For nearly ten years, the Stanford Brain Organogenesis Program has been redefining how scientists study the human brain. Instead of relying on intact brain tissue from humans or animals, researchers in the program grow three-dimensional brain-like structures in the lab using stem cells. These tiny models, called human neural organoids and assembloids, allow scientists to explore the brain's development and function in entirely new ways.
Launched in 2018 as part of Stanford's Wu Tsai Neurosciences Institute through its Big Ideas in Neuroscience initiative, the program unites experts from neuroscience, chemistry, engineering, and other disciplines. Together, they investigate neural circuits related to pain, genes linked to neurodevelopmental disorders, and new methods for studying brain connectivity.
One challenge has persisted throughout the program's progress: scaling up production. To deeply understand brain development, study developmental disorders, or test potential therapies, researchers need to produce thousands of organoids that are uniform in size and shape. However, these delicate structures tend to stick together, making it difficult to grow large, consistent batches.
A team led by Wu Tsai Neuro affiliates Sergiu Pasca, the Kenneth T. Norris, Jr. Professor of Psychiatry and Behavioral Sciences, and Sarah Heilshorn, the Rickey/Nielsen Professor of Engineering, recently found an unexpectedly simple fix. As reported in Nature Biomedical Engineering, the key to preventing organoids from clumping was xanthan gum, a widely used food additive.
"We can easily make 10,000 of them now," said Pasca, the Bonnie Uytengsu and Family Director of the Stanford Brain Organogenesis Program. In keeping with the program's commitment to making their techniques widely available, they've already shared their approach so others can take advantage of it. "This, as with all of our methods, is open and freely accessible. There are already numerous labs that have implemented this technique."
So few you could name them
That level of productivity was once unimaginable. Around twelve years ago, Pasca had just developed a way to turn stem cells into three-dimensional tissues that would later be known as regionalized neural organoids. At the time, he could only make a handful of them.
"In the early days, I had eight or nine of them, and I named each of them after mythological creatures," Pasca said.
But Pasca's goal was much larger: to uncover how the developing brain can go awry in conditions such as autism or Timothy syndrome, and to explore how drugs might affect that development. "We needed to produce thousands of organoids, and they should all be the same," he said.
He also recognized that success would require a diverse team of specialists. "I thought, 'This is an emerging field and there are a lot of problems we're going to face, and the way we're going to face them and solve them is by implementing innovative technologies,'" Pasca said.
To achieve that vision, Pasca collaborated with Wu Tsai Neuro affiliate Karl Deisseroth, a neuroscientist and bioengineer, assembling an interdisciplinary group that officially launched the Stanford Brain Organogenesis Program with support from the Wu Tsai Neuro Big Ideas in Neuroscience grant.
The nonstick solution
The stickiness problem reared its head soon after. Organoids were fusing together, resulting in smaller numbers of organoids of different shapes and sizes.
"People in the lab would constantly say, 'I made a hundred organoids, but I ended up with twenty,'" Pasca said.
That was both a blessing and a curse. On the one hand, it suggested that researchers could stick two different kinds of organoids together -- say, a tiny cerebellum and spinal cord -- to study the development of more complex brain structures. Indeed, these assembloids are now a key part of Pasca and his colleagues' work.
On the other hand, the team still needed to be able to create large numbers of organoids so they could gather precise data on brain development, screen drugs for growth defects, or carry out any number of other projects at scale.
One possibility would be to grow each organoid in a separate dish, but doing so is often inefficient. Instead, the lab needed something to keep organoids apart while growing them in batches, so Pasca worked with Heilshorn, a Stanford Brain Organogenesis Program collaborator and materials engineer, to try out some options.
The team ultimately looked at 23 different materials with an eye toward making their methods accessible to others.
"We selected materials that were already considered biocompatible and that would be relatively economical and simple to use, so that our methods could be adopted easily by other scientists," Heilshorn said.
To test each one, they first grew organoids in a nutrient-rich liquid for six days, then added one of the test materials. After another 25 days, the team simply counted how many organoids remained.
Even in small amounts, xanthan gum prevented organoids from fusing together, and it did so without any side effects on organoid development. That meant that researchers could keep the organoids separated without biasing their experimental results.
Scaling up at last
To demonstrate the potential of the technique, the team used it to address a real-world issue: Doctors often hesitate to prescribe potentially beneficial drugs to pregnant people and babies because they don't know whether those drugs might harm developing brains. (Although FDA-approved drugs go through extensive testing, ethical concerns mean they are generally not tested on pregnant people or babies.)
To show how organoids address that problem, co-lead author Genta Narazaki, a visiting researcher in Pasca's lab at the time the research was done, first grew 2,400 organoids in batches. Then, Narazaki added one of 298 FDA-approved drugs to each batch to see if any of them might cause growth defects. Working closely with co-lead author Yuki Miura in the Pasca lab, Narazaki showed that several drugs, including one used to treat breast cancer, stunted the growth of the organoids, suggesting they could be harmful to brain development.
That experiment shows that researchers could uncover potential side effects -- and do so very efficiently, Pasca said: "One single experimenter produced thousands of cortical organoids on their own and tested almost 300 drugs."
Pasca and his Stanford Brain Organogenesis Program colleagues are now hoping to use their technique to make progress on a number of neuropsychiatric disorders, such as autism, epilepsy, and schizophrenia. "Addressing those diseases is really important, but unless you scale up, there's no way to make a dent," Pasca said. "That's the goal right now."
