Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Turin, Italy; POLITO BIOMedLAB, Politecnico di Torino, Turin, Italy; Interuniversity Center for the promotion of the 3Rs principles in teaching and research, Italy.
Department of Mechanical and Aerospace Engineering, Politecnico di Torino, Turin, Italy; POLITO BIOMedLAB, Politecnico di Torino, Turin, Italy; Interuniversity Center for the promotion of the 3Rs principles in teaching and research, Italy; CNR-IPCF, National Research Council-Institute for Chemical and Physical Processes, Pisa, Italy.
Biomater Adv. 2023 Nov;154:213620. doi: 10.1016/j.bioadv.2023.213620. Epub 2023 Sep 8.
Alveoli are the functional area of respiratory system where the gaseous exchanges take place at level of the alveolar-capillary barrier. The development of safe and effective therapeutic approaches for treating lung disease is currently limited due to the lack of realistic preclinical models for their testing and validation. In this work, tissue engineering approaches were exploited to develop a biomimetic platform that provide an appropriate mimicking of the extracellular environment and the multicellular architecture of human alveoli. Here, we propose the implementation of two biomimetic in vitro models to reproduce the features of the main anatomic portions of the physiological alveolar-capillary barrier. First, a co-culture barrier model was obtained by integrating an electrospun polycaprolactone-gelatin (PCL-Gel) membrane in a modified transwell insert (PCL-Gel TW) to mimic the alveolar basement membrane (coded as thin model). Alveolar epithelial (A549) and lung microvascular endothelial (HULEC-5a) cells were cultured on the apical and basolateral side of the PCL-Gel membrane, respectively, under physiologic air-liquid interface (ALI) conditions for 7 days. The ALI condition promoted the expression of type I and type II alveolar epithelial cell markers and the secretion of mucus in A549 cells. Increased cell viability and barrier properties in co-cultures of A549 and HULEC-5a compared to mono-cultures revealed the effectiveness of the model to reproduce in vitro physiological-relevant features of the alveolar-capillary barrier. The second portion of the alveolar-capillary barrier was developed implementing a tri-culture model (coded as thick model) including a type I collagen (COLL) hydrogel formulated to host lung fibroblasts (MRC-5). The thick barrier model was implemented by seeding HULEC-5a on the basolateral side of PCL-Gel TW and then pouring sequentially MRC-5-laden COLL hydrogel and A549 cells on the apical side of the electrospun membrane. The thick model was maintained up to 7 days at ALI and immunofluorescence staining of tight and adherent junctions demonstrated the formation of a tight barrier. Lastly, the ability of models to emulate pathological inflammatory conditions was validated by exposing the apical compartment of the PCL-Gel TW to lipopolysaccharide (LPS). The damage of A549 tight junctions, the increase of barrier permeability and IL-6 pro-inflammatory cytokine release was observed after 48 h exposure to LPS.
肺泡是呼吸系统的功能区域，在这里进行气体交换，位于肺泡-毛细血管屏障的水平。由于缺乏用于测试和验证的现实临床前模型，目前治疗肺部疾病的安全有效的治疗方法的发展受到限制。在这项工作中，利用组织工程方法开发了一种仿生平台，为人类肺泡的细胞外环境和多细胞结构提供了适当的模拟。在这里，我们提出实施两种仿生体外模型，以重现生理肺泡-毛细血管屏障的主要解剖部分的特征。首先，通过将静电纺丝聚己内酯-明胶（PCL-Gel）膜集成到改良的 Transwell 插入物（PCL-Gel TW）中，获得共培养屏障模型，以模拟肺泡基底膜（编码为薄模型）。肺泡上皮（A549）和肺微血管内皮（HULEC-5a）细胞分别在 PCL-Gel 膜的顶侧和基底外侧进行培养，在生理气液界面（ALI）条件下培养 7 天。ALI 条件促进了 A549 细胞中 I 型和 II 型肺泡上皮细胞标志物的表达和粘液的分泌。与单核培养相比，A549 和 HULEC-5a 的共培养中细胞活力和屏障性能的增加表明该模型能够有效地再现肺泡-毛细血管屏障的体外生理相关特征。肺泡-毛细血管屏障的第二部分通过实施三培养模型（编码为厚模型）来开发，该模型包括一种旨在容纳肺成纤维细胞（MRC-5）的 I 型胶原蛋白（COLL）水凝胶。通过将 HULEC-5a 接种在 PCL-Gel TW 的基底外侧，然后将负载 MRC-5 的 COLL 水凝胶和 A549 细胞依次注入到静电纺丝膜的顶侧，来构建厚模型。厚模型在 ALI 条件下维持长达 7 天，免疫荧光染色显示紧密和粘附连接形成了紧密的屏障。最后，通过将 PCL-Gel TW 的顶侧暴露于脂多糖（LPS）来验证模型模拟病理性炎症条件的能力。在 LPS 暴露 48 小时后，观察到 A549 紧密连接的损伤、屏障通透性的增加和促炎细胞因子 IL-6 的释放。
