What is Cell Painting? 

Microscopy images are an incredibly rich source of data, and this idea has led to the development of image-based profiling. The Cell Painting assay, first described in 2013 by Bray et al and Gustafsdottir et al represents a simple and powerful technique that has been widely adopted in the field to enable image-based profiling by researchers across academia and industry. 

Cell Painting relies on the systematic use of fluorescent dyes to stain organelles and cellular compartments  in a standardized manner, allowing for phenotypic profiles to be generated for each condition. Those phenotypic profiles can then be compared to profiles from other compounds or treatments, and similarities and differences used to assess similarities and differences. 

Traditional high content assays like nuclear translocation or neurite outgrowth have focused on a specific endpoint (e.g. nuclear:cytosolic intensity ratio), and a common analogy is that they are similar to trout fishing in which you use a very specific bait to catch a certain type of fish, like a pink Powerbait. Cell Painting can be thought of as more fishing with dynamite, where you’re capturing all the fish in a given area in a fairly target agnostic manner. 

How is Cell Painting done? 

Sample Prep and Imaging

Like many other imaging-based assays, cells of interest are seeded into multi-well imaging plates and allowed to grow for a number of days. Next cells are exposed to chemical or genetic perturbations that may include small molecule libraries, RNAi or CRISPR/Cas9 targets for a certain period of time (typically between 6-48 hours). Following this exposure period, cells are fixed and then stained with markers listed below: 

• Hoechst (DNA stain, labels nuclei)

• Concanavalin A (ER stain, labels ER structure)

• SYTO14 (RNA stain, labels cytoplasmic RNA and nucleoli)

• Phalloidin (F-actin stain, labels cytoskeleton)

• Wheat Germ Agglutinin (plasma membrane stain, labels Golgi and plasma membrane)

• MitoTracker (mitochondrial stain, labels mitochondria)

Following fixation and staining, the cells can then be visualized using fluorescence microscopy platforms. This allows scientists to detect the different dyes within the cells and to determine the specific locations of the labeled cell components, sensing changes to the distribution or intensity of the dyes (Figure 1). These images are then analyzed to quantify the amount and distribution of the labeled components within the cells.

Figure 1: Examples of staining results for untreated (A) and treated (B and C) samples, stained with common Cell Painting dyes. Figure from Gustafsdottir SM et al 2013, used under Creative Commons License.

Feature Extraction and Analysis

Following image acquisition, the cells, nuclei and organelles are detected and segmented using image analysis software and measurements are extracted. These measures quantify the amount and distribution of each of the labeled components within the cells. Following this, measurements are then viewed as aggregated or single cell data and turned into what is called a phenotypic profile. Then, scientists can compare the profiles to known compounds, and decisions can be made about whether to further study specific compounds, pathways or targets. 

Cell Painting in the Drug Development Process

Cell Painting can be utilized across the drug development process, from being used as the basis of phenotypic screening to identifying potential targets through derisking compounds  for safety assessment and toxicity testing. 

Phenotypic Screening

Cell Painting can be used in the initial phases of compound library screening, by allowing scientists to identify specific changes in cell morphology that are associated with specific diseases. Following feature extraction and dimensionality reduction, the assay can cluster hits into groups that show biologically similar effects. These profiles can then be compared to reference compounds and/or healthy cells to determine if the disease state was overcome or restored to the healthy phenotype. 

Mechanism of Action (MOA) Assessments

Following the identification of potential drug candidates, Cell Painting can be used to elucidate the compound’s mechanism of action. By comparing the phenotypic cell profile to those with known MOA, scientists can better understand how the compound is interacting with cells. Additionally, by comparing candidate profiles to those of reference compounds, potential off-target activation can be identified for later assessment. 

Safety and Toxicity Testing

A growing area of interest in application of Cell Painting is in the area of safety - specifically in toxicity testing. As mentioned previously, potential off-target activation can be assessed utilizing Cell Painting methods. Additionally, phenotypic changes in cells can be compared to a compound database including those with known toxic effects, eg. in the area of hepatotoxicity. For example, if exposure to a potential candidate generates a profile similar to that of a known hepatotoxic compound, it is highly likely that the candidate compound will exhibit similar effects.