Abstract
We have developed a novel 3D cell culture model that uses mouse inner-medullary collecting duct (mIMCD3) cells to generate epithelial spheroids. This model is amenable to efficient siRNA knockdown and subsequent rescue with human patient-derived alleles. Spheroids develop apicobasal polarity and complete lumens, and they are consequently an ideal model for polarity defects seen in renal ciliopathies such as nephronophthisis. Briefly, mIMCD3 cells are transfected and subsequently passaged to a Matrigel mixture, which is seeded in chamber slides and covered in growth medium. Once the spheroids are formed, Matrigel is dissolved and immunocytochemistry is performed in the chamber slides. The technique is amenable to semiautomatic imaging analysis, and it can test multiple genes simultaneously, gene-dosing effects and a variety of therapeutic interventions. The spheroid technique is a unique and simple 6-d in vitro method of interrogating ex vivo tissue organization.
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Acknowledgements
We thank C.J. Westlake and L. Sang for their help in setting up the protocol, and M.A. Jewett for expert urological support. R.H.G. is supported by the Dutch Kidney Foundation grants CP11.18 (KOUNCIL) and 13A3D103, and EU FP7/2009 241955 (SYSCILIA) and 305608 (EURenOmics). H.A. is supported by the Anna-Liise Farquharson Kidney Cancer Research Fund, Princess Margaret Foundation, Toronto, Canada.
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R.H.G. and P.K.J. designed the protocol, which was independently validated by H.A. R.H.G. and H.A. drafted the manuscript.
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Supplementary information
Real-time movie of mIMCD3 cells in a spheroid transfected with somatostatin receptor 5-HTTP:GFP.
Cilia and basolateral membranes are green fluorescent. (MOV 2867 kb)
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Giles, R., Ajzenberg, H. & Jackson, P. 3D spheroid model of mIMCD3 cells for studying ciliopathies and renal epithelial disorders. Nat Protoc 9, 2725–2731 (2014). https://doi.org/10.1038/nprot.2014.181
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DOI: https://doi.org/10.1038/nprot.2014.181
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