Cryo sfm freezing medium

JPCs derived from three patients were included in this study in accordance with the local ethical committee (approval number 074/2016BO2, 17.05.2016) and after obtaining written informed consent. Jaw periosteal tissue was cut in small pieces with a scalpel and incubated in DMEM/F12 (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% hPL (ZKT Tübingen gemeinnützige GmbH), 100 U/mL penicillin-streptomycin (Lonza, Basel, Switzerland), 2.5 µg/mL amphotericin B (Biochrom, Berlin, Germany), 50 µg/mL gentamicin (Lonza), and 10 µg/mL ciprofloxacin (Sigma-Aldrich, St. Louis, USA) for 1–2 weeks. Outgrowing cells were passaged using TrypLE Express (Thermo Fisher Scientific) and expanded and frozen in passage one using Cryo SFM freezing medium (Promocell, Heidelberg, Germany). From passage two onward, JPCs were grown in hPL5-medium (DMEM/F12 containing 5% hPL, 100 U/mL penicillin-streptomycin, and 2.5 µg/mL amphotericin B).

Normal human epidermal keratinocytes (nHEKs) derived from neonatal foreskin were purchased from Cell Systems and handled as previously described⁴⁸ . Briefly, nHEKs were grown in Dermalife K Medium Complete without TGFα (Cell Systems) in the presence of penicillin-streptomycin (100 µg.mL⁻¹). Cells were passaged using Trypkit (Cell Systems) for trypsinization and were frozen at different passages in Cryo-SFM freezing medium (Promocell). nHEKs were routinely used for experiments at passage P3 corresponding to approximatively 10 population doublings. Cells were seeded at a density of 5 000 cells per cm² either on 24 mm diameter high precision glass coverslips (#1.5 from Marienfeld) for high resolution confocal microscopy, on 12 mm diameter coverslips (#1.5 from ThermoScientific) for structured illumination microscopy or on 35 mm diameter glass bottom dishes (MatTek) for live-cell imaging. Surfaces were coated by incubation with either 17 mg.L⁻¹ fibronectin solution (VWR; low coating density) or 33 mg.L⁻¹ fibronectin (high coating density) for 30 min at 37 °C each in a total volume of 1.5 mL. Microscopic imaging was done 2 days after seeding, when cells were still not confluent (less than 20% confluency).