Forcyte Biotechnologies, Inc. is a drug discovery startup performing phenotypic screening of contractile cell force as a HTS assay endpoint. The company manufactures and utilizes unique microplates with soft-film bottoms embedded with fluorescent micropatterns to which thousands of cells simultaneously adhere and then contract (or squeeze). Optical measurements of micropattern contraction in the presence of drug provides functional data on whether a drug modulates cellular force generation, enabling discovery of novel mechano-modulating medicine (mechano-medicine). For example, a drug found to safely reduce the forces generated by airway smooth muscle cells may comprise a novel therapeutic candidate for treating asthma via bronchodilation. This screening approach also uniquely enables target-agnostic discovery of drugs for conditions like hypertension, preterm labor, spastic bladder, cardiac failure, or any other conditions exhibiting abnormal cellular force generation (mechanical disease).
After fabricating these microplates called FLECS-plates, with the appropriate adhesive molecule to support adhesion of the target cells of choice, and stiffness to provide enough flexibility to observe contraction, Forcyte will seed up to four distinct cellular populations together into each well of the plate. This is distinguishable by a unique optical barcode consisting of the presence or lack of 2 cytoplasmic dyes. The cells will then be incubated with a drug until it is time to image the plate and read the results of the screen.
The team needed an imaging system that could:
- Rapidly image an entire 384-well plate in under 20 mins per plate
- Image in 4 fluorescent channels: micropatterns, nuclear dye, cytoplasmic dye 1, cytoplasmic dye 2
- Maintain an incubator environment to prevent loss of mechanical activity
- Provide exceptionally high-quality and rapid auto-focus
- Organize terabytes worth of images
Forcyte accomplished its goal of executing 4-way multiplexed screens of cellular contraction (in 4 distinct optically-barcoded human cell types simultaneously) using the Molecular Devices ImageXpress® Micro 4 system with environmental control add-ons. The laser-based autofocus enabled rapid but robust focusing on our micro-patterned features, the cell nuclei, and the cytoplasmic dyes at a milli-second time scale. Coupled with its swift stage mobility, this capability facilitated image acquisition of a full 384-well FLECS-plate including all four channels at sufficient exposure times in under ~16 mins. Importantly, the system acquires images of entire wells (full 384-well area in one image) with sufficient spatial resolution to observe minute differences in micro-pattern size changes. Learn more about the 384-well FLECS-plates on the Forcyte Biotechnologies website.
The ImageXpress® Micro 4 High-Content Imaging System is a high-throughput, widefield imager that can acquire images of whole organisms and cellular or intracellular events. It features proprietary fast frame rate technology, allowing for the capture of fast biological processes such as calcium oscillations in cardiomyocytes. It is highly configurable and adaptable to changing research needs. Available options include confocal, brightfield, phase contrast, liquid handing, and environmental control.
Forcyte has successfully used this system, together with automation components, to screen up to 30,000 cell-drug combinations per day, with results showing that despite the old-school belief that contractile pathways are conserved across tissues and cell types, there exist inhibitors of cell contraction that are highly tissue-specific. In other words, using the system in multiplexed format, depicted in the figure below, we discover hits that significantly inhibit contraction in one cell type, but not in at least three others. This method is enabling Forcyte to perform the world’s largest mechano-medicine discovery campaign, with built-in specificity counter-screens towards developing first-in-class drugs for a range of mechanical diseases.