A three-dimensional collagen scaffold cell culture system for screening anti-glioma therapeutics

Abstract

Three-dimensional (3D) culture, which can simulate in vivo microenvironments, has been increasingly used to study tumor cell biology. Since most preclinical anti-glioma drug tests still rely on conventional 2D cell culture, we established a collagen scaffold for 3D glioma cell culture. Glioma cells cultured on these 3D scaffolds showed greater degree of dedifferentiation and quiescence than cells in 2D culture. 3D-cultured cells also exhibited enhanced resistance to chemotherapeutic alkylating agents, with a much higher proportion of glioma stem cells and upregulation of O6-methylguanine DNA methyltransferase (MGMT). Importantly, tumor cells in 3D culture showed chemotherapy resistance patterns similar to those observed in glioma patients. Our results suggest that 3D collagen scaffolds are promising in vitro research platforms for screening new anti-glioma therapeutics.

Keywords: MGMT; chemosensitivity; collagen scaffold; glioma stem cells; three-dimensional culture.

Figure 1. Comparison of glioma cell morphology by H&E staining and SEM

Primary glioma cells in 2D and 3D culture with H&E staining A and B. Scale bar = 100 μm. U87 cells in 2D and 3D culture in SEM image C and D. Scale bars = 100 μm and 10 μm. Primary glioma cells in 3D scaffolds and human glioma tissue imaged by SEM E and F. Scale bars = 100 μm and 10 μm. Red arrow indicates glioma cells.

Figure 2. U87 cell proliferation and dedifferentiation in 3D collagen scaffolds

U87 cell proliferation in 2D and 3D culture assessed at different time points A. 3D culture induces accumulation of cells in G0/G1 phase with concomitant reduction of cells in S phase B. Levels of Ki67, caspase-3, cleaved-PARP and GFAP in U87 cells in 2D and 3D culture as measured using flow cytometry C. Results are shown as the means ± SD. *P<0.05.

Figure 3. Glioma cell responses to chemotherapeutics

U87 and primary glioma cell viabilities in 2D and 3D culture after exposure to DDP A and D. CCNU B and E. and TMZ C and F. Data represent the mean percentage viability (2D and 3D; left axis) ± SD normalized against untreated control cells.

Figure 4. Chemotherapy resistance mechanisms shown by 3D-cultured glioma cells

Levels of drug resistance-related genes were examined by qRT-PCR in U87 A. and primary glioma cells C. Expression data was normalized against GAPDH. MGMT and CD133 expression in U87 B. and primary D. cells via Western blotting. *P<0.05.

Figure 5. U87 cell stemness in 3D culture

CD133 expression shown by confocal microscopy A and B. and flow cytometry C. Scale bar = 100 μm. Relative Nanog, Oct4 and Sox2 mRNA D. and protein E. levels in cultured cells as measured by qRT-PCR and Western blotting. qRT-PCR data was normalized against GAPDH. Colony and sphere formation by U87 cells in 3D culture F and G. Data represent the means ± SD. *P<0.05.

Figure 6. Inhibition of growth of U87 and primary glioma cells by DDP, CCNU and TMZ in 2D vs. 3D culture

*P<0.05.