Why brain organoids are changing neuroscience

Human cerebral organoids are becoming increasingly valuable tools for studying aspects of human brain development that are difficult — or impossible — to investigate using traditional experimental models. By recreating key features of the developing brain in a 3D system, these stem cell-derived models are helping researchers explore how diseases emerge, evaluate new therapeutic approaches, and answer biological questions that cannot be addressed using animal models alone.

To learn more about the opportunities and remaining challenges of this rapidly evolving field, DDN spoke with Rodrigo Cristofoletti, Director of the Center for Pharmacometrics and Systems Pharmacology at the University of Florida. In this Q&A, he discusses the promise of cerebral organoids, the role of imaging in advancing these complex 3D models, and the technical innovations that could further accelerate their use in studying neurotropic viral infections and beyond.

Can you describe your research focus?

I would say that, in a nutshell, my research integrates stem cell technology, organoids, microphysiological systems, and quantitative in silico modeling to study human disease in physiologically relevant settings. The goal is to improve our mechanistic understanding of disease itself, while also supporting better translation during drug development, especially in the early stages.

What is the biggest limitation of traditional animal models for understanding brain development?

Animal models are very important — we've learned a lot from them — but I would say the key limitation is related to species-specific biology.

Let's take the brain as an example. We have differences in neurodevelopment, differences in immune responses, and even differences in pathogen tropism. Those differences raise the question of whether animal models can fully reproduce the mechanisms underlying certain diseases, especially brain infections.

So, we may not be able to fully reproduce the effects of infections on the developing human brain. That's why it's quite important to have human-relevant models that can complement ongoing research with animal models.

Cerebral organoids are often described as "mini brains," but that can be misleading. How would you explain what they actually represent?

It's really not fair to say that cerebral organoids are mini brains because that can change the public's perception. Some people may think we have a fully functional miniature brain growing in the lab, and that could even lead to the misconception that these tissues are conscious. That's definitely not the case.

Brain organoids, or cerebral organoids, are stem cell-derived 3D tissues that reproduce some selective features of early human brain development, including cellular diversity and aspects of spatial organization. However, they are not able to fully recreate brain function, and they are certainly not conscious.

When studying neurotropic infections such as Zika virus, what structural or developmental changes only become apparent at the tissue level?

We know that Zika virus likes to infect neural progenitor cells, so much of its pathology is related to its ability to kill those cells. However, that's only part of the story.

When you study Zika infection using a 3D tissue such as a brain organoid, you have the opportunity to answer additional biological questions. For example, is the infection changing the organization of the tissue itself?

When it comes to the brain, especially the cortex, we have a very specialized organization with different layers of neurons — a process called lamination. Only when we have a 3D construct can we really ask whether a disease is affecting that lamination process.

What are the biggest technical challenges when working with cerebral organoids compared to traditional 2D cell cultures?

Since there are many different types of brain organoids, I'll answer with cerebral organoids in mind. Cerebral organoids are what we call unguided brain organoids because we don't force the cells to differentiate into a specific lineage — we let nature work, and then we study how the cells make decisions.

That's a very good model for developmental biology, but because the cells are making decisions in real time, we can expect a lot of batch-to-batch variability. Even within the same batch, one organoid can be different from another.

Another limitation is that brain organoids can grow quite large. They can reach the millimeter range, so you can actually see them without a microscope. That's interesting, but it comes with a caveat. Once they become very large, it's difficult to feed the cells in the core of the organoid. As a result, it's quite common to develop necrotic cores. It's almost like having a pool of dead cells inside the organoid. Those dead cells send signals, and that can disrupt the ability of the in vitro system to reproduce the biology we're trying to study.

How do you preserve organoid integrity during long-term infection studies?

It's not easy. It's very important to be gentle with the organoids. We standardize the handling and try to minimize unnecessary manipulation. We only remove the organoids from the incubator when it's really necessary.

For infection studies, we first co-incubate the organoids with the pathogen for a defined period of time. After that, we wash the wells to remove any remaining pathogen. That way, only pathogens that successfully entered the intracellular compartment remain in the system.

That's important because later we collect the supernatant. If we detect pathogens there, they're coming from a productive infection inside the organoid. Some pathogens are released back into the medium, and that provides an indirect measurement of sustained infection over time.

How have advances in imaging changed the way you design organoid experiments?

In my lab we're mainly interested in infectious diseases and oncology. For both of those areas, one of our major readouts is whether the cells are alive or dead. In the past, we mostly performed live-dead analysis as an endpoint. At the end of an experiment, we'd fix the cells and count how many were alive and how many were dead.

Now we have access to non-toxic dyes that allow us to design longitudinal experiments. We can track the same organoids over time and measure, on a daily basis, how they're responding to a treatment or to a pathogen.

I think technologies like the LICORbio Atlas imager really give us the ability to move beyond looking at a single cell. It's almost like moving from studying one specific tree in a forest to looking at the whole forest.

—Rodrigo Cristofoletti, University of Florida

Another important point is that we're not trying to look at one small portion of an organoid. We want to see the whole organoid, and not only that — we want to see all the organoids in the well so we can generate quantitative measurements. I think technologies like the LICORbio Atlas imager really give us the ability to move beyond looking at a single cell. It's almost like moving from studying one specific tree in a forest to looking at the whole forest.

Have improvements in imaging simply increased confidence in existing data, or have they enabled entirely new scientific questions?

I would say both. Better imaging increases confidence in established measurements while also enabling new questions about spatial relationships.

There are questions that really require us to go down to the cellular level, while others require us to look at the tissue level. Improved imaging helps us do both. It gives us more confidence in the readouts while allowing us to ask questions that simply weren't possible before.

What technical advances would most accelerate organoid-based studies of neurotropic infections: better biological models, better imaging, or better analytical pipelines?

I would say it's really a combination of all three. Brain organoids are great, but they're still avascular. If we can introduce vasculature, we'll be able to better feed the cells in the core of the organoid, which will help address the necrotic core issue.

At the same time, because many labs are moving toward longitudinal studies, we need better imaging technologies that don't expose organoids to excessive phototoxicity. So, we still need improvements in the biological component of the model, but better imaging technology will also improve our analytical pipelines and help us generate more consistent and robust longitudinal studies.