.”

Key Takeaways

• Do thorough research before conducting experiments using extracellular matrices (ECMs). Consider the unique environment cells experience in the body and choose the appropriate ECM, media, growth factors, and biochemical composition to promote successful cell attachment, proliferation, and differentiation.
• Understand the reciprocal relationship between cells and ECM. Cells produce, remodel, and interact with the ECM, while the ECM transmits signals to influence cell characteristics and activities. Consider both the biochemical and physical properties of the ECM to create physiologically relevant models.
• Be aware of variability between different lots of ECMs. Biological-derived products like ECMs can exhibit inherent variability. Normalize each experiment for the ECM's protein concentration and try to reserve vials from the same batch to reduce inconsistencies.
• Handle temperature-sensitive ECMs, such as Matrigel and collagen, with care. Pre-chill all materials that come into contact with the gel, thaw ECM slowly overnight, and store it on ice during use. Aliquot the ECM into single-use vials to preserve its quality and efficiency.
• Consider factors beyond the ECM ingredient list. Thin coats of ECM are better for cell proliferation, while thicker coats are suitable for 3D culture. Determine whether standard ECM or growth factor reduced ECM is required for specific experiments.
• Stay informed about advancements in ECM technology. Synthetic ECMs are being studied and developed to address issues of inherent variability in animal-derived products.