Stanford University scientists are growing thousands of miniature human brains using common food additives

Launched in 2018 as part of Stanford University’s Wu Tsai Neuroscience Institute through the Big Ideas in Neuroscience initiative, the program brings together experts from neuroscience, chemistry, engineering and other disciplines. Together they investigate neural circuits related to pain, genes associated with neurodevelopmental disorders, and new ways to study brain connectivity.

One challenge has persisted throughout the program’s progress: increasing production. To deeply understand brain development, study developmental disorders, or test potential treatments, researchers need to produce thousands of organelles that are uniform in size and shape. However, these delicate structures tend to stick together, making it difficult to grow large, consistent groups.

A team led by Wu Cai Neuro’s Sergio Basca and Professor Kenneth T. Norris Jr. School of Psychiatry and Behavioral Sciences, and Sarah Heilshorn, the Reiki/Nielsen Professor of Engineering, recently came up with an unexpectedly simple solution. As stated in Nature of biomedical engineeringThe key to preventing the organoids from clumping was xanthan gum, a widely used food additive.

“We could easily make 10,000 of them now,” said Pasca, Bonnie Uyetengso, director of the Organogenesis Program at Stanford University. In keeping with the program’s commitment to making their technology widely available, they have already shared their approach so that others can benefit from it. “This, as with all our methods, is open and freely accessible. There are already many laboratories that have applied this technique.”

Very few people you can name

This level of productivity was once unimaginable. About a dozen years ago, Baska had just developed a way to turn stem cells into 3D tissues that would later become known as regional neural organoids. At that time, he could only make a handful of them.

“In the early days, I had eight or nine of them, and I named them all after mythical creatures,” Paska said.

But Paska’s goal was much bigger than that: to uncover how the developing brain can go awry in conditions like autism or Timothy syndrome, and to explore how drugs affect that development. “We needed to produce thousands of organisms, and they all had to be identical,” he said.

He also realized that success requires a diverse team of professionals. “I thought this was an emerging field and there were a lot of problems we were going to face, and the way we were going to confront them and solve them was by applying innovative technologies,” Paska said.

To realize this vision, Paska collaborated with Wu Tsai Neuro affiliate neuroscientist and bioengineer Carl Deisseroth, in assembling an interdisciplinary group that officially launched the Stanford Brain Organogenesis Program with support from the Wu Tsai Neuro Big Ideas Grant in Neuroscience.

Non-stick solution

The stickiness problem reared its head soon after. The organelles were fusing together, giving rise to smaller numbers of organelles of different shapes and sizes.

“People in the lab kept saying, ‘I made a hundred organoids, but I ended up with 20,’” Paska said.

That was a blessing and a curse at the same time. On the one hand, it suggested that researchers could glue two different types of organelles together—for example, a small cerebellum and a spinal cord—to study the development of more complex brain structures. In fact, these gatherings are now a central part of the work of Paska and his colleagues.

On the other hand, the team still needs to be able to create large numbers of organoids so they can collect accurate data on brain development, screen drugs for developmental defects, or carry out any number of other large-scale projects.

One possibility is to grow each organoid in a separate dish, but doing so is often ineffective. Instead, the lab needed something to keep the organoids separate from each other while growing them in batches, so Paska worked with Helshorn, a materials engineer and collaborator in Stanford’s organogenesis program, to try out some options.

The team ultimately looked at 23 different materials with the goal of making their methods accessible to others.

“We chose materials that are truly biocompatible and that will be relatively economical and easy to use, so that other scientists can easily adopt our methods,” Heilshorn said.

To test each one, they first cultured the organoids in a nutrient-rich liquid for six days, then added one of the test substances. After another 25 days, the team simply counted the number of organelles remaining.

Even in small amounts, xanthan gum prevented the organelles from fusing together, and it did so without any side effects on the development of the organelles. This means that researchers can keep the organelles separate without biasing their experimental results.

Finally expanding

To demonstrate the potential of this technology, the team used it to address a real problem: doctors are often reluctant to prescribe medications that might be beneficial to pregnant women and children because they don’t know whether these medications might harm developing brains. (Although FDA-approved drugs undergo extensive testing, ethical concerns mean they are generally not tested on pregnant women or children.)

To show how organoids address this problem, co-lead author Genta Narazaki, a visiting researcher in Paska’s lab at the time of the research, grew 2,400 organoids in batches. Next, Narazaki added one of 298 FDA-approved drugs to each batch to see if any might cause developmental defects. Working closely with co-lead author Yuki Miura in Baska’s lab, Narazaki showed that several drugs, including one used to treat breast cancer, inhibited the growth of the organoids, suggesting that they may be harmful to brain development.

This experiment shows that researchers can detect potential side effects — and do so very efficiently, Paska said: “One experimenter produced thousands of cortical organoids on his own and tested nearly 300 drugs.”

Paska and his colleagues in the Organogenesis Program at Stanford now hope to use their method to make progress in a number of neuropsychiatric disorders, such as autism, epilepsy and schizophrenia. “Tackling these diseases is really important, but unless they scale up, there’s no way to make a difference,” Paska said. “That’s the goal now.”