Cell-based assays form an important part of the biological researcher’s repertoire and are used for screening all types of compounds as well as increasing our understanding of biological targets and pathways. As compared to in vitro high-throughput screening assays, cell-based assays are considered more faithful to physiology. Cell-based assays assume no prior knowledge of a direct molecular target. Rather, they focus on the pathway of interest, including regulatory networks and feedback control mechanisms. This approach expands the range of potential targets within the assay and adds functions to the screening criteria for a compound, like an ability to cross cellular membranes and cytotoxicity.
In order to execute a successful cell-based screen, there are a number of practical strategies that can be applied.
Choosing an assay system
Choosing the correct cell culture system is crucial to achieving good results in a screening assay. The screening strategy should create the correct biological context, while also being feasible in terms of availability of reagents and compatibility with automation technology.1
Considerations for a cell-based assay system include:
- Cell type—primary cell, native, engineered cell line, or model organism
- Assay approach—functional assay, reporter-gene assay, phenotypic assay
- Follow-up assay—target identification, validation, secondary assays
- Detection method—fluorescent, luminescent, or spectrophotometric
The best way to sort out the right system from many options is to run a prototype assay, evaluating a full range of experimental conditions. Once initial choices are made for cell type, assay approach, and detection method, the assay can be optimized for other variables including cell density, incubation times, amount of reagent, and concentration of assay components. Stability of reagents and readout signals should also be evaluated in the prototype assay, because scale up to a high-throughput platform may introduce longer times for each step of the assay.
The best way to sort out the right system from many options is to run a prototype assay, evaluating a full range of experimental conditions.
Health of cells
One source of difficulty with cell-based assays is unfamiliarity with the basics of cell culture such as recognizing when cells are healthy versus when they are not. According to Jane Lamerdin, head of research and development at DiscoverX, “A large part of success with cell-based assays is being good with cells. You’re going to get a lot more out of your assays if your cells are taken care of properly.”
There are a couple of obvious signs of poor health in cell cultures, Lamerdin explains. One is the color of the media. If it’s yellow or very pink, that’s a sign the cells aren’t doing well. A proper cell split ratio is also important for cell health and optimal performance in cell-based assays. When engineered cells (such as U-2OS) are seeded too sparse or too dense, they generally will not perform well in cell-based assays, as expression of the desired protein is not optimal under these conditions. Ideal seeding and plating densities maintain the cells in log phase growth and do not exceed a maximum cell confluency of 85-90%.
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Correct regulation of temperature and carbon dioxide is also critical for maintaining healthy cell cultures. In one example of an assay developed to study regulation of heat shock response, researchers chose a HeLa cell line stably transfected with a heat shock protein promoter fused to a firefly luciferase reporter gene.2,3 Difficulties arose during the experiment when unexpectedly high background signal was observed seemingly at random in some plates. The cause of the high background turned out to be variations in the amount of time plates spent at room temperature before being placed in the 37°C incubator. Researchers corrected the problem by minimizing the amount of time at room temperature. This case study illustrates the sensitivity of cell cultures to variations in temperature.
The same experiment also showed that cells were very sensitive to CO2 concentrations. The concentration of CO2 was varied from 4.5 to 6%. For concentrations above 6%, there was no measurable luciferase activity in the cells.
Contamination
Contamination is a common and costly problem in cell-based assays. Contaminants can enter a culture through media, reagents, the cells themselves, or unknown routes. There are three primary types of cell culture contaminants: chemical, biological, and cell-line cross contamination. Biological contamination can take the form of bacteria, molds, yeasts, viruses, protozoa, or mycoplasma.
A clean laboratory and good aseptic technique are the primary and most important methods for preventing contamination in cell cultures. Beyond that, sourcing cell lines, serums, and reagents makes a difference. Purchasing quality materials from reputable vendors can minimize contamination issues.
For researchers who are not confident building a cell-based assay screen from scratch, there are many off-the-shelf assay systems available. Enzo, for example, offers a range of cell-based screening products and kits. A commercial assay kit takes the guesswork out of developing a system and protocol, and many include proprietary technologies like unique dyes and reagents that offer specific advantages for your study. When using one of its kits, Enzo’s product manager, Vanessa Fonte, says the best advice she can offer is, “Follow the instructions in our protocol. It is provided to be very straightforward and accurate. Our protocols have been validated to provide optimal results.”
Powerful new applications
With the advent of gene-editing systems like CRISPR/Cas9, cell-based screening assays have never been more relevant. CRISPR/Cas9 is rapidly transforming the field of biotechnology as a gene-editing tool. Its usefulness for functional genomic screening is just starting to be realized.
The Cas9 protein, which is used in gene editing to target and cleave a DNA sequence, can also target functionality to a specific genomic position.
Illustration of a typical cell-based assay workflow for pooled genomic screening. Image courtesy of Horizon Discovery.
“If you think about what you’re trying to do with functional genomics, CRISPR is a really powerful new tool for this. You can do small scale, or really high throughput,” says Benedict Cross, team leader for discovery screening at Horizon Discovery. CRISPR screening is one of the services the company offers, and Cross says interest in the technology has exploded. “There are lots of really innovative kinds of screens being conducted. People start to engage their imaginations.”
In a typical pooled screen, Cas9 is expressed in a target cell line or encoded in a viral vector and expressed in cultured cells. The phenotype of interest is screened in a cell-based assay, and then deep sequencing of PCR-amplified genomic DNA reveals sequences that are enriched or depleted after selection.4
A combination of pooled CRISPR screening with single-cell RNA sequencing, known as CRISPR droplet sequencing (CROP-seq), enables high-throughput functional assay of regulatory mechanisms in heterogenous cell populations.5 The advantage of this method is being able to rapidly sequence a large library with single-cell transcriptome resolution.
Conclusion
Cell-based screening is a powerful method that continues to find new applications and new technological innovations. Successful cell-based screening relies on healthy mammalian cell cultures and sterile, high-quality materials. Screening results may be adversely affected by poor quality cell cultures, variations in temperature and CO2, and contamination by microorganisms, chemicals, or other cell lines. With new technologies such as CRISPR/Cas9 gene editing spurring advanced applications for cell-based assays, the effort required to master cell-based screening is worthwhile.
References
1. An, W, Tolliday, N, “Introduction: cell-based assays for high-throughput screening,” Methods Mol Biol, 486:1-12, 2009. [PubMed ID: 19347612]
2. Maddox, C, et al., “Adapting cell-based assays to the high throughput screening platform: problems encountered and lessons learned,” JALA Charlottesv Va, 13(3):168-173, 2008. [PubMed ID: 19492073]
3. Westerheide, S, et al., “Celastrols as inducers of the heat shock response and cytoprotection,” J Biol Chem, 279(53):56053-60, 2004. [PubMed ID: 15509580]
4. Agrotis, A, Ketteler, R, “A new age in functional genomics using CRISPR/Cas9 in arrayed library screening,” Front Genet, 6:300, 2015. [PubMed ID: 26442115]
5. Datlinger, P, et al., “Pooled CRISPR screening with single-cell transcriptome readout,” Nat Methods, 14(3):297-301, 2017. [PubMed ID: 28099430]
Image: Starvation induces an increase in green fluorescence intensity as demonstrated by the presence of punctate cytoplasmic structures (top) compared to control cells (bottom). Nuclei counterstained with Hoechst dye (blue). Image courtesy of Enzo Life Sciences.