Primary human bronchial epithelial cells grown from explants.
Primary human bronchial epithelial cells grown from explants.
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从外植体中生长的原代人支气管上皮细胞。
DOI：
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发表时间：
2010
期刊：
影响因子：
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通讯作者：
M. Dolovich
中科院分区：
文献类型：
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作者：
A. Yaghi;A. Zaman;M. Dolovich
人类支气管上皮细胞是疾病的细胞模型和研究辅料和药物对人类上皮细胞功能和结构的影响所必需的。在这里，我们详细描述了从支气管气道组织中生长支气管上皮细胞的方法，这些组织是由外科医生在肺部手术（例如肺癌或肺减容手术）时收集的。在获得伦理许可和知情同意后，外科医生根据病理需要，为我们提供远离病变区域的支气管部分。然后将该组织用作外植体的来源，用于培养原代支气管上皮细胞。用冷EBSS冲洗长0.5-1cm、直径<或=1cm的支气管段，去除多余的实质组织。切片被切开，切成2-3mm(3)的组织片。这些碎片被用作原代细胞的来源。用胶原蛋白（30 μ g/ml）、纤维连接蛋白（10 μ g/ml）、BSA （10 μ g/ml）组合包覆100mm培养板1-2小时后，在培养板上划痕4-5处，将组织片置于划痕处，然后加入适合上皮细胞生长的培养基（含添加剂的DMEM/Ham F-12），将培养板置于37℃5% CO(2)湿化空气培养箱中。每3-4天更换一次培养基。上皮细胞在3-4周内从碎片中生长出来，形成直径约1.5 cm的环。通过将外植体移到新的预涂膜板上，外植体可以重复使用多达6次。细胞用胰蛋白酶/EDTA提取，汇总，计数，并重新镀于T75细胞结合瓶中以增加细胞数量。在T75烧瓶中接种2-3百万个细胞，4周内达到80%的融合度。扩增的原代人上皮细胞可在气液界面上培养并分化。本文描述的方法提供了从新鲜分离的组织中获得的丰富的人支气管上皮细胞来源，并允许将这些细胞作为疾病模型进行研究，以及进行药理学和毒理学筛选。
Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and < or =1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm(3) pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 microg/ml), fibronectin (10 microg/ml), and BSA (10 microg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37 degrees C in 5% CO(2) humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.