Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens

Xuefeng Liu, Ewa Krawczyk, Frank A Suprynowicz, Nancy Palechor-Ceron, Hang Yuan, Aleksandra Dakic, Vera Simic, Yun-Ling Zheng, Praathibha Sripadhan, Chen Chen, Jie Lu, Tung-Wei Hou, Sujata Choudhury, Bhaskar Kallakury, Dean G Tang, Thomas Darling, Rajesh Thangapazham, Olga Timofeeva, Anatoly Dritschilo, Scott H Randell, Christopher Albanese, Seema Agarwal, Richard Schlegel

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.