Randell Scott H, Fulcher M Leslie, O'Neal Wanda, Olsen John C
Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Methods Mol Biol. 2011;742:285-310. doi: 10.1007/978-1-61779-120-8_18.
When primary human airway epithelial (hAE) cells are grown in vitro on porous supports at an air-liquid interface (ALI), they recapitulate in vivo morphology and key physiologic processes. These cultures are useful for studying respiratory tract biology and diseases and for testing new cystic fibrosis (CF) therapies. This chapter gives protocols enabling creation of well-differentiated primary CF and non-CF airway epithelial cell cultures with non-proprietary reagents. We also discuss the production of retroviral and lentiviral vectors, the derivation of hAE cell lines, reporter gene assays, and the evolving science of gene overexpression and knockdown in ALI hAE cultures.
当原代人呼吸道上皮(hAE)细胞在气液界面(ALI)的多孔支持物上进行体外培养时,它们会重现体内的形态和关键生理过程。这些培养物对于研究呼吸道生物学和疾病以及测试新的囊性纤维化(CF)治疗方法很有用。本章提供了使用非专利试剂创建分化良好的原代CF和非CF呼吸道上皮细胞培养物的方案。我们还讨论了逆转录病毒和慢病毒载体的生产、hAE细胞系的衍生、报告基因检测以及ALI hAE培养物中基因过表达和敲低的不断发展的科学。