238 Chapter 9 Table 7: Patient characteristics of the fibroblasts donors used in this study. *: tested with Mann-Whitney test. †: tested with Fisher’s exact test. ES: Ex-smoker, F: Female, IPF: Idiopathic pulmonary fibrosis, M: Male, NS: Non-smoker, Control IPF P value Age (median, (min-max)) 65, (59-72) 63.5, (61-68) 0.688 * Sex (M/F) 6/0 6/0 >0.999† Smoking status 5 EX, 1 NS 4 EX, 2 NS >0.999† Seeding primary lung fibroblasts in lung ECM-derived hydrogels Primary human lung fibroblasts (n = 6 for both control and IPF, all at passage 5) were harvested using 0.25% Trypsin-EDTA (Gibco) and centrifuged at 500 x g for 5 minutes. Afterwards, the supernatant was discarded and the pellet resuspended using 10 mL full complete growth media to count the cells using an automated cell counter NC-200 (Chemometec, Denmark). Then, the cell suspension was transferred to new tubes at a concentration of 2.5 x 106 cells per tube and centrifuged again. The supernatants were discarded and the pellets were resuspended using 2.5 mL pregel of each type. After ensuring the proper dispersal of the cell pellet in the pre-gel solution, 200 μL cell suspension was cast per well of 48-well plates and incubated for 2 hours. For each experimental set, empty hydrogels from control or IPF pregels were also cast and used as fibroblast-free controls. After observing hydrogel formation, the empty and fibroblast-seeded hydrogels were supplemented with 400 μL complete growth media for the culture period. The gels were incubated for 7 and 14 days with complete growth media in a CO2 incubator (5% CO2, 37°C), with half change of growth media at days 4, 7 and 11. Paraffin Embedding, Sectioning, and Deparaffinization ECM-derived hydrogels (both fibroblast-seeded and empty) were fixed using 500 μL 2% paraformaldehyde (PFA; Sigma-Aldrich) for 30 minutes at room temperature after removing the growth media from the wells at the end of 7- or 14-day culture period. Afterwards, they were embedded in 1% agarose (Invitrogen, Waltham, MA, USA) solution to prevent dehydration. Agarose-embedded hydrogels then were fixed with 4% formalin and embedded in paraffin. Five μm sections from the paraffinembedded samples were cut and placed on Star Frost (Knittel Glass, Braunschweig, Germany) glass slides and incubated at 65°C for 1 hour to ensure retention of the sections on the slides. Then, the slides were deparaffinized using serial ten minute incubations in xylene (Klinipath BV, Duiven, Netherlands) solution, 100% ethanol (Fresenius Kabi), 96% ethanol, 70% ethanol and distilled water.
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