Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications, including regenerative medicine, drug sensitivity testing, gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue, and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d, the technique is directly applicable to diagnostic and predictive medicine. Moreover, the epithelial cells can be propagated indefinitely in vitro, yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment.