L gln

Amino acid-free (AA−) medium was prepared by RPMI 1640 powder (R8999–04A, US Biological Life Science) and sodium phosphate dibasic (5.6 mM, the same concentration as commercially available RPMI 1640 medium, US Biological Life Science), supplemented with 10% (v/v) dialyzed FBS (Thermo Fisher Scientific). Amino acid-sufficient (AA+) medium was prepared by adding proper volumes of MEM amino acids solution (essential amino acids, EAA, 50×), MEM non-essential amino acids solution (NEAA, 100×), and 200 mM L-Gln (all from Sigma-Aldrich) to AA− medium to reach a final concentration of 1×EAA, 1×NEAA, and 2 mM Gln. The medium was supplemented with 10% (v/v) dialyzed FBS. Medium containing single amino acids (Ala, Leu, Gln, or Arg) or their combinations was prepared with AA– medium (prepared to the same concentrations present in the AA+ medium). All media were adjusted to pH7.5 and filter-sterilized (0.2 μm) before use.

The skin was isolated from the dorsal of the bovine fetus and rinsed 6–10 times in PBS, and digested for 12 h at 4°C using 0.25% collagenase type II. After rinsing the digested skin tissues 6–10 times in PBS, the epidermis tissues were gently scraped off, and rinsed 3–5 times in PBS with 1% (w/v) penicillin and streptomycin (Bioss). The remaining derma was cut into about 1 mm³ pieces using an ophthalmic scissors, and digested for 15 min at 37°C with 0.25% (w/v) Tyrisin (Gibco) [containing 0.01% (w/v) EDTA]. Then DMEM (Dulbecco's modified Eagle's medium) (Gibco) containing 10% (v/v) FBS (fetal bovine serum, Hyclone) was added to terminate the reaction. The cell suspension was centrifuged at 100 g for 8 min, the cells were resuspended with complete medium [(DMEM/F12+ 10% FBS +10 ng/ml bFGF (basic fibroblast growth factor, Peprotech)+2 mM/ml L-Gln (Sigma)] glutamine and seeded in a cell culture dish. Cells were cultured in a 5% (v/v) CO₂ incubator at 37°C for 2 h, and then the cell suspension was transferred to 6-well plates, and continued to culture at 37°C in 5% CO₂. When the cells reached 80–90% confluence, 0.25% trypsin and 0.02% EDTA were added to the digested cells and subcultured at a ratio of 1:1. The morphology and growth situation of cattle DMSCs was observed by an inverted microscope.

Primary culture follows published methods for chondrocytes^{38 (link),39 (link)}. In short, MCC was collected from 10–20 day old mice in CO2 independent collection medium (Gibco, 18045–088), washed in 1x sterile PBS supplemented with 25 mg/ml Plasmocin (InVivoGen, ant-mmp), 50 U/ml penicillin and 0.05 mg/ml streptomycin (Sigm, P0781) for 5 minutes, transferred to a 3 mg/ml type II collagenase (Worthington Biochemical, LS004174) in DMEM (Gibco, 11966–025) digestion medium for one hour, and then transferred to a 1 mg/ml collagenase digestion medium overnight in 5% CO2 at 37 °C. All cell medium and washes were supplemented with 25 mg/ml Plasmocin (InVivoGen, ant-mmp), 50 U/ml penicillin and 0.05 mg/ml streptomycin (Sigm, P0781), and 2mM _L-Gln (Sigma, G7513). After digestion, cells were dispersed by agitation with a serological pipette, filtered through a 40 µm cell strainer, centrifuged at 10,000 g for 15 minutes at room temperature, the pellet was re-suspended in supplemented sterile 1x PSB, and centrifuged again. The resulting pellet was re-suspended in 10 ml of FBS supplemented advanced DMEM (Gibco 12492–013). Cell density was calculated with a hemocytometer, and cells were plated at 10 × 10⁴ cells/cm² with a glass cover slip. Culture medium was replaced every two days until confluence at approximately 8 days.

BCLC9 cells were generated as previously described¹². Standard culture medium for BCLC9 cell lines is Dulbecco's Modified Eagle Medium (DMEM) high glucose (Sigma-Aldrich, St Louis, MO) and HAM F12 (Sigma-Aldrich, St Louis, MO) (1:1). Medium is supplemented with: 1% Na Pyruvate 100 mM (Sigma-Aldrich, St Louis, MO), 1% Pen/Strep 10000 U/mL (Lonza Group Ltd, Switzerland), 1% L-Gln 200 mM (Sigma-Aldrich, St Louis, MO), 1% Non-Essential Aminoacids (NEAA) (Lonza Group Ltd, Switzerland), 10% FBS (Life Technologies). BCLC9-miR122 and BCLC9-AKT3 KD culture medium is supplemented with neomycin (G418 disodium salt, Sigma-Aldrich, Germany; at a final concentration of 500 μgr/mL) and puromycin (Sigma-Aldrich, Germany; at a final concentration of 10 μgr/mL) respectively, once a week to keep selective pressure.