Dmem ham s f12

Human gingival fibroblasts were purchased from Provitro AG (HFIB-G, Berlin, Germany). Fibroblasts were maintained in DMEM/Ham’s F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS (fetal calf serum; Bovine Calf Serum Iron Supplemented, VWR International, Radnor, PA, USA) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37 °C in 5% CO₂. Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins, as described earlier [31 (link)]. In short, HUVECs were retrieved by Collagenase H digestion (Roche Diagnostics, Mannheim, Germany) of perfused human umbilical veins and seeded into cell culture plates with a growth area of 25 cm² coated with 1% gelatin. HUVECs were cultured in specialized medium for endothelial cells (Endopan 3, PAN-Biotech GmbH, Aidenbach, Germany) and antibiotics. Cells were not used for experiments for longer than passage 10. Their typical morphology was closely monitored to exclude contamination and undesired effects due to cellular senescence. Tests to exclude mycoplasma contamination (Venor^® GeM Classic, Minerva Biolabs GmbH, Berlin, Germany) were carried out regularly.

The established and characterized BCEC line [52 (link)] was used in the study. BCECs were isolated from maternal placentomes of a pregnant cow [52 (link)]. Cells were stored in cryotubes at −150 °C at the Institute for Anatomy, University of Veterinary Medicine Hannover, Foundation, Germany. For cultivation, cells were thawed and seeded into T75 flasks (TPP Techno Plastic Products AG, Transadingen, Switzerland) filled with Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F12 (Sigma-Aldrich^® Chemie GmbH, Taufkirchen, Germany). DMEM was supplemented with 10% fetal calf serum (FCS) (Biochrom, Berlin, Germany), Penicillin (100 µU/mL, PAA, Coelbe, Germany), Streptomycin (2 µg/mL, PAA, Coelbe, Germany) and L-Glutamine (2 mM, PAA, Coelbe, Germany). This culture medium will be called full medium (FM) in the following. Cells were cultivated in an incubator at 5% CO₂ and 37 °C, and culture medium was replaced every other day. Subculturing was performed at 90% confluency via Trypsinisation for 5 min at 5% CO₂ and 37 °C using 0.5% Trypsin (Sigma-Aldrich^® Chemie GmbH, Taufkirchen, Germany) in PEM (EDTA 2 mM in phosphate-buffered saline (PBS)). For the experiments, cells were prepared individually. More detailed information will follow in the respective chapters. Cells between cell passage 18 and 32 were used in the experiments.

Human thymidine-kinase-negative osteosarcoma cells, 143B (TK–) were a gift by Dr. David Tscharke (John Curtin School of Medical Research, ANU), and human HCC1806 breast cancer cells were a gift by Dr. Jeff Holst (Centenary Institute, Sydney, NSW, Australia). Both cell lines were either cultured in DMEM/Ham’s F12 (Sigma 6124 supplemented with 2-mM glutamine) or in BME medium (Thermo 21010) supplemented with 10% dialyzed fetal bovine serum (FBS, Life Technologies), non-essential AAs (Table 1), and 0.5-mM sodium pyruvate at 37°C in a humidified atmosphere of 5% CO₂ in air. For sub-culturing, cells were detached by trypsinization (0.05 or 0.25% trypsin–EDTA, GIBCO). Cell counting was performed using a Scepter cell counter (Millipore, United States) or a hemocytometer. All complete cell culture media were supplemented with 2-mM L-glutamine (GIBCO). Cell viability after trypsinization was generally ≥95% as evaluated by trypan-blue exclusion.

Tumours from Pdgfrb-Cre, p53^R172H/R172H mice were freshly processed by rinsing in PBS and then mincing to ∼1 mm³ pieces using two scalpels. After transferring to a 15 ml Falcon tube containing 10 ml digestion media [maintenance medium omitting foetal bovine serum (FBS) and including 300 U/ml collagenase I (Worthington) and 100 U/ml hyaluronidase (Fisher)], cells were subjected to shaking incubation at 37°C for 18 h. After pelleting at 1300 rpm (340 g) for 5 min, cells were resuspended in 2 ml maintenance media [DMEM/Ham's F12 (Sigma-Aldrich), 10% FBS (Gibco), amphotericin (Gibco) and penicillin-streptomycin (Sigma-Aldrich)] before being transferred to a six-well plate. Cells were left undisturbed for 5-7 days in a humidified incubator at 37°C with 5% CO₂ before refreshing media and moving on to flasks when confluent. Tumours from Cdh5-CreER^T2, Trp53^fl/fl mice were manually minced using scalpels and left undisturbed for 5-7 days before transferring to flasks when confluent. Cells were maintained in Ham's F-12K Medium (Gibco), 10% FBS (Gibco), amphotericin B (Gibco), penicillin-streptomycin (Sigma-Aldrich), 0.1 mg/ml heparin (Sigma-Aldrich) plus 20 µg/ml endothelial cell growth supplement (Sigma-Aldrich, E2759) and checked routinely for mycoplasma infection.

The chondrogenic progenitor ATDC5 cells were cultured in DMEM:Hams F‐12 (1:1; Sigma‐Aldrich) supplemented with 1% penicillin/streptomycin (Gibco Laboratories, Gaithersburg, MD) and 10% FBS (Invitrogen, Carlsbad, CA) and cultured at 37 °C in a humidified incubator containing 5% CO₂. ATDC5 cells were then treated with rSPP1 (200 ng mL⁻¹, #763602; BioLegend, San Diego, CA) or anti‐CD44 antibodies (2.15 µg mL⁻¹, #C2368; Leinco Technologies, Fenton, MO) at 37 °C for 72 h.

A total of 10 uteri from mares in early luteal phase of the estrous cycle were used. The equine epithelial and stromal cells were isolated following the methodology recently described [22 (link)]. Cells were cultured at 38°C in a humidified atmosphere of 5% CO₂. The culture medium was Dulbecco's modified Eagle's medium/nutrient F-12 Ham (DMEM/Ham's F-12; Sigma-Aldrich, Madison, USA; D8900) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, Madison, USA; #C6278) and antibiotic and antimycotic solution (Sigma-Aldrich, Madison, USA; #A5955); it was changed every 2 to 3 days. After reaching 90 to 95% confluence (5 or 7 days of the incubation of stromal or epithelial cells, resp.), cells were trypsinized [22 (link)].Further, cells were seeded at a density of 5 × 10⁵ viable cells/mL for epithelial cells and 2 × 10⁵ viable cells/mL of stromal cells in 24 or 96-well plates, regarding the experiment. Both cell types viability were over 90%.The cell culture homogeneity was evaluated using immunofluorescent staining for epithelial and stromal cell specific markers (cytokeratin, vimentin, resp.) as described before [22 (link)]. The epithelial and stromal cell homogeneity was around 97%.

Collected amniotic fluid, at a 0.1–5 mL volume, was transferred from syringes to tubes under sterile conditions of a laminar chamber. The content of the syringes was inspected macroscopically for contamination, such as residual blood. Then, the tubes were centrifuged at 350×g for 10 min, the supernatant was discarded, and the precipitate was suspended in Dulbecco’s Modified Essential Medium (DMEM/Ham’s F12, Sigma, Germany) supplemented with 20% fetal bovine serum (FBS, Sigma, Germany), 10 ng bFGF (Sigma, Germany), and 1% antibiotic solution (penicillin/streptomycin, amphotericin B, Sigma, Germany). The viability of the cells was determined by the trypan blue test. Then, the cells were transferred to 35 mm Petri dishes. The dishes were incubated at 37°C with 5% CO₂ and constant humidity. The culture medium was replaced every 48 h. The cells were cultured until they reached confluence and then transferred to new Petri dishes.