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Nat Protoc. 2014 Dec;9(12):2725-31. doi: 10.1038/nprot.2014.181. Epub 2014 Oct 30.

3D spheroid model of mIMCD3 cells for studying ciliopathies and renal epithelial disorders.

Author information

1
Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, the Netherlands.
2
1] Research Oncology, Genentech, South San Francisco, California, USA. [2] Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.

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.

PMID:
25356583
DOI:
10.1038/nprot.2014.181
[Indexed for MEDLINE]

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