Design of a vascularized in vitro skin model to mimic metastatic melanoma

Melanoma is a skin cancer that arises from genetically altered or activated epidermal melanocytes. The prognosis of patients who reach the metastatic phase (stage IV) is grim, presenting a 5-years survival rate between 5% and 19%, depending on the site and extent of the metastatic disease. Currently, there is no definitive cure for metastatic melanoma, and existing treatments are hindered by drug resistance and significant side effects. Moreover, advanced treatment options often fail because of transport limitations, poor targeting capacity, and side effects. The traditional drug testing models mainly based on oversimplified 2D or 3D cell cultures failed in supporting the drug development process, because of their lack of similarity with the complex pathophysiology of the metastatic disease. This work aims at developing an in vitro three-dimensional dynamic model of metastatic melanoma through microfluidic technology. A custom-made microfluidic chip composed by three chambers connected in series was designed. A primary melanoma model was cultured in the first chamber which is linked to the other chambers through a microfluidic channel. The two additional chambers can host matrices mimicking frequent targets of melanoma metastases, such as brain, lung or bone. To obtain the primary melanoma model, first a dermis mimic was prepared using a collagen/hyaluronic acid based hydrogel seeded with human dermal fibroblasts (Hff-1), covered with a layer of keratinocytes (HaCaT), to reproduce the epidermis. In details, the obtained hydrogel was composed of type I bovine collagen and methacrylate hyaluronic acid (HAMA), able to successfully mimic the mechanical and biological properties of the dermis. The hydrogel successfully reflected the native extracellular matrix proprieties, as confirmed by rheological characterization and viability test. The model, cultured for up to 21 days, was able to host different cell populations, as confirmed by immunofluorescence analysis. An endothelial cells layer was cultured in the basal surface of the hydrogel to mimic the endothelial layer of blood vessels and to induce the development of vessel sprouts within the dermal compartment. A spheroid of melanoma cells (SK-MEL) was incorporated in the dermal compartment to mimic the initial stage of the pathology. To achieve a mature endothelial layer the model was exposed to dynamic flow provided by a pump, supplying an essential stimulus for the development of the vasculature. Confocal microscopy analysis confirmed the presence of a dense and aligned endothelial layer, as well as the development of a capillary network within the hydrogel. The obtained 3D melanoma model represents a physiologically relevant environment for studying tumour progression in a more ethical and cost-effective way. Future developments include the optimization of the metastatic compartment and the employment of the model as a validation platform for innovative drug-delivery systems.