Tips for successful Single-Cell Cloning

Mastering Single-Cell Cloning

Achieving efficient and consistent single-cell cloning requires awareness of various factors that can make or break its outcome. Here, we share important tips and considerations for succeeding in single-cell cloning, covering culture medium selection, choice of culture dishes, cell health, contamination prevention and more.

Culture medium

The obvious one – a common approach is using the exact same cell culture medium as for bulk culture, which sounds all too plausible. Occasionally, researchers opt to add ‘conditioned culture medium’. Such medium has already been exposed to cells and ‘conditioned’ with secreted factors. Adding conditioned medium aims to provide single cells with growth conditions and secreted factors mirroring bulk culture. While this can occasionally be of benefit, alternative tweaks may be better suited and result in a notable increase in efficiency. For example, in the case of cells growing in serum-containing medium, single-cell cloning can get a significant boost when increasing serum concentration slightly (15% – 20%).

Bulk Cell Culture Density

Cells adapt their physiology and proliferation in response to the density of the culture. High-density cultures can result in strong cell-cell interactions and lowered cell proliferation. Preparing single-cell suspension from such cultures might require excessive force to dislodge cell clumps which can impair cell viability. Consider splitting cultures 1-2 days before starting single-cell workflows to stimulate proliferation and breaking cell clumps. Experiment with different cell densities to find the optimal balance for your specific cell type.

Choice of Culture Dishes

If cloning efficiency is low, it might be worth to use culture dishes from different vendors. Though dishes might look alike from the outside, their surfaces may well differ which has a significant impact on cell behaviour. Indeed, our own experiences revealed strong differences in single-cell cloning outcome across two types of TC-treated dishes.

Cell Health and Viability

Ensure that cells used for single-cell cloning are in a healthy state and free from contamination. Healthy, proliferating cells have a higher likelihood of growing out as single cells and survive the cloning process. Assess cell morphology, check for signs of stress, and maintain optimal culture conditions to promote cell health.

Gentle Handling Techniques

Single-cell cloning demands a delicate touch during cell isolation and manipulation. Gentle handling minimizes cell stress and increases the chances of successful cloning. Be mindful of pipetting forces and avoid excessive agitation that could compromise cell integrity.

Cell Adhesion Molecules

Consider the use of cell adhesion molecules or substrates that mimic the natural extracellular matrix. These can promote cell adhesion and enhance the likelihood of successful single-cell attachment and growth.

Regular Quality Control Assessments of Reagents

Implement regular quality control assessments for all reagents and culture materials used in the cloning process. This includes checking the quality of the culture medium, and other supplements to ensure consistent and reproducible results. Avoid using expired reagents.

Be patient

Single-cell cloning can be time-consuming, especially utilizing slow-growing cells. Unless cells need their medium exchanged on a regular basis, such as in the case of pluripotent stem cells, it is best to leave them in their optimum growth environment. Be patient and only assess single-cell outgrowth intermittently. Excessive handling with shifts in CO2, temperature and humidity can add unnecessary stress to single cells and very low-density cultures.


Mastering single-cell cloning requires a combination of optimized culture conditions, careful technique, and a thorough understanding of your specific cell type. By implementing tips outlined in this article, researchers can enhance the success rate of single-cell cloning, leading to the generation of homogeneous cell populations for various applications in cell biology and biotechnology.