Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, the Netherlands.
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.
Nat Protoc. 2014 Dec;9(12):2725-31. doi: 10.1038/nprot.2014.181. Epub 2014 Oct 30.
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.
我们开发了一种新颖的 3D 细胞培养模型，该模型使用小鼠内髓集合管（mIMCD3）细胞生成上皮球体。该模型适用于有效的 siRNA 敲低，随后用人源患者衍生等位基因进行挽救。球体形成顶底极性和完整的腔，因此它们是肾纤毛病变中所见极性缺陷的理想模型，例如肾单位肾病变。简要地说，将 mIMCD3 细胞转染，随后传代到基质胶混合物中，该混合物播种在腔室载玻片上，并覆盖在生长培养基中。一旦形成球体，就溶解基质胶并在腔室载玻片上进行免疫细胞化学分析。该技术适用于半自动成像分析，并且可以同时测试多个基因、基因剂量效应和各种治疗干预措施。球体技术是一种独特而简单的 6 天体外方法，可以研究离体组织组织。
