BMSCs were isolated from bone marrow washouts as described previously [22 (link),23 (link),24 (link)]. In short, bone marrow was collected in a heparinized syringe and cells were fractioned on a Ficoll-Paque Plus density gradient (GE Healthcare Europe, Freiburg, Germany). The fraction of mononuclear cells containing the BMSCs was washed in PBS (Life Technologies, Darmstadt, Germany) and seeded in several 0.1% gelatin-coated (Sigma-Aldrich, Steinheim, Germany) T75 cell culture flasks (Nunc, Roskilde, Denmark).
Cells were cultivated in expansion medium composed of DMEM high glucose supplemented with 12.5% FCS, 1% penicillin/streptomycin, 1% L-glutamine (Life Technologies, Darmstadt, Germany), 1% non-essential amino acids (NEAA; Life Technologies, Darmstadt, Germany), 0.1% β-mercaptoethanol (Life Technologies, Darmstadt, Germany) and 4 ng/mL fibroblast growth factor 2 (Abcam, Cambridge, U.K.). To discard non-adherent cells, medium was changed after the first 24 h and subsequently twice weekly. Cells were passaged at 80% confluency and stored in liquid nitrogen until further use in passage 4.
Cells were cultivated in expansion medium composed of DMEM high glucose supplemented with 12.5% FCS, 1% penicillin/streptomycin, 1% L-glutamine (Life Technologies, Darmstadt, Germany), 1% non-essential amino acids (NEAA; Life Technologies, Darmstadt, Germany), 0.1% β-mercaptoethanol (Life Technologies, Darmstadt, Germany) and 4 ng/mL fibroblast growth factor 2 (Abcam, Cambridge, U.K.). To discard non-adherent cells, medium was changed after the first 24 h and subsequently twice weekly. Cells were passaged at 80% confluency and stored in liquid nitrogen until further use in passage 4.
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