(582a) Investigating Glioblastoma Stem Cell Behaviors in Three-Dimensional Hyaluronic Acid Hydrogels
In this study, U87 cell line and patient derived D456 cells were cultured adherently (in the presence of serum) and/or as suspension culture (serum-free). To prepare HA hydrogels, HA was functionalized with methacrylate groups to form HA methacrylate. A 5% w/w solution of HA methacrylate was made in Neurobasal-A medium (for suspension cells) and Eagleâs Minimum Essential Medium (EMEM) with serum (for adherent cells), to which cells and cross-linker dithiothreitol (DTT) were added to encapsulate the cells in the resulting HA hydrogels. We observed that all the seeded single cells (serum grown U87, serum-free U87 and serum-free D456) expanded and formed spheres, and the size of the spheres increased with time (from day 7 to day 14) indicating the proliferation of GBM cells in the HA hydrogels. The addition of integrin binding peptide (RGD) in the HA hydrogels did not significantly affect the sphere sizes of GBM cells as compared to only HA hydrogels. Further, increasing the initial cell seeding density of serum-free U87 and D456 cells influenced the sphere distribution in HA hydrogels. Interestingly, clonal expansion of serum-free U87 and D456 cells was observed in HA hydrogels as compared to when grown as suspension culture on tissue culture polystyrene (TCPS). Evaluation of GBM stemness markers when cultured in HA hydrogels as compared to adherent and/or suspension culture indicated higher expression of Nestin and Sox2. In addition, CD133, Nanog and OCT-4 expression were also altered. Finally, we demonstrated that HA hydrogels can support long term GSC culture (up to 60 days) with retention of stemness markers. Overall, such biomimetic culture systems could further our understanding of microenvironmental regulation of GSC phenotypes.