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Cell line development

The revolution in single cell cloning for pharmaceutical cell line development is here

Are you tired of seeding thousands of cells in large stacks of cell culture plates only to find out they are either no single cells, viable or productive? Tired of screening hundreds of plates for the one clone that has it all and will fulfill your projects titer goal? Why leave your pharmaceutical cell line development to chance? With the CellCelector™ single cell and colony picking platform you can now effectively assess and verify your clones before deciding which ones to choose. Within under a week you will get from a pool of single cells to plates full of clones that have proven to be monoclonal, viable and productive. Instead of relying on large quantities of plates to produce a winner you can now use actual data reliably predicting the future of your clones. This saves consumable and media costs, incubator space but most of all valuable time to reach your project goals by avoiding missteps, a second cloning round and unnecessary procedures.

One-round single cell cloning workflow with integrated monoclonality proof and viability assay

The CellCelector can now be used for high-throughput single cell cloning allowing fast generation of clonal pharmaceutical production cell lines. Clones are generated within just one cloning round while providing robust in-process image-verified monoclonality proof (HT-NIC method for High Throughput Nanowell based Image verified Cloning).

With this integrated monoclonality and viability assessment of clones as well as industry leading outgrowth rates after clone transfer into 96 or 384 well plates the CellCelector single cell cloning technology represents the next generation in single cell cloning approaches. As such it goes far beyond the traditional methods and provides a superior alternative to limiting dilution, FACS sorting or single cell printing techniques. This patent-pending method has been developed and validated in collaboration with ProBioGen AG and other CellCelector customers.
Nanowell with one single cell

CellCelector™ nanowell cell culture plates

24 well Nanowell plate
The HT-NIC method is based on the CellCelector™ Nanowell plates (available from ALS). These plates are available as 24- and 6-well cell culture plates featuring thousands of tiny nanowells at the bottom of each well. Depending on the plate type this results in between 4,300 to 22,000  nanowells per well or between 100,000 to 130,000 nanowells per plate. Cells sitting inside the nanowells are efficiently separated from each other. As they are covered by the same media they are however not isolated from each other which leads to an effective co-culturing of cells. All cells in the pool will contribute to the outgrowth of the cell line while maintaining their monoclonality. This leads to industry leading outgrowth rates of single cells even for extremely difficult to grow cell lines.

CellCelector™ nanowell-based single cell cloning workflow

Cell seeding is performed in a way similar as in conventional cell culture plates. After seeding the cells they are captured randomly inside the nanowells following the classic Poisson distribution. Automated scanning of the seeded wells followed by an automated identification of all nanowells containing a single cell provides a robust and documented image-based monoclonality proof. Depending on the number of cells seeded 400 to 600 single cells are captured per well and can be analyzed. Seeding multiple wells allows to start with up to ~14,000 nanowells containing a single cell - all within just one nanowell plate.
Pipetting into the nanowell plate
After monoclonality has been documented cells will grow 3 to 6 days in an incubator up to 20 to 75 cells per clone. Contrary to a traditional methylcellulose-based approach the CellCelector nanowell-based method allows the colony growth in liquid media. Clones are sharing the same media but are effectively separated from each other by the nanowell walls. After the cells have grown into single cell clones the nanowell plate is scanned again and monoclonal, viable clones are automatically selected and transferred into 96 or 384 well plates for further analysis and upscaling.
Cell growing in nanowell

Automated monoclonality proof: robust single cell detection in nanowells

In a conventional single cell cloning workflow single cells are seeded in 96 well plates making reliable automated single cell detection in bright field difficult as cells are often settled at the very edge of the well. Thus the monoclonality status of a given clone at day 0 is usually checked manually or retroactively once the clone has grown. But why search a single cell within a well area that is more than 100 times larger than the surface occupied by a cell? With the CellCelector™ HT-NIC approach the cells are separated and clearly visible within 200 µm large nanowells and can therefore be identified reliably and automatically by software. Even just after seeding or when they touch the nanowell border.
Monoclonality assessment: single cell detection in nanowells

Forget limiting dilution, CellCelector™ offers 100% monoclonality

Limiting dilution, a well known traditional method for single cell cloning, is based exclusively on statistical probabilities for monoclonality which depend on the average number of cells seeded per well according to the Poisson distribution. In order to limit the percentage of polyclonal wells seeding is usually done with not more than 0.2 cells per well. Unfortunately it results in only ~16 % wells being monoclonal (i.e. seven eighths of the plate remain empty). Thus to be able to get 400 single-cell wells more than 25 96 well plates need to be seeded. It’s possible to double the number of single cell wells by seeding 0.5 cells/well at the cost of a significant increase in non-monoclonal wells.
By automated identification of single cell nanowells, tracking of their growth into clones and cross-contamination free transfer of grown clones into 96 well plates the CellCelector™ HT-NIC cloning method provides 100 % monoclonal wells independently of sample preparation, cell type or cell density used for seeding into the nanowell plate.
The efficiency in obtaining monoclonal wells with the CellCelector™ nanowell-based cloning method compared to that of limiting dilution used with two seeding densities.
To obtain 400 monoclonal wells using the limiting dilution method 26 or 13 96 well plates for 0.2 cells/well or 0.5 cells/well seeding density need to be used. To arrive at the same number of single cell nanowells just one well of theCellCelector™ nanowell plate would be sufficient.

Get the highest percentage of viable single cell clones

The limiting dilution method leads to only 10 % to 20 % of the seeded wells with viable clones grown from a single cell. Other traditional methods like FACS single cell sorting or single cell printing provide a much higher percentage of single cells seeded into a well. High shear stress usually associated with flow cytometry and the fact that the deposited single cells sit in a large volume of media on their own leads to uncertain outgrowth at rates between 30 % and 64 %. Also this requires specialized and expensive cloning media with external growth factors and is unsuitable for difficult to grow cell lines that simply won’t grow from single cells.

In the CellCelector™ HT-NIC method however all seeded cells share the same media and contribute to each other’s growth by releasing natural growth factors without compromising clonality. Only after a clone has been proven to be monoclonal and viable it will be selected and gently transferred into a 96 well plate. Since it is now a viable and monoclonal small clone instead of a single cell that is transferred it will continue to grow in a large well.

Disposable consumables that eliminate sample-to-sample contamination

ALS offers affordable disposable CellCelector™ glass capillaries and nanowell plates preventing any cross contamination between cloning experiments. One glass capillary and one nanowell plate (or even a few wells of the latter) are sufficient to perform a single cell cloning experiment without any limitation of the number of destination microplates used for clone transfer.
Based on budget constraints a used capillary can be automatically rinsed and sterilized by the system after the experiment for later usage.  
Nanowell plate - fluorescence

Reliable single cell cloning in a controlled sterile environment

Incubator flowbox with ALS CellCelector
To run the assay and clone transfer in sterile conditions the CellCelector™ system can be placed inside a biosafety cabinet. This can be a standard hood or a customized box offered by ALS together with the system. For further improvements in cell viability and assay stability the CellCelector™ can also be installed in the ALS Incubator FlowBox which combines a compact laminar flow cell culture hood with temperature, humidity and CO2 control as well as UV lamps for sterilization.
Control panel incubator flowbox

Benefits of CellCelector™ single cell cloning for cell line development

Easy to use

  • Simple sample preparation

  • User-friendly software with features developed specifically for single cell cloning

  • No routine maintenance necessary


  • Getting 100 % of clones originating from single cells

  • Single cloning round with integrated image-verified monoclonality proof and clone viability assessment

  • Cell viability after clone transfer into 96 well plates up to 95 %


  • High quality images for robust automated label-free single cell detection and monoclonality proof to the FDA

  • 100 % selective clone recovery without clone-to-clone contamination

  • Disposable single-use capillaries and nanowell plates to avoid cross-contamination between projects

  • Integrated traceability of all clones from single cell to clone to transfer

  • Automated clone transfer quality control (Picked/Failed)

Time, material and space saving

  • Getting proven viable, monoclonal clones in less than 6 days

  • Significant cost savings on consumables, media and incubator storage space

  • One well of 24 well nanowell plate results in 200 to 500 target clones

  • No need of an additional imaging system for a monoclonality assessment


  • Compatibility with various cell lines (CHO, HEK293; NS0, PER.C6 etc.)

  • No limitation on cell size

  • Easy cell density or media optimization by running up to 24 samples within one plate

  • Compatible with a variety of other workflows (single B-cell screening, Hybridoma screening, gene editing, rare single cell isolation etc.)

  • Compatible with a large range of standard or custom source and destination consumables (microplates, dishes, slides, PCR plates/tubes etc.)
Want to learn more?
CellCelector for single cell cloning
CellCelector™ Flex
Incubator FlowBox for single cell cloning
Incubator FlowBox
Nanowell plate for single cell cloning
Nanowell plates
glass capillaries for single cell cloning
CellCelector™ glass capillaries
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