Customizable and Open-Source 3D Printed Inserts for Controlled Release and Cell Culture Experiments

Cell culture inserts are a key tool for in vitro experimentation in both cell-based assays and general in vitro analytical experiments. However, during and following the COVID-19 pandemic, a combination of increased demand and disruptions to global supply chains saw massive reductions in their availability. Many researchers faced long lead times and experienced significant research delays as a result. In addition, the high cost and limited customizability of commercial inserts make them inaccessible to researchers with limited funding resources and inappropriate for specialized research applications, respectively. We sought to develop a low-cost, open-source, and customizable 3D printed insert platform to address these issues of availability, accessibility, and customizability. Hanging-type 3D printed inserts (3DPIs) were designed as two components─the insert cradle and the donor well─and assembled into a complete insert following 3D printing via fused-filament fabrication. The performance and versatility of the 3DPIs were demonstrated through a series of exemplar in vitro experiments. In a conventional permeability assay, 3DPIs supported cell monolayer formation and successfully restricted large molecule transport. In a T cell migration assay using a hydrogel biomaterial, 3DPIs (20 μm pore size) enabled evaluation of the cell encapsulation and release properties of the biomaterial. Finally, solvent-resistant 3DPIs were used to develop a drug diffusion assay for an in situ forming implant formulation. The 3DPIs exhibited comparable performance to commercial inserts when conducting the permeability assay. However, only the 3DPIs could be used in the cell migration and drug diffusion assays due to the limited range of membrane pore sizes offered by commercial inserts (≤8 μm) and their poor solvent resistance. The 3DPIs could be easily customized to serve the requirements of the target experiments. Importantly, due to the use of open-source hardware and software, the 3DPIs could be locally manufactured at extremely low cost. As a result, the 3DPIs invariably have greater accessibility and improved availability, which could make their use more resistant to funding challenges, or supply chain disruptions, and provide a viable alternative to commercial cell culture inserts.