A 3D printed device for in vitro generation of stratified epithelia at the air-liquid interface

Air-liquid interface (ALI) cultures are used to produce stratified epithelial tissues in vitro, notably for the production of oral mucosal equivalents. Currently, there are few purpose-built devices which aim to enhance the ease and reproducibility of generating such tissue. Most ALI cultures utilise stainless steel grids or cell culture inserts to elevate the matrix or scaffold to the surface of the culture media. Here, a novel buoyant epithelial culture device (BECD) was designed to both contain a fibroblast-seeded collagen hydrogel and float in culture media, thereby automatically maintaining the ALI without further user intervention. BECDs aim to mitigate several issues associated with ALI culture; reducing the chance of media flooding the epithelial layer from physical disturbance, reducing technique-sensitivity for less experienced users, and improving the reproducibility of the epithelia generated. H400 oral squamous cell carcinoma cells cultured in BECDs for 7, 14 and 21 days showed continuous increase in epithelial tissue thickness with expected localisation of epithelial differentiation markers: cytokeratin 5, involucrin and E-cadherin. Fused filament fabrication 3D printing with polypropylene used in BECD production allows for rapid turnover and design iteration, presenting a versatile, adaptable and useful tool for application in in vitro cell culture.