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As regulations for cell line development become increasingly more stringent, researchers will be required to perform single-cell cloning and provide evidence that a cell line is derived from a single cell (proof of clonality). Until recently, there were two main methods to prove monoclonality. In this study, we conducted six sets of experiments, cloning two CHO cell lines with animal-component-free (ACF) cell culture media and supplements, and compared the performance a CloneSelect™ f.sight single cell printer versus two other accepted cloning methods (FC and LD).