1 glutamine

The method used for MSC culturing was previously described^{25 (link)}. Briefly, bone marrow was obtained from femoral bones of adult Sprague–Dawley rats or GFP-expressing Sprague–Dawley rats (W-Tg [CAG-GFP]184Ys), then diluted with 15 ml DMEM (Sigma) supplement, 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific Inc.), 2 mM 1-glutamine (Sigma), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Thermo Fisher Scientific Inc.), and incubated for 3 days in an atmosphere containing 5% CO₂ at 37 °C. Following confluence, adherent cells were detached with trypsin-ethylenediaminetetraacetic acid solution (Sigma) and subcultured at 1 × 10⁴ cells/ml. In our previous study, morphological features of the bone marrow-derived MSCs, including characteristically flattened and spindle-shaped cells that appeared as plastic adherent cells, were reported^{26 (link)}. We also performed a phenotypic analysis of surface antigens, which revealed cluster of differentiation (CD)45−, CD73+, CD90+, and CD106− on MSCs, and confirmed that MSCs gave rise to mesenchymal derivatives, including osteocytes, adipocytes, and chondrocytes^{27 (link),28} . For the present study, MSCs were used after three passages.

The Hela cell line was purchased from the Cell Resource Center in the Shanghai Institutes for Biological Sciences (SIBS, Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM/high glucose), fetal bovine serum (FBS), phosphate buffered saline (PBS), trypsin-EDTA, and penicillin/streptomycin were provided by Hyclone (Thermo Scientific, Waltham, MA, USA ). The Hela cell line was cultured in standard culture flasks using DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% 1-glutamine (Sigma-Aldrich, St. Louis, MO, USA). After the third generation, the cells were collected via trypsin, and then a cell suspension at a density of ∼ ${10}^6$ cells/mL was made.