Van den Bossche Sara, Ostyn Lisa, Vandendriessche Valerie, Rigauts Charlotte, De Keersmaecker Herlinde, Nickerson Cheryl A, Crabbé Aurélie
Laboratory of Pharmaceutical Microbiology, Ghent University, Ottergemsesteenweg 460, Ghent 9000, Belgium.
Centre of Advanced Light Microscopy, Ghent University, Ottergemsesteenweg 460, Ghent 9000, Belgium; Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ottergemsesteenweg 460, Ghent 9000, Belgium.
Eur J Pharm Sci. 2023 Nov 1;190:106567. doi: 10.1016/j.ejps.2023.106567. Epub 2023 Aug 24.
In vitro models of differentiated respiratory epithelium that allow high-throughput screening are an important tool to explore new therapeutics for chronic respiratory diseases. In the present study, we developed in vivo-like three-dimensional (3-D) models of bronchial epithelial cell lines that are commonly used to study chronic lung disease (16HBE14o, CFBE41o and CFBE41o 6.2 WT-CFTR). To this end, cells were cultured on porous microcarrier beads in the rotating wall vessel (RWV) bioreactor, an optimized suspension culture method that allows higher throughput experimentation than other physiologically relevant models. Cell differentiation was compared to conventional two-dimensional (2-D) monolayer cultures and to the current gold standard in the respiratory field, i.e. air-liquid interface (ALI) cultures. Cellular differentiation was assessed in the three model systems by evaluating the expression and localization of markers that reflect the formation of tight junctions (zonula occludens 1), cell polarity (intercellular adhesion molecule 1 at the apical side and collagen IV expression at the basal cell side), multicellular complexity (acetylated α-tubulin for ciliated cells, CC10 for club cells, keratin-5 for basal cells) and mucus production (MUC5AC) through immunostaining and confocal laser scanning microscopy. Results were validated using Western Blot analysis. We found that tight junctions were expressed in 2-D monolayers, ALI cultures and 3-D models for all three cell lines. All tested bronchial epithelial cell lines showed polarization in ALI and 3-D cultures, but not in 2-D monolayers. Mucus secreting goblet-like cells were present in ALI and 3-D cultures of CFBE41o and CFBE41o 6.2 WT-CFTR cells, but not in 16HBE14o cells. For all cell lines, there were no ciliated cells, basal cells, or club cells found in any of the model systems. In conclusion, we developed RWV-derived 3-D models of commonly used bronchial epithelial cell lines and showed that these models are a valuable alternative to ALI cultures, as they recapitulate similar key aspects of the in vivo parental tissue.
能够进行高通量筛选的分化呼吸道上皮体外模型是探索慢性呼吸道疾病新疗法的重要工具。在本研究中，我们构建了常用的支气管上皮细胞系的类体内三维（3-D）模型，用于研究慢性肺病（16HBE14o、CFBE41o和CFBE41o 6.2 WT-CFTR）。为此，将细胞接种在旋转壁式生物反应器（RWV）中的多孔微载体珠上进行培养，这是一种优化的悬浮培养方法，与其他生理相关模型相比，它允许进行更高通量的实验。将细胞分化情况与传统的二维（2-D）单层培养以及呼吸道领域当前的金标准即气液界面（ALI）培养进行比较。通过评估反映紧密连接形成（闭合蛋白1）、细胞极性（顶端侧的细胞间粘附分子1和基底细胞侧的IV型胶原表达）、多细胞复杂性（纤毛细胞的乙酰化α-微管蛋白、杯状细胞的CC10、基底细胞的角蛋白-5）和黏液分泌（MUC5AC）的标志物的表达和定位，在这三种模型系统中评估细胞分化情况，通过免疫染色和共聚焦激光扫描显微镜进行观察。结果通过蛋白质免疫印迹分析进行验证。我们发现，对于所有三种细胞系，紧密连接在2-D单层、ALI培养和3-D模型中均有表达。所有测试的支气管上皮细胞系在ALI和3-D培养中均表现出极化，但在2-D单层中未表现出极化。黏液分泌杯状样细胞存在于CFBE41o和CFBE41o 6.2 WT-CFTR细胞的ALI和3-D培养中，但在16HBE14o细胞中不存在。对于所有细胞系，在任何模型系统中均未发现纤毛细胞、基底细胞或杯状细胞。总之，我们构建了常用支气管上皮细胞系的基于RWV的3-D模型，并表明这些模型是ALI培养的有价值替代物，因为它们概括了体内亲本组织的类似关键特征。
