Despite a long history, research with cell-based assays is on the rise. The articles on PubMed from 1998 to 2017 that turned up from a search of “cell-based assay” increased by almost 20 times in that decade. Moreover, advances in technology continually improve cell-based assays (CBAs).
At the pharmaceutical company H. Lundbeck, neurobiologist Gunnar Dietz and his colleagues developed a CBA to study how a form of nerve growth factor (NGF) binds to the protein sortilin. Binding of sortilin to the pro form of NGF (NGFpro) may play a role in nervous system diseases, including Alzheimer’s and Parkinson’s disease, spinal cord injury, and many others.
“The development of the cell-based assay was the critical bottleneck of a large drug-discovery campaign—for both small molecular entities and blocking antibodies—that was initiated to find entities that block the Sortilin-NGFpro domain interaction,” Dietz explains. Initial compound hits were identified by high-throughput screen using a cell-free binding assay. Structures identified in such a primary screening step may have very different binding properties in a biologic context. A CBA serves to narrow down and optimize the number of positive compounds to be tested in costly whole-organism disease models.
Several earlier attempts to develop a cellular assay based on sortilin-NGFpro interaction had failed. Eventually, the team created a CBA that can test compounds or antibodies in 96-well plates. Dietz calls it “a medium-throughput format to validate initial hits,” and it might be possible to push it to a 384-well format. He adds, “The image collection and data acquisition are fully automated using an array scanner, which not only saves time, but also renders more objective readouts.”
It’s rarely easy to develop a new CBA.
It’s rarely easy to develop a new CBA. The main challenges are generating an assay with a reproducible readout with a high signal-to-noise ratio, which is necessary to achieve sufficient selectivity (few false-positives) and sensitivity (few false negatives), plus high stability. When asked about additional challenges in developing this sortilin CBA, Dietz says, “We had to be careful to weed out artefacts that a fluorescence-based assay is prone to.” He points out that intelligent positive and negative controls in each assay are a must in that regard. In the end, Dietz says, that a CBA should “ideally provide a mechanistic readout that is also disease-relevant.” Moreover, the reagents used in each assay should not be forbiddingly expensive: If each reaction consumes US$10, it would not be suitable to test a million compounds in triplicate.
That collection of challenges and objectives reveals some of the complexity of developing an accurate, reliable, and useful CBA.
Driving drug discovery
Researchers create a variety of approaches to drug discovery with CBAs. The assay developed by Dietz and his colleagues provides one example.
From Charles River Laboratories, director of biology Omar Aziz says, “In its primary function, cellular assays enable us to assess the efficacy of compounds in a cellular environment, which is crucial to understanding compound behaviors in a biological system.” He adds, “The other distinct advantage of cellular assays is that they also allow us to align our readouts with a translatable biomarker—without this it becomes very challenging to determine the in vitro to in vivo correlation for efficacious dose predictions.”
The in vitro to in vivo correlation plays a crucial role in developing a drug. Scientists need this, Aziz explains, to take “an informed approach to study dosing outside of running the actual efficacy studies, which are generally time consuming and expensive to run and repeat if necessary.”
Image: High content imaging of αSMA expression in bronchial fibroblasts. Image courtesy of Charles River.
Although the advantages of CBAs are clear, hurdles can be just as apparent. “The main challenges of running cell-based assays are to identify and access the most appropriate cell type for the study,” Aziz says. “In an ideal world, this would be human primary cells, however, in most cases this simply is not possible.” Instead, scientists often rely on cell lines—looking for one, he says, “that has the required properties and target expression needed to resemble the in vivo system.” But that always leaves the problem of how the in vivo model differs from a real-world application in humans.
Biocompare’s Assay Kits Search Tool
Find, compare and review assays
from different suppliers Search
In many cases, addressing the challenges of using CBAs in drug discovery can be eased with technology, such as flow cytometry. This technology “is a powerful cell-based approach that enables multi-parameter analysis and sorting of single cells,” says Matthew Miceli, global product manager for research reagents at BD. “This analysis can help researchers study and discover biomarkers and pathways related to disease and identify new drug targets.”
Flow cytometry can be applied to drug research in many ways. For one thing, scientists can use flow cytometry to look for how a compound interacts with various types of cells. This technology, Miceli says, can also be used for “cell phenotyping for the study of distinct cell populations, monitoring cell cycle and apoptosis, and quantification of proteins with bead-based assays.”
Although flow cytometry offers many advantages for CBA-based drug discovery, including high throughput, selecting the optimal reagents for multi-parameter flow is one key to running a successful experiment. “As multi-parameter panels become more complex, it is critical that researchers select the fluorescent reagents that minimize spectral overlap and compensation and effectively resolve rare cell populations,” Miceli notes. “In addition, when studies are performed across multiple sites, standardization of instruments and experiments are key to obtaining reproducible data, a challenge that labs can overcome by selecting the right instruments and instituting proper protocols.”
Combining skill sets
NeuroSGC is developing cell-based assays for neurological diseases, such as Parkinson’s disease and ALS,” says Thomas Durcan, group leader of the iPSC (induced pluripotent stem cell) platform at the Montreal Neurological Institute (MNI) at McGill University. “Using the Open Science principles, NeuroSGC will share reagents, assays, and results with the research community, in order to spark further discovery and accelerate new drug discovery programs.”
In describing the hopes of this partnership, Durcan says that one goal “is to use iPSCs to understand the molecular basis of disease and identify drug treatments that can ameliorate key disease characteristics in cell-based assays.” For example, he says, “The iPSC-derived cells used in the high-throughput, small-molecule screens represent the neuronal cell types that are affected in the disease of interest.”
Like other applications of CBAs, iPSC-based approaches face hurdles. “Using human iPSC-based assays to mimic disease-relevant phenotypes in vitro poses unique challenges,” Wolfgang Reintsch of the MNI iPSC platform explains. “In many cases, it requires culturing cells over weeks to produce the neural cell type of interest.” Sometimes, three-dimensional culturing of more than one type of cell is also required.
Expanding CBAs to new levels, such as 3D culturing of multiple cell types, requires advances in methodology and analysis. Nonetheless, moving CBAs closer to in vivo conditions also increases the value of the results from any drug testing. Consequently, CBAs can enhance the productivity of drug discovery and, potentially, lower the cost. That will benefit healthcare around the world.