ABSTRACT
Recovery of live cells from three-dimensional (3D) culture would improve analysis of cell behaviors in tissue engineered microenvironments. In this work, we developed a temperature responsive hydrogel to enable transient 3D culture of human glioblastoma (GBM) cells. N-isopropylacrylamide was copolymerized with hydrophilic grafts and functionalized with the cell adhesion peptide RGD to yield the novel copolymer poly(N-isopropylacrylamide-co-Jeffamine(®) M-1000 acrylamide-co-hydroxyethylmethacrylate-RGD), or PNJ-RGD. This copolymer reversibly gels in aqueous solutions when heated under normal cell culture conditions (37°C). Moreover, these gels redissolve within 70 s when cooled to room temperature without the addition of any agents to degrade the synthetic scaffold, thereby enabling rapid recollection of viable cells after 3D culture. We tested the efficiency of cell recovery following extended 3D culture and were able to recover more than 50% of viable GBM cells after up to 7 days in culture. These data demonstrate the utility of physically crosslinked PNJ-RGD hydrogels as a platform for culture and recollection of cells in 3D.
Subject(s)
Cell Culture Techniques/methods , Hydrogels/pharmacology , Temperature , Acrylamides/chemical synthesis , Acrylamides/chemistry , Cell Survival/drug effects , Chromatography, Gel , Elasticity , Humans , Hydrogels/chemical synthesis , Hydrogels/chemistry , Magnetic Resonance Spectroscopy , Rheology , Tissue Scaffolds/chemistry , Tumor Cells, Cultured , ViscosityABSTRACT
The invasion of malignant glioblastoma (GBM) cells into healthy brain is a primary cause of tumor recurrence and associated morbidity. Here, we describe a high-throughput method for quantitative measurement of GBM proliferation and invasion in three-dimensional (3D) culture. Optically clear hydrogels composed of thiolated hyaluronic acid and gelatin were chemically crosslinked with thiol-reactive poly(ethylene glycol) polymers to form an artificial 3D tumor microenvironment. Characterization of the viscoelasticity and aqueous stability indicated the hydrogels were mechanically tunable with stiffness ranging from 18 Pa to 18.2 kPa and were resistant to hydrolysis for at least 30 days. The proliferation, dissemination and subsequent invasion of U118 and U87R GBM spheroids cultured on the hydrogels were tracked in situ with repeated fluorescence confocal microscopy. Using custom automated image processing, cells were identified and quantified through 500 µm of gel over 14 days. Proliferative and invasive behaviors were observed to be contingent on cell type, gel stiffness, and hepatocyte growth factor availability. These measurements highlight the utility of this platform for performing quantitative, fluorescence imaging analysis of the behavior of malignant cells within an artificial, 3D tumor microenvironment.
