3D CUSTOM-PRINTED INSERTS FOR THE DEVELOPMENT OF IN VITRO MODELS OF THE HUMAN ESOPHAGEAL EPITHELIUM IN HYDROGEL

In vitro models of epithelia have been frequently obtained by air-liquid interface (ALI) culture on commercially available transwell plates. Transwell inserts are standardized, have high costs and restrains regarding choice of materials. Based on that, the thesis was aimed at designing an in vitro model of esophageal epithelium by in vitro air-liquid interface (ALI) based on cellularized hydrogel, casted in a custom 3D printed inserts with tailorable dimensions to fit into different sizes of well plates and grid porosity to use different cell culture strategies. The inserts were fabricated using fused deposition modeling (FDM) from the biocompatible polymer polycaprolactone (PCL). The hydrogel matrix was based on gelatin methacryloyl (GelMA), a biocompatible derivative of gelatin. Different batches of GelMA were prepared by changing the degree of methacrylation, to find out the most suitable in mimicking the stiffness of the extracellular matrix of the esophageal submucosa. UV-mediated photocrosslinking, using lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as initiator was employed, and its optimal concentration was determined minimizing curing time and providing biomimetic stiffness (both assessed through photorheological characterization). Further tests were conducted to analyze hydrogel degradation kinetics and permeability in ALI conditions. Cytocompatibility was evaluated using human esophageal epithelial cells (HEEC). The obtained results demonstrated that the custom 3D printed inserts are easy to produce and are considerably less expensive compared to commercial Transwell inserts. GelMA hydrogels displayed adequate permeability and showed no cytotoxicity towards HEEC in elution tests but proved to be softer than ex vivo esophagus.