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Nat Protoc. 2017 Feb;12(2):439-451. doi: 10.1038/nprot.2016.174. Epub 2017 Jan 26.

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

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Department of Pathology, Georgetown University Medical Center, Washington, DC, USA.
Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC, USA.
Department of Oncology, Georgetown University Medical Center, Washington, DC, USA.
Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, Texas, USA.
Department of Dermatology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.
Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC, USA.
Department of Cell Biology and Physiology, The University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.


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.

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