An Ex Vivo Acid Injury and Repair (AIR) Model Using Precision-Cut Lung Slices to Understand Lung Injury and Repair

Q1 Agricultural and Biological Sciences Current protocols in mouse biology Pub Date : 2020-11-20 DOI:10.1002/cpmo.85

Sally Yunsun Kim, Róisín Mongey, Mark Griffiths, Matthew Hind, Charlotte H. Dean

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引用次数: 5

Abstract

Recent advances in cell culture models like air‒liquid interface culture and ex vivo models such as organoids have advanced studies of lung biology; however, gaps exist between these models and tools that represent the complexity of the three-dimensional environment of the lung. Precision-cut lung slices (PCLS) mimic the in vivo environment and bridge the gap between in vitro and in vivo models. We have established the acid injury and repair (AIR) model where a spatially restricted area of tissue is injured using drops of HCl combined with Pluronic gel. Injury and repair are assessed by immunofluorescence using robust markers, including Ki67 for cell proliferation and prosurfactant protein C for alveolar type 2/progenitor cells. Importantly, the AIR model enables the study of injury and repair in mouse lung tissue without the need for an initial in vivo injury, and the results are highly reproducible. Here, we present detailed protocols for the generation of PCLS and the AIR model. We also describe methods to analyze and quantify injury in AIR-PCLS by immunostaining with established early repair markers and fluorescence imaging. This novel ex vivo model is a versatile tool for studying lung cell biology in acute lung injury and for semi-high-throughput screening of potential therapeutics. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Generation of precision-cut lung slices

Basic Protocol 2: The acid injury and repair model

Basic Protocol 3: Analysis of AIR-PCLS: Immunostaining and imaging

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一个体外酸损伤和修复(AIR)模型使用精确切割的肺切片来了解肺损伤和修复

近年来，细胞培养模型如气液界面培养和离体模型如类器官的研究进展促进了肺生物学的研究;然而，这些模型和代表肺部三维环境复杂性的工具之间存在差距。精确切割的肺切片(PCLS)模拟体内环境，弥补了体外和体内模型之间的差距。我们建立了酸损伤和修复(AIR)模型，用盐酸滴剂联合Pluronic凝胶损伤组织的空间限制区域。损伤和修复通过免疫荧光检测，使用强大的标记物，包括用于细胞增殖的Ki67和用于肺泡2型/祖细胞的前表面活性蛋白C。重要的是，AIR模型可以在不需要初始体内损伤的情况下研究小鼠肺组织的损伤和修复，并且结果具有高度可重复性。在这里，我们提出了生成PCLS和AIR模型的详细协议。我们还描述了通过免疫染色建立早期修复标记和荧光成像来分析和量化AIR-PCLS损伤的方法。这种新型离体模型是研究急性肺损伤肺细胞生物学和半高通量筛选潜在治疗方法的多功能工具。©2020 Wiley期刊公司基本方案1:生成精确切割的肺切片基本方案2:酸损伤和修复模型基本方案3:分析空气- pcls:免疫染色和成像

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Current protocols in mouse biology Agricultural and Biological Sciences-Animal Science and Zoology

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet, all too often, nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. The aim of Current Protocols in Mouse Biology is to provide the clearest, most detailed and reliable step-by-step instructions for protocols involving the use of mice in biomedical research. Written by experts in the field and extensively edited to our exacting standards, the protocols include all of the information necessary to complete an experiment in the laboratory—introduction, materials lists with supplier information, detailed step-by-step procedures with helpful annotations, recipes for reagents and solutions, illustrative figures and information-packed tables. Each article also provides invaluable discussions of background information, applications of the methods, important assumptions, key parameters, time considerations, and tips to help avoid common pitfalls and troubleshoot experiments. Furthermore, Current Protocols in Mouse Biology content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Mouse Biology brings together resources in mouse biology and genetics and provides a mouse protocol resource that covers all aspects of mouse biology. Current Protocols in Mouse Biology also permits optimization of mouse model usage, which is significantly impacted by both cost and ethical constraints. Optimal and standardized mouse protocols ultimately reduce experimental variability and reduce the number of animals used in mouse experiments.