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Shaking Up 3D Culture: Novel Systems Agitate Cells to Facilitate Cell-Based Screening

For life sciences discovery and technology professionals, working with 3D culture has numerous advantages, including emulation of the in vivo environment; high cell yields from less laboratory space; and a significant reduction in costs of labor and consumables. 

That’s the word from Robin Felder, Ph.D., professor and associate director of clinical chemistry and toxicology at the University of Virginia School of Medicine. Felder, a longtime contributor to the Society and the founding editor of SLAS’s Journal of Laboratory Automation (JALA), recently presented an SLAS Webinar on automated 3D cell culture systems for high-throughput screening, cell-based assays and tissue engineering, in which he discussed the benefits of such systems, now and in the future.

“Several issues need to be addressed when building and/or working in 3D systems, including how cells will grow and differentiate, how to ensure and maintain cell quality, how to attain high yields and how to automate production to provide both convenience and standardization,” Felder says. His group and others are making progress in each of these areas.

Making Cells Feel at Home

“Ninety-nine percent of the cells used in drug discovery are eukaryotic and anchorage-dependent, so they have to attach to a surface to differentiate and express in vivo-like phenotypes. That’s been done in the old-fashioned Petri dish or T-flask since time began,” Felder says. “You put the cells on plastic and they create their own basement membrane, where they grow and essentially make a carpet of cells.”

To use those cells in an assay, they must be removed from the plastic and moved into a microplate. But adding substances to a carpet of cells to get them to detach from the plastic is “the Achilles’ heel of cell culture,” according to Felder. Most researchers use trypsin, which eats up proteins on the surface of the cell, he explains. “Basically, you damage the cells enough to get them off, but then you have to wash them, put them in the next process and let them recover. That takes time and a lot of hands-on effort.”

In contrast, by continually bathing cells on a lifelike surface, in a medium that satisfies all cell needs, “you trick them into thinking they’re in the body,” which is the basis of 3D culture, Felder says. “The  medium has to be easy to use and manipulate.” One solution is to place cells in suspension, where they can grow until needed—something often done with cancer-derived cell lines, Felder explains.

“But we purists want to use cells obtained directly from the human body, expand them in a way that mimics their in vivo environment , and analyze them appropriately—and do it in such a way that it’s fully automatable or at least significantly reduces the labor component.” That, he says, also requires   placing the cells under shear stress, which doesn’t happen when cells are sitting in gel.

Why is shear stress important? Felder points to a study in which researchers stretched a rubber membrane over a plastic lab-on-a-chip in which lung cells were growing. “As they stretched the membrane, they noticed that the cells began to resemble lung cells better than any cells on a Petri dish or in gel or any other medium, because there were moving in a cycle that mimicked how people breathe in the real world.” Donald Ingber, founding director of the Wyss Institute for Biologically Inspired Engineering at Harvard University, detailed this study at SLAS2015.

To create that “movement” aspect of the living human body, Felder’s group took alginate beads and “spritzed them with tiny ferromagnetic particles so we could manipulate them by an externally applied magnetic field,” he says. “Then we coated them with various biomimetic surfaces such as fibronectin, laminin, polylysine and gelatin—things you can buy off the shelf. We know that cells love these surfaces, but not all cells love all surfaces. You have to find the right match.”

“Now we have alginate spheres, or beads, that are manipulable by magnetic particles, coated with something the cells of interest like, and the cells grow,” Felder continues. His next step was to put the cells in tubes and keep them moving, providing shear stress with the BioWiggler™, a programmable eight-position stirrer that’s “like eight little washing machines that provide gentle agitation. The type of cell you’re trying to grow determines the amount of agitation.”

In addition, the agitation allows the beads, which hover in a liquid medium, to become oxygenated, whereas cells grown on a Petri dish end up “starving for oxygen,” Felder says. “We’re finding that just by giving cells constant access to a uniform oxygen field, we’re getting fantastic phenotypes. What’s more, they’re expressing millions of cell surface microvilli. In a Petri dish, you get almost none.”

Another pressure facing cell culture biologists is the need to genotype a cell of interest to ensure what’s growing in a dish is actually the cell of interest. When cells are grown on Petri dishes, “you keep passaging and passaging as you expand them and there’s a good deal of evidence to show that with each passage, the cell becomes a bit different from the original lineage,” Felder says. To keep the line consistent with the original cell, researchers set some cells aside from the early passage and keep checking back, something that “involves a lot of labor and cost.”

With his group’s 3D system, “we can go directly from primary cells, put them in a container with the support beads, and each time a cell lands on a bead, it creates a clone of itself—no passaging involved,” Felder explains. “The result is billions of cells in a 50mL centrifuge tube, and we’ve kept those cells alive, happily thinking they’re in the body, for up to nine months, continuously drawing on them to do our experiments.”

Personalized Diagnostics

Felder’s 3D system also enables precision diagnostics. The group developed a “virtual renal biopsy” using renal proximal tubule cells to determine how an individual handles salt—and, as a result, how their salt consumption might affect their blood pressure. “One graph in the study is really telling. It shows a line going from salt sensitive to salt resistant to inverse salt sensitive in response to a controlled diet,” Felder says. “We showed in a small study that the cells tested in our system had a correlation of .88 to what’s actually happening in the body.” The team recently received a $10.5 million grant from the US National Institutes of Health to expand that work to a larger population.

When pharmaceutical companies use 3D biology to create panels of drugs that handle “the spectrum of human diseases,” says Felder, drugs are likely to be effective for many more people. Urine-based diagnostics are particularly attractive, he suggests, because urine is “one of the world’s easiest specimens to get.” 

That said, “other researchers are pulling cancer cells out of circulating blood, expanding those, and looking at their response to various chemotherapeutic agents,” he notes. “The challenge with cancer is that tumors are complex, with many different cell types, and they’re anoxic, so it’s hard to reproduce the tumor environment precisely.”

His group’s effort to sell a product that mimicked the tumor microenvironment, as well as providing culture maintenance and scalability “is gaining slow acceptance because it’s a little too far out for most people and probably a decade too early,” he says.

Stem Cells in 3D

Lee L. Rubin, Ph.D., Harvard Department of Stem Cell and Regenerative Biology, Cambridge, MA, has been studying stem cells in 3D culture. “Stem cell researchers have gotten into 3D in two different ways,” Rubin explains. “Some have been trying to grow a developing gut or lung, starting from stem cells that give rise to those organs. Others are starting from pluripotent stem cells—embryonic stem cells (ES) or induced pluripotent stem cells (IPS)—to do the same kind of thing, while retaining the ability to generate cells from multiple types of tissues.”

Researchers have discovered that by starting with stem cells in 3D culture, they see “a remarkable amount of neural structural organization” without doing anything more than adding a few factors to the cells. “They differentiated, grew and organized themselves,” Rubin says. From there, investigators have gone on to develop organoids or mini-brains in gels that resemble the extracellular matrix or even just as aggregates growing in suspension.

“The downside of organoids for our group, which does a great deal of small molecule screening, is that every one of them is different, and this variability makes screening more difficult. We want to start with good, healthy neurons with mature properties, but grown in a more reproducible format,” Rubin explains. Therefore, he adapted a method developed by his Harvard colleague, Douglas Melton, Ph.D., for making pancreatic islets. Like Felder’s method, it takes advantage of agitation. 

The method is “really simple,” Rubin says. “We take IPS cells and grow them in suspension in a kind of bioreactor, which is really a sophisticated spinner flask, where they form highly uniform spheres. Then we add factors to the medium. The advantages are that we can make billions of cells that are consistent from one sphere to the next. And we can make lots of different kinds of cells, depending on what factors we add.” The system is particularly good if a researcher needs large numbers of cells from a small number of lines, he notes.  

Rubin and his team have used the system to make motor neurons involved in diseases such as amyotrophic lateral sclerosis, cortical neurons involved in psychiatric disorders such as schizophrenia and dopaminergic neurons involved in Parkinson’s disease. And he’s used it to produce mature neurons on a large scale. Like Felder, he discovered that spinning keeps the cells “well oxygenated and well fed, with good nutrient penetration”—and growing to about half a millimeter in diameter with no necrosis at the center (often a problem with cells grown on a static system, such as a Petri dish).

Next Steps

Felder and Rubin are continuing to refine their processes for large-scale cell-based screening using 3D culture. For example, Felder is working with materials that are United States Food and Drug Administration-approved for direct injection into the body. “We want to set up a process in which we can grow cells from an individual and provide clones in bulk, with a good laboratory practice/good manufacturing process overlay on the cell culture. Cell culture is an art right now. We want to make it into a science, and then into a manufacturable product, so we can have true regenerative medicine—from your body back into your body—with high quality control.”

Rubin has found that stem cells seem to be able to mature longer with a 3D approach. “We can keep them for quite some time—months, or even more than a year,” he says. This opens up the possibility of studying changes in older neurons that are associated with neurodegenerative diseases. “We’re hopeful that these 3D methods, which are more in vivo like and enable cells to live longer, will allow us to see changes that have been difficult to see so far.”

In addition, Rubin is working with Harvard colleague Ingber, a pioneer in organ-on-chip technology, to create ways to vascularize the 3D-cultured cells so they can grow larger without necrosing. “It’s a long-term goal, but we would then be able to produce a supply of neurons to constitute a brain-on-a-chip,” he says.

Transitioning to 3D Culture

What does it take for laboratory science and technology professionals to make the transition to 3D culture? “The first thing you need to do is give up the normal cues you’ve been using to determine whether your cells are healthy,” Felder says. “If you grow cells on a Petri dish, they get all stretched out because they’re seeking something, and then they find each other and they go to confluence. If you look in a dish and see cells that are all stretched out with spindles, you think, ‘Oh, my cells are happy today.’ But they’re really unhappy because they’re trying to find a place to get comfortable.”

When working with 3D culture, “you’re working with cells that are already happy. They just show up with appropriate shapes,” Felder says. “Then you have to find new ways to stain them to study the 3D nature of the cells. For example, we use a microvilli stain and if it’s positive, we know our cells are happy. Basically, you need to study new cues to determine the health of your cells and ensure that you’re on the right track.”

“You also have to better understand the animal or human physiology of what you’re trying to study,” Felder continues. “Working with 3D systems is more of an integrative science, as opposed to focusing solely on the biochemistry.”

From a management perspective, the transition is simple because there’s no need to revamp existing laboratory equipment, according to Felder. “Fortunately, 3D culture currently works in standard cell culture rooms. Everything’s the same except you reduce the amount of plastics waste substantially, and you reduce the labor component by about 75%,” he says. People who were previously dedicated to working solely with cell culture “can expand their activities into the laboratory and actually measure the cells,” he suggests, adding that some pharmaceutical companies now have a full-time cell culture group that satisfies the needs of all high-throughput screening processes.

Learn More  

Felder is a session chair in the SLAS2017 Automation and High-Throughput Technologies Track, which will include information on 3D cell culture systems. In April 2016, he presented the SLAS Webinar, “Fully Automated 3D Cell Culture Provides Standardized, Biologically Relevant and High Production for Human Cells,” which is archived and free for dues-paid SLAS members. In addition, SLAS will publish a Special Issue on 3D Cell Culture, Drug Screening and Optimization in 2017 in SLAS Discovery (previously known as JBS). Richard Eglen, Ph.D., of Corning Life Sciences and Jean-Louis Klein, Ph.D., of GlaxoSmithKline are special issue guest editors.

August 22, 2016