Rabata Anas, Hampl Ales, Koledova Zuzana
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625 00, Brno, Czech Republic.
International Clinical Research Center - Center of Biomolecular and Cellular Engineering, St. Anne's University Hospital Brno, Brno, Czech Republic.
Methods Mol Biol. 2017;1612:149-165. doi: 10.1007/978-1-4939-7021-6_11.
Lung epithelium contains distinctive subpopulations of lung stem/progenitor cells (LSPCs) that are essential for lung epithelial maintenance and repair in vivo. Hence, LSPCs are in the center of interest of lung biology due to their promising therapeutic applications. To reach this goal, proper characterization of LSPCs, understanding of their proliferation and differentiation potentials and elucidation of mechanisms that control them are necessary. Therefore, development of reliable in vitro clonogenic assays has been needed. We established lungosphere assay, an in vitro sphere-forming 3D culture assay that enables to evaluate stem/progenitor cell activity, self-renewal and differentiation capacity of LSPCs and to conveniently test the effect of various treatments on LSPCs. Here we provide a detailed description of procedures for isolation of adult mouse lung epithelial cells, their culture in non-adherent conditions to form LSPC-derived spheroids (lungospheres) and for embedding of lungospheres into 3D extracellular matrix to model processes of lung tissue maintenance in a physiologically relevant microenvironment.
肺上皮包含独特的肺干细胞/祖细胞亚群（LSPCs），这些细胞对于体内肺上皮的维持和修复至关重要。因此，由于其有前景的治疗应用，LSPCs成为肺生物学研究的焦点。为实现这一目标，对LSPCs进行恰当的表征、了解其增殖和分化潜能以及阐明控制它们的机制是必要的。因此，需要开发可靠的体外克隆形成试验。我们建立了肺球形成试验，这是一种体外成球的三维培养试验，能够评估LSPCs的干细胞/祖细胞活性、自我更新和分化能力，并方便地测试各种处理对LSPCs的影响。在这里，我们详细描述了分离成年小鼠肺上皮细胞的程序、在非贴壁条件下培养以形成LSPCs来源的球体（肺球）以及将肺球嵌入三维细胞外基质以在生理相关微环境中模拟肺组织维持过程的方法。
