Contract Pharma - Structure vs. Function in Cerebral Organoids: How and Why to Study Both

Authors: David Ferrick, Ph.D., Chief Scientific Officer of Axion BioSystems

Contract Pharma, 2022

Cerebral organoids offer highly relevant, three-dimensional in vitro models that allow scientists to replicate key attributes of the human brain without the use of animals. Neurons and glial cells are notoriously difficult to culture, and in traditional cases, their morphology differs widely from neurons in vivo . As scientists aim to develop organoids that more accurately reflect the pathophysiology of neurological diseases, they must monitor both the function and the structure of the cells in cerebral organoids. Researchers can do this using a combination of live-cell imaging and bioelectronic assays. 

Scientists derive cerebral organoids from induced pluripotent stem cells (iPSCs), which can be made from adult skin cells. Essentially, researchers can replicate a person’s physiology to study neurological diseases such as epilepsy, ALS, autism spectrum disorder, and Parkinson's disease. 

Because cerebral organoids are so diverse in structure and function, many researchers want to examine both. Neurons and their supporting cells work in networks to transmit information, making connections called synapses with multiple cells to create an intricate web of activity, which is hard to replicate in a dish. Furthermore, neurons are plastic, meaning they can form and remove synapses as they mature to create more efficient networks. Because cerebral organoids can be grown in dishes for weeks or longer, scientists are able to observe how neural structure and activity change during development or in response to genetic mutations, toxin exposures, or treatment candidates. Scientists can simultaneously study how cell morphology changes and can monitor electrical activity using a combination of live-cell imaging and a bioelectronic assay, respectively.  

The Benefits and Limitations of Traditional Live-Cell Imaging Techniques

Live-cell imaging involves using microscopy to observe and quantify events in specimens ranging from organelles to cellular networks. Researchers can use this technique to study morphology within a cerebral organoid, providing information about cells’ maturity, identity, and plasticity in real-time.

However, live-cell imaging techniques are not immune to the fundamental challenges of studying any cell culture. Cultured neurons are famously fickle, and they are greatly impacted by exogenous signals. Since researchers' most essential findings might only come after observing neurons for days or weeks, the cells' in vitro environment must reflect human physiology as closely as possible.

In addition, the imaging technique itself can alter plasticity and cell health. Typically, the method involves visualizing proteins using fluorescent tags, which can disturb cell behavior and health. The light from the microscope also causes the dyes to react with oxygen, producing free radicals that can damage and kill cells. As a result, cells can only be studied for a short time before they die.

To overcome these limitations, researchers can visualize cells directly in their incubator using microscopy techniques that do not require fluorescent labels. Today, researchers can find automated brightfield microscopes able to visualize entire surfaces of cell culture vessels and operate inside standard CO₂ incubators, biological safety cabinets, or on a benchtop.

Visualizing neurons only tells part of the story, however. The health of the nervous system is dictated not only by neuron morphology but also by activity. To address questions related to organoid function, scientists can use a bioelectronic assay.

Functional Testing of Cerebral Organoids with a Bioelectronic Assay

Bioelectronic assays capture changing activity during neural development using noninvasive multielectrode sensor arrays (MEAs) embedded in the bottom of a multiwell plate. Because these assays do not rely on visualizing specimens, they do not require any dyes, probes, or labels that could interfere with cells' natural behavior. Instead, the MEAs use tiny electrode-based sensors to record the timing and spatial distribution of the cells’ electrical activity in real-time, over days to weeks. 

Researchers are increasingly using this technique to better characterize neurological diseases and disorders and identify potential treatments. For example, Evangelos Kiskinis, Ph.D., of Northwestern University used cerebral organoids to model KCNQ2-associated epilepsy. Yang Yang, Ph.D., of Purdue University predicted effective treatments for inherited erythromelalgia. Finally, Alysson Muotri, Ph.D., of the University of California San Diego recently modeled a form of autism spectrum disorder called Pitt-Hopkins syndrome. 

Remotely Monitoring Cell Viability

Typically, obtaining real-time data requires significant hands-on time, but some bioelectronic assays come with remote monitoring capabilities that allow users to collect more data without the extra work, freeing them up to do other things. This capability could eliminate the need for scientists who work with hazardous reagents or pathogenic viruses to wear protective gear every time they need to check on their experiments. It could also benefit those who need to work remotely due to pandemic restrictions, travel, or personal activity.
 
Scientists are limited in what they can accomplish in a day, in part because much of the work must be done in person. For scientists seeking to monitor cells in real-time, remote monitoring capabilities will enable them to reduce hands-on time and dedicate more time to other research projects. 

Bioelectronic Assays and Live-Cell Imaging Work Together

Measuring the structure and function of the same cells over time was once technically challenging. But bioelectronic assays can now provide critical functional data about cellular dynamics in real-time, in the context of spatial and morphological changes, by employing imagers that fit in an incubator to continuously track and monitor the cells. Together, these new techniques allow researchers to explore unique aspects of their in vitro models and collect data that could help researchers understand the dynamic activity in the brain better than ever before.