Cancer therapeutics are developed through extensive screening; however, many therapeutics evaluated with 2D in vitro cultures during pre-clinical trials suffer from lower efficacy in patients. Replicating the in vivo tumor microenvironment in vitro with three-dimensional (3D) porous scaffolds offers the possibility of generating more predictive pre-clinical models to enhance cancer treatment efficacy. We developed a chitosan and hyaluronic acid (HA) polyelectrolyte complex 3D porous scaffold and evaluated its physical properties. Chitosan-HA (C-HA) scaffolds had a highly porous network. C-HA scaffolds were compared to 2D surfaces for in vitro culture of U-118 MG human glioblastoma (GBM) cells. C-HA scaffold cultures promoted tumor spheroid formation and increased stem-like properties of GBM cells as evidenced by the upregulation of CD44, Nestin, Musashi-1, GFAP, and HIF-1α as compared with 2D cultures. Additionally, the invasiveness of GBM cells cultured in C-HA scaffolds was significantly enhanced compared to those grown in 2D cultures. C-HA scaffold cultures were also more resistant to chemotherapy drugs, which corresponded to the increased expression of ABCG2 drug efflux transporter. These findings suggest that C-HA scaffolds offer promise as an in vitro GBM platform for study and screening of novel cancer therapeutics.

Despite recent advances in the understanding of its cell biology, glioma remains highly lethal. Development of effective therapies requires a cost-effective in vitro tumor model that more accurately resembles the in vivo tumor microenvironment as standard two-dimensional tissue culture conditions do so poorly. Here we report on the use of a three-dimensional (3D) chitosanalginate (CA) scaffold to serve as an extracellular matrix that promotes the conversion of cultured cancer cells to a more malignant in vivo-like phenotype. Human U-87 MG and U-118 MG glioma cells and rat C6 glioma cells were chosen for study. In vitro tumor cell proliferation and secretion of factors that promote tumor malignancy, including VEGF, MMP-2, fibronectin, and laminin, were assessed. The scaffolds pre-cultured with U-87 MG and C6 cells were then implanted into nude mice to evaluate tumor growth and blood vessel recruitment compared to the standard 2D cell culture and 3D Matrigel matrix xenograft controls. Our results indicate that while the behavior of C6 cells showed minimal differences due to their highly malignant and invasive nature, U-87 MG and U-118 MG cells exhibited notably higher malignancy when cultured in CA scaffolds. CA scaffolds provide a 3D microenvironment for glioma cells that is more representative of the in vivo tumor, thus can serve as a more effective platform for development and study of anticancer therapeutics. This unique CA scaffold platform may offer a valuable alternative strategy to the time-consuming and costly animal studies for a wide variety of experimental designs.

Purpose-Hepatocellular carcinoma (HCC) is a prevalent solid malignancy. Critically needed discovery of new therapeutics has been hindered by lack of an in vitro cell culture system that can effectively represent the in vivo tumor microenvironment. To address this need, a 3D in vitro HCC model was developed using a biocompatible, chitosan-alginate (CA) scaffold cultured with the human HCC cell lines. Methods-The correlation between the cell function such as secretion of growth factors and production of ECM in vitro, and the tumor growth and blood vessel recruitment in vivo was investigated.Results-HCC cells grown on 3D CA scaffolds demonstrated morphology characteristic and increased expression of markers of highly malignant cells. Implantation of CA scaffolds cultured with human HCC cells in mice showed accelerated tumor growth. Histology revealed marked differences in morphology and organization of newly formed blood vessels between tumors produced by different pre-cultured conditions. Resistance to doxorubicin was significantly pronounced in CA scaffold-cultured HCC cells compared to 2D or Matrigel cultured HCC cells.Conclusions-This 3D model of HCC, with its ability to more closely mimic the in vivo tumor behavior, may serve as an invaluable model for study and application of novel anticancer therapeutics against HCC.

Osteochondral tissue engineering poses the challenge of combining both cartilage and bone tissue engineering fundamentals. In this study, a sphere-templating technique was applied to fabricate an integrated bi-layered scaffold based on degradable poly(hydroxyethyl methacrylate) hydrogel. One layer of the integrated scaffold was designed with a single defined, monodispersed pore size of 38 μm and pore surfaces coated with hydroxyapatite particles to promote regrowth of subchondral bone while the second layer had 200 μm pores with surfaces decorated with hyaluronan for articular cartilage regeneration. Mechanical properties of the construct as well as cyto-compatibility of the scaffold and its degradation products were elucidated. To examine the potential of the biphasic scaffold for regeneration of osteochondral tissue the designated cartilage and bone layers of the integrated bi-layered scaffold were seeded with chondrocytes differentiated from human mesenchymal stem cells and primary human mesenchymal stem cells, respectively. Both types of cells were co-cultured within the scaffold in standard medium without soluble growth/differentiation factors over four weeks. The ability of the integrated bi-layered scaffold to support simultaneous matrix deposition and adequate cell growth of two distinct cell lineages in each layer during four weeks of co-culture in vitro in the absence of soluble growth factors was demonstrated.