Organoids add fresh dimensions to cell, tissue and organ research

Tissue engineering, gene editing, organs-on-chips: There’s no question cell cultures and cell lines have transformed biology and medicine.

But as science approaches the limits of what one or two cell types can do, researchers are turning to organoids — a newer tool that can imitate bodily environments and mimic not just cells, but tissues and organs.

“One of the most remarkable things in the last 10 years is the discovery, through the cooperative work of literally hundreds of labs around the world, that we can culture essentially any cell type outside the body using this organoid toolkit,” said Andrew Ewald, who directs the Department of Cell Biology at Johns Hopkins School of Medicine.

For more than a century, researchers have grown cells in labs to study diseases, toxins and treatments. But, like photocopying a single page of a book, culturing cells only tells part of the story.

Organoids, which contain multiple, interacting cell types, are more like a synopsis. The distinction between cultures and organoids is the difference between a cucumber and a salad, or a spark plug and an engine.

“They’re organ-like: The structural features of the cells in these three-dimensional arrangements is very similar to how they would look inside the body,” said Ewald.

Ewald’s research builds upon work by the field’s pioneers, who showed in the late 1980s that a breast’s epithelial cells, grown on a 3-D protein scaffold, could form ducts and even make milk — a process difficult to study in living humans.

“We're able to watch new tubes initiate, elongate, bifurcate and polarize to their mature differentiation state, all within a few days in the laboratory,” said Ewald.

Ewald also studies cancer and metastasis. But cancer cells can take weeks or years to form new tumors in vital organs, so Ewald uses organoids to model the different stages of the process in the lab.

This window that organoids offer into complex cellular processes could help scientists see how cells develop, change and die — even in the intricate, specialized world of the brain.

“We really use these to study aspects of neurodevelopment and neurodegenerative disease, with a focus on Alzheimer's disease,” said ASU molecular biologist David Brafman.

Researchers can grow organoids from either adult stem cells, which make cells specific to their own tissue type, or Induced pluripotent stem (iPS) cells which — with the right nudge involving certain cultures and growth factors — can become any cell type in the body.

Which type researchers choose to use depends on their needs and goals.

Brafman’s lab introduces different disease risk factors into iPS cells taken from a healthy individual. Because these cells are genetically identical, they provide a clearer causal link between risk factors and diseases.

“That provides a lot more control, rather than taking a set of iPS cells from a group of healthy patients and a group of diseased patients, where you're not only comparing the disease state to healthy state, but then you're also comparing differences in genetic background,” said Brafman.

Organoids’ usefulness lies partly in their ability to construct whole neighborhoods housing diverse, interacting cell types. To make sense of this complex environment, researchers use special dyes to stain cell types with different colors.

“And when we actually look at them, we can see that they have all cell types of the tissue that we collected from,” said UA cancer cell biologist Curtis Thorne.

Picture an organoid as a gelatinous blob peppered with raisin-like spots, not unlike an unsavory ambrosia salad. The recipe is straightforward, if not simple: Start with a healthy or diseased sample; add enzymes; chop to separate desired cells; place them in a 3-D protein scaffold; add nutrient soup; and leave to grow in a climate-controlled chamber.

“Over a few weeks, we'll see these small structures grow out that we call organoids,” said Thorne. “And when they get big enough, we can break them up and resuspend them in the scaffold, and they continue to grow and amplify.”

The scaffolding itself consists of typical proteins secreted by the body’s connective tissue cells, such as laminins, collagens and fibronectins.

“If you take cells from different parts of the body, and you put them in an appropriate three-dimensional environment, they'll organize into tissues that very closely resemble those that they would form in the body,” said Ewald.

Without scaffolding, organoids have as much structure as Jell-O without a mold; with it, cells can organize into veritable tissue tiramisus. Pancreatic cells will form pancreatic ducts; neuronal cells will form brain structures. And they’ll do it surrounded by familiar cellular faces.

“Think of cells as living a social life: They’re not one cell sitting in a petri dish becoming a pancreas; it's a group of cells touching each other with molecular Velcro,” said Ewald. “They're making and breaking contacts with their neighbors, and they're sending signals inside their own cells and to their neighbors based on those contacts.”

Those neighbors could include blood vessels, immune cells, or fibroblasts or other epithelial cells.

“This has been important even in areas of research where there were already a lot of ways to culture and maintain cells,” said Ewald. “But it's been a revolution for rare diseases, and for diseases where it's difficult to get cells out of the body to study in the first place.”

Rare diseases often lack a sufficient market to attract pharmaceutical research. One example is Cushing’s disease, a progressive syndrome in which the body overproduces the stress hormone cortisol. Cushing’s can cause weight gain, hypertension, heart attack and stroke, and can significantly shorten patient’s lives.

“The lack of progress is due to low funding and low interest in these diseases, despite the havoc that they can wreak on patients,” said neurosurgeon Dr. Andrew Little, director of Barrow Neurological Institute’s Pituitary Center. “However, we now have a new model that we can use to study these diseases.”

Cushing’s most commonly stems from a benign tumor that makes the pituitary gland pump out far too much ACTH (adrenocorticotropic hormone). This overstimulates the adrenal glands, causing them to boost cortisol production.

Little performs transsphenoidal surgeries on Cushing’s patients to remove these pituitary tumors. The process entails using an endoscope to access the gland though the nose and sphenoid bone.

“Fortunately, the pituitary gland lies just behind the back of the nose,” said Little. “It dangles down from the brain like a cherry on a stem, so we can sneak underneath the brain through the nose and access the pituitary gland.”

As a side benefit, and with his patent’s consent, Little can send the removed tumors to colleagues like Yana Zavros, director of UA’s Tissue Acquisition and Cellular/Molecular Analysis Shared Resource.

Zavros studies Cushing’s disease by growing three different kinds of organoids: One from the patient's tumor tissue; one from normal human pituitary tissue, for comparison; and one made by nudging peripheral blood cells with growth factors so they turn into pituitary tissue.

Little said the third technique offers unique advantages.

“It's very plentiful, and we can easily obtain that tissue just through a blood draw through a peripheral vein,” he said. “Whereas the other two types require me to perform a brain surgery to obtain.”

Thorne uses organoids to study how gut tissues maintain, repair and defend themselves. The intestine comprises a wide array of stem cells that must identify each other and know when to regenerate tissue, but these signals can go wrong when cancer enters the picture.

“We can interrogate them in all different ways — probe them with different drugs or growth factor treatments — and they recapitulate the way the tissue behaves in the in the body, but they do it in a dish,” said Thorne.

Thorne’s lab focuses on the surface cells of the small and large intestine — a veritable jungle of mucus-secreting goblet cells, hormone-secreting hormone cells, and nutrient-absorbing colonocytes and enterocytes.

“It’s a tissue that's made up of many cells, and organoids recapitulate that heterogeneity,” said Thorne.

Before organoids, cellular research relied largely on cell lines, often derived from cancers.

Some were so durable and prolific they were practically immortal — the most famous and controversial example being the HeLa cells acquired from Henrietta Lacks by Johns Hopkins researcher Dr. George Gey in 1951, prior to current informed consent practices.

Though expedient, such cell lines can be a bit zombie-like: they multiply, but don’t separate into different cell types, and they didn't function like cells in the body.

“And so it was easy to grow them, but they don't represent the growth behaviors of cells in the body, and they don't represent the function of cells in the body,” said Thorne.

Pathologists have had it even worse, relying for their diagnoses on dead tissue biopsied and fixed in formaldehyde.

“So pathology is going to go from a from an endpoint or dead tissue discipline to a living tissue discipline, which I think will be a major paradigm shift for the field of medical pathology,” said Thorne.

Thorne says organoids already play a lead role in testing new drugs and identifying which ones should advance to clinical trials. But he also believes organoids are poised to move from the lab bench to the clinic in the next few decades and advance personalized medicine in the process.

The hope is that researchers could then study both heathy and cancerous tissues collected from specific patients by growing them into organoids, which they would genetically profile and expose to different therapies.

“When you go into the clinic, your tissue will be collected, and it'll be kept alive, and it'll be profiled in a way that helps to direct the clinician for how he treats your own disease,” he said.

Brafman feels less bullish about such doctor-patient interactions with organoids. He sees them remaining a preclinical tool that affects patients indistinctly by providing more effective or cheaper treatments.

Either way, organoids aren’t a panacea; they’re just another tool in the kit, better suited than others to their particular uses.

“So, again, the key word there is ‘better,’” said Brafman. “It's not anywhere close to the same sort of environment as in neurodevelopment, but it's better than 2-D.”

Organoids remain fiddly, though — hard to scale up, decipherable only via state-of-the-art imaging and genomics, and vexingly intricate.

“The more complex you make the model system, the less scalable it is,” said Thorne.

Until research clears that bottleneck, scientist must rely on simpler organoids and studies they can automate.

Meanwhile, some worry fear, ignorance and mounting government red tape will further curtail progress.

Concerns persist, too, that neither patients nor hospitals grasp organoids well enough for the informed consent process to work as it should — especially given assertions that some folks play by different rules.

“Within the law, patients are always protected,” said Thorne. “But some medical institutions are more aggressive at acquiring tissue — under consent — but acquiring tissue and doing interesting things with that tissue.”

Organoids are exciting tools shedding new light on cellular development and disease within 3-D structures mimicking tissues and organs. But only time will tell whether these cellular soufflés rise to their full potential.

“Science is a slow, laborious process,” said Brafman. “That doesn't mean that we shouldn’t fund it or be excited about it; that's just sort of the process.”

“Anybody who wants to donate cells or tissues or organs after they pass away, I would highly encourage them to do it,” he said.