Msc nutristem xf supplement

A bone marrow aspirate totaling 50 ml was obtained from the posterior superior iliac spine of 10 healthy donors (mean age 41.7 years, range 28 to 55 years; 10 males) undergoing bone marrow puncture. All participants provided written informed consent. The study followed the ethical guidelines and was approved by the Ethics Committee of Affiliated Cancer Hospital of Zhengzhou University (approval no. 2020239). Then, the marrow was mixed gently with heparin to prevent coagulation. After dilution with phosphate-buffered saline (PBS), the mixture was added to a tube containing the same amount of lymphocyte separation solution and centrifuged at 2000 rpm for 20 min. Then, the mononuclear cells in the white membrane were collected, washed and centrifuged twice with PBS. The mononuclear cells were cultured in culture medium (NutriStem MSC XF Basal Medium, Biological Industries, 05–200-1A; NutriStem MSC XF Supplement, Biological Industries, 05-201-1U) and incubated in a 5% CO₂, 37 °C incubator. After incubation for 48 h, the media was refreshed, and the nonadherent cells were discarded with the exchanged culture medium. The remaining adherent cells were MSCs.

The human bone marrow-derived mesenchymal stem cells (hMSCs) were purchased from the Cell Bank of the Chinese Academy of Sciences (SCSP-405; Shanghai, China) and cultured in complete medium containing 500 ml NutriStem®MSC XF Basal Medium (Biological Industries, Israel), 3 ml NutriStem^® MSCXF Supplement (Biological Industries, Israel) and 5 ml penicillin and streptomycin (ThermoFisher Scientific, Waltham, MA, United States) in an atmosphere of 5% CO₂ and 95% humidity at 37°C. The culture medium was changed every 2 days. Cells beyond passage 10 were not allowed for experiments. For osteoblast differentiation, hMSCs at an appropriate confluency were induced by the addition of 100 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μg/ml ascorbic acid. Cell experiments were repeated three times (n = 3).

Colony-forming unit-fibroblast (CFU-F) assay has been set up by coating the 6-well cell culture plates with 0.1% gelatin (Sigma, 9391) and seeding the BM-MSCs of the donors as to be 40 × 10³ cells/well. The plates were prepared as duplicates for all WT, HMZ and HTZ groups. The mix of MSC Nutristem XF medium (Biological Industries, 05-200-1A) and MSC Nutristem XF Supplement was freshened in every 3 days as prewarmed before using for 10 days. For colony detection, the colonies were stained with Giemsa stain stock solution (Sigma G5637-5G) diluted with 1X DPBS in 1:10 ratio. The cells in each well were washed with 1X DPBS twice. Ice cold 99.9% methanol (0 °C) was added for fixation for 10 min. After incubation time is over, methanol was aspirated and the plates were left uncovered to let the remaining methanol to evaporate. Giemsa working solution (1:10 diluted stock solution) was added at a volume of 2 mL to cover the surface of the cells in each well and incubated for 10 min at RT. The wells were washed with plenty of ddH₂O for 3 times until all the dye residues were removed. For the quantitation, the wells were imaged to calculate colony numbers and sizes by using ImageJ (v. 1.53a) and the results were statistically analyzed with GraphPad Prism.