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High-content Cellular Imaging Assays Designed to Enable Discovery and Development of Safe, Efficacious Therapeutics

Scientist on November 3, 2022
in Tech Snapshot

Tech Snapshot® captures today’s cutting-edge tools and technologies that will help drive drug discovery tomorrow. This installment was written by PhenoVista Biosciences.

PhenoVista was founded in 2014 with a mission to leverage the power of high content imaging to enable deep interrogation of physiologically relevant cell models. To date, we have fulfilled this mission by providing bespoke assay services to our clients whose highly complex assays require outside-of-the-box thinking to deliver impactful results. However, throughout this journey we have noted that more mainstream, out-of-the-box assays could significantly benefit from our outside-the-box approach and have designed a suite of Ready-2-Go assay services accordingly that will help extend our original mission further. In general, we focused on three areas of improvement, which we will highlight in this article: the dyes/stains used, the analysis performed and the cell model.

Ready-2-Go Cell Health Assay Service

Evaluating the impact of drug candidates on cell proliferation and viability is essential to the success of any drug-discovery or drug-development campaign. Depending on the disease of interest, drug developers may seek to enhance cell repopulation (e.g., degenerative diseases) or inhibit it (e.g., cancers). Additionally, this is often the first step in characterizing new therapeutics, as assessment of candidate drugs for potential toxicity is critical before advancing into clinical trials.

Researchers commonly opt for a straightforward measurement of cell viability (i.e., live vs. dead); however, cell health is far more nuanced. In addition to cell survival and proliferation, the mechanism(s) of cell death (i.e., apoptosis vs. necrosis) can also vary depending on therapeutic activity. That said, apoptotic cells may classify as viable in a standard live-dead assay, although they are dying.

A multiplexed imaging approach can provide a more comprehensive view of cell health yet can be challenging to perform. Particularly, as cells senesce in vitro, they often detach from the growth surface, rendering accurate measurement of their viability and proliferation using standard antibody-based detection difficult or impossible due to potential cell loss exacerbated by the required pipetting and wash steps. Considering these challenges, PhenoVista has developed Ready-2-Go Cell Health Assay Service, enabling robust, accurate, simultaneous quantitation of cell viability, apoptosis and proliferation using a homogeneous assay workflow with dye-based detection. This approach maximizes data quality by minimizing pipetting steps and eliminating wash steps that could exacerbate loss of dead or dying cells.

How it Works

Figure 1: Ready-2-Go Cell Health assay. Cells are seeded into 384-well plates and incubated in the presence of compound for 4, 24, and 48 hours, at which point they are fixed and stained in preparation for imaging and analysis. In the images below, A549 cells were treated with Paclitaxel and staurosporine for 24 hours. Nuclei were stained with Hoechst (blue), apoptotic cells with CellEvent (green), and dead cells with DRAQ7 (red). This palette affords gating of 4 populations of cells - alive, alive & apoptotic, dead & apoptotic, and dead. Proliferation is determined by the comparing nuclear counts at time zero and the endpoint.

Ready-2-Go (R2G) Mitochondrial Health Assay Service

Mitochondria play a fundamental role in maintaining cellular health by converting sugars and oxygen into energy (ATP), using a process that relies on their membrane potential. Perturbations in mitochondrial function are detrimental to cell health and, therefore, are critical to monitor in any drug development campaign, whether desirable (e.g., in cancer treatment) or not (e.g., off-target toxicity). Additionally, mitochondrial health is a valuable early indicator of drug toxicity which is, again, essential to address in order to ensure clinical success.

Accurate quantitation of therapeutic treatment impact upon mitochondrial health requires a high-resolution imaging approach coupled with single-cell level analysis. PhenoVista’s Ready-2-Go Mitochondrial Health Assay Service screens for changes in mitochondrial membrane potential using a fluorescent dye that accumulates within the mitochondria at a rate that depends upon its membrane potential. We measure the fluorescence intensity of mitochondria from images at the single-cell level and then report out the quantitative results at the population-level.

Additionally, we have integrated measurements of cell count (via nuclear staining) as well as necrosis (via a dead cell dye) to deliver a more comprehensive picture of how drug treatment impacts overall cell health. Our Ready-2-Go Mitochondrial Health Assay Service enables us to assess the effects of your drug candidates as a continuum of cell health and to better understand the variability of the response.

How it Works

Figure 2: Ready-2-Go MitoHealth assay service. The representative images of A549 cells shown above were captured following 24 hours of treatment with vehicle (left image), 15 μM Rotenone (middle), or 50 μM Rotenone (right). Mitochondrial staining intensity (shown in green) is indicative of healthy mitochondria and is highest under vehicle control and decreases in a dose-dependent manner upon rotenone treatment, as expected and similar to what we observe for U-2 OS and HEP G2 cells under similar conditions.

Ready-2-Go Lung Fibrosis Assays

Fibrosis is a pathological state that arises from abnormal tissue repair, whereby connective tissue replaces healthy issue to an extent that disrupts normal functioning of the organ. Accumulation of proteins in the extracellular matrix, which results from prolonged activation of myofibroblasts following diverse types of severe or repetitive tissue damage, is a hallmark of this disease. Prolonged myofibroblast activation induces the formation of alpha smooth muscle actin (α-SMA)-containing stress fibers, a common marker in fibrosis assays including PhenoVista’s Ready-2-Go Lung Fibrosis Assay Service.

Most current fibrosis assays are challenging to integrate into the initial stages of drug discovery, where higher throughput approaches may offer limited biological relevance, yet the more complex, relevant models are not easily scalable. Specifically, simpler models of fibrosis often fail because standard cell-culture procedures tend to induce fibrosis-like phenotypes, making it difficult to distinguish between healthy and diseased states. In contrast, while 3D models of fibrosis can distinguish the two phenotypes, they are not amenable to high throughput screening, which limits the potential and efficiency of drug-candidate identification.

Keeping these points in mind, PhenoVista had developed a Ready-2-Go Lung Fibrosis Assay Service that uses primary, normal human fibroblasts (NHLF) cells cultured using a protocol that limits basal induction of fibrosis (often observed with routine, cell-culture procedures) to generate a physiologically relevant, 2D model of fibrosis. This not only allows for enhanced control of fibrotic induction, but also affords a format amenable to higher-throughput therapeutics profiling.

How it Works

Figure 3. Ready-2-Go Lung Fibrosis assay service. The representative images of NHLF cells shown above were taken following 48-hour exposure to either vehicle (PBS) (left), TGF-β (middle), or TGF-β + inhibitor ALK5i (a.k.a. Galunisertib) (right). The images show a visible increase in the levels of actin filaments and α-SMA-containing fibers with TGF-β treatment, a phenotype not observed when cells were pre-treated with the inhibitor ALK5i. This observation was quantified by measuring the total phalloidin intensity at different concentrations of TGF-β, with and without ALK5i inhibitor present, and is shown on the graph on the right.

We have extended our mission to reduce barriers between the use of physiologically relevant cell models and high-content imaging with the launch of our Ready-2-Go assay services. We hope to enable better translation of drugs to the clinic using these services, with the ultimate aim of improving the safety and efficacy of drug treatments. If you would like to speak with us, please contact us at bd@phenovista.com

assay services cell models drug discovery