Tips for successful Single-Cell Cloning

Introduction

Biology is inherently complex. No single cell is exactly like any other with diversity and heterogeneity being a hallmark and driving force of life itself. However, often it is vital to separate cells with desirable traits from the rest of the pack. Single-cell cloning is a technique used in biology to isolate single cells from a diverse mix of cells and propagate them into clonal populations. This allows researchers to establish and amplify genetically homogenous cell populations with desirable traits for research and production applications. In this article, we discuss some frequently asked questions about single-cell cloning. Why is single-cell cloning important in biological research? Single-cell cloning is crucial for several reasons. For example, in the context of cancer research, it facilitates a better understanding of co-existence, and interdependency of cell clones in tumours. For drug discovery projects, clonal cultures with desired sensitivities or resistances to particular drugs can help elucidate drugs mechanisms of action in the absence of confounding factors. Moreover, single-cell cloning is essential for creating homogenous cell lines for robust and consistent production workflows for antibodies. Another main application is the isolation and generation of clonal cell lines as part of gene-editing workflows, for example CRISPR-Cas9. What are common methods for performing single-cell cloning? Several methods exist for isolating and cloning individual cells, including manual and automated solutions. Manual processes, such as limiting dilution only require a relatively simple laboratory setup. Automated processes, such as fluorescence-activated cell sorting (FACS), and microfluidics-based techniques offer the prospect of streamlining the workflow. Main considerations for any method include the assessment of clonality, associated costs and complexity of the workflow. What are some challenges of single-cell cloning? Single-cell cloning poses challenges such as single-cell viability, assurance of clonality, contamination, and the time-consuming nature of the process, especially when performing manual workflows. Different types of cells have different requirements for successful single-cell propagation. Retaining cell viability while isolating individual cells can be challenging, particularly when working with delicate cell types. How can I improve the success of single-cell cloning experiments? Several factors can influence the success of single-cell cloning. These include the density and quality of the starting culture as well as the culture medium. We have compiled a list of tips for successful single-cell cloning for further insights. How can researchers address issues related to clonal instability in derived cell lines? Cell culture processes continuously pose a selection environment. It is therefore important to be aware of  changes in cell behaviour over time. Once single-cell-derived cell lines with a desired trait have been established, we recommend cryopreserving early passages as master bank. This master cell bank can further be expanded into a working cell bank. The latter serves as a reservoir to supply cells for respective applications. It is good practice to periodically restart the culture from a low-passage vial to minimize the accumulation of genetic and phenotypic variations. Additionally, optimizing culture conditions, including media composition and growth parameters, can contribute to the stability of single-cell-derived cell lines. Are there alternatives to single-cell cloning for obtaining homogenous cell populations? Depending on the downstream application, alternatives to single-cell cloning exist. Bulk population selection involves isolating cells based on a general characteristic or a selection marker. Subcloning, on the other hand, involves isolating and expanding a subset of cells from an existing culture, allowing for the enrichment of specific phenotypes. These approaches still result in a genetically mixed population and therefore may not be compatible with downstream procedures.