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Important considerations for optimising outgrowth from single cells in cloning plates during cell line development

When developing biotherapeutics there are many parts of the process that require optimisation. Cell line development is a key part of this process and there are many aspects where changes can have a dramatic effect on timelines and outcomes.

One key area which is challenging to many of our customers is outgrowth of clones in their plates following single cell seeding. If conditions haven’t been optimised a high seeding efficiency will not often translate through into a high number of resultant clones for screening.

Key Factors

1. Cell line choice

This is an important primary consideration and the options can be as follows:

  1. Public source of CHO;
  2. Proprietary cell line e.g. MilliporeSigma, Horizon, Lonza, Selexis; or
  3. Develop in-house from scratch.

Big pharma will generally go for option 3. Smaller and medium-sized companies will generally go for option 2. The advantage of buying/licensing in a cell line is that it usually comes with defined protocols. These models have also become less financially onerous with much lower access fees and reduced royalty-based burdens.

For the proprietary cell lines to work effectively for in-house customers, the vendors need to provide not only the cell lines, but also the reagents and protocols for a robust and reproducible approach. The handover of methods can be made even more robust with the use of recommended and turnkey instrumentation for the key steps such as single cell cloning and growth monitoring.

2. Choice of gene editing technology

Prior to seeding, the cell line must be known to be stable; the chosen methodology must allow for accurate insertion of the GOI into the host genome. For example, we have discussed ATUM’s transposase technology in a previous blog. However, successfully placing a single, edited, high-producing clone in each well of a plate is just the start of a technically challenging process.

3. Single cell cloning method

For instruments that can seed single cells, a major consideration should be the dispensing pressure when single cells are transferred into wells. If too high, the cells will be damaged on arrival which can impact cell survival. Many cell types are indeed FACS-sensitive and hence Solentim’s VIPS single cell cloning platform, which uses a much lower pressure than FACS systems when dispensing cells, can result in higher cell survival rates, increasing outgrowth and cloning efficiency.

Janssen Clone Outgrowth using Solentim VIPS For Janssen’s use of the VIPS, as published in a recent case study, it was shown to have the same outgrowth rate as for gentle manual limiting dilutions, however the number of wells available in which growth was observed increased by 68% [see table].


VIPS has shown to work well in practice with customers using Horizon’s and MilliporeSigma’s commercial CHO cell lines.

4. Growth media and supplements

Depending on the seeding method, single cells can either be dispensed into wells pre-filled with media (e.g. FACS or SCP), or single cells can be dispensed into dry wells and media then added on top i.e. the VIPS. Commercial growth media is available from several vendors. Further optimisation using supplements can potentially boost cell survival and outgrowth.

Some customers also dispense cells into a smaller volume initially and then after a few days top-up the media as part of a feeding strategy. Additionally, the top-up can be a different media to the cloning media, which supports the cells growth once they are through the first cell divisions.

There is a basic requirement that the media and any supplements are animal component-free and at this stage the media will be different to the final production media. For outgrowth, customers also often employ conditioned media.

However, there is not one method that fits all and conditions need to be tried independently for the specific proteins to be expressed. Even with recommended media, modifications of some key ingredients and addition of supplements may be able to obtain excellent yields.

5. Well plate volume

The size of the well and volume of liquid in the well is also a consideration. We have found a good number of customers have moved from 96 to 384 well plates for cloning as the smaller well volume seems to encourage cell recovery growth, thus giving rise to better outgrowth.

We have also noted instances where single cells start to divide over the first few days after cloning and then cease to divide further for unknown reasons.

6. Imagers for monitoring cell outgrowth

Whole well imagers provide crucial information on which wells to progress further based on outgrowth and single cell origin.

Well Plate optimising outgrowth from Single Cell
Whole well imagers provide crucial information

Outgrowth can be assessed initially by measuring either confluence or ‘total cell numbers’ in the well plates, and then subsequently after expansion by ‘viable cell numbers’ (for titer measurements) in shaking plates, tubes and scale-down mini bioreactors.

Instruments such as the Cell Metric facilitate imaging during cell line development, allowing scientists to monitor outgrowth as whole wells well in situ, providing a growth rate curve and percentage confluence for each well, plus tracking back to the single cell clone. It is no good having high seeding efficiency without most of these clones actually growing on into colonies.

Good outgrowth metrics enables scientists to make decisions earlier in the process and they can confidently progress potentially fewer clones in fewer plates.

Summary Suggestions

This blog is not exhaustive and only touches on a number of the key aspects of cell survival and optimising outgrowth from single cells.

The best advice is to consider using one of the main commercial CHO cell providers as they will provide recommendations and protocols to get you started in-house quickly.

However, to get best results given changes in expression for different molecules, using equipment that is gentle for dispensing cells to give high survival rates (e.g. the VIPS) along with media optimisation and addition of supplements can literally double the outgrowth and production of these cell lines.

Solentim will shortly publish results to show the improvements in outgrowth for one of the commercial cell lines through modulating changes in background media and addition of a novel commercially available animal component-free supplement.

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