Hybridoma & CHO clone picking from semi-solid media
Screening and isolation of high-expression clones for antibody production
A fundamental step in the development of new cell lines for antibody production is the identification of single cell-derived clones that produce high and consistent levels of the target protein. Compared to the time-consuming and labor-intensive steps of conventional techniques, productivity screening with the CellCelector™ can be performed in a more efficient way.
Selection and isolation of high-producer clones
The antibody generation of hybridoma clones can vary dramatically between different clones. The proteins produced by the clones and secreted into the medium can be made visible by fluorescence-labelled secondary antibodies. As the viscosity of the semi-solid media prevents the antibody from diffusing further into the media the signal will appear as a fluorescent halo around the cell clone that produced it. To select specifically high-producer clones the CellCelector™ software compares the size of the cell colony visible in bright field with the size of fluorescence halo surrounding the cell colony and calculates the rate of the antibody productivity of each clone. This allows ranking of cell clones across multiple source plates for best antibody production.
Schematic view of hybridoma clones (light blue) and the detection of secreted antibodies (dark blue) by fluorochrome labelled secondary antibodies (green with yellow circle) (A-C)
Microscopic view (overlay of bright field and fluorescence illumination) of hybridoma clones after incubation with fluorochrome labelled secondary antibodies (D-F).
The intensity of antibody production correlates with the diameter of the fluorescent halo around the colony: colony producing no antibodies (A and D), colony producing medium amounts of antibodies (B and E); colony producing high amounts of antibodies = high producer (C and F). The size of the colonies is similar (see enlarged area in bright field observation D-F).
Antibody producing CHO cell colony; QC images before and after picking with the semi-solid media module of the ALS CellCelector™
CHO cell colony harvested with the semi-solid media module cultured in a 96 well destination plate (at day 1 and day 5 after harvesting).
Benefits of CellCelector™ monoclonal colony picking from methylcellulose and other semi-solid media
High resolution optics and intelligent imaging system
- Detection of small colonies or single cells near larger clones ensuring monoclonal isolation of target clones.
- The structure of colonies can be analyzed and morphological parameters (e.g. for sphericity and shape) can be defined to exclude suspicious (e.g. elongated) colonies from picking.
- Microscopic images for quality control (before and after picking images) and live image during picking.
Customization and flexibility
- Up to 6 fluorescence channels; filter sets can be customized based on customer application requirements.
- Standard as well as customized source and destination plate types usable.
- Upstream and downstream automation integration possible.
- Long-term experiments possible with integration in our Incubator FlowBox
Use of disposable tips
- Use of disposable plastic tips that are automatically exchanged between picks avoid cross-contamination and clogging of nozzles.
- No washing steps necessary for tip decontamination.
- Different diameters for various clone sizes available.
Features
- Selection of top ranked clones from a batch of plates: first scanning of all plates then picking plate by plate.
- Controlled low aspiration volumes and speeds prevent simultaneous picking of neighboring clones.
- Automated verification of neighboring clone movements during picking ensures that all target clones are re-centered automatically before aspiration.
- Automated analysis of the area surrounding the target clones right before picking avoids picking of clones which are too close to neighboring clones.
- User-definable sorting and ranking strategy, e.g. ranking for clone quality, size or productivity.
RELATED PUBLICATIONS
- Zoldan, K. et al. Automated clonal selection of high-producing hybridoma colonies from methylcellulose-based, semi-solid medium using the cell separation robot CellCelectorTM nature methods application notes (2010)
- Caron A.W. et al. Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins (CHO) BMC Biotechnol. 9: 42 (2009)
- Julien, A. et al. Antigen specific antibody-producing clones selected by isotypes Biosystems International Application Note
- Gilbert, R. Fluorescent labeling in Semi-Solid Medium to Identify High Expressing Clones for Recombinant Protein Production (CHO) CHI's Bioprocessing Summit Presentation, Boston 2011
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