Lung Institute, Department of Medicine.
Regenerative Medicine Institute, and.
Am J Respir Cell Mol Biol. 2022 Dec;67(6):623-631. doi: 10.1165/rcmb.2021-0507MA.
The epithelium lining airspaces of the human lung is maintained by regional stem cells, including basal cells of pseudostratified airways and alveolar type 2 (AT2) pneumocytes of the gas-exchange region. Despite effective techniques for long-term preservation of airway basal cells, procedures for efficient preservation of functional epithelial cell types of the distal gas-exchange region are lacking. Here we detail a method for cryobanking of epithelial cells from either mouse or human lung tissue for preservation of their phenotypic and functional characteristics. Flow cytometric profiling, epithelial organoid-forming efficiency, and single-cell transcriptomic analysis were used to compare cells recovered from cryobanked tissue with those of freshly dissociated tissue. AT2 cells within single-cell suspensions of enzymatically digested cryobanked distal lung tissue retained expression of the pan-epithelial marker CD326 and the AT2 cell surface antigen recognized by monoclonal antibody HT II-280, allowing antibody-mediated enrichment and downstream analysis. Isolated AT2 cells from cryobanked tissue were comparable with those of freshly dissociated tissue both in their single-cell transcriptome and their capacity for organoid formation in three-dimensional cultures. We conclude that the cryobanking method described herein allows long-term preservation of distal human lung tissue for downstream analysis of lung cell function and molecular phenotype and is ideally suited for the creation of an easily accessible tissue resource for the research community.
人类肺的气腔衬里上皮由区域干细胞维持，包括假复层气道的基底细胞和气体交换区域的肺泡 II 型（AT2）型肺细胞。尽管有有效的长期保存气道基底细胞的技术，但缺乏有效保存远端气体交换区域功能性上皮细胞类型的方法。在这里，我们详细介绍了一种从鼠或人肺组织中冷冻保存上皮细胞的方法，以保持其表型和功能特征。流式细胞术分析、上皮类器官形成效率和单细胞转录组分析用于比较从冷冻保存组织中回收的细胞与新鲜分离组织中的细胞。从酶消化的冷冻保存的远端肺组织的单细胞悬液中分离出的 AT2 细胞保留了泛上皮标记物 CD326 的表达以及单克隆抗体 HT II-280 识别的 AT2 细胞表面抗原，从而允许抗体介导的富集和下游分析。从冷冻保存组织中分离出的 AT2 细胞在其单细胞转录组及其在三维培养物中形成类器官的能力方面与新鲜分离的组织中的细胞相当。我们得出结论，本文描述的冷冻保存方法允许长期保存人类远端肺组织，用于下游肺细胞功能和分子表型分析，非常适合为研究界创建易于访问的组织资源。
