Image on left: InSphero 3D InSight tumor microtissue acquired using the PerkinElmer Opera High Content Screening System. Other images courtesy of InSphero.
"The future of therapy is a molecule/cell therapy combination," says Sitta Sittampalam, Ph.D., senior scientific officer and project manager at the US National Institutes of Health (NIH)'s National Center for Advancing Translational Sciences, and co-chair of the SLAS Stem Cells and 3D Microtissues Special Interest Group. "In 20 years, if you have a failing kidney, you won't just get a pill and hope it works; you will receive an injection of stem cells and a drug that makes those stem cells go to your kidney to rebuild it."
Sittampalam's vision is fueled by work underway at NIH and elsewhere at the intersection of stem cell research and 3D screening. The use of 3D culture systems for regenerative medicine is advancing exponentially, Sittampalam observes. A recent example is the use of mouse embryonic stem cells in 3D culture to generate inner ear sensory epithelia. The resulting cells transformed into epithelia in a self-organized process that mimicked normal development and exhibited the functional properties of native mechanosensitive hair cells.
Using other 3D cell culture strategies, researchers also have succeeded in creating "mini-kidneys"; vascularized and functional livers; and cerebral organoids that model both the healthy brain and microcephaly. "Clearly, we're making progress in producing viable tissue and organoids for regenerative medicine purposes," Sittampalam says. "We know that mixing stem cells with other cells in a 3D environment will make it easier to create functional organs, and that we then have the possibility of screening tissue from those organs for regenerative pharmacology. We're still in very early stages, but the screening field is beginning to move—and will continue to move—in this direction."
In nature, stem cells grow in a 3D environment, Sittampalam explains. Embryonic development involves fertilization of an egg by single cells, followed by the formation of a spherical blastula containing an inner cell mass (ICM) made up of stem cells. "The blastula looks like a basketball—definitely 3D," he says. During gastrulation, the stem cells that make up the ICM give rise to three germ layers—ectoderm, mesoderm and endoderm—that go on to develop into organs. "If you take those cells from the ICM and put them in a 2D environment, they're not going to develop the same way they do in the real world. They have to be in a 3D structure where they encounter and interact with other cells they would respond to in the natural environment," he emphasizes. "Trying to differentiate them as a single layer on a plate just isn't the same. They don't form tissues as efficiently."
Advances made in the past few years have convinced Sittampalam that 3D culturing of stem cells will be "the next wave" for drug discovery. "Until recently, we didn't know how to make stem cells, how to culture them and how to produce them in large quantities. We also didn't have the growth media that could actually sustain these cells once they were produced," he says. "Now we have those ingredients—the ability to produce enough stem cells that we can begin to work efficiently with them, and the unique media in which they can thrive. This opens the door to developing physiologically relevant models for learning about health and disease and, ultimately, for screening for small molecules."
The option is vital, according to Sittampalam, because so many drugs developed through screening in two-dimensional confluent layers "don't work very well in animals or in vivo," he observes. "The reality is, the success rate is very low. What we would like to do now is screen in tissue mimics or organoids, which have all the different types of cells that are found in the actual organ—for example, we could test a new compound for toxicity in a functional liver model."
Three-dimensional tumor models could improve the discovery of effective oncology drugs. When Sittampalam was deputy director of the Institute for Advanced Medical Innovation at the University of Kansas Medical Center, his group showed that whenever tumor cells are grown in 3D rather than in a flat, confluent layer, "their response to standard care drugs is very different. The 3D cells were less responsive and survived at much higher drug concentrations than those cultured in 2D. The response we saw in 3D is likely to be very similar to how the tumor would respond to the drug in the body."
Earlier efforts to use stem cells to create "diseases in a dish"—diseased tissues that could serve as models for drug screening and testing—"didn't work because researchers were still trying to use a 2D culture method," Sittampalam says. "People finally realized that stem cells need a 3D environment, just like what happens in the embryo, to fulfill this promise. It was an 'aha' kind of moment, and it's why we now are seeing papers showing the use of 3D culture to produce physiologically relevant tissues."
Very recent work shows that exposing stem cells to specific proteins can cause them to recapitulate features associated with certain diseases, and so researchers are beginning to use stem cells to create relevant disease models. For example, researchers have generated heart muscles that behave like hearts of people with the inherited disorder known as arrhythmogenic right ventricular dysplasia/cardiomyopathy and nerve cells that recapitulate features associated with Parkinson's disease. Taken together, these advances demonstrate that "stem cell technology is advancing on multiple fronts, and screening is not far behind," Sittampalam says.
"If you look at the current 3D literature, you'll see that most researchers are not focusing on screening; they're thinking about how they can grow functional tissues and organoids," Sittampalam says. "At NIH and in some other labs, we are going to the next level. We are working on producing organoids in 96- and 384-well plates, so we can screen them and scale them up. The technology is not here yet, but it will be soon, because it is already appearing in Europe." He points to InSphero, based in Germany, which is producing 3D tumors in 96-well plates. The company won a New Product Award at SBS 2011 for its 3D microtissue technology, and also is involved in a Eurostar project to produce livers in 3D.
"What needs to be developed now are high-content imaging technologies that can image these three-dimensional tissues in depth," Sittampalam says. "Then we will need informatics solutions to analyze the large amounts of resulting data. So, not only do we need to develop these tissues and organoids in 3D, we have to have the accompanying technologies to actually measure what's going on in those tissues, in large quantities."
On the automation side, scale-up technologies are needed to bring stem cell 3D screening into the mainstream, says Kelvin Lam, Ph.D., founder and president of Simplex Pharma Advisors, and a presenter in the SLAS2014 short course, Introduction to the Derivation and Maintenance of Human Induced Pluripotent Stem Cells. "Until recently, making a 3D neuronal stem cell or organoid has been considered an art. Now the field is trying to standardize the result by using a machine to do that part of the work," Lam says. "At the same time, robots are being produced to do screening in 3D environments with non-stem cells, and that same technology could be used with stem cells."
A potential downside, Lam notes, "is that while analysis of 3D cell systems will produce richer, more complex data, there will be lower throughput because you can't do hundreds of plates at the same time. Also, you need expertise in stem cell manipulation—particularly human stem cell manipulation—and data interpretation is more complex than for 2D. Reproducibility could also be an issue for 3D stem cell culture, because simply changing one thing could change a lot."
That said, "a scientist who understands the 3D culture system is more marketable today," Lam says. Moreover, Sittampalam adds, "3D is a disruptive technology that is gaining momentum. It will be an addition to current automated screening technologies and assays—and will probably replace the simple use of single cells in a confluent, artificial, single-layer model. So drug-discovery scientists need to be watching this field, and learn how to work in it."
"The Stem Cells and 3D Microtissues SIG took place on Wednesday afternoon, near the end of SLAS2014, so we did not expect much participation," Sittampalam says. "But the room was full, with more than 80 people attending." The session was co-organized by Marcie Glicksman, co-director of Laboratory for Drug Discovery at the Harvard NeuroDiscovery Center and Brigham and Women's Hospital. Presentations by Sharon Presnell, Ph.D., executive vice president and chief scientific officer at Organovo Holdings in San Diego, and Leslie Mathews, Ph.D., senior research scientist at the NIH, both addressed aspects of stem cells in 3D microtissues, and "the response was incredible," Sittampalam notes. "There were a tremendous number of questions, and clearly a lot of people are thinking in this direction."
The main issue discussed during the SIG was the possibility of adding a stem cell component to the mix when developing physiologically relevant tissues in 3D culture. "Many people are building microtissues for screening using diseased cells and co-culturing them with the cells normally found in those tissues," Sittampalam says. "But adding stem cells could conceivably add to the tissue cells that might be missing." He gives this example: "If you cut your skin, right away you get inflammation, then you clean the cut and put an antibiotic gel on it. But it's the stem cells in the skin that actually repair it. Those cells are dormant until there is an injury; then they spring to life," he explains. "Similarly, if you are building microtissues to put in wells and screen, you need to have stem cells there so they will fill in the gaps, adding whatever cells you haven't included in the microtissue that are normally found there." For now, "we're not even sure what's missing," Sittampalam acknowledges. "But the environment has to be what it's really like in the body for that to emerge."
March 3, 2014