从外植体生长的首要人权支气管上皮细胞
Summary
在这里，我们描述一个人支气管气道组织的外植体，包括气 – 液界面上的区别的增长为发展初级人支气管上皮细胞的具体方法。这种方法提供了一个丰富的原代细胞源调查在人的肺部健康和疾病的气道上皮细胞的作用。
Abstract
人支气管上皮细胞需要为疾病的细胞模型，并探讨人类上皮细胞的功能和结构上的辅料和药物制剂的效果。在这里，我们详细描述了支气管气道组织是由外科医生在肺部手术（如肺癌或肺减容手术）的次收获的支气管上皮细胞生长的方法。伦理委员会批准和知情同意，医生拿了什么是需要病理，并为我们提供了一个支气管部分，是从病变区域的远程。该组织是作为一个日益增长的文化原发性支气管上皮细胞，可用于使用的植源。支气管段约0.5 – 1cm长和≤直径1cm冲洗用冷水EBSS和除去多余的实质组织。段切开，成2 – 3mm的肉末<sup> 3</sup>组织块。这些作品是用来作为原代细胞源。经过1-2小时100毫米文化涂层板的胶原蛋白相结合（30微克/毫升），纤维连接蛋白（10微克/毫升），与牛血清白蛋白（10微克/毫升），在4-5个地区和组织的钢板被划伤件放置在划痕地区，然后加入培养基（DMEM / HAM用添加剂的F – 12）适用于上皮细胞的生长和板被放置在一个孵化器在37 ° C，5％CO<sub> 2</sub>加湿空气。改变培养基每隔3-4天。上皮细胞的生长，形成约1.5厘米，直径3-4周环件。植，可重新使用多达6次转移到新的预涂层钢板。细胞被取消，使用胰蛋白酶/ EDTA，汇总，计算，在T75细胞绑定烧瓶中重新镀金，以增加它们的数量。 T75烧瓶种子2-3万细胞生长在4个星期到80％汇合。扩大初级人类的上皮细胞是可以培养的气 – 液界面上区分。这里描述的方法提供了丰富的新鲜分离的组织的人支气管上皮细胞源，并允许作为疾病模型，为研究这些细胞和药理学和毒理学筛选。
Protocol
Discussion
在这项研究中，我们提出了详细的文化和扩大初级人支气管上皮细胞的方法。我们演示了如何植和支气管组织，促进上皮细胞生长的媒体培养，移植可以提供连续无差异（淹没）和差异（气 – 液界面上的）的细胞模型研究了人类呼吸道上皮细胞源。在药物检测系统，更接近其真正的生理环境中的细胞，这些细胞可以利用。这是非常重要的，你看从植/移植的细胞生长，并确认的鹅卵石形态。如果细…
Acknowledgements
作者非常感谢理查德Inculet博士提供的支气管组织。获得圣若瑟医疗汉密尔顿和西安大略/伦敦健康科学中心（博士大卫马克），组织和档案馆委员会，病理科大学研究伦理委员会的批准。我们也感谢提供一些对免疫所需的材料提供电信设备制造商，我们的ALI的文化和丹妮拉法卡斯厄尼 – 斯皮策（电子显微镜，加拿大麦克马斯特大学）。这项工作是由一个从加拿大安大略省胸科协会座长期授予;阿斯玛亚吉博士FSORC奖学金，圣若瑟医疗保健，加拿大安大略省汉密尔顿，的支持。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Albumin from bovine serum, powder | Sigma | A4919 | Use for coating solution | |
| COLLAGEN TYPE I 0.1% Solution Sterile-filtered | Sigma | C8919 | Use for coating solution | |
| Fibronectin from human plasma | Sigma | F2006 | Use for coating solution | |
| Earle’s Balanced Salt Solution (EBSS, sterile) | Sigma | 28888 | Use for rinsing tissue and for coating solution | |
| DMEM/Ham F-12 | Sigma | D8437 | ||
| FBS | Sigma | F1015 | Inactivate, then aliquot and freeze (-20°C); use fresh when needed | |
| Antibiotic-Antimycotic Stabilized | Sigma | A5955 | Aliquot into 2 ml vials and freeze (-20°C); use fresh when needed | |
| Albumin solution from bovine serum | Sigma | A8412 | BSA | |
| Pituitary Extract Bovine | Sigma | P1476 | BPE | |
| Epidermal growth factor | Sigma | E9644 | EGF | |
| (±)-Epinephrine | Sigma | E1635 | ||
| Insulin | Sigma | I6634 | ||
| Tranferrin Human | Sigma | T8158 | ||
| Triiodo-L-thyronine | Sigma | T6397 | ||
| Hydorcortisone | Sigma | H0888 | ||
| Retinoic Acid | Sigma | R2625 | ||
| Trypsin | Sigma | T9935 | ||
| EDTA | Sigma | E6758 | EDTA | |
| Phosphate buffered saline | Sigma | P5368 | PBS | |
| 70% ethanol | Use for disinfecting and cleaning | |||
| 24 well Transwells | Corning | 3470 | ||
| Cell Culture Flasks (T75) CLLBND from Corning | Fisher Scientific | 05-539-104 | These flasks have a Cell Bind coating which promotes cell attachment and growth | |
| Monoclonal Anti-Cytokeratin, pan-FITC antibody | Sigma | F3418 | Permeabilization: 0.2% TRITON X-100/PBS; Use 1:250 dilution | |
| Antibody: E-Cadherin (H108) | Santa Cruz | sc-7870 | Do not permeabilize; Use 1:250 dilution and A21207 (Invitrogen) as secondary antibody | |
| Alexa Fluor 594 donkey anti-rabbit IgG | Invitrogen | A21207 | Use 1:400 dilution | |
| Antibody: Monoclonal mouse Anti- α-Smooth Muscle Actin-Cy3 | Sigma- Aldrich | C6198 | Permeabilization: 0.2% TRITON X-100/PBS; Use 1:100 dilution | |
| Antibody: Vimentin (RV202) | Santa Cruz | Sc-32322 | Permeabilization: 0.2% TRITON X-100/PBS; Use 1:100 dilution and T2402 (Sigma- Aldrich) as secondary antibody | |
| Rabbit anti-mouse TRITC | Sigma- Aldrich | T2402 | Use 1:400 dilution | |
| Antibody: CD31 or PECAM-1 (M-20) | Santa-Cruz | Sc-1506 | Do not permeabilize; Use 1:200 dilution and Donkey anti-goat IgG-FITC as secondary antibody | |
| Donkey anti-goat IgG-FITC | Santa-Cruz | Use 1:100 dilution | ||
| Vectashield Mounting medium with DAPI | Vector Laboratories | H-1200 | Refrigerate in the dark; stains nuclei and retains fluorescence during prolonged storage | |
| Vectashield Mounting medium | Vector Laboratories | H-1000 | Refrigerate in the dark; retains fluorescence during prolonged storage | |
| Hoechst Stain solution | Sigma | H6024 | Stains nuclei; use 1:10 dilution and Vectashield H-1000 |
Other requirements: incubator, biological safety cabinet (BSC), centrifuge, 100mm culture plates, sterile tubes (15 ml, 50 ml, and 2 ml), sterile pipette tips, scalpel handle and blades, small sharp scissors, lab coats and gloves. These can be obtained from your preferred suppliers.
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