Geometry and mechanical characteristics of the environment surrounding the Engineered Heart Tissues (EHT) affect their structure and function. Here, we employed a 3D tissue culture platform fabricated using two-photon direct laser writing with a high degree of accuracy to control parameters that are relevant to EHT maturation. Using this platform, we first explore the effects of geometry based on two distinct shapes: a rectangular seeding well with two attachment sites, and a stadium-like seeding well with six attachment sites that are placed symmetrically along hemicylindrical membranes. The former geometry promotes uniaxial contraction of the tissues; the latter additionally induces diagonal fiber alignment. We systematically increase the length of the seeding wells for both configurations and observe a positive correlation between fiber alignment at the center of the EHTs and tissue length. With increasing length, an undesirable thinning and "necking" also emerge, leading to the failure of longer tissues over time. In the second step, we optimize the stiffness of the seeding wells and modify some of the attachment sites of the platform and the seeding parameters to achieve tissue stability for each length and geometry. Furthermore, we use the platform for electrical pacing and calcium imaging to evaluate the functional dynamics of EHTs as a function of frequency.
