Generation of Clonal Cultures of Adherent or Suspension Cells Using Flat Sessile Drops for Assurance of Monoclonality

Single-cell isolation is an essential step in many biomedical workflows, including genetic analyses and drug-based assays. It is commonly attempted through limiting dilution into microtiter wells. However, dark optical edge effects at the well periphery make it difficult to confirm which wells contain just one cell. Consequently, statistical methods are used to obtain the probability that a well contains a single cell. Sessile microdrops can be deposited in the center of wells away from obscuring walls. If these drops have low contact angles, optical edge effects are minimal. A dilute cell suspension can be infused into such drops, which are then imaged to confirm the presence of a single cell with certainty. Subsequently, wells are flooded with media and incubated to allow clonal growth. The fraction of single cells yielding colonies then provides an accurate and non-probabilistic measure of cloning efficiency. We demonstrate average cloning efficiencies between 62% and 78% with human embryonic kidney, cancer, and induced pluripotent stem cells, as well as Chinese-hamster suspension cells. We verify that stem cells continue to express pluripotency markers after cloning and incorporate the method into a gene-editing workflow for cell-line development. This demonstrates the seamless integration of sessile microdrops into established protocols, providing assurance of monoclonality with high cloning efficiency.