Human MSCs (Lonza; PT-2501; hMSC) were expanded in low glucose-DMEM growth medium supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (pen/strep). Cells between passage 5–7 were used for all the experiments.
To determine the optimal genipin concentration to crosslink cell-laden 3D CMA hydrogels, hMSCs were first cultured in 2D tissue culture plates and exposed to different concentrations of genipin (0 mM, 0.25 mM, 0.5 mM, 1mM, 5mM and 10 mM). Briefly, hMSCs were seeded in a 24-well plate at a density of 25,000 cells/cm2 and cultured for 24 h to allow for cell attachment. Following this, cells were incubated with different concentrations of genipin in 50 mM HEPES buffer for 1 h at 37 °C. Post genipin exposure, cells were washed three times and cultured in alpha-MEM culture medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 mM β-glycerophosphate for an additional 24 h. At the end of culture, cells were washed with 1x PBS and stained with calcein AM and ethidium homodimer (Life Technologies) for 15 min at 37 °C and imaged under a fluorescence microscope (Zeiss) to assess the effect of genipin concentration on hMSC viability.
To determine the optimal genipin concentration to crosslink cell-laden 3D CMA hydrogels, hMSCs were first cultured in 2D tissue culture plates and exposed to different concentrations of genipin (0 mM, 0.25 mM, 0.5 mM, 1mM, 5mM and 10 mM). Briefly, hMSCs were seeded in a 24-well plate at a density of 25,000 cells/cm2 and cultured for 24 h to allow for cell attachment. Following this, cells were incubated with different concentrations of genipin in 50 mM HEPES buffer for 1 h at 37 °C. Post genipin exposure, cells were washed three times and cultured in alpha-MEM culture medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 10 mM β-glycerophosphate for an additional 24 h. At the end of culture, cells were washed with 1x PBS and stained with calcein AM and ethidium homodimer (Life Technologies) for 15 min at 37 °C and imaged under a fluorescence microscope (Zeiss) to assess the effect of genipin concentration on hMSC viability.