Dmem ham s f12

Untransfected human brain neuroglioma H4 cells (H4, LGC Standards GmbH/ATCC, Wesel, Germany), H4 cells stably transfected with human APP 751 (H4 APP 751) or H4 APP 751 (see below) with and without bi-allelic deletion of CTSB via CRISPR/Cas9 (H4 APP754 CTSB –/–) were maintained in DMEM medium supplemented with 10% superior fetal bovine serum (FBS, Biochrom, Berlin, Germany), 100 IU/ml penicillin and 100 μg/ml streptomycin (Biochrom) with or without 500 μg/ml G418 (Thermo Fisher Scientific/Roche, Grenzach-Wyhlen, Germany) and/or 2 μg/ml Puromycin (Santa Cruz Biotechnology, Heidelberg, Germany) respectively. A complete change of the medium was performed every two to three days. For the assessment of Aβ in conditioned supernatants, the medium was changed to serum-free DMEM/Ham's F12 (Biochrom) with G5 supplement (Thermo Fisher Scientific/Gibco, Darmstadt, Germany) and 10 mM Hepes (Biochrom).

Human BMSCs were purchased from PromoCell GmbH, Heidelberg, Germany at passage two. They were cultured in flasks (COSTAR, Cambridge, USA) by using MSC growth medium with supplemented cytokines (PromoCell GmbH, Heidelberg, Germany), in an incubator with a humidified atmosphere maintained at 37°C and 5% CO₂. The media were changed twice weekly. At 80–90% confluence, cells were trypsinized (Trypsin/EDTA, PAA, Pasching, Austria) and cultured further as per the recommendation of the company. They were sub-cultured at 80% confluence until passage 5. For all the experimental protocols, passage 5 cells were trypsinized and used directly. Tri-lineage differentiation (osteogenic, adipogenic, chondrogenic) was demonstrated in 2D conditions for BMSCs prior to their use (Figure S1). The cells are found to be highly positive for CD73, CD90, CD105 and negative for CD14, CD20, CD34, CD45 after passage 5 [10] (link) using flow cytometry (FACS Calibur, BD biosciences, Heidelberg, Germany) using a human MSC phenotyping kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), as recommended by the International Society for Cellular Therapy (ISCT).
The scaffold-cell constructs and MSCs in 2D were cultured in basal media (DMEM/Ham’s F-12 (1∶1) with 10% fetal calf serum, 2 mg/L of L-glutamine -all purchased from Biochrom AG, Berlin, Germany).

Primary endometrial epithelial cell culturing was carried out as described before [18 (link),23 (link)]. Briefly, bovine uteri were collected from a local slaughterhouse, and small tissue pieces (approximately 0.1 cm²) from the area between the caruncles were collected. The collected tissue was minced with scalpels and then digested for 2 h at 37 °C in a solution containing 150 U/mL of collagenase, 150 U/mL of hyaluronidase, 200 U/mL of penicillin and 20 μg/mL of streptomycin (Sigma-Aldrich) in Hank’s balanced salt solution (Biochrom). Cells were then centrifuged, and the cell pellet was washed with cell culture medium (DMEM/Ham’s F-12 and 10% FBS, gentamicin and amphotericin B; all from Biochrom). The cells were seeded in 25 cm² flasks at 37 °C + 5% CO₂ for 18 h. During this time, fibroblast cells had already attached, while the medium containing non-attached cells was transferred to a new 25 cm² flask in order to obtain a pure (>99%) epithelial cell culture. Cells were transferred to a 75 cm² flask when a confluence level of >80% was achieved. Cells were allowed to grow and finally passaged into a 24-well plate at a final density of 2 × 10⁵ cells per well.

Stem-like glioma cells (SLGCs) were cultured in N-medium (DMEM/Ham’s F12 (Biochrom, Berlin, Germany) containing 20% BIT 9500 serum-free supplement (PELOBiotech, Planegg, Germany), 2% of a 200 mM Glutamine solution, 1% Amphotericin, 1% standard Penicillin/Streptomycin mix, and 20 ng/ml of recombinant human EGF and bFGF (Promo Cell, Heidelberg, Germany)). In order to induce differentiation, SLGC lines were transferred into DMEM/Ham’s F12 containing 10% FCS but lacking EGF and bFGF (referred to as F-medium). For limited dilution assays, 500–800 cells were plated in 96 well plates, as only 5–10% of the cells from our SLGC lines survive this condition. Typically, primary clones formed spheres, which were enzymatically dissociated, followed by stepwise propagation in larger formats. Before further use in research, the expression of stemness markers was monitored by immunocytochemistry.–The established human glioblastoma cell line U87-MG, referred to as U87 in the manuscript, and the established human colon cancer cell line CaCo2 were grown in DMEM (supplemented with 1% Amphotericin, 1% standard Penicillin/Streptomycin mix) containing 10% and 20% FCS, respectively. All cell cultures were incubated at 37°C in a water-saturated atmosphere in the presence of an air-carbon dioxide (5% CO₂) mixture.

HyClone Leibovitz L-15 was purchased from GE Healthcare Life Sciences, Marlborough, MA, USA, DMEM/Ham’s F12 and DMEM (4.5 g/L glucose) were purchased from Biochrom AG, Berlin, Germany, and RPMI, penicillin-streptomycin (P/S), and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) were purchased from Sigma-Aldrich, St. Louis, MO, USA. L-glutamine (L-glut), insulin-transferrin-selenium (ITS), epidermal growth factor (EGF), and fetal bovine serum (FBS) were purchased from Gibco™, Life Technologies, Carlsbad, CA, USA.
DON (Biopure, Romer Labs®, Tulln, Austria) was dissolved in distilled water (6.75 mmol/L) and further diluted with the respective media. DOM-1 (Biopure, Romer Labs®, Tulln, Austria) was obtained in acetonitrile (180.1 μmol/L), evaporated with nitrogen, and further diluted with the respective media. Contaminations of 0.1–0.2% DON and 3% iso-DOM-1 were detected via LC-MS/MS. The calculated solubility (Marvin Software Version 17.9.0, 2017, ChemAxon, (http://www.chemaxon.com)) in water at pH 7 was 14.51 mg/mL (~49 mmol/L) for DON and 11.15 mg/mL (~40 mmol/L) for DOM-1, thus, used concentrations were far below the solubility threshold.