AbstractThere is increasing pressure for high-throughput development and production of cell and gene therapies as many are progressing through clinical trials for eventual patient use. To ensure high quality of gene therapies from cell lines such as HEK293, which is often used to produce AAV and lentiviral vectors, it is important to prove that the resulting products were derived from a clonal cell line. This is often a bottleneck, however for the first time a single instrument provides a solution to this challenge. The UP.SIGHT combines single-cell cloning with a new imaging technology for 3D full well imaging to generate cell lines for gene therapies with probability of monoclonality of >99.99%. This workflow can reduce the time required to determine high titer clones and improve the overall process of gene therapy development.