Crosslinked Chitosan‐PEG Hydrogel for Culture of Human Glioblastoma Cell Spheroids and Drug Screening

2D monolayer cell cultures are routinely utilized for preclinical cancer drug screening, but the results often do not translate well when drugs are tested in vivo. To address this limitation, a biocompatible chitosan‐PEG‐genipin hydrogel (CSPG gel) is synthesized to create a gel that can be easily dispensed into 96‐well plates at room temperature and neutral pH. The stiffness of this gel is tailored to be within the range of human glioblastoma tissue to promote the formation of tumor spheroids. Differences in cell morphology, proliferation rate, and dose‐dependent drug cytotoxicity are compared among cell spheroids grown on CSPG gels, cells in monolayer culture on tissue culture polystyrene, and cells cultured on Matrigel. Tumor spheroids on CSPG gels display statistically significantly greater resistance to chemotherapeutics than in the conditions where cells do not form spheroids. Gene expression analysis suggests that resistance of cells on CSPG gels to the therapy may be partially attributed to upregulation of ATP‐binding cassette transporters and downregulation of DNA mismatch repair genes stimulated by spheroid formation. These findings suggest CSPG gel generates tumor spheroids that better reflect the malignant behavior of glioblastomas and provides a cost‐effective substrate for preclinical, high‐throughput screening of potential cancer therapeutics.