Cell culture flask

0.1 × 10⁶ cells were seeded into cell culture flasks (25 cm², Greiner bio-one) for gene expression studies, while 10,000 cells were seeded into 10 cm dishes for colony formation assays (CFA). Drugs were added after 24 h and remained on the cells for 24 h prior to protein or RNA extraction and 4 days prior to measuring of colonies in CFA. Stocks of Temsirolimus (Sigma) in ethanol and Simvastatin (Sigma) in DMSO were prepared. Final solvent concentrations after treatment was limited to 0.1% (vol/vol). Controls were treated with the solvent equivalent of the highest drug concentration.

Cell experiments were conducted using HCEC-B4G12, a subpopulation from the parental cell line HCEC-12 (Leibnitz Institute, Braunschweig, Germany). Cultivation of HCECs was performed in a mixture of HAM’s F12 and F199 culture medium (Lonza Group Ltd, Basel, Switzerland) supplemented with fetal bovine serum (15%, Gibco, Thermofisher Scientific, Waltham, MA, US), penicillin/streptomycin (1%), l-glutamine (2 mM), bFGF (2 ng/ml) and L-ascorbic acid 2-phosphate (0.3 mM, all purchased from Sigma Aldrich, Saint Louis, MO, USA)^{57 (link)}. Cells were kept in an incubator at 37 °C, 5% CO₂ and 21% O₂. HECEs were cultured in cell culture flasks (Greiner Bio-One International GmbH, Germany) until 80–90% confluency before the initial cell seeding.
The nanofibrous scaffolds were clamped into MINUSHEET® tissue carrier (Minucells and Minutissue, Bad Abbach, Germany) and placed in a 24-well plate. Before cell seeding, the scaffolds were washed for 30 min in ethanol (70%) and rinsed with PBS three times afterwards. Then, the scaffolds were incubated in 1 ml of medium and stored in the incubator for at least 12 h. On each scaffold, 20,000 cells were seeded yielding an initial cell density of about 700 cells per mm². The PTFE cylinder was removed at the earliest 6 h after cell seeding. Medium was replaced every second day, starting at day 1.

Leukocyte concentrates from freshly withdrawn peripheral blood of healthy adult human donors were provided by the Institute of Transfusion Medicine (University Hospital Jena, Germany). The experimental protocol was approved by the ethical committee of the University Hospital Jena. All methods were performed in accordance with the relevant guidelines and regulations. PBMC were isolated using dextran sedimentation and Ficoll-Histopaque 1077-1 (Sigma-Aldrich, Taufkirchen, Germany) centrifugation. PBMC were seeded in PBS containing 1 mM Ca²⁺ and 0.5 mM Mg²⁺ in cell culture flasks (Greiner Bio-one, Frickenhausen, Germany) for 1.5 h at 37 °C and 5% CO₂ for adherence of monocytes. For differentiation and polarization of monocytes towards M1- and M2-MDM, published criteria [27 (link)] were used. Thus, M1-MDM were generated by incubating monocytes with 20 ng/mL GM-CSF (Peprotech, Hamburg, Germany) for 6 days in RPMI 1640 supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine (Biochrom/Merck, Berlin, Germany), and penicillin-streptomycin (Biochrom/Merck), followed by 100 ng/mL LPS (Sigma-Aldrich) and 20 ng/mL IFN-γ (Peprotech) treatment for another 48 h. M2-MDM were obtained after differentiation of monocytes with 20 ng/mL M-CSF (Peprotech) for 6 days, and then with 20 ng/mL IL-4 (Peprotech) for additional 48 h of polarization.

Primary HUVECs (human umbilical vein endothelial cells) were isolated from human umbilical cord provided by Department of Gynaecology and Obstetrics of the University Hospital Aachen in accordance with the human subjects approval of the local ethics committee of the RWTH Aachen University Hospital (votum #EK 2067) after obtaining written consent. The approval includes the use of umbilical cord-derived cells for in vitro studies in tissue engineering. The umbilical cord was washed with 37°C Phosphate Buffered Saline (PBS) and was cannulated. 2.4 IU/mL Dispase-solution (Roche^®) was filled into the vein. The vein was incubated for 30 min at 37°C and 5% CO₂. Detached HUVECs were flushed with 37°C pre-warmed Phosphate Buffer Saline (PBS) into a 50 mL falcon tube (Falcon BD) and centrifuged at 500 g for 5 min. The supernatant was discarded and the cell pellet was resuspended in EBM-2 (Lonza^®) supplemented with EGM-2 SingleQuot Kit Suppl. & Growth Factors (Lonza^®) and 1% antibiotic/antimycotic solution (Gibco^®). The resuspended cells were plated into cell culture flasks (75 cm³, Greiner bio-one), pre-coated with gelatine (Gelatine Type B, Sigma) and were maintained in a humidified incubator at 37°C and 5% CO₂ and cultivated to 80% confluency. For all experiments, pooled cells from four different donors were used in passage four.

The stem cells were obtained from donors undergoing surgical treatment at either the Department of Cardiac Surgery or the Department of Oral Surgery at the University of Rostock. This study conforms to the Declaration of Helsinki, and all cell donors gave their informed written consent. The experimental protocol and further experiments were reviewed and approved by the Ethics Committee of the University of Rostock (No. A 2011 119 and No. A 2011 91).
Ca9-22 was provided by the Japanese Collection of Research Bioresources Cell Bank Osaka, Japan. The cells were cultured in DMEM supplemented with 10% FCS in cell culture flasks (Greiner Bio-one, Frickenhausen, Germany) (5% CO₂, 37°C).
HGiF were cultured in DMEM supplemented with 10% FSC (10% CO₂, 37°C). Isolation and culturing of hBMSC was performed as described by Gaebel et al.[18] (link), and hDFSC as described by Haddouti et al.[19] . The authenticity of the stem cells was confirmed by plastic adherence, and flow cytometric analysis (monoclonal antibodies against CD29, CD44, CD45, CD73 and CD90 using a FACS scan flow cytometer LSRII with CellQuest-Software, Becton Dickinson). Multipotency of the cells was shown through induction into osteoblasts, chondroblasts, and adipocytes as previously described [20] (link).