Single-Cell Cloning: Enabling Precision Biology

Understanding Single-Cell Cloning

The ability to work with cultures derived from single cells enables researchers to mitigate potential confounding factors in the form of genetic or behavioural heterogeneity, establishing a foundation for reproducible experimental outcomes.
Moreover, cell-based production processes often utilizes cultures that originated from a single cell. This supports a comparably robust and consistent production run of antibodies and other reagents.

Hence, single-cell cloning is a crucial technique involving the isolation and subsequent propagation of individual cells from a genetically (or otherwise) diverse cell population. Various methods, ranging from manual dilution in well-plates to fully automated processes and miniature culture formats, can be employed, each with their own pros and cons.

Applications of Single-Cell Cloning

Genome Engineering
Genome-editing techniques, exemplified by CRISPR-Cas9, often yield a diverse mix of cells, with only a subset displaying accurate edits. The key to precision lies in cultivating separate clonal populations from this edited pool, enabling the extraction and identification of lines with the desired modifications.
In cases where cell lines exhibit significant diversity, such as genetically unstable cancer cells, establishing and characterizing clonal lines before delving into genome engineering can prove highly beneficial for reproducible and interpretable research.

Monoclonal Antibody Production
Monoclonal antibody production requires the isolation and cloning of individual antibody-producing cells, either as part of antibody discovery workflows utilizing hybridomas, or pipelines employing production cell lines, such as CHO and HEK systems. Clonality of respective cultures is key to ensure the consistent production of antibodies with distinct specificity, crucial for both research and therapeutic use.

Stem Cell Research
Stem cell research relies on single-cell cloning to isolate and propagate specific cell types. Whether studying developmental pathways or generating differentiated cell lines for transplantation research, precise control often starts with individual cells.

Cancer Research and Drug Discovery
In cancer research, single-cell cloning is vital to isolate and study individual cancer cells, aiding in the understanding of intratumoral heterogeneity. In drug discovery, cloning cells with specific drug-resistant or sensitive phenotypes aids the development of targeted therapies and interrogation of underlying molecular mechanisms.

Manual Methods for Single-Cell Cloning

Serial Dilution
Cells are plated and successively diluted within well-plates via a dilution series. Starting from a known cell number and dilution factor, the well-plate will end up with some wells containing single cells. Both types of cells – suspension as well as adherent cells – can be used.

Limiting Dilution
Cells are diluted to a suitable concentration prior to plating, such that the probability of plating one cell per well is favoured over plating more than one cell per well. Each well is monitored over time for colony formation. Like serial dilution (see above), this approach is applicable to suspension as well as adherent cells. Often the concentration of the single-cell suspension will be chosen to accommodate 0.3-0.5 cells/well.

Cloning Rings
These cloning cylinders can be used to ringfence single cells and or colonies to separate them from the rest of the culture on round dishes. This method allows for a fairly precise isolation of chosen cells for cloning but is restricted to adherent cells.

Despite the relatively simple laboratory setup required for manual cloning workflows, they come with several critical drawbacks. Common culture formats used for manual workflows are incompatible with an easy visual confirmation of single cells. It is therefore recommended to perform a second round of cloning to increase the probability of obtaining true clonal lines. In addition, manual workflows are work-and time-intense and require comparably high amounts of culture reagents.

Advances in Automation

Recent years have seen significant advancements in automating single-cell cloning workflows, enhancing efficiency and reducing variability associated with manual processes.

However, obtaining automation systems come with an initial expenditure and running cost can be comparably expensive to manual workflows. This is a particularly consideration for scientists requiring expensive reagents for their cell models, such as induced pluripotent stem cells. Novel miniature culture formats using only nanolitre volumes per clone enable a significant reduction in required reagents and associated cost savings.

Benefits of Automation

Precision and Reproducibility: Automation mitigates human error, ensuring consistent and reproducible results.
High Throughput: Automated systems handle large numbers of cells simultaneously, expediting the cloning process and increasing experimental throughput.
Time Efficiency: Automation significantly reduces the time required for single-cell cloning workflows with continuous monitoring and streamlined processes.


Single-cell cloning facilitates applications that demand genetic homogeneity and reproducibility. Automation and novel miniature culture formats have been reshaping the landscape. This offers researchers powerful and cost-effective tools to accelerate the process. As technology advances, the future promises even greater precision and control in the study and manipulation of biological systems.

Explore our frequently asked questions about single-cell cloning and review tips for successfully propagating single cells – happy cloning!