Bronchial Epithelial Cell Culture Protocol

TPU Estane 58311 NAT 028 (Brussel, Belgium) and PLA NatureWorks^®, 2003D were purchased from NatureWorks LLC, Plymouth, MI, USA, with a specific gravity of 1.24 and melt index of 5.0–7.0 g/10 min (2.16 kg loads at 210 °C). The cell line normal human bronchial epithelial (BEAS-2B) cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). BEAS-2B is the most common cell line used to evaluate tracheal scaffolds in vitro [48 (link),49 (link),50 (link)]. The cells were cultured in alpha minimum essential medium (α-MEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS) (Thermo Fischer Scientific, Waltham, MA USA) and 1% penicillin/streptomycin (Sangon, Shanghai, China). Cell growth was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide test, also known as MTT assay (Sigma Aldrich, St. Louis, MI, USA). Other materials used were Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA), KaryoMAX^® Colcemid^TM (Thermo Fischer Scientific, Waltham, MA USA), ketamine/xylazine cocktail (Troy Laboratories Pty Limited, Glendenning, Australia), xylene (Bendosen), Harris haematoxylin (Sigma-Aldrich, Gillingham, UK), ammonia water (Merck), eosin (Sigma-Aldrich, Gillingham, UK), and hexamethyldisilazane (HMDS) (Sigma-Aldrich, Gillingham, UK).