Long Term Culture of the A549 Cell Line Promotes Differentiation Towards an Alveolar Type II (ATII) Phenotype; Comparison of A549 cells with Primary ATII Cells

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line. The A549 Cell Line was harvested in log phase growth. The gene expression profile of the triplicate cultures of A549 cells in this state was compared to cells from replicate primary alveolar type cultures obtained from macroscopically normal areas of lung tissue from a single donor with lung cancer undergoing lung resection surgery.

肺部研究需要能够模拟肺泡上皮生理学特征的模型，但由于原代细胞或干细胞在使用中存在可重复性、一致性以及技术与伦理层面的挑战，目前已广泛采用连续传代的癌细胞或其他永生化细胞系。A549“肺泡”细胞系问世已有四十余年，但学界对其作为原代肺泡II型（ATII, alveolar type II）细胞的合适模型的适用性尚未达成统一共识。鉴于多数针对A549细胞的研究均采用增殖细胞的短期培养方案，我们推测，能够抑制该癌细胞增殖的培养条件，可促进其向更成熟的ATII细胞表型转化。我们先检测了A549细胞在不同培养基中长期培养时的生长状态，随后通过微阵列分析（microarray analysis）探究了细胞周期与ATII分化相关通路的时序调控规律，并与新鲜分离的人源ATII细胞的基因表达情况进行了对比。分析结果显示，在Ham’s F12培养基中长期培养可显著调控细胞周期相关基因，最终得到静息细胞群体，该群体的自噬、分化及脂生成通路均出现显著上调。同时，该群体中与原代细胞共有的差异表达基因数量有所增加，提示其具备ATII细胞的特征并出现板层小体（MLB, multilamellar body）的形成。后续通过油红O染色（Oil Red-O staining）与透射电子显微镜（Transmission Electron Microscopy）检测，进一步证实了分化后的A549细胞确实表达板层小体。本研究明确了一套可在A549细胞中诱导ATII分化特征的培养条件，该条件或将为该细胞系的相关研究提供助力。本研究所用的A549细胞系取自对数生长期。我们将该状态下A549细胞的三次重复培养样本的基因表达谱，与从一名接受肺切除术的肺癌患者的肺组织肉眼观正常区域中分离得到的原代肺泡型细胞培养重复样本进行了对比。