Isolation and culturing of inferior turbinate stem cells (ITSCs)

Human nasal inferior turbinates were obtained by biopsy during routine surgery after informed consent according to local and international guidelines. The ethics board of the medical faculty of the University of Münster approved of all the procedures described in this article (No. 2012–015-f-S). All experiments were performed in accordance with these approved guidelines and regulations. ITSCs were isolated from adult human inferior turbinate tissue and pre-cultured as previously described by Hauser et al.²⁰. The isolated ITSCs were cultured within a 3D blood plasma matrix as described by Greiner et al.²¹. Blood plasma was kindly provided by the Institut für Laboratoriums- und Transfusionsmedizin (Herz- und Diabeteszentrum NRW, Bad Oeynhausen, Germany). Briefly, Dulbecco’s modified Eagle’s medium/Ham’s F-12 (1:1) (DMEM/F-12; Biochrom, Berlin, Germany) containing L-Glutamin (L-Glu; 200 mM; Sigma-Aldrich, St. Louis, USA), epidermal growth factor (EGF; 20 ng/mL; R&D Systems, Wiesbaden, Germany), basic fibroblast growth factor (bFGF/FGF-2; 40 ng/mL; lab-made) and B27 supplement²⁸ was applied, hereinafter referred to as standard medium. The cells were cultured in tissue culture flasks (TPP, Trasadingen, Austria) in standard medium containing 10% blood plasma at 37 °C, 5% O₂ and 5% CO₂ in a humidified incubator (Binder, Tuttlingen, Germany) and fed every two to three days. For passaging, collagenase I (Serva Electrophoresis, Heidelberg, Germany) was applied for one hour at 37 °C followed by harvesting by centrifugation at 300 × g (Universal 320, Hettich GmbH, Tuttlingen, Germany) and cultivation in standard medium with 10% blood plasma as described above.