Cell-based assays have become fundamental and irreplaceable tools in drug discovery and life science, providing increasingly accurate models for normal and pathological human biological processes. However, these tools come with limitations. As new cutting-edge assay formats that use only a fraction of the typical volume of cells and reagents become available, researchers can overcome some of the major limitations associated with cell-based assays.
The challenge begins with limited access to precious cell lines. Assays in multiwell formats can quickly expend cells. Primary cells, or human tissue cell-derived iPS (induced pluripotent stem) cells, such as cardiomyocytes, hepatocytes, and neuronal cells are still not broadly used in a high-throughput manner, mainly due to their high cost.
Although wells are an efficient and simple strategy to segregate experimental conditions in various formats, they have major restrictions when using suspension, loosely adherent and, in some cases, fully adherent cells—especially with high-throughput formats such as 96-, 384-, and 1,536-well plates.
Suspension cells such as the U-937 line or PBMCs are challenging to use in cell-based assays as they may be washed away in assays involving repeated washing steps. This concern is magnified in laboratories employing automated plate-washing equipment.
The answer may be found in a microscale solution offered with a bioassay platform recently introduced by Curiox Biosystems. The DropArray System was designed to miniaturize cell-based assays in a format that is practical and easy to integrate into today’s labs.
A key benefit to miniaturizing cell-based assays is the proportional reduction in cell and reagent volumes required to complete an assay. Using standard liquid-dispensing equipment, cell sample drops containing as few as 100 cells are deposited onto DropArray microplates (Figure 1). The cell samples reside on small hydrophilic spots in 96 or 384 array patterns on the flat, hydrophobic surface of the plate. DropArray Liquid Lid, an immiscible, gas-permeable fluid is used to cover and protect samples from evaporation or loss due to repeated washing steps, yet still allows gas exchange (Figure 1).
DropArray Washing Stations automate the task of gentle washing procedures. Plates are positioned in the liquid-tight chamber of the system’s washing station, and an aqueous washing buffer is automatically applied. Minimal cell loss is achieved with the station’s gentle, low velocity shaking motion.
It is these key features that enable assays to be performed using adherent, weakly adherent, and even nonadherent cells for long-term on-plate incubations without the risk of evaporation. Consumption of cells and reagents per data point is reduced at least 3 to 10 times.
Low Cell and Reagent Volumes
iPS-derived cells such as hepatocytes, cardiomyocytes, and neural cells are increasingly popular for various types of assays. However, the cost of these cells prohibited their broad use in drug discovery or tests. The DropArray plate provides a reduction of cell and reagent consumption with no cell loss during washing.
In a comparison of a conventional 384-well plate with a DropArray plate, the difference in cell and reagent volumes is sizable (Table). In this example comparison, a conventional 384-well system uses 9,000 μL of media, while the well-less DropArray system uses 900 μL. Similarly, if the conventional plate uses 5,000–7,500 iPS cardiomyocytes, for instance, the DropArray plate requires only 1,200–1,500 cells. Researchers can see the immediate benefit in reducing the number of cells involved in their assays.
Retention of Cells During Washing
It is difficult to run nonadherent or semi-adherent cell-based assays in a high-throughput manner due to extensive cell loss during washing steps. The DropArray Automated Washers perform plate washing using a gentle, low-velocity liquid exchange that does not detach the cells. It allows for high-throughput handling of semi-adherent and suspension cells in exactly the same manner as adherent cells with minimal cell loss.
To demonstrate, a cell-based extracellular protein-protein interaction assay (Figure 2) was used to compare the cell retention of a standard 384-well plate with that of a DropArray plate after 4 automated washings —equivalent to 16 washings by hand (Figure 2A). COS7 cells transfected with either HNT or OPCML expression constructs were fixed and then incubated with NEGR1-hFc bait protein. The cells were then washed and stained with antihuman AF488 (green) and scanned on the IN Cell Analyzer 2000 (GE Healthcare).
The same reagents for transfection and staining were used on a standard 384-well microtiter plate (Aurora, Brooks Automation) and a DropArray plate for a side-by-side comparison of the two formats. Hoechst staining (blue) of the nuclei shows the same cell density on both plate formats. Scale bar = 70 μm. Figure 2B shows the quantification of the mean intensity of the green channel from the images acquired.
Using three different cell types, equivalent intensity values are seen when NEGR1-hFc binds to cells expressing three different expression constructs of known NEGR1 binding partners. The same values are generated from the standard 384-well format, but only for the adherent cell line (COS7), while the weakly adherent (293T) and suspension adapted (293S) cell lines were not retained during the washing procedure on the standard 384-well plate format.
The Curiox DropArray platform allows scientists to perform cellular assays using primary or other precious cells in lower volumes for greater efficiency. DropArray is suitable for use with adherent, semi-, and nonadherent cells. Long term, on-plate incubation is achieved without edge effect and evaporation is minimized by applying a “liquid lid” of immiscible sealing fluid. Researchers are finally able to run cell-based assays as biology dictates, not based on the availability of cells or cost of reagents.