114 Chapter 5 Table 4: Characteristics of donors included in the study. Non-IPF (n=4) IPF (n=4) P value Sex 3M/1F 3M/1F >0.999# Smoking history 0.0285# Former 3 0 Never 1 4 Age (median (min-max)) 55 (36-58) 61 (27-68) 0.3143† FEV1% (Pred.) (median (min-max)) 96.5 (70-111) 42.0 (17-64) 0.0286† #Indicates p value as assessed by the Chi-square test. † indicates p value as assessed by the MannWhitney test. IPF: Idiopathic pulmonary fibrosis, FEV1 (pred.): Forced expiratory volume in 1 second (predicted). Tissue dissociation Unfractionated cell suspensions were obtained from parenchymal lung tissue without any visible airways present as previously described by Kruk et al [7]. Briefly, lung tissue was cut into small sections (1 cm3) and treated overnight at 4 °C with Trypsin/ EDTA (0.25%; Gibco, Waltham, MA), supplemented with 1% penicillin (100 U/mL) / streptomycin (100 µg/mL) (P/S) (Gibco), Collagenase A (2 mg/mL; Roche, Basel, Switzerland) and DNase (0.04 mg/mL; Sigma-Aldrich, Burlington, VT). Subsequently, EpCAM (CD326)+ epithelial progenitors were isolated from this unfractionated cell suspension by negative selection for CD31 and CD45 to deplete endothelial cells and hematopoietic cells [8], followed by a positive selection for CD326 using magnetic beads (human anti-CD31, human anti-CD45, human anti-CD326) (Miltenyi Biotec, Bergisch Gladback, Germany) according to manufacturer’s instructions. The cell suspensions were then resuspended in Small Airway Growth Medium (SAGM) (PromoCell, Heidelberg, Germany), counted manually and kept on ice until use. Organoid culture MRC-5 human foetal lung fibroblasts (ATCC, Manassas, VA) were cultured in Ham’s F12 medium (Gibco) containing 10% foetal bovine serum (FBS) (Sigma-Aldrich) and 1% P/S. When confluent, cells were treated with Mitomycin C (0.01 mg/mL) (SigmaAldrich) for 2 hours to inhibit proliferation. Subsequently, cells were trypsinized and resuspended in SAGM, counted manually and kept on ice until use. To generate organoids, unfractionated lung cell suspensions or EpCAM+ epithelial cells were mixed in a 1:1 ratio with MRC-5 cells. This cell mixture, diluted 2:1 with SAGM, was seeded in 100 µL Matrigel (8.4 mg/mL; Corning, New York, MA) on top of 6.5 mm Transwell inserts (0.4 µm pore size; Corning) and cultured for 7 days in SAGM medium
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