Isolation and culture of primary equine tracheal epithelial cells

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• 作者：Workineh Shibeshi (1)
  Getu Abraham (1)
  Carsten Kneuer (1) (4)
  Christin Ellenberger (2)
  Johannes Seeger (3)
  Heinz-Adolf Schoon (2)
  Fritz R. Ungemach (1)
  
• 关键词：Primary epithelial cells ; Horse ; Trachea ; Cytokeratin
• 刊名：In Vitro Cellular & Developmental Biology - Animal
• 出版年：2008
• 出版时间：August 2008
• 年：2008
• 卷：44
• 期：7
• 页码：179-184
• 全文大小：341KB
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• 作者单位：Workineh Shibeshi (1)
  Getu Abraham (1)
  Carsten Kneuer (1) (4)
  Christin Ellenberger (2)
  Johannes Seeger (3)
  Heinz-Adolf Schoon (2)
  Fritz R. Ungemach (1)
  
  1. Institute of Pharmacology, Pharmacy and Toxicology, University of Leipzig, An den Tierkliniken 15, 04103, Leipzig, Germany
  4. Federal Institute for Risk Assessment, Thielallee 88-92, 14195, Berlin, Germany
  2. Institute of Pathology, University of Leipzig, An den Tierkliniken 33, 04103, Leipzig, Germany
  3. Institute of Veterinary Histology & Embryology, University of Leipzig, An den Tierkliniken 43, 04103, Leipzig, Germany
  

文摘

Culture of airway epithelial cells is a useful model to investigate physiology of airway epithelia and airway disease mechanisms. In vitro models of airway epithelial cells are established for various species. However, earlier published method for isolation and culture of equine tracheal epithelial cells requires significant improvements. In this report, the development of a procedure for efficient isolation, characterization, culture, and passage of primary equine tracheal epithelial cells are described. Epithelial cells were isolated from adult equine trachea by exposing and stripping the mucosal epithelium from the adjacent connective tissue and smooth muscle. The tissue was minced and dissociated enzymatically using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution for 2?h at 37° C. Cells were collected by sieving and centrifugation, and contaminating fibroblasts were removed by differential adhesion. This procedure resulted in a typical yield of 1?×-07 cytokeratin-positive epithelial cells per gram tracheal lining tissue. Viability was 95% by trypan blue exclusion and isolates contained approximately 94% cytokeratin-positive cells of epithelial origin. Cells seeded at a density of 6.9?×-04 cells/cm2 in serum-free airway epithelial cell growth medium formed monolayers near confluency within a week. Confluent cells were dissociated using dispase II and first passages (P1) and second passages (P2) were successfully established in serum-free medium. Collagen coating of tissue culture flask was not required for cell adhesion, and cultures could be maintained at the level of P2 over 30?d. In the present study, we could establish a high-yield protocol for isolation and culture of equine tracheal epithelial cells that can serve for in vitro/ex vivo studies on the (patho-)physiology of equine airway disease as well as pharmacological and toxicological targets relevant to airway diseases.