A Workflow to Produce a Low-Cost In Vitro Platform for the Electric-Field Pacing of Cellularised 3D Porous Scaffolds

[Image: see text] Endogenous electrically mediated signaling is a key feature of most native tissues, the most notable examples being the nervous and the cardiac systems. Biomedical engineering often aims to harness and drive such activity in vitro, in bioreactors to study cell disease and differentiation, and often in three-dimensional (3D) formats with the help of biomaterials, with most of these approaches adopting scaffold-free self-assembling strategies to create 3D tissues. In essence, this is the casting of gels which self-assemble in response to factors such as temperature or pH and have capacity to harbor cells during this process without imparting toxicity. However, the use of materials that do not self-assemble but can support 3D encapsulation of cells (such as porous scaffolds) warrants consideration given the larger repertoire this would provide in terms of material physicochemical properties and microstructure. In this method and protocol paper, we detail and provide design codes and assembly instructions to cheaply create an electrical pacing bioreactor and a Rig for Stimulation of Sponge-like Scaffolds (R3S). This setup has also been engineered to simultaneously perform live optical imaging of the in vitro models. To showcase a pilot exploration of material physiochemistry (in this aspect material conductivity) and microstructure (isotropy versus anisotropy), we adopt isotropic and anisotropic porous scaffolds composed of collagen or poly(3,4-ethylene dioxythiophene):polystyrenesulfonate (PEDOT:PSS) for their contrasting conductivity properties yet similar in porosity and mechanical integrity. Electric field pacing of mouse C3H10 cells on anisotropic porous scaffolds placed in R3S led to increased metabolic activity and enhanced cell alignment. Furthermore, after 7 days electrical pacing drove C3H10 alignment regardless of material conductivity or anisotropy. This platform and its design, which we have shared, have wide suitability for the study of electrical pacing of cellularized scaffolds in 3D in vitro cultures.