Construction of human three-dimensional lung model using layer-by-layer method.
Construction of human three-dimensional lung model using layer-by-layer method.
复制标题
采用逐层法构建人体三维肺模型。
DOI：
10.1089/ten.tec.2022.0184
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Akashi M.
中科院分区：
文献类型：
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作者：
Akamatsu Y;Akagi T;Sumitomo T;Takahara Y;Akiyama S;Kawabata S*;Akashi M.
呼吸道是生物防御的前线屏障之一。肺上皮细胞间粘附提供保护免受细菌和病毒感染，并防止病原体侵入深部组织。由病原体引起的肺上皮细胞间粘附功能障碍与几种疾病的发生有关，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，通常使用二维细胞培养和动物模型，尽管对于评价宿主特异性或人体生物学反应没有用。随着严重急性呼吸综合征冠状病毒2型的迅速发展和全球范围内的传播，人们越来越感兴趣的是开发一个三维（3D）的体外模型来分析病原体和宿主之间的相互作用。然而，一些模型具有不清楚的上皮极性或屏障功能不足，需要使用复杂的技术，成本高，培养时间长。我们先前报道了使用细胞外基质蛋白、纤连蛋白（FN）和明胶（G）的逐层（LbL）细胞包被技术制造3D细胞多层膜。在本研究中，利用这种LbL细胞包被技术构建人3D肺模型，其中将单层的人下气道上皮腺癌细胞系Calu-3细胞置于由FN-G包被的人原代肺成纤维细胞组成的3D细胞多层上。由此构建的3D肺模型显示了上皮成纤维细胞层，其保持均匀的厚度直到孵育7天。免疫组化染色观察上皮细胞E-cadherin、ZO-1和粘蛋白的表达。使用跨上皮电阻值评价上皮屏障完整性。结果表明，本研究构建的人肺组织三维模型与人肺组织相似，具有上皮极性和屏障功能，可用于评价肺炎和多种病原体相关的感染和病理机制。影响声明建立了一种新的肺组织体外模型，采用逐层细胞包被技术，建立了一个三维培养的肺模型，该模型具有上皮细胞极性和化学屏障功能，可用于研究病原体相互作用和人类生物学。
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)in vitrolung 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 statementA novelin vitromodel 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.