Mem alpha

Bone marrow was isolated from femurs and tibias of 8 Sprague-Dawley’s rats. MSC were isolated by plastic adhesion, amplified up to 80–90% confluence in a complete medium containing MEM alpha (Biological Industries) supplemented with 10% fetal calf serum (FCS, HyClone) + 1% penicillin streptomycin (Gibco) and 1 ng/mL of bFGF (Peprotech), then pooled at the same ratio for each donor. The cells were frozen in MEMα + 10% Albumin (Vialebex, LFB) + 1% penicillin streptomycin + 10% Dimethylsulfoxide (Sigma Aldrich) and finally preserved in liquid nitrogen. For in vivo experiments, MSC were thawed and seeded at 3000 to 5000 cells/cm². Medium was changed one day after thawing. When MSC reached 90 to 100% of confluence, they were harvested using trypsine-EDTA (Gibco) and suspended in a sterile syringe at a concentration of 2.10⁶ cells/mL of Ringer lactate solution (Fresinus).

Human PDLSCs were isolated from the periodontal tissues of premolars extracted from the Department of Oral Maxillofacial Surgery, School of Stomatology, Shandong University (Jinan, China). Primary PDLSCs were obtained from periodontal tissues which were cut into 2 mm² pieces. PDLSCs were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ in MEM-alpha (Biological Industries, Israel) with 10% fetal bovine serum (FBS, Lonsera, China) at 37 °C in a humidified atmosphere with 5% CO₂. Selected surface markers of the PDLSCs, including CD31, CD34, CD45, CD90, CD146, and Stro-1, were analyzed by flow cytometry.

Human-bone-marrow-derived mesenchymal stem cells (hBM-MSCs) (ATCC, Manassas, VA, USA) were cultured in a dedicated complete growth medium: MEM-alpha (Biological Industries, Beit HaEmek, Israel), supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin.

GC tissues were obtained from GC patients treated at the Affiliated People's Hospital of Jiangsu University (Zhenjiang, Jiangsu, China). The study protocol was approved by the Ethics Committee of Jiangsu University. Informed consent forms were obtained from all subjects. GCMSCs were isolated from human GC tissues as previously described 25 (link). Briefly, fresh GC tissues were cut into approximately 1 mm³-sized pieces, which were then adhered to 60 mm cell culture dishes (Corning, USA) and were cultured in MEM-ALPHA (Biological Industries, Israel) supplemented with 10% fetal bovine serum (FBS, Biological Industries) at 37°C with 5% CO_2. The culture medium was replaced every 3 days. When the fibroblast-like cells reached 85% confluence, the cells were trypsinized and expanded for up to five passages.
The human GC cell lines SGC-7901, MGC-803, HGC-27, and AGS were obtained from the Chinese Academy of Sciences Type Culture Collection Committee Cell Bank (Shanghai, China). SGC-7901, MGC-803, and HGC-27 were cultured in RPMI-1640 (Biological Industries) with 10% FBS. AGS were cultured in DMEM/F-12 (Biological Industries) with 10% FBS at 37°C with 5% CO_2.