Penicillin streptomycin

Human UC was obtained from full term deliveries (38–40 weeks) of healthy mothers after written informed consent, in accordance with the standards of the Hannover Medical School Ethics Committee and with the Helsinki Declaration of 1975, as revised in 1983. UC pieces (UCP) were prepared as previously described [19 (link)] and cultivated in MSC15 (MEM α, GlutaMAX™ Supplement, no nucleosides (Thermo Fisher Scientific, Waltham, MA, USA), 15% hAB-serum (C.C.Pro GmbH, Oberdorla, Germany), 1% penicillin/streptomycin (PAN-Biotech, Aidenach, Germany)) or human serum replaced by 5% platelet lysate (PL BioScience GmbH, Aachen, Germany). After approximately two weeks, the 12-wells (Sarstedt, Nuembrecht, Germany) contain around 0.5–1.2*10⁵ adherent cells. To expand cells from 12 to 6-well plates (Sarstedt) for subsequent analysis, we removed the UCP and passaged the MSC culture with trypsin (PAN-Biotech). The MSC monolayer cultures were maintained in MSC10 (MEM α, GlutaMAX™ Supplement, no nucleosides (Thermo Fisher Scientific), 10% hAB-serum (C.C.Pro GmbH), 1% penicillin/streptomycin (PAN-Biotech)) or with 5% platelet lysate (PL BioScience GmbH). Mesenchymal origin was confirmed by antibody staining and was characterized as CD34- (Biolegend, San Diego, USA), CD45- (Miltenyi, Bergisch Gladbach, Germany), CD73 + (Biolegend), CD90 + (Biolegend) and CD105 + (Miltenyi).

The Caco-2/PD7 clone was kindly provided by Edith Brot-Laroche (Unité de Recherches sur la Différenciation Cellulaire Intestinale, Villejuif Cedex, France) (Mahraoui et al., 1994 (link)). Caco-2/PD7 cells were maintained in high-glucose DMEM (PAN Biotech GmbH) supplemented with 20% (v/v) fetal bovine serum (FBS,Thermo Fisher Scientific, life technologies™, Darmstadt, Germany) and 1% penicillin/streptomycin (PAN Biotech, Aidenbach, Germany).
HeLa-GLUT4-myc-GFP cells were maintained in RPMI 1640 medium (2,000 μg/ml NaHCO₃, stable glutamine, low endotoxin) supplemented with 10% FBS, 1% penicillin/streptomycin and 1% G418 (PAN-Biotech, Aidenbach, Germany). All cells were grown at 37°C in a humidified atmosphere with 5% CO₂.

Human CS cell lines SW1353 (American Type Culture Collection, Manassas, VA, USA) and CAL-78 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany) were propagated in Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing stable glutamine, 1.2 g/L NaHCO₃, 5% fetal bovine serum, and 1% penicillin/streptomycin (SW1353) or RPMI 1640 containing 20% fetal bovine serum and 1% penicillin/streptomycin (all PAN Biotech, Aidenbach, Germany) in a humidified atmosphere at 5% CO₂ and 37 °C.

Human endothelial cell line HDMEC (Promocell, Heidelberg, Germany) were propagated in a humidified atmosphere at 5% CO₂ and 37 °C in ECGM-MV medium (Endothelial Cell Growth Medium MV 2; Promocell, Heidelberg, Germany) with the following medium supplements: Fetal Calf Serum 0.05 mL/mL; Epidermal Growth Factor 5 ng/mL, Basic Fibroblast Growth Factor 10 ng/mL; Insulin-like Growth Factor (Long R3 IGF) 20 ng/mL; Vascular Endothelial Growth Factor 165 0.5 ng/mL; Ascorbic Acid 1 μg/mL; Hydrocortisone 0.2 μg/mL. Further, 1% v/v penicillin/streptomycin (PAN Biotech, Aidenbach, Germany) was then added to this medium.
CCL-93 cells (ATCC, Manassas, VA, USA), the non-malignant fibroblast cells of cricetulus griseus, were propagated in a humidified atmosphere at 5% CO₂ and 37 °C in DMEM containing 4.5 g/L glucose with 10% v/v FCS and 1% v/v penicillin/streptomycin (all from PAN Biotech, Aidenbach, Germany).

The rat liver epithelial cell line WB-F344 and its tumorigenic counterpart WB-ras were chosen for this study [41 (link)]. Both were obtained from J. E. Trosko, Michigan State University, East Lansing, MI, USA. WB-ras cells are WB-F344 cells, which were transfected with the oncogene ras, enabling to compare the response of similar cell types with either normal or tumorigenic characteristics. Cells were cultured in low-glucose DMEM (#P04-01500), supplemented with 2 mM L-glutamine, 5% fetal calf serum and 1% penicillin/streptomycin (#P06-07300) (all purchased from PAN-Biotech GmbH, Aidenbach, Germany) in a humidified CO₂ (5%) incubator. A number of 4∙10⁵ cells were seeded in each well of a 12-well plate and incubated for 48 h to form confluent monolayers.
The human skin cell lines HaCaT and SK-MEL-28 were cultured in RPMI (#P04-17500, PAN-Biotech), supplemented with 2 mM L-glutamine, 8% fetal calf serum and 1% penicillin/streptomycin. Cells were seeded in 12-well plates and grown until a confluent monolayer was formed to conduct the experiments.