Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia.
Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium: a simple methodological approach for ex vivo studies of primary ciliary dyskinesia.
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DOI：
10.1186/s13630-017-0049-5
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发表时间：
2017-01-01
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影响因子：
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通讯作者：
Nielsen, Kim Gjerum
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文献类型：
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作者：
Marthin, June Kehlet;Stevens, Elizabeth Munkebjerg;Nielsen, Kim Gjerum
背景：三维外植体球体形成是以前用于研究呼吸道上皮离子和水运输的一种体外技术。移植的细胞和片状鼻上皮形成完全分化的球体，包裹着部分充满液体的管腔，纤毛尖面朝向外部，可用于纤毛功能分析。方法：我们对原发性睫状体运动障碍(PCD)患者和健康对照组鼻道上皮来源的球体中的纤毛搏动模式和纤毛搏动频率进行了两组比较研究。在第1天通过鼻刷活检取出鼻毛细胞和纤毛薄片，并使用高速视频成像进行纤毛搏动模式和频率的分析，以获得标准参考值。同一天，通过孵育来自不同刷检的细胞和薄片，在同一个体中启动了三维外植体球体的形成。对采集的球体进行分析，并将球体纤毛搏动模式和频率与第1天起相应的参考值进行比较。结果：在无血清培养液中球体形成速度快。在18个抽样个体中，有15个(82%)成功形成。因此，在7名健康对照组和8名PCD患者中，形成成功，而在3例因感染而发生PCD的患者中，形成失败。在收获球体之前，健康组培养的中位数(范围)为4(1-5)天，而PCD组为2(1-5)天。与相应的第1天标准参考值相比，椭圆形纤毛搏动的模式和频率没有变化。正常对照组(9.3赫兹)与PCD患者(2.4赫兹)之间的椭球纤毛搏动频率差异有非常显著意义(P
BACKGROUND: Three-dimensional explant spheroid formation is an ex vivo technique previously used in studies of airway epithelial ion and water transport. Explanted cells and sheets of nasal epithelium form fully differentiated spheroids enclosing a partly fluid-filled lumen with the ciliated apical surface facing the outside and accessible for analysis of ciliary function.METHODS: We performed a two-group comparison study of ciliary beat pattern and ciliary beat frequency in spheroids derived from nasal airway epithelium in patients with primary ciliary dyskinesia (PCD) and in healthy controls. Nasal ciliary cells and sheets were removed on day 1 by nasal brush biopsy and analyzed with regard to ciliary beat pattern-and frequency using high-speed video imaging for standard reference values. Three-dimensional explant spheroid formation was initiated in the same individual on the same day by incubation of cells and sheets from a separate brush biopsy. Harvested spheroids were analyzed earliest possible and values of spheroid ciliary beat pattern and frequency were compared to the corresponding reference values from day 1.RESULTS: Spheroids formed fast in serum-free culture medium. Formation was successful in 15 out of 18 (82%) sampled individuals. Thus, formation was successful in seven healthy controls and eight PCD patients, while unsuccessful in 3 with PCD due to infection. Median (range) number of days in culture before harvesting of spheroids was 4 (1-5) in healthy versus 2 (1-5) in PCD. Spheroid ciliary beat pattern and frequency were unchanged compared to their corresponding day 1 standard reference values. Spheroid ciliary beat frequency discriminated highly significant between healthy controls (9.3Hz) and PCD patients (2.4Hz) (P