Amphotericin b

The explant cultures were prepared from ITB and ACL tissues. Surrounding the connective tissue, in the case of ACL, the synovial membranes were also removed. The pure ligament tissue was cut into 2–3 mm² slices and incubated in T-25 culture flasks with a growth medium (Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12, 1:1) containing 10% fetal calf serum (FCS), 1% penicillin-streptomycin, 2.5 µg/mL amphotericin B, 1% nonessential amino acids (all from Biochrom AG, Berlin, Germany), and 25 µg/mL ascorbic acid (Sigma-Aldrich) at 37 °C and 5% CO₂. After 1–2 weeks, the ITB and ACL fibroblasts started to migrate from the tissue slices (Graphical abstract). Subsequently, fibroblasts were harvested using 0.05% trypsin/0.02% EDTA (Biochrom AG) and expanded in T-75 and T-175 culture flasks (CellPlus, Sarstedt AG, Nümbrecht, Germany) (Figure 1E,F) for further characterization, spheroid, and scaffold cultures. The explants were cultured for 8–10 weeks. For analyzing the marker protein expression profile by immunocytochemistry, ITB as well as ACL fibroblasts derived from three different donors each were seeded on poly-l-lysin-coated cover slides at 1 × 10⁴ cells/cm². For the gene expression analysis, three human ITB and three human ACL samples obtained from different donors were cultured in a monolayer (passages 3–4).

MSCs were harvested and cultivated as published previously, following standardized protocols [58 (link),59 (link)]. Prior to use in the experiments, frozen cells were thawed at 37 °C and resuspended in culture medium. After centrifugation and aspiration of the supernatant medium, cells were expanded in cell culture flasks (Thermo Fisher Scientific, Dreieich, Germany) containing expansion medium (ESM; 83% Dulbecco’s modified Eagle’s medium high glucose, 14% fetal calf serum, 2 mM l-glutamine, 1% non-essential amino acids, 50 µM β-mercaptoethanol (all Thermo Fisher Scientific, Dreieich, Germany), 100 µg/mL penicillin/streptomycin, 2.5 mg/mL amphotericin B (both Biochrom, Berlin, Germany), and 4 ng/mL fibroblast growth factor 2 (Abcam, Berlin, Germany)). Cells in Passage 2 were used for the experiments.

To prepare inocula, tissue samples (prescapular lymph nodes, liver or kidney; 200 mg) were homogenized using a handheld microtube homogenizer, in which tissue in the microtube was triturated evenly using a plastic pestle by simple pushing in and pulling out the pestle in 1 ml of cold phosphate-buffered saline (PBS) with 2% penicillin and streptomycin and 5 µg/ml of amphotericin B (Biochrom) and incubated for 10 min at 4°C (52 (link)). Prepared tissue homogenates were frozen at −70°C and thawed to 4°C. Solid debris was pelleted by centrifugation at 7,000 rpm for 10 min at 4°C. Clarified homogenates were used as an inoculum, applied over different cell lines, incubated at 37°C, and observed daily for the development of cytopathic effect (CPE). Each inoculated cell line was subjected to three blind passages.

We acknowledge the gift of the pTRAF plasmid from Elias Arnér, Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. Colorectal carcinoma cell line HCT116 transformed with a pTRAF plasmid [18 (link),33 (link)] were cultured in Dulbecco’s modified Eagle’s (DMEM) supplemented with 10% fetal bovine serum at 37°C in 5% CO₂, 1% of penicillin/streptomycin and 0.1% of amphotericin B (Biochrom, United Kingdom). Cells were seeded in 96-well culture plates at a 1.0 x10⁴ cells/cm², and adhesion allowed for 24 hours. Portoamides were tested on a concentration-response assay (up to 10 μM) and incubated on an Incucyte® ZOOM Live-Cell Analysis System (Essen Instruments, Ann Arbor, MI). For the duration of 48h, Nrf2 activity was read at λex = 585 nm, emission filter: 625–705 nm, and auranofin (6 μM) was used as a positive control to confirm Nrf2 activity [18 (link)].

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.