Clone Screening, Assurance and Hit Picking

The important point here is that regardless of what cell dispensing method was used, you need to show that the single cell successfully arrived in the well.

From a regulatory perspective, the whole well image is fundamentally important as assurance of clonality.

With the advent of high quality automated imaging in the last 5 years, many customers now only perform a single round of cloning which significantly reduces their timelines for the whole cell line development process (read Systimmune case study)

The numbers of plates screened can vary enormously between customers from a handful to several hundred per project. Part of this can be related to the nature of insertion (random versus targeted) and the efficiency of the seeding methods.After seeding, cells are allowed to settle to the bottom of the well or can be centrifuged.

Cells are then imaged by the Cell Metric which is a dedicated imager for assurance of clonality.

The regulator has set out the expectations for the imaging and that it should be carried out on a whole well basis and should be done on Day 0 and then subsequent days of growth and division.

The imager should also be used to validate your cloning method (FACS, LD, VIPS etc). This is only feasible if the imager can guarantee focus 100% for every well in the plate. The Cell Metric is uniquely able to do this by adjusting focus in each well for the curvature and distortion of each plate.

Validation methods can be designed for bright field and fluorescence modes. These experiments are essential to generate some statistical support to your cloning methods and the chances of a second cell being in a well which you believe to be clonal.

It is generally not recommended or encouraged to use fluorescence labels in the real experiments as they have been indications of toxicity and slowing in cell growth, and given that customers are developing therapeutics for patients then extraneous fluorescence should be avoided.

Imaging is carried out for Day 0 and subsequent days of growth and ultimately colony formation. This can be 10-21 days depending on cell types.

A library of images is created for each well and it is possible to track back in time from the colony all the way back to Day 0 and confirm if it started from 1 cell or not (see images below).

Cell Metric Colony

Cell Metric 2 Cells

Cell Metric 1 Cells

It can vary from customer to customer when they do this review of wells. Some customers do it immediately after colony formation and then hit pick the wells which formed a colony and started from a single cell. This could involve picking and re-arraying several hundred clones.

Other customers re-array all the wells that grew into 24 DWP which are then shaken in fed-batch mode. Once they have found the best clones based on titre, they will take this smaller subset and review the original well images for assurance of clonal origin.

For the clonal wells of choice, the Cell Metric can write a hit picking list which can be exported to a third-party liquid handling robot for re-compiling into full 96 well plates of clones.

Finally, a report can be rapidly generated for the well history of the top clones. This documentation package is called the Clonality Report and is importantly something which customers can include as part of their IND submission to the regulator to support their clonality claims.

To find out more

Next Steps