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Sarah Webb, Ph.D.
BioTechniques, Vol. 62, No. 3, March 2017, pp. 93–98
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Sarah Webb explores the expanding tools and technologies for 3-D cell culture—from hydrogels to organs-on-a-chip.

Petri dishes pervade cell biology, providing flat surfaces on which cells can attach and grow. But researchers are increasingly recognizing that introducing a third dimension into cell culture changes cell behavior and more closely matches the natural environments of living cells.

However, that’s not the sole advantage of 3-D culture systems. They also allow researchers to study the effects of physical and mechanical stress on cells and even provide a look at physiologically relevant processes such as fluid flow.

Aside from informing basic science, more complex 3-D cell culture systems are also paving the way for realistic in vitro systems to study normal organ function as well as disease pathology. Organs-on-a-chip, which integrate cells in 3-D culture within microfluidic systems, could soon play a vital role in drug discovery.

Cultural tools

The majority of cells in our bodies reside in 3-D environments. Compared with cultures atop a 2-D surface, these 3-D microenvironments are more complex in terms of interactions between cells and the extracellular matrix, cell–cell communication, and transport of soluble factors.

For decades, researchers have realized that changes in the cell microenvironment can lead to profound differences in cell behavior and pathology. Even relatively simple differences, such as whether a cell is mounted on glass or plastic, can change its response to drugs, notes chemical engineer Steven Caliari of the University of Virginia. Twenty-five years ago, Mina Bissell’s group at Lawrence Berkeley National Laboratory demonstrated that normal breast epithelial cells plated in two dimensions exhibited tumor-like characteristics. But when cultured in a 3-D environment that closely resembled glandular structures, these same cells adopted a “healthier” phenotype (1).

“That’s a good example of a healthy cell going down a disease path when you culture them in the wrong environment,” Caliari explains.

Caliari’s group is focused on designing new materials that researchers can use to probe fundamental questions in cell biology. Today, there are an array of options for culturing cells in 3-D, many of which are commercially available (2). Collagen, the primary protein constituent of many tissues, is a good model material for 3-D culture. “You can buy sources of collagen where you can simply adjust the pH and the temperature to a physiological range, and that collagen will self-assemble into a hydrogel,” Caliari says. A downside of collagen hydrogels, however, is that they’re often very soft, so they aren’t always the best choice when a test system needs to be mechanically robust.

In some cases, a research question dictates hydrogel choice. Also at the University of Virginia, Jennifer Munson studies brain cells and the environmental factors that affect their behavior. Since hyaluronic acid (HA) is abundant in the brain, it’s the basis for many of her experiments rather than collagen, which doesn’t occur in that tissue type.

Because one of 3-D cell culture’s selling points is the ability to test the effects of physical stresses on cell behavior, tuning the mechanical properties of 3-D culture hydrogels is often of great interest to researchers. But this feature can be hard to control using natural biopolymers. That’s a primary reason for choosing synthetic hydrogels, Caliari says, where researchers can adjust the mechanical properties and also change ligand presentation, adhesive motifs, or growth factor availability. Although polyacrylamide is ubiquitous in 2-D culture, its precursors are toxic, so it can’t be used to completely encapsulate cells. Polyethylene glycol (PEG) hydrogels, on the other hand, are popular for 3-D culture because the material has been widely used with many available protocols.

PEG is also a simple platform for introducing chemical modifications that can allow researchers to create formulations that gel when mixed or when exposed to light. One downside to the use of synthetic polymers can be cell recovery. To address this, some PEG kits include peptide-based degradation sequences that can be cleaved with matrix metalloprotease (MMP). With a natural polymer hydrogel, a variety of enzymes can provide a relatively gentle way to recover cells.

Harvard University researcher Ali Khademhosseini is trying for the best of both worlds by combining gelatin with photocrosslinkers to exploit the biocompatibility of natural polymers with a programmable chemistry (3). With 3-D printing or other layering strategies, it is possible to build structures using patterns of light to lock in the gel architecture. According to Khademhosseini, the combination of tunable mechanics and hydrogel degradation provides the opportunity to modify the environment with growth factors or other molecules. His team has now extended this idea to elastins, which they use to create more rubbery gels that have better mechanics for epithelial cells or that might even be suitable for use as surgical sealants (4).

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