In vitro model of vascularised skin tissue for drug testing

Skin models are useful in vitro tools to study the development of skin pathologies and to screen and develop new specific drugs. They are becoming an alternative to animal models which are often ineffective in reproducing features of human skin due to difference in the evolution. Additionally, the animal testing ban rise the necessity to develop models that closely mimic in vivo-like environment in vitro. Skin has a very complex structure and physiology, therefore, challenges associated to composition, architecture and cellular organization are to be addressed if aiming at replicating its native properties. Among the organisational requirements of a vascularised in vitro skin model, endothelial cells must be incorporated in the dermal compartment together with fibroblasts. For this, hydrogels capable to mimic native extracellular matrix composition are exploited for housing the cells by providing a hydrated environment to favor cell proliferation and functionality. In this work, a new vascularised 3D skin model is engineered by combining core concepts in tissue engineering, CAD design and additive manufacturing, and fluido-dynamics to study drug extravasation through the endothelial barrier in drug delivery approaches. For this, a biomimetic hydrogel based on collagen, hyaluronic acid and fibrinogen as ECM-like materials with the addition of 4S-star PEG and thrombin as crosslinkers are developed and characterized in terms of its physico-chemical, mechanical and biological. The materials evidenced storage and loss moduli in the range of 10-100 kPa quite similar to those values of the native skin tissue, with sol/gel transition within 180±30 s at 37°C. Furthermore, morphological analysis by Scanning Electron microscopy demonstrated suitable porosity with average pore diameter of 120 um for cells to proliferate and proper function within the matrix. Cellular tests (i.e. Live/Dead, metabolic activity) and nutrient diffusion tests demonstrated an ideal environment for fibroblasts and endothelial to thrive. Next, an in-house fluidic chamber was designed using a CAD software and manufactured by 3D-printing to establish a dynamic culture and the perfusion through the vessel. The design enables to obtain a 10mm diameter dermal model and create an open channel with a diameter of 500 um in the bottom which can be seeded with endothelial cells to obtain a vessel. The perfusable chambers were tested by flushing colored water, revealing the importance of hydrogels stiffness and confirming the suitable final compositions of collagen, alginate and fibrin based hydrogel. Once obtained the open channel, endothelial cells (HUVECs) were injected into it, the device was submerged in media and incubated overnight to allow HUVECs to completely adhere to the channel walls and form a uniform endothelial lining. The cells were transfected with a fluorophore to evaluate in real time their location and the successfully formation of the vessel by confocal/fluorescence microscope imaging. The feasibility to create a vessel with known dimensions and locations to be integrated into a perfused 3D skin model was demonstrated in this project, making it a suitable model to study sprout formation and the maintenance of the endothelial barrier integrity. Future work would evaluate the use of fluorescent nanoparticles and their passage from the flow to the surrounding tissue, towards investigating the tumor angiogenesis and cancer invasion pathways in melanoma cell spheroids at a pre-established distances from the vessel.