Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, Osaka, Japan.
Division of Special Care Dentistry, Osaka University Dental Hospital, Osaka, Japan.
Tissue Eng Part C Methods. 2023 Mar;29(3):95-102. doi: 10.1089/ten.TEC.2022.0184.
The respiratory tract is one of the frontline barriers for biological defense. Lung epithelial intercellular adhesions provide protection from bacterial and viral infections and prevent invasion into deep tissues by pathogens. Dysfunction of lung epithelial intercellular adhesion caused by pathogens is associated with development of several diseases, such as acute respiratory distress syndrome, pneumonia, and asthma. To elucidate the pathological mechanism of respiratory infections, two-dimensional cell cultures and animal models are commonly used, although are not useful for evaluating host specificity or human biological response. With the rapid progression and worldwide spread of severe acute respiratory syndrome coronavirus-2, there is increasing interest in the development of a three-dimensional (3D) lung model for analyzing interactions between pathogens and hosts. However, some models possess unclear epithelial polarity or insufficient barrier functions and need the use of complex technologies, have high cost, and long cultivation terms. We previously reported about the fabrication of 3D cellular multilayers using a layer-by-layer (LbL) cell coating technique with extracellular matrix protein, fibronectin (FN), and gelatin (G). In the present study, such a LbL cell coating technique was utilized to construct a human 3D lung model in which a monolayer of the human lower airway epithelial adenocarcinoma cell line Calu-3 cells was placed on 3D-cellular multilayers composed of FN-G-coated human primary pulmonary fibroblast cells. The 3D lung model thus constructed demonstrated an epithelial-fibroblast layer that maintained uniform thickness until 7 days of incubation. Moreover, expressions of E-cadherin, ZO-1, and mucin in the epithelial layer were observed by immunohistochemical staining. Epithelial barrier integrity was evaluated using transepithelial electrical resistance values. The results indicate that the present constructed human 3D lung model is similar to human lung tissues and also features epithelial polarity and a barrier function, thus is considered useful for evaluating infection and pathological mechanisms related to pneumonia and several pathogens. Impact statement A novel model of lung tissue was established. Using a layer-by-layer cell coating technique, a three-dimensional cultured lung model was constructed. The present novel model was shown to have epithelial polarity and chemical barrier functions. This model may be useful for investigating interaction pathogens and human biology.
呼吸道是生物防御的第一道防线。肺上皮细胞间的黏附提供了对细菌和病毒感染的保护，防止病原体侵入深部组织。病原体引起的肺上皮细胞间黏附功能障碍与多种疾病的发生有关，如急性呼吸窘迫综合征、肺炎和哮喘。为了阐明呼吸道感染的病理机制，常使用二维细胞培养和动物模型，但这些模型不能用于评估宿主特异性或人类生物学反应。随着严重急性呼吸综合征冠状病毒 2 的快速传播和全球蔓延，人们越来越关注开发用于分析病原体与宿主相互作用的三维（3D）肺模型。然而，一些模型上皮极性不明确或屏障功能不足，需要使用复杂的技术，成本高，培养时间长。我们之前报道了使用细胞外基质蛋白纤维连接蛋白（FN）和明胶（G）的层层（LbL）细胞包被技术构建 3D 细胞多层的方法。在本研究中，利用 LbL 细胞包被技术构建了一种 3D 人肺模型，即在由 FN-G 包被的人原代肺成纤维细胞组成的 3D 细胞多层上放置一层人下呼吸道上皮腺癌细胞系 Calu-3 细胞。构建的 3D 肺模型在孵育 7 天内保持均匀的上皮-成纤维细胞层厚度。此外，通过免疫组织化学染色观察到上皮层中 E-钙黏蛋白、ZO-1 和粘蛋白的表达。通过跨上皮电阻值评估上皮屏障完整性。结果表明，本研究构建的人 3D 肺模型与人体肺组织相似，具有上皮极性和屏障功能，因此可用于评估肺炎和几种病原体相关的感染和病理机制。
