Organotypic brain slices preserve the complex 3D cellular structure and are the most potent ex vivo cultures, similar to in vivo conditions. However, their use is limited because of expensive membrane inserts and because the slices cannot be used for live-cell imaging. This study aimed to develop a low-cost and easy “ring-insert” to culture brain slices that can also be used for live-cell imaging. In this study, we created a “ring-insert” by gluing a low-cost (roll) membrane on plastic rings. These “ring-inserts” can be cultured with organotypic coronal mouse brain slices (150 µm) on top with the well-established interface method. In addition, these “ring-inserts” can be easily inverted and visualized under an inverse fluorescence microscope for live-cell imaging. We provide evidence that cholinergic (septum) and dopaminergic (mesencephalon) neurons survive on the “ring-inserts” for at least two weeks. Furthermore, we microcontact printed nerve growth factor and glial cell line–derived neurotrophic factor and visualized the outgrowth of cholinergic and dopaminergic neurons. Their activity was tested using fluorescent cell tracking and calcium imaging with Rhod-4/Fluo-4 after depolarization and calcium stimulation. In summary, a semipermeable 0.4-µm-pore membrane (Biopore Membrane BGCM0010, Merck Millipore) was glued onto 26 mm silicone O-rings by using Sylgard 184 polydimethylsiloxane prepolymer (Sylgard 184 Silicone Elastomer Kit, Dow Europe, Germany). Organotypic brain slices (150 µm) from postnatal day 10 mice were placed on top of these rings and cultured with the respective media underneath. The membrane has a hydrophilic polytetrafluoroethylene surface and becomes transparent when wet, thus allowing for the visualization of brain slices under a fluorescence microscope. The “ring-insert” represents a low-cost and easy model for culturing organotypic brain slices and allows for subsequent live-cell imaging.