Escaping the Cage
Researchers have begun to grow what’re known as organoids – tiny, three-dimensional models of organs composed of the same kind of cells as real organs. Some have heralded this as a potential way to end animal testing. But the technology – even when fully developed – can’t do everything. These technologies will never entirely replace animal research, say experts. But that is not their purpose. As these new technologies develop, it might become increasingly less necessary, and maybe even detrimental, to keep relying on animal models, especially as scientists identify how inaccurate animal models can be.
There’s a catch, though: animal testing is deeply baked into the FDA’s drug and treatment approval processes.
Animal testing works, but just barely
Animal testing, for better or worse, is an unavoidable reality. For now, at least, scientific research – especially in pharmaceuticals – restricts human trials until after a company can show a drug is non-toxic and stands a decent chance of working as intended. That requires animal testing. Rats and mice are two of the most common research subjects, but many animals, ranging from fruit flies to rhesus monkeys, are a crucial part of scientific research.
“Everyone knows [animals are] not optimal, at best,” said Don Ingber, the founding director of the Wyss Institute for Biologically Inspired Engineering at Harvard University. “They're poor predictors of what you see when you get to clinical trials.”
In fact, about 90% of all drugs that begin clinical trials in the United States are never approved by the U.S. Food and Drug Administration (FDA). This is despite the fact that drug companies are required to do preclinical studies to show that their drugs are not toxic and may work in people by testing them in animals. A 2016 analysis found that most of these drugs failed because they didn’t work like they should in people, either because they didn’t produce the intended effect (40-50% of the time), they were too toxic (30%), or they weren’t absorbed or excreted well enough in patients (10-15%). Only about 10% of drugs weren’t approved because of other factors, like lack of interest and poor planning.
Clearly, drugs don’t work the same in animals as they do in people. But for a long time, there was no alternative. In 1937, a drug to treat strep called sulfanilamide, dissolved in poisonous diethylene glycol, killed over 100 people, including about 30 children. The law forming the basis of the modern FDA regulation of drugs, including toxicology testing on animals, was passed the next year. In the 1960s, thalidomide, a drug used to treat morning sickness across Europe, caused children to be born with shortened or missing limbs. Scientists and legislators concluded that animal testing was the only way to keep drugs from harming people.
To the rescue
Organoids present an alternative to animal testing that’s attractive both morally and scientifically. “They're kind of a middle ground in between the simple cell monolayer cultures and the animal system,” said Benjamin Freedman, an associate professor in the division of nephrology at the University of Washington School of Medicine who does organoid research.
Freedman started to become interested in kidney organoid research after several members of his family were affected by kidney disease. Originally a stem cell researcher, he was interested in finding new ways to potentially regenerate damaged kidneys. But he also knew that growing kidney tissue, and eventually organoids, could lead to new insights on how to treat kidney disease.
Like all organoids, Freedman’s kidney organoids start with stem cells – cells that have the ability to eventually develop into any type of cell given the right environment. Scientists like Freedman have come up with ways to create exactly the right type of environment to cause stem cells to become kidney cells.
“What's great is that the cells actually know how to do this by themselves – they have this program built in to make kidney structures,” said Freeman. “We just have to get them to, you know, to follow that program.”
Shay Soker, a professor of regenerative medicine, biology, and bioengineering at Wake Forest School of Medicine, started his work on liver organoids intending to grow fully functioning, full-sized livers from stem cells. But his team kept running into technical problems – the blood vessels and scaffolding needed to support such a large biological structure were difficult to engineer in the lab. Even smaller versions were hard to create. So Soker came up with another idea. Using liver from mice, detergents were used to dissolve the cells, leaving the vascular and supporting structures behind. Soker could then replace the mouse liver cells with human liver cells, using the mouse liver’s natural “scaffolding” as a base.
Unfortunately, this process was inefficient if they wanted to grow a large amount of organoids, which they could use to model liver cancer and to study drug metabolism, a process that starts in the liver. So the team decided to go even smaller. They devised a way to combine hepatocytes, the main type of liver cell, endothelial cells, and stellate cells, which Soker described as supporting cells, in way that they will self-assemble in tiny, spherical liver organoids about 200 micrometers, or 0.2 mm, wide. Though you can see them with the naked eye if you look carefully, said Soker, it’s best to use a microscope.
A revolutionary approach
Not all organoids are grown from human cells – many are from animals. David Breault, an associate professor of pediatrics at Harvard Medical School and a principal faculty member at the Harvard Stem Cell Institute, first started working with stem cells from mouse intestine. Research on these early organoids focused on studying the stem cells that made them, and eventually Harvard took notice. The Harvard Digestive Disease Center asked Breault to form an organoid core, training other scientists to grow and work with organoids. The new program also led to an explosion of collaboration between researchers, said Breault, and eventually, they were able to grow human intestinal organoids.
“We began building a bio repository of organoids from human samples from both children and adults who would donate a small biopsy,” said Breault. While having the human organoids was useful, he said it was the combination of the mice and human organoids that has led to some of the most interesting research.
“It has just sort of revolutionized our approach [and] our ability to go back and forth between the mouse and the human to ask translationally relevant questions,” he said. For instance, Breault said, they have used organoids to help discover a new disease – a genetic condition that impacts both the intestine and the adrenal glands, Breault’s other area of study.
The adrenal glands, located above the kidneys, produce stress hormones. Human organoids allowed Breault’s team to grow intestinal cells with this condition in the lab, enabling them to characterize the condition in detail and giving them reason to look for impacts in the adrenal glands. When the team studied a mouse model with this condition, they found effects in the adrenal glands not previously known, and could show that human patients with the same intestinal condition had the same effects in their adrenal glands.
Translational work is especially important for organoid research, and not only comparing from mice to humans, but comparing human organoids to living humans, said Magdalena Kasendra, the director of research and development at the Center for Stem Cell and Organoid Medicine (CuSToM) at Cincinnati Children's Hospital. The center works with pharmaceutical and biotech companies to validate organoid models for drug testing, mostly for liver organoids.
“We work very closely with pharma, we work very closely with various biotechs who develop, for example, different types of drugs that could be tested in organoids,” said Kasendra. “And we do all of that, to de-risk those platforms, to validate them, to make sure that indeed, they could be useful as predictive models of human biology.”
Organ chips and human implants
Not all organoids are intended to simply be used as models, either for normal biology or to model a disease like cancer. Like other stem cell technologies, some organoids are being developed as treatments themselves. In addition to its work on liver organoids, CuSToM is working to develop intestinal organoids that can be implanted in someone whose body is rejecting or at risk of rejecting an intestinal transplant. The intestinal tissue grown from a patient’s own intestine could be implanted in their intestine. When tested in rats, the intestinal tissue heals injured intestines. If this can be done with apatient’s own cells, there’s no risk of rejection. Kasendra and her colleagues’ job is to make sure that this approach is safe to try in people.
While Kasendra said that some of their testing is done directly on the intestinal tissue, the final verification of the technique still has to be done in animals. She said that with organoids and other technologies improving, that might not always be true.
At the same time, organ chips – bearing tiny channels coated with biological tissues – are making headway too. Sometimes they leverage organoids by using them to source their tissues, said Don Ingber, the Wyss Institute founding director. Ingber’s work focuses on organ-on-chip technology, and he is also the founder of Emulate, which produces these organ chips.
“We’re able to recreate the three-dimensional structures of tissues as well as the mechanical environment, like breathing motions, or peristaltic-like motions, vascular perfusion, or blood flow, air in the lungs, and tissue-tissue interfaces, which is what makes an organ, where two different types of tissues come together,” said Ingber. Organ chips also have certain advantages over organoids, he said. For example, they have several different tissue types and can recreate tissue barriers, immune cells, and the physical microenvironment. They can also model how a patient might respond to changes in drug levels over time in their body.
The FDA gets in the way
Though these technologies are advancing quickly, it might be a long time before they make much of a dent on testing in animals. Unlike animal models, Kasendra said that these alternatives need to be validated on a case-by-case basis – it’s unlikely that any one organoid model will see universal use.
Until recently, there were also seemingly insurmountable regulatory barriers, specifically in the form of FDA-mandated preclinical testing on animals. The general rule was that companies had to have tested their drug on two types of animals before proceeding to clinical trials, said Margaret Riley, director of the animal law program at the University of Virginia School of Law. But on September 29, the FDA Modernization Act 2.0, a bill introduced by the unlikely alliance of Senators Cory Booker and Rand Paul, passed the Senate. The House has yet to vote on this legislation but it has bipartisan support.
There will likely always be some types of experiments that are difficult or impossible to do without animals. Kasendra said vaccination research might be difficult to replicate, since the immune system is so complicated. Likewise, research on things like cognition is impossible to do without a living animal, said Ingber.
At the same time, Ingber said, it is not possible to use animals as models to test the effects of many drugs. Of drugs being developed now, over 40% are biologics, he said – things like monoclonal antibodies or RNA therapies so specific to humans that testing them in animals would not make sense. The animals would not have the specific drug targets needed for these drugs to work.
Animal testing raises ethical questions
As it becomes more likely that we will live in a world where testing on animals might be less necessary, it might become useful to adopt a new framework for thinking about ethical testing on animals. Philosophers have long disagreed about whether it is ethical to do scientific research on animals, said David DeGrazia, the Elton professor of philosophy at George Washington University.
People who justify animal research “would appeal to the social benefits of animal research and would claim that at least some animal research offers very important benefits that cannot be gotten any other way,” said DeGrazia. “But that kind of statement is actually a lot more likely to come from animal researchers or other members of the biomedical community.”
It is true, though, that there are numerous avenues of research – particularly those involving the brain and processes that involve more than one organ system – that will be impossible to study without the use of animals.
One framework of thinking about reducing harm to animals is the “three Rs” framework. This involves replacing animal subjects with alternatives, refining the practices used in research, and reducing the number of animals used to only what is necessary for the study. But DeGrazia thinks the framework could do with some updates.
DeGrazia said that the three Rs framework has some inherent problems, like the fact that “refining” doesn’t establish any kind of upper limit to animal suffering. But particularly applicable to this new technology is the fact that the framework doesn’t have a way to determine if the experiment should be done with animals in the first place. The “no alternative” principle, a harsher stance on “replacement,” hinges on the fact that there might be alternatives available. Increasingly, there are.
In a paper written with Tom L. Beauchamp, an emeritus professor of Philosophy at Georgetown University, he suggests a new framework. The two suggest three principles of social benefit, one of the key motivations of animal research, and three principles of animal welfare. When thinking about animal welfare, they argue, animals should not be unnecessarily harmed unless scientific purposes justify that harm. Animals should also have their basic needs met and not be subject to severe suffering for a long time. When thinking about social benefit, the experiment must have an expected net benefit, must provide sufficient value to justify harm, and there must be no alternative to performing tests on animals.
“I think the public would generally agree if you can do it without using sentient animals – animals who can feel – you should,” DeGrazia said.
Though DeGrazia said he hasn’t heard many animal researchers express enthusiasm for reducing animal testing, Soker said he was all for it. But he was quick to add that researchers’ motivation might not primarily be about ethics, though he said he would be “more than happy” if organoid research reduced animal testing. He said he was more concerned with creating accurate scientific models, enabling better drug development and more productive research.
“The issue of animal research is extremely important,” he said. “But you have to understand that we're actually motivated mostly by science and understanding biology.”