Tumor models have advanced over the years as scientists have made several improvements. In the past, typically 2D cultures were used. Due to their artificial nature, their results are often not transferable to the human pathophysiology. 3D models are a promising approach for a more in vivo-like microenvironment. Here, the upcoming field of biofabrication makes it possible to develop more sophisticated models with spatially arranged components, which can be used for in vitro and in vivo applications.
With this study, we developed a printable hydrogel consisting of alginate, hyaluronic acid, and gelatin (Alg/HA/Gel) for extrusion-based bioprinting. We analyzed the bioink’s printability, mechanical properties, and suitability as basis for in vitro melanoma models that mimic the tumor microenvironment. In vivo, we characterized its effect on tumor progression, vascularization, and metastasis in a defined and isolated arteriovenous (AV) loop model in the rat.
The hydrogel showed mainly elastic properties with a storage modulus of 10.5 kPa at 1 rad/s using dynamic mechanical analysis. Its stiffness was to a great extent tunable when increasing the alginate content. The bioink showed good printability and shape-fidelity. The human melanoma cell line Mel Im had high survival rates during the printing process. Cell cycle analysis with the fluorescent-based cell cycle indicator (FUCCI) of these cells did not reveal an impact on cell cycle populations over one week, neither due to the printing nor in beads. Adipose-derived stem cells were able to survive within the hydrogel and differentiate into the adipogenic and the osteogenic lineage over 21 days. This was demonstrated via Oil Red O and Alizarin red stainings and qPCR. A Gaussia princeps Luciferase fusion protein producing HEK293 cell line revealed the feasible diffusion of proteins with a size of 150 kDa through the hydrogel. In the in vivo AV loop model, the hydrogel facilitated good tumor progression, vascularization and reliable lung metastases over four weeks. This closely resembles the human pathophysiology and morphology. Whole-mount light sheet fluorescence microscopy (anti-CD31) and histological sections (HMB-45) of the explants and explanted lungs supported these findings.
In summary, this Alg/HA/Gel bioink is a versatile tool for basic and applied cancer research. In combination with the AV loop model, it is a unique in vivo model to study melanoma pathophysiology and possible therapies.
1. Schmid, R. et al., Adv. Funct. Mater. 32, 2107993 (2021)