Encapsulation and Viability of hMSCs in PEG Hydrogels

hMSCs, provided by the Tulane Center for Gene Therapy through a grant from NCRR of the NIH (grant P40 RR0 17 447) were used at passage 3 for all experiments. hMSCs were expanded using growth media (low-glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). hMSCs were encapsulated by resuspension in monomer solution containing a 5 wt% PEG and peptide in a stoichiometrically balanced ratio and 2.2mM (0.05 wt%) I2959 in PBS at a density of 300000 cells mL⁻¹. Suspensions, in 6mm × 1mm circular molds, were exposed to 7–10 mW cm⁻² 352-nm centered light (40W black-light blue lamp from Sankyo Deiki) for 5 min. Following polymerization, hydrogel discs were removed and placed into growth media. For morphology and viability experiments, gels were incubated for 30 min in PBS with 2µM calcein 4mM ethidium homodimer (Live/Dead cytotoxicity kit from Invitrogen). Confocal images were taken using a Zeiss 510 laser scanning confocal microscope. Image analysis and cell-area calculations were performed using MetaMorph software for 3D stacks flattened to 2D images. A minimum of three spots on three different hydrogels was used and standard error is reported relative to individual cells. A one-way ANOVA and Tukey’s test with α=0.05 were used to determine statistical differences among the data sets.