A Fast and Reliable Method to Generate Pure, Single Cell-derived Clones of Mammalian Cells

Stable cell cloning is an essential aspect of biological research. All advanced genome editing tools rely heavily on stable, pure, single cell-derived clones of genetically engineered cells. For years, researchers have depended on single-cell dilutions seeded in 96- or 192-well plates, followed by microscopic exclusion of the wells seeded with more than or without a cell. This method is not just laborious, time-consuming, and uneconomical but also liable to unintentional error in identifying the wells seeded with a single cell. All these disadvantages may increase the time needed to generate a stable clone. Here, we report an easy-to-follow and straightforward method to conveniently create pure, stable clones in less than half the time traditionally required. Our approach utilizes cloning cylinders with non-toxic tissue-tek gel, commonly used for immobilizing tissues for sectioning, followed by trypsinization and screening of the genome-edited clones. Our approach uses minimal cell handling steps, thus decreasing the time invested in generating the pure clones effortlessly and economically. Graphical abstract: A schematic comparison showing the traditional dilution cloning and the method described here. Here, a well-separated colony (in the green box) must be preferred over the colonies not well separated (in the red box).