Culturing aortic valvular interstitial cells in an environment that models the aortic valve is an essential step towards understanding the progression of calcific aortic valve disease. Here the adaption of a 3D stacked paper-based culture system is presented for analyzing valve cells in a thick collagen gel matrix. Filter paper layers, modeled after a 96-well plate design, were printed with a wax well-plate template and then seeded with valve cell and collagen mixtures that quickly gelled into 3D cultures. Stacking these layers permitted extensive customization of culture thickness and cell density profiles to model the full thickness of native valve tissue.