Organoids in Drug Development: Evolving and Expanding

How Charles River scientists are leveraging the power of these miniaturized in vitro systems as a potential alternative to animal testing

Seven years ago, cancer expert Julia Schueler spent an eight-week sabbatical at Boehringer Ingelheim that involved a lot of pipetting. Chemists and biologist know this to be a tedious task, but for Schueler, a trained veterinarian and therapeutic area lead for oncology at Charles River Laboratories, it was worth it because she got a chance to see up close how BI's global cancer hub in Vienna was using 3D cell cultures known as organoids.

Schueler's lab in Freiburg, Germany, which specializes in patient-derived xenografts or PDX models-mice that bear tissue or cells from a patient's tumor, creating an environment for continued growth-are now developing organoids from these mouse avatars. "The idea is to have an in vitro tool that is as heterogeneous as the PDX, but still animal-free," said Schueler. "The project we are currently pushing is to have a lung cancer organoid panel that comprises seven cold tumors [those unlikely to trigger a strong immune response] and seven hot tumors, where we know how these tumors behave in vivo." Schueler add that the classification of tumors into hot and cold has a direct impact on the treatment regimen of the individual patient. "Having both groups represented in a preclinical study increases the predictive strength of which patient population might benefit most from the new drugs."

Freiburg also plans to add breast cancer organoids, and its sister site in North Carolina's Research Triangle is looking into developing colon cancer organoids. The ultimate goal, says Schueler, is to be able to offer clients an effective way of identifying human-specific drug targets and disease mechanisms, and even find the best treatment for individual patients.

Cancer is not the sole focus of Charles River's organoid research. The Leiden site, whose target discovery programs straddle multiple disease areas, has developed intestinal organoids that can assess drug toxicity in the GI tract, and they recently started a program to generate liver organoids to evaluate liver toxicity, the organ where most drugs pass through and are metabolized. Meanwhile, the Portishead site is developing a tonsil organoid platform to test vaccines and immunomodulators.

Buti says one of the most important roles right now for organoids is to be an early predictor of a drugs success or failure, which ultimately saves the client money and reduces downstream clinical liabilities. "We want to catch early on the 'bad guy,' but we also don't want to filter out what could be actually a really good drug. Basically, having this [organoid] system that better mimics the patient's physiology, well, we have more control over the decisions we make," says Ludovico Buti, PhD, a Senior Research Leader in Leiden.

What is an organoid?

Organoids are 3D cell cultures derived from stem cells (human, and both fetal or adult) that self-organize and originate organ-specific cell types that aim to mimic the structure, function, and cellular complexity of human organs, making them, in some ways, a more translatable tool than animals that are similar but not identical to the genetic makeup of humans. Organoids range in size from less than the width of a hair to five millimeters, and because they come from stem cells can expand in their cultures over time. These in vitro miniaturized versions of organs are a good fit for studying complex organs, such as the brain, lung, kidney, or pancreas and are now widely used in academic labs to study organ development.

Organoids are sometimes confused with organ chips, but the platforms are actually quite different. While organoids grow and self-assembly much like human organs do, organ chips are heavily engineered man-made systems in which the biological components (cells) are subjected to a tissue-specific microarchitecture (engineered part). Organoids lack the microfluidic environment and stretch capacity that organ chips do yet contain most of the biological components. It's no surprise given the advances of both that scientists are exploring ways of using organoids and organ chips together to achieve better outcomes.

To create organoids scientists embed pluripotent cells into a large extracellular network of protein and other molecules that give structure to cells and tissues. Specific growth factors and proteins maintain the stem cell's phenotype or function. Based on the initial stem cell population used, and the specific growth factors added, the cells in the matrix will begin to assemble into 3D organoid structures that behave like the specific tissue targeted.

Organoids were first developed in 1987 by labs at UC Berkeley and in Denver, following two important scientific successes: the application of an enzyme important for breaking down collagen in damaged tissue and helping healthy tissue grow, and the isolation of a gelatinous protein mixture called Matrigel that supports the maintenance of induced pluripotent stem (iPS) cells. Both the accuracy and reproducibility of organoids have improved over time, and today organoids are a valid substitute for two-dimensional cell models. In 2009 a research group in the Netherlands led by Hans Clevers (now head of Pharma, Research and Early Development at Roche) developed a modified culture condition for human intestinal organoids that improves the culture efficiency and maintains their long-term multi-differentiation capacity. Two years later, scientists from the University of Cincinnati were able to start from iPSC and recapitulate the full differentiation into intestinal organoids.

In 2016, researchers made organoids from the cell samples of patients with cystic fibrosis, and used the 3D models to predict which patients would response to new drugs. During the COVID pandemic, lung organoids were used to study questions of cellular microbiology and to screen new therapies and vaccines.

How are organoids being used commercially?

With organoids recognized as one of the New Alternative Methods in drug development, the door is now open for companies to use them in their preclinical drug assessments. In fact, a number of large pharma companies, such AstraZeneca, Roche and GlaxoSmithKline investing in organoid research. Although organoids and other non-animal models are still in their infancy, there is growing evidence that they can help decrease the cost and time of preclinical research, and improve the odds of success in the clinic, where currently nine out of every 10 drugs fail due to low efficacy or unexpected toxicities.

Intestinal organoid grows up in 7 days. (Qun Wang)

Buti's team in Leiden first began experimenting with organoids as part of OrganoVIR, a consortium funded by the European Union to study human viral infections in organoids. "We mainly wanted to understand what the translational aspects of this 3D model were," said Buti. "We know that the high attrition rate in drug discovery is no longer sustainable. What we wanted to understand was what the added value of using organoids compared to cell lines was, and then to benchmark this to what was happening in patients."

They did this by teaming up with an academic group of biologists who specialize in enteroviruses, RNA viruses that typically occur in the GI track but can also strike the central nervous system and other organs. Buti said they recapitulate infections in the organoids with the strain enterovirus A71, a common cause of hand, foot, and mouth disease. There is no approved drugs to treat this infection, and the few experimental compounds that reached clinical trials performed poorly. The researchers took these failed compounds and tested them against an intestinal organoid and a 2D cell line. The findings from this project, which were published last year in the journal Cells, were telling. "In the cell line, all three compounds worked really well, but when we did the same exercise using the organoid, two out of the three compounds showed they were either not effective enough or were too toxic, or both," said Buti. "This only exemplifies that with an organoid we have introduced an additional filter early on during the drug discovery process. Basically, having this [organoid] system, which mimics the patient's physiology, gives us more control over the decisions that we make."

The work with OrganoVIR established proof-of-concept for using organoids to assess the toxicity of antiviral drugs in the intestine, says Buti. But he adds the value of intestinal organoids goes beyond this. "Especially when you think about the oncology field, chemotherapy often has toxicity in the intestine," he says. "Those can now be tested in the organoids, and help you make an informed decision on how toxic the drug will be before developing it further."

Tonsil organoids

The use of tonsil organoids is another hot area of research that is inching closer to commercial viability. The platform was bolstered three years ago when a study led by Stanford University demonstrated how you can use human tonsils to develop an organoid that recapitulates in vitro several key features of germinal centers -- structures that form in lymph nodes upon infection or vaccination. The research team showed how they were able to use this organoid system to define the essential cellular components necessary to produce an influenza vaccine response. Human tonsil epithelial organoids were also used by a Korean research team as an ex vivo model for SARS-CoV-2 infection.

Charles River's Portishead site, which specializes in Discovery research involving immunology, immuno-oncology, and virology, is experimenting with tonsil organoids on several fronts, says Dan Rocca, PhD, a Research Leader in the Biotherapeutics Division at Charles River and based in Portishead. These areas, he says, include using organoids to be a better predictor of human response to therapeutic antibodies, gene therapies or cell therapies in much the same way the Stanford team used tonsil organoids to predict responses to influenza vaccines. Another key area of research being pursued by Portishead uses human tonsil organoids to generate therapeutic antibodies.

"You can use mouse models or phage display platforms to discover antibodies," says Rocca. "But with mice you have to use very sophisticated genetic models, like a humanized mouse, and if you take the phage approach you have to have super optimized systems, which are available. What would really complement these approaches is if you had an antibody discovery in human system. The tonsil organoids potentially could be used to do exactly that."

For instance, Rocca says, if you take a cancer protein that is a therapeutic target and put that into the tonsil organoids you should theoretically be able to generate an antibody response against it. "You could then clone that antibody by isolating some of the B cells generated by the tonsil organoids and have a human antibody that matures the same as it does in a real person. It would also represent a more physiologically relevant system in which to test drugs which aim to modify the immune system, particularly T or B cell responses. We are just starting to explore its' potential uses really."

So, are organoids ready for prime time?

How quickly organoids are folded into preclinical drug development will come down to how confident both users and regulators are in incorporating data from the tool into IND packages. Advances in organoids will also require a solid financial commitment by biopharma and partners, and continued improvements in the technology. Full characterization using multi-omics to understand both drug target expression and physiological relevance, which can be a large and expensive task.

But the window is definitely open. Schueler says her group has been approached by company's interested in the PDX lung organoids. And Buti says Charles River is now able to offer its intestinal organoids platform for early drug assessments, as well for disease modeling and target validation. With so many large pharma companies already investing money and time on organoid models, Buti says there will be value for clients to outsource the science to contract partners who have the expertise.

Moreover, new tools are afoot to make organoids easier to use. In April, the δypha Consortium, which aims to bring real-life patient pharmacokinetics to the lab, received a grant from the European Innovation Council and SMEs Executive Agency for €2.5 million to validate Sync Biosystems technology with its international end users, including Charles River. The Sync system is designed to reduce stress and turbulence caused by the automatic pipettes use in culturing cells. This kind of wind shear is a major challenge for users of organoids and other types of 2D and 3D cell systems; the Sync system minimizes that disruption and at the same time will allow to combine the organoids with a microfluidic system enabling pharmacodynamic testing.

While the growth of organoids is evident, whether they ever fully replace animals remains to be seen. "I hope so, but for now I see this model more to test different conditions that otherwise you wouldn't be able to test in vitro, and then basically narrow down to one or two options that will be tested in the animal model," says Buti. "In this sense, in the short terms I see more the potential to reduce the use of animals in research, because organoids will help researchers, early on, go for the [drug] that looks the most promising."

Rocca agrees, and says it is also important to make sure the hype doesn't get ahead of the science. "I think the problem with all these new technologies is that although they show promise, they are still being developed by the scientific community, particularly for use in drug development, so considerable effort needs to be put into validation and testing before we'd be in a position to completely remove animal testing - despite the fact this is something we all strive for."

Public link

Please use the above public link if you want to share this noodl on another website.