Transferrin

Tracheal RECs were isolated from Lewis rats using methods we described previously (Zang et al., 2012 (link); Zang et al., 2013 (link)). Briefly, RECs were dissociated from the tracheal mucosal lining with 2 mg/mL pronase (Roche Applied Science, Indianapolis, IN) in Ham’s F-12 medium overnight (12 h) at 4°C. Cells were collected by centrifugation at 500 g for 10 min. Cell pellets were re-suspended in DMEM/Ham’s F-12 medium (Sigma) supplemented with 10 μg/mL insulin (Sigma), 0.1 μg/mL hydrocortisone (Sigma), 0.1 μg/mL cholera toxin (Sigma), 5 μg/mL transferrin (BD, Franklin Lakes, NJ), 50 μM phosphoethanolamine (Sigma), 80 μM ethanolamine (Sigma), 25 ng/mL epidermal growth factor (BD), 70 μg/mL bovine pituitary extract (United States Biological, Swampscott, MA), 3 mg/mL bovine serum albumin (Sigma), and 5 nM retinoic acid (Sigma). Epithelial cells at passage 0 were centrifuged with a Shandon Cytospin (GMI, Inc., Ramsey, MN) and stained with antibodies against cytokeratin peptide 17, beta tubulin IV, mucin 5AC, and P63 (all at 1:200; Sigma) to identify their components. We found that the percentage of cells expressing the markers of rat respiratory tract epithelial differentiation decreased during subculture. Because most cells became fibroblast-like cells in passage 2, we used only freshly isolated RECs for cell seeding in the ensuing experiments.

Collagen type I aqueous solution was kindly supplied by Nitta Gelatin Co. (Osaka, Japan). Human pulmonary fibroblasts (HPFs), fibroblast growth medium 2 (FGM2), and fetal bovine serum (FBS) were purchased from Takara Bio Inc. (Shiga, Japan). Cell Count Reagent SF and HEPES were purchased from Nacalai Tesque Inc. (Kyoto, Japan ). A mouse lung fibroblast cell line (Mlg2908), a human alveolar adenocarcinoma cell line (A549), and a mouse alveolar epithelial cell line (MLE-12) were purchased from ATCC (VA, USA). F12K media were purchased from ATCC (VA, USA). L-Glutamine, solution (200 mM) was purchased from (Thermo Fisher Scientific Inc., MA, USA). Insulin, sodium selenite and β-estradiol were purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan). A Transferrin was purchased from BD Japan (Tokyo, Japan). A hydrocortisone was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). A mixture of 10,000 U penicillin and 10 mg/mL streptomycin (Pen-Strep) and poly(vinyl alcohol) (PVA; average molecular weight: 85,000–124,000; 87%–89% hydrolysed) were purchased from Merck KGaA (Darmstadt, Germany). Dichloromethane (DCM), DMEM/Ham’s F-12 (DMEM/F12), PLGA-5005, and 4% paraformaldehyde in phosphate-buffered saline (4% PFA/PBS) were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Fragmin was purchased from Kissei Pharmaceutical Co. Ltd. (Tokyo, Japan).

For chondrogenic differentiation, a micromass culture system was used. MSCs (in 5 μl) at a centration of 1.6 × 10⁷ cells/ml were dropped in the centers of 24-well plates. The plates were placed in incubator at 37 °C, 5% CO₂ without culture medium for 2 hours. These cells were then cultured in chondrogenic induction medium (CIM), which is basal medium supplemented with 10 ng/ml transforming growth factor-β3 (R&D Systems), 500 ng/ml bone morphogenetic protein-2 (R&D Systems), 10^–7 M dexamethasone, 50 mg/ml ascorbate-2-phosphate, 40 mg/ml proline, 100 mg/ml pyruvate (all from Sigma-Aldrich), and 1:100 diluted ITS + Premix (6.25 mg/ml insulin, 6.25 mg/ml transferrin, 6.25 mg/ml selenous acid, 1.25 mg/ml bovine serum albumin, and 5.35 mg/ml linoleic acid) (Becton Dickinson). The chondrogenic medium was changed every 3 days.