Notas de aplicación

Cell line development (CLD) is a critical process in biopharmaceutical production involving the isolation and expansion of single cells with desirable traits to generate stable, monoclonal cell lines. Traditional methods like limiting dilution and fluorescence- activated cell sorting (FACS) both rely on placing a single cell in a well for monoclonal expansion, which is highly inefficient even for the most robust cell lines due to the low viability of single cells. The unique design of the CellRaft Array enables thousands of cells to be cultured in a shared media reservoir where the cells can grow as effectively as they do in bulk culture, but with maintained spatial segregation as each cell sits on an encoded microscale polystyrene growth surface called a CellRaft (Figure 1A). Using the CellRaft AIR System, the array is serially imaged to capture a complete growth record of single cells as they form monoclonal colonies in the microwells (Figures 1B, C). CellRaft technology enables complete tracking and traceability of thousands of single cells while improving cell viability and monoclonal expansion by eliminating the need for fluidic manipulation or harsh single-cell culture methods. Using CellRaft Cytometry, software analysis tools enable users to identify CellRafts with their cells of interest at each timepoint, including algorithms to identify single cells, colonies, fluorescence intensity, and morphology characteristics. Once monoclonal colonies of interest are identified, the CellRaft AIR system performs the automated isolation of the CellRafts into 96-well collection plates for monoclonal expansion.

The CellRaft AIR System performs automated imaging of cells grown on the CellRaft Array in brightfield and 3-channel (blue, green, red) fluorescence. Using temporal image analysis, CellRaft Cytometry identifies monoclonal colonies using automated analysis algorithms for identifying single cells, dividing cells, and confluent colonies, all rooted in brightfield analysis (Figure 1C). In addition, users can employ fluorescent analysis tools, whether using fluorescently labeled cells, passive dyes, or antibody staining, to further interrogate heterogenous populations to isolate monoclonal colonies with their specific phenotype of interest (Figure 2).

Following isolation of monoclonal cell lines and transfer to a 96-well plate, the growth and expression levels of each colony of interest were monitored over the course of multiple days. Within the NIS-Elements software, a protocol was created that would automatically image the entire 96-well plate and measure a variety of statistical parameters such as total well coverage and mean colony fluorescence in multiple channels. At each timepoint, each well of the plate harboring 16 representatives from varying fluorescent protein expression level groups (Figure 4A) was imaged and a whole-plate overview image was produced to allow easy monitoring of continued experimental fidelity (Figure 4B). Automated

Maintaining an end-to-end chain of custody from a single cell proliferating into a microcolony is not a trivial problem. Pairing the powerful CellRaft Technology for culturing, identifying, and isolating monoclonal cells of interest with automated imaging and analysis workflows, powered by the Eclipse Ji with NIS-Elements enables the collection of an unbroken monoclonal history that provides new insights and validation for a wide variety of biological and pharmaceutical research.