Developing Homogenous 3D Neural Cultures for High Throughput Screening

Drug use disorders are increasing worldwide, with few individuals receiving treatment for these conditions.¹ In particular, abuse of prescription opioids and the rise in popularity of the synthetic opioid fentanyl has led to an opioid crisis in North America.² 

To battle this epidemic, the National Institutes of Health (NIH) launched the Helping to End Addiction Long-term (HEAL) Initiative in 2018. As part of this effort, scientists at the NIH National Center for Advancing Translational Sciences (NCATS) led by Marc Ferrer and Emily Lee developed a new way to screen for opioid use disorder (OUD) treatments, which they described in a recent Communications Biology paper.³ “We were looking for [3D] models that we could repurpose for screening compounds to target this initiative,” said Lee. “We realized quickly that the existing models were not adaptable for high throughput screening.” 

To quickly test tens of thousands of compounds, the researchers needed to measure hundreds of wells in minutes with a plate reader. They found that brain organoids, which grow from stem or progenitor cell populations and differentiate into complex clusters that recapitulate in vivo morphology, were too heterogenous and took too long to mature for use in their high throughput screens. 

As an alternative, Lee and Ferrer’s team came up with an innovative way to make brain region-specific spheroids suitable for their needs. Spheroids are simple 3D cell clusters that mature in a matter of weeks. Previously, researchers developed neural spheroids similarly to how organoids are made—by allowing pluripotent cells to differentiate within wells, which creates heterogenous populations. The NCATS team instead formed prefrontal cortex (PFC)-like and ventral tegmental area (VTA)-like spheroids by mixing together already differentiated neural cells at ratios that mimic each brain region’s in vivo composition. 

“One of the interesting and different things that they did was aggregating a discrete number of cells and defining that [population] from the get go,” said Madeline Andrews, a neuroscientist at Arizona State University who was not involved in this study. “It demonstrates a new and interesting approach to using stem cell-derived populations to get a slightly more mature kind of cell type faster.”

The researchers confirmed that their starting ratios stayed consistent as the spheroids matured. They developed a plate reader assay that used a fluorescent dye to track intracellular calcium oscillations, which indicate neuronal activity.⁴ To model OUD, the team grew their spheroids in 384-well plates and exposed them to an opioid receptor agonist over 10 days, which mimicked neuronal stimulation from drug use. The treatment lowered calcium activity in the PFC-like spheroids, which was reversed upon application of a drug used clinically to treat opioid overdoses. In the future, they could expose their spheroids to a collection of chemicals called a compound library to identify additional therapies for OUD.

The brain region-specific spheroid models also proved promising for studying neurodegenerative diseases. Lee and Ferrer’s team assembled PFC-like spheroids with GABAergic neurons that contained an Alzheimer’s disease (AD) risk allele. These spheroids exhibited defective calcium activity, which was reversed when treated with compounds used to improve cognition in patients with AD, including the clinically-approved drugs Memantine and Donepezil. “We’re currently working on expanding to additional diseases, looking at things like epilepsy and amyotrophic lateral sclerosis,” Lee said. 

The researchers also plan to automate the manual steps in the spheroid maturation process and develop a high throughput screening method for testing assembloids made from their spheroids, as the VTA and PFC regions interact. 

“I think [this model] is going to expedite discovery and also the understanding of what these therapeutics can actually do,” said Andrews. “Anytime we can push the timeline a little bit faster, it helps science proceed more quickly.”

References

1. UNODC. World drug report 2023. Accessed January 18, 2024. https://www.unodc.org/unodc/en/data-and-analysis/world-drug-report-2023.html
2. Ciccarone D. The rise of illicit fentanyls, stimulants and the fourth wave of the opioid overdose crisis. Curr Opin Psychiatry. 2021;34(4):344.
3. Strong CE, et al. Functional brain region-specific neural spheroids for modeling neurological diseases and therapeutics screening. Commun Biol. 2023;6(1):1-18.
4. Ali F, Kwan AC. Interpreting in vivo calcium signals from neuronal cell bodies, axons, and dendrites: a review. Neurophotonics. 2019;7(1):011402.