Abstract Patient-derived xenografts (PDX) are widely recognized as a more physiologically relevant preclinical model to standard cell line xenografts. PDX models faithfully recapitulate the original patient genetic profile, gene expression patterns and tissue histology. Despite their benefits, PDX models are limited by their inherent variability, lower throughput and lack of growth in vitro. The ability to generate cell lines from PDX models would enable high throughput chemosensitivity screens, ex vivo genetic manipulation and the development of novel orthotopic models. Development of stable PDX cell lines remains a challenge due to murine stromal outgrowth, lineage commitment and limited differentiation potential. Conditional reprogramming (CR) cell technology is a novel cell culture system facilitating the generation of stable cultures without genetic manipulation (Liu, Am J Pathol, 2012). The success of CR cell technology is dependent upon the combination of feeder cell-derived factors and Rho Kinase (ROCK) inhibitor. CR cells, therefore, represent a new class of progenitor-like cells, distinct from the phenotype of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. The purpose of this study was to identify the advantages, limitations and potential applications of CR technology for derivation of PDX explant cell lines. Early passage human lung and ovarian PDX tumors were cultured in CR conditions to create stable explant cell lines. Cell lines were established from 5/8 (63%) PDX tumors and were expanded over 6 months in culture with varying morphologies and growth kinetics. Due to normal outgrowth of murine stromal cells, early CR cultures contained mixed populations and required murine depletion to enrich for human cells. Key oncogenic mutations in a model of ovarian papillary serous adenocarcinoma were preserved in the enriched tumor cell population. While purified CR PDX cell lines were amenable to high throughput chemosensitivity screens, in vitro chemosensitivity did not consistently predict response in in vivo murine models. The CR PDX cell lines were additionally assessed for genetic manipulation and ability to form tumors in vivo. Collectively, these results demonstrate the applications of CR technology for the generation of stable explant cell lines from PDX models for preclinical studies. Citation Format: Alexandra Borodovsky, Travis J. McQuiston, Brian Dougherty, Ambar Ahmed, David Whitston, Daniel Stetson, Gretchen K. Hubbard, Sharon S. Challberg, Brian A. Pollok, Celina M. D’Cruz. Use of conditional reprogramming to develop and characterize cell cultures from patient-derived xenograft (PDX) models of human lung and ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 641.