Improving Clone Isolation and Screening In Hybridoma Cells – Moving away from Limiting Dilution

By on September 24, 2014
Handling of cell culture plates for cultivation of cells in life science

In successful hybridoma production, identifying hybridoma cell lines that have good manufacturing potential is key. To accomplish this, transfected or fused cell pools must be separated and cloned into monoclonal cultures. Thorough fusion screening and clone selection are crucial to an efficient and successful hybridoma program. The best clones, often called interesting clones, are stable and good producers that demonstrate specific attributes based on the product specifications.

The most prevalent traditional cloning technique, limiting dilution, has been used for many years, but it is both time- and labor-intensive. While this method has many drawbacks, it is still commonly used, particularly with hybridoma cells. Its widespread use is due primarily to the low cost of materials and low capital investment. However, the method is limited and with advancements in cell line development and successful alternatives available, many groups are replacing limiting dilution with more advanced technologies.

Methods for Clone Isolation and Screening

Limiting Dilution

In limiting dilution cloning, a mixed population of cells is diluted in liquid media and is dispersed into 96-well plates or other culture vessels. The goal of this method is to isolate individual cells into single wells or vessels. In order to accomplish this, the wells are seeded at an average density of less than one cell per well. While low seeding density increases the chance of isolating single cells into single wells, it also means that there will be more empty wells. Empty wells represent a waste of personnel time, consumables and lab space. Nonetheless, no matter how low the seeding density, some wells will still contain multiple cells.

Conversely, if the cells are seeded at a higher density, you will obtain fewer empty wells, but you will also increase the chance of having more than one cell per well. Because of the number of empty wells and wells with multiple cells, the percentage of wells containing one cell can be quite low, with the number of wells containing interesting clones even lower. It therefore becomes a balancing act where seeding density is adjusted to accommodate reasonable efficiency of the experiment as well as acceptable results with respect to isolating a truly clonal population.

Due to the inherent limitations in the traditional limiting dilution technique, achieving a high probability of monoclonality (POM) quickly and efficiently can be extremely challenging. Simply defined, probability of monoclonality (POM) means the probability that a population of cells is made up of genetically identical cells. The inefficiency of limiting dilution means that in order to achieve a good producing cell line with a high POM, many rounds of subcloning and screening are required.

Semi-Solid Cloning

An alternative to limiting dilution in liquid media is to employ the use of semi-solid media. Using semi-solid cloning permits a large number of cells to be plated at the same time, while the high viscosity of the medium ensures individual cells grow into discrete colonies. The colonies are then isolated using a pipette manually or through the use of automation and moved to liquid media in 96 well plates or other vessels for expansion and screening.

Using semi-solid media offers several advantages over limiting dilution. First, for many labs, a much higher POM is easier to achieve. It also enables visual identification of some cell characteristics, i.e. larger colonies will mean faster-growing clones and smaller colonies mean slower-growing clones. This means a variety of clones can be isolated with diverse characteristics, thus improving the chance of finding an interesting clone. Semi-solid cloning also supports culture survival during expansion, as thousands of cells per colony are seeded into each well. This seeding density for expansion ensures a high rate of survival and very few to no empty wells.

Another advantage is that you don’t have specific cell types dominating the culture. In liquid media you have a mixed population of fast-growing and slow-growing cells and in this environment, the fast-growing cells, which are not typically the most productive, dominate the culture. This provides fewer slow-growing clones for screening; losing comparatively slow-growing clones during cloning can be problematic as they are often better producers. In semi-solid media you do not have a domination of the culture by fast-growing cells, and this presents more opportunity to find that elusive high-yielding clone.

With higher POM, fewer rounds of subcloning and screening are required, resulting in shorter development times with semi-solid media.


Another way to advance clone selection is to employ automation in the process. Automated systems couple software with imaging systems; this allows scientists to program the criteria for the cells they would like to select. The machine then identifies which cells meet the set criteria and, through the use of robotics, isolates and plates these cells of interest without the need for manual pipetting. There are many automated screening systems that use different methods for identifying clones of interest.

Automating the system enables high throughput, so more clones can be screened overall, increasing the probability of finding a high-producing clone. Automation also makes the process more reliable and requires fewer employee resources. It is also regulatory friendly as it minimizes the amount of manual handling and reduces the introduction of human error. In addition, the implementation of bar codes and other tracking devices improves traceability throughout the process. For these reasons, automation is becoming more commonplace in CHO cell line development, however it is less frequently used in other cell lines, including hybridomas. Automation offers very attractive benefits, but these systems can be costly and may not be an option for all labs or all cell lines.


Antibody production in hybridoma cells is an important step in biopharmaceutical manufacturing, as well as being a crucial tool for large-scale antibody production for myriad applications including research, diagnostic tools and industrial applications. Isolating a high-producing cell line can speed product development by reducing the number of runs required to obtain initial testing material and can even speed time to a go/no go decision. Hence, there is a good argument for using a more advanced technology for isolation and screening of hybridoma cells.

Improvements in cloning compared with limiting dilution, like semi-solid cloning and automation, allow the overall process from fusion to final clone selection to be accomplished much faster. This time savings allows companies to cut overall drug discovery time and get to clinic faster.


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  1. Pingback: Cloning Dilution | The Gene Therapy Fountain

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