Box 1

Techniques for Improving Imaging of Single Cells during Cloning

A variety of techniques have been developed and employed to improve the reliability of images for the detection of single cells as part of the cloning process. Methods to ensure cells are in the appropriate focal plane of the imager include cell settling, requiring typically several hours for cells after plating prior to taking a day 0 image, and plate centrifugation, which allows a rapid method to ensure all cells are pulled to the bottom of the well. Additionally, the application of animal component–free fluorescent dyes coupled with fluorescence enabled imagers can also dramatically improve the ability of an imager to identify cells but also to distinguish viable cells from cell-associated and other debris. Configurations of imagers including numbers of images taken, image resolution and magnification, and image stitching are all critical parameters in defining an imaging process to support the cloning process. Prior to single-cell plating it is possible to characterize the number of doublets present in the cell suspension via manual inspection using a microscope. This technique allows for evaluation of the quality of the suspension for cloning and can be used to reject undesired pools with a high number of doublets. Process modifications including addition of anti-clumping agents and use of cell strainers can also be applied to reduce the incidence of clumps of cells. Imaging across multiple time points (e.g., day 0, 1, 2, etc.) can provide additional evidence for the presence of only a single cell through the observation of growth from only the progenitor cell identified on day 0. These methods can be applied alone or in combination to enhance the overall imager performance as needed for the degree of confidence and assurance required from a clonal image.