Stem Cells in 3D

Cell morphology and physiology arise from a wide range of factors, and the environment plays a fundamental role. To grow cells in culture, scientists started with two dimensions, growing sheets of cells, but now some researchers use three dimensions, such as culturing cells on 3D scaffolds. In organisms, cells build 3D structures. So, culturing them in ways that allow 3D arrangements of cells can build a more realistic environment. Combining 3D culturing with stem cells, which can turn into various cell types, teaches us more about the basics of biology, and how to fix it when things go wrong.

In general, culturing cells in 3D should be more realistic. As Mike Valley, senior research scientist at Promega, says, “The morphology, gene expression, and overall biological response of cells assessed in 3D culture models are frequently more physiologically relevant than that of cells studied in standard 2D culture formats.”

Adding a dimension to cultures provides other benefits, as well. For instance, Thomas Bartosh Jr., assistant professor of medical physiology at the college of medicine at Texas A&M University, Temple, says that culturing cells in 3D provides “better flexibility, permitting cellular organization into discrete regions, similar to natural tissues,” and it “recapitulates better cell-to-cell and cell-to-matrix interactions, which are important for regulating cellular functions.” In short, a 3D cell culture is more like a tissue than a 2D one.

In short, a 3D cell culture is more like a tissue than a 2D one.

A 3D structure of cells can also tell scientists more about medicine. As Christopher Moraes, Canada Research Chair in Advanced Cellular Microenvironments at McGill University, says, “My lab and others have shown that on flat plastic tissue culture dishes, such as those often used to grow cells in the lab, cancer cells will readily respond to low doses of chemotherapies. However, once those cells are enclosed within a 3D scaffold, they become over 1,000 times more resistant to those chemotherapies.”¹

Significance of shape

The more that scientists study stem cells in 3D cultures, the more we will learn. “Stem cells are well-known to respond to their surroundings, and making their culture environment more realistic through the use of 3D cultures allows us to better predict how they will behave when used as a therapy in the real-world human body,” Moraes explains.

Putting stem cells in 3D cultures even enhances some medicinal features. As Bartosh says, “Stem cells are activated in 3D cultures to secrete a variety of therapeutic factors, which could be a potential way to improve their therapeutic utility.”

Moreover, some cellular mechanisms only appear in 3D. For example, Bartosh and his colleagues “found that human mesenchymal stem cells can be readily eaten or ‘cannibalized’ by breast cancer cells, mimicking cell cannibalism in vivo, or in tumors,” he points out. “This phenomenon is rare or absent in 2D cultures, making it difficult to study without a 3D system.”²

Economics and other obstacles

Despite the obvious benefits of culturing stem cells in 3D, nothing is perfect. For one thing, it costs more. “3D culture scaffolds and materials are orders of magnitude more expensive than 2D tissue culture plastic,” Moraes notes. “While this is acceptable for many situations, drug discovery in the pharmaceutical industry typically requires several million experiments, leading to prohibitively high 3D culture costs.”

Despite the obvious benefits of culturing stem cells in 3D, nothing is perfect. For one thing, it costs more.

The thickness of a 3D culture can also make some steps in assays more complicated. “One limitation for studying 3D cell cultures is the ability of assay reagents to penetrate the multiple cell layers of 3D microtissues to accurately measure their biology,” Valley says. Carefully engineered systems may be applied to address these issues. For example, Moraes’ lab has developed advanced 3D culture printing techniques that can be applied to generate microscale cultures. The small size of these cultures allows rapid transport and penetration of drugs into the tissues.³

Examining cells in 2D is also easier. To analyze how cells behave in 3D cultures, says Moraes, “we either have to get the cells out of 3D culture in order to conduct gene- or protein-expression analyses, or we need to look at them while they are in 3D, which requires specialized and expensive microscopes.”

In addition, some things just don’t need 3D culturing. “The gold standard methods for culturing and propagating stem cells do not use 3D methods,” says Vanessa Ott, global strategic marketing manager at Promega. “The differentiation of stem cells into terminal cell types may benefit from 3D culture, depending on the cell type.”

Nonetheless, Ott adds: “Where 3D may have the most impact is assay endpoints, which may better recapitulate human biology if the cells are cultured in 3D.” This is especially true if you are talking about more complex systems that include multiple cell types—for example, co-culture of neurons and astrocytes, skeletal muscle cells and motor neurons, or hepatocytes and macrophages. Co-culture of multiple cell types in a 3D environment may best represent cellular behavior in vivo.”

Starting to build a brain

As populations age around the world, Alzheimer’s disease could become more feared than cancer—and it already is in some families. Some hope, though, could come from what is being learned in building 3D brain-like structures from stem cells.

“The 3D culture system, especially the organoids, better capture the structural complexity of the human brain,” says Li-Huei Tsai, Picower professor of neuroscience at the Massachusetts Institute of Technology. “Neurons also seem to mature better in this system.” For example, Tsai and her colleagues turned stem cells into 3D neural tissue, and “this system proved to support the development of certain pathological features of brain disorders, including Alzheimer’s disease, where amyloid aggregates and Tau pathology have been demonstrated.”⁴

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To get more from this technique, Tsai plans to add other elements to her structures. “First, we need a system to support the development of the brain network and circuits,” she explains. “To this end, it would be nice to integrate the vasculature system so that nutrients can be infused into the 3D culture.” After that, she also plans to add other cell types, including astrocytes, microglia, and oligodendrocytes. As Tsai reminds us, these cells “are very abundant in human brains.”

What we don’t know

Although the first guess is that 3D is more natural, questions remain. “Are all 3D environments the same?” Moraes asks. “Are cells responding to being in 3D, or is some other parameter also being changed?”

In some cases, scientists know of some things that change in 3D. For example, results must be interpreted carefully because 3D cultures have diffusion limitations of oxygen and nutrients, which could produce experimental artifacts.

Clearly, much more work remains before we will see just how much scientists can learn from culturing stem cells in 3D. “We’re only beginning to scratch the surface on all these issues, which will change how we interpret experiments in which stem cells behave in 3D culture,” Moraes concludes. For some of us, these research tools will reveal amazing answers to intriguing questions; for other people, this technology might one day save our loved ones from disappearing into a neurodegenerative disease or other devastating illness.

References

1. Leung, BM, et al. Microscale 3D collagen cell culture assays in conventional flat-bottom 384-well plates. Journal of Lab Automation 20:138–145. 2015. [PMID:25510473]

2. Bartosh TJ, et al. Cancer cells enter dormancy after cannibalizing mesenchymal stem/stromal cells (MSCs). PNAS 113:E6447–E6456. 2016. [PMID: 27698134]

3. Moraes C, et al. Aqueous two-phase printing of cell-containing contractile collagen microgels. Biomaterials 34:9623–9631. 2013. [PMID: 24034500]

4. Raja WK, et al. Self-organizing 3D human neural tissue derived from induced pluripotent stem cells recapitulate Alzheimer’s disease phenotypes. PLoS One 11:e0161969. 2016. [PMID: 27622770]