Air-liquid interface culture of mouse tracheal-bronchial epithelial cells_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

CHEN Si ¹ ^# , XIAO Hua ¹ ^# , ZHANG Wei ¹ , KONG Chen ¹ , CHEN Changming ² , Venkataramana Sidhaye ³ , LI Qiang ⁴ ,  BAI Chong ¹

1. Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of the Naval Medical University, Changhai Hospital, Shanghai 200433, P. R. China;
2. Department of Emergency Medicine, The 948^th Hospital of PLA, Wusu, Xinjiang 833000, P. R. China;
3. Division of Pulmonary and Critical Care Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA;
4. Department of Respiratory and Critical Care Medicine, Dongfang Hospital, Tongji University, Shanghai 200120, P. R. China;

Corresponding author：

BAI Chong, Email: baic_hx@hotmail.com

Keywords：

Airway surface liquid layer; Pseudostratified ciliated columnar epithelium; Air-liquid interface; Tracheal-bronchial epithelial cells; Primary culture; Trans-epithelium electrical resistance; Ciliary beat frequency

DOI：

10.7507/1671-6205.201903074

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Objective To establish a method of air-liquid interface culture and ciliary beat frequency measurement of mouse tracheal-bronchial epithelial cells to simulate the physiological function of airway epithelium.Methods BALB/c mouse tracheal-bronchial epithelial cells were obtained by digestion with 1 mg/mL protease in cold temperature overnight, and the digestion time was optimized to ensure the quantity and viability of the obtained cells. After removing fibroblasts by differential velocity adhesion method, the cells were cultured into collagen coated Transwell inserts. Proliferating phase and air-liquid interface culture were promoted with different culture media.Results Cell numbers obtained by cold protease overnight digestion for 12 h, 14 h and 16 h were (1.78±0.33)×10⁵, (1.93±0.26)×10⁵ and (2.01±0.28)×10⁵, respectively. Cell viability by trypan blue staining were (96.86±0.25)%, (94.73±1.63)% and (86.87±5.95)%, respectively. Cells were 100% confluent in Transwell chamber after 1-week proliferation, and the ciliary beat frequency was observed under microscope after 2 - 3 weeks of air-liquid interface culture. The cilia structure was confirmed by hematoxylin-eosin staining, electron microscopy and immunofluorescence. Ciliary beat frequency of the cells obtained by this method was consistent with that of mouse trachea in vivo, which further demonstrated its capacity in simulating the physiological function of airway epithelium. Conclusions The separation and air-liquid interface culture system as well as the ciliary beat frequency measurement method established in this experiment is simple, stable, efficient and reliable, which establishes a substantial foundation for exploring the pathogenesis and treatment mechanism of airway diseases. It can also provide reference for the culture of epithelium in the airway of other species and/or other organs.

Citation： CHEN Si, XIAO Hua, ZHANG Wei, KONG Chen, CHEN Changming, Venkataramana Sidhaye, LI Qiang, BAI Chong. Air-liquid interface culture of mouse tracheal-bronchial epithelial cells. Chinese Journal of Respiratory and Critical Care Medicine, 2019, 18(4): 362-368. doi: 10.7507/1671-6205.201903074 Copy

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3.	Rock JR, Onaitis MW, Rawlins EL, et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci U S A, 2009, 106(31): 12771-12775.
4.	You Y, Brody SL. Culture and differentiation of mouse tracheal epithelial cells. Methods Mol Biol, 2013, 945: 123-143.
5.	Delmotte P, Sanderson MJ. Ciliary beat frequency is maintained at a maximal rate in the small airways of mouse lung slices. Am J Respir Cell Mol Biol, 2006, 35(1): 110-117.
6.	文秀芳, 周向东. 气道上皮杯状细胞化生和分泌调控. 国际病理科学与临床杂志, 2008, 28(1): 59-63.
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16.	徐登城, 张德军, 王烨, 等. 小鼠呼吸道原代上皮细胞分泌三磷酸腺苷的特征研究. 西南国防医药, 2013, 23(9): 936-938.
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20.	田丹, 王曦, 陈文书, 等. GSK3 在人、鼠、猪的肺组织及培养的猪支气管上皮细胞中的表达. 中国组织化学与细胞化学杂志, 2005, 14(6): 598-602.
21.	祝洁, 朱颜鑫, 曹慧军, 等. 重组甲型流感病毒基质蛋白 1 和 2 通过 ERK 信号因子诱导小鼠气管上皮细胞产生 IFN-γ. 病毒学报, 2018, 34(5): 489-495.
22.	高元妹, 徐军. 小鼠气管上皮细胞的气液相界面培养. 广东医学, 2008, 29(2): 217-219.
23.	颜建辉, 黄丽娟, 刘剑萍, 等. RNA 干扰基因表达对哮喘小鼠支气管上皮细胞凋亡的影响. 基因组学与应用生物学, 2017, 36(9): 3516-3520.
24.	Franzdottir SR, Axelsson IT, Arason AJ, et al. Airway branching morphogenesis in three dimensional culture. Respir Res, 2010, 11(1): 162-171.
25.	Shamir ER, Ewald AJ. Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat Rev Mol Cell Biol, 2014, 15(10): 647-664.
26.	Zhu Z, Hofauer B, Heiser C. Improving surgical results in complex nerve anatomy during implantation of selective upper airway stimulation. Auris Nasus Larynx, 2018, 45(3): 653-656.
27.	Tan Q, Choi KM, Sicard D, et al. Human airway organoid engineering as a step toward lung regeneration and disease modeling. Biomaterials, 2017, 113: 118-132.
28.	Butler CR, Hynds RE, Gowers KH, et al. Rapid expansion of human epithelial stem cells suitable for airway tissue engineering. Am J Respir Crit Care Med, 2016, 194(2): 156-168.
29.	Hynds RE, Butler CR, Janes SM, et al. Expansion of human airway basal stem cells and their differentiation as 3D Tracheospheres. In: Clifton NJ. Methods in Molecular Biology. New York: Humana Press, 2016.

1. Ball M, Bhimji SS. Anatomy, Airway. Treasure Island (FL): StatPearls, 2018.
2. Davidson DJ, Gray MA, Kilanowski FM, et al. Murine epithelial cells: isolation and culture. J Cyst Fibros, 2004, 3(Suppl 2): 59-62.
3. Rock JR, Onaitis MW, Rawlins EL, et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci U S A, 2009, 106(31): 12771-12775.
4. You Y, Brody SL. Culture and differentiation of mouse tracheal epithelial cells. Methods Mol Biol, 2013, 945: 123-143.
5. Delmotte P, Sanderson MJ. Ciliary beat frequency is maintained at a maximal rate in the small airways of mouse lung slices. Am J Respir Cell Mol Biol, 2006, 35(1): 110-117.
6. 文秀芳, 周向东. 气道上皮杯状细胞化生和分泌调控. 国际病理科学与临床杂志, 2008, 28(1): 59-63.
7. 张涛, 王向东, 张罗. 跨膜蛋白 16A 在呼吸道黏膜上皮细胞的表达和功能研究. 国际耳鼻咽喉科头颈外科杂志, 2018, 42(2): 67-72.
8. Srinivasan B, Kolli AR, Esch MB, et al. TEER measurement techniques for in vitro barrier model systems. J Lab Autom, 2015, 20(2): 107-126.
9. Nieto MA. Epithelial-Mesenchymal Transitions in development and disease: old views and new perspectives. Int J Dev Biol, 2009, 53(8-10): 1541-1547.
10. Turley EA, Veiseh M, Radisky DC, et al. Mechanisms of disease: epithelial-mesenchymal transition--does cellular plasticity fuel neoplastic progression?. Nat Clin Pract Oncol, 2008, 5(5): 280-290.
11. Szymaniak AD, Mahoney JE, Cardoso WV, et al. Crumbs3-mediated polarity directs airway epithelial cell fate through the hippo pathway effector Yap. Dev Cell, 2015, 34(3): 283-296.
12. Joskova M, Sutovska M, Durdik P, et al. The role of ion channels to regulate airway ciliary beat frequency during allergic inflammation. Adv Exp Med Biol, 2016, 921: 27-35.
13. Raidt J, Wallmeier J, Hjeij R, et al. Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia. Eur Respir J, 2014, 44(6): 1579-1588.
14. Sedaghat MH, Shahmardan MM, Norouzi M, et al. Effect of cilia beat frequency on muco-ciliary clearance. J Biomed Phys Eng, 2016, 6(4): 265-278.
15. 刘海沛, 华丽, 刘全华, 等. 冷刺激诱导哮喘小鼠支气管上皮细胞炎症反应的研究. 临床和实验医学杂志, 2017, 16(11): 8-11.
16. 徐登城, 张德军, 王烨, 等. 小鼠呼吸道原代上皮细胞分泌三磷酸腺苷的特征研究. 西南国防医药, 2013, 23(9): 936-938.
17. 段慧菡, 蒋义国. 苯并[a]芘暴露的小鼠原代支气管上皮细胞中 miR-320 对细胞周期的影响. 环境与健康杂志, 2010, 27(5): 386-389.
18. 高静韬, Ahmed Hegab, 黑田葵, 等. 几种影响小鼠气管上皮基底干细胞体外增殖及分化潜能因素的研究. 国际呼吸杂志, 2017, 37(15): 1121-1131.
19. 王曦, 吴人亮, 郝春荣, 等. 吸烟小鼠呼吸道上皮细胞上皮钙粘附素表达的研究. 中国组织化学与细胞化学杂志, 1998, 7(3): 302-305.
20. 田丹, 王曦, 陈文书, 等. GSK3 在人、鼠、猪的肺组织及培养的猪支气管上皮细胞中的表达. 中国组织化学与细胞化学杂志, 2005, 14(6): 598-602.
21. 祝洁, 朱颜鑫, 曹慧军, 等. 重组甲型流感病毒基质蛋白 1 和 2 通过 ERK 信号因子诱导小鼠气管上皮细胞产生 IFN-γ. 病毒学报, 2018, 34(5): 489-495.
22. 高元妹, 徐军. 小鼠气管上皮细胞的气液相界面培养. 广东医学, 2008, 29(2): 217-219.
23. 颜建辉, 黄丽娟, 刘剑萍, 等. RNA 干扰基因表达对哮喘小鼠支气管上皮细胞凋亡的影响. 基因组学与应用生物学, 2017, 36(9): 3516-3520.
24. Franzdottir SR, Axelsson IT, Arason AJ, et al. Airway branching morphogenesis in three dimensional culture. Respir Res, 2010, 11(1): 162-171.
25. Shamir ER, Ewald AJ. Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat Rev Mol Cell Biol, 2014, 15(10): 647-664.
26. Zhu Z, Hofauer B, Heiser C. Improving surgical results in complex nerve anatomy during implantation of selective upper airway stimulation. Auris Nasus Larynx, 2018, 45(3): 653-656.
27. Tan Q, Choi KM, Sicard D, et al. Human airway organoid engineering as a step toward lung regeneration and disease modeling. Biomaterials, 2017, 113: 118-132.
28. Butler CR, Hynds RE, Gowers KH, et al. Rapid expansion of human epithelial stem cells suitable for airway tissue engineering. Am J Respir Crit Care Med, 2016, 194(2): 156-168.
29. Hynds RE, Butler CR, Janes SM, et al. Expansion of human airway basal stem cells and their differentiation as 3D Tracheospheres. In: Clifton NJ. Methods in Molecular Biology. New York: Humana Press, 2016.

Chinese Journal of Respiratory and Critical Care Medicine

Air-liquid interface culture of mouse tracheal-bronchial epithelial cells

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