Low-cost Ring-Inserts Optimize Culture and Imaging of Organotypic Brain Slices

A team from the Psychiatric Laboratory at the Medical University of Innsbruck has developed a low-cost “ring-insert” system for long-term culture and imaging of living brain cells. Published in Biofunctional Materials, the work offers a practical, scalable method to grow and study neurons directly in brain tissue slices, creating new opportunities for drug testing, live-cell microscopy, and disease research.

The model builds on organotypic brain slice cultures—150 µm-thick sections from young mice that preserve the brain’s natural structure and organization. From a single mouse brain, 50–100 slices can be obtained, greatly reducing animal use under the 3R principle (reduce, refine, replace). Traditional commercial membrane inserts for these slices can cost up to €20 each. The Innsbruck team’s self-made ring-inserts cost only €2–€3 each.

Each ring-insert consists of a small silicone ring with a thin permeable membrane. Brain slices rest on the membrane above nutrient-rich medium, staying flat and stable for direct microscopic imaging. This stability enables detailed observation of neuron growth, connectivity, and reactions over time. According to Univ. Prof. Dr. Christian Humpel, senior author of the study, “Despite their simplicity, [the inserts] support high-quality brain tissue cultures and allow us to observe living neurons in real time, including how they grow, form connections and respond to stimulation.”

The research targeted cholinergic neurons involved in memory and attention, which are lost in Alzheimer’s, and dopaminergic neurons controlling movement, which deteriorate in Parkinson’s. The team used microcontact printing (µCP) to apply growth factors in precise patterns on the membranes—nerve growth factor (NGF) for cholinergic neurons and glial cell line-derived neurotrophic factor (GDNF) for dopaminergic neurons. These patterns guided nerve fiber extension, and both cell types survived for two weeks in culture.

To confirm functional activity, calcium imaging with fluorescent dyes showed neurons responding to potassium chloride stimulation with bursts of fluorescence, indicating active communication. As first author Alessa Gern notes, “We can monitor live neuronal activity in real time. It gives us direct insight into how these cells function, respond to stimulation and potentially degenerate. This was not possible before.” 

Beyond basic research, the model could be adapted for drug testing, genetic modification, viral delivery, or combined with other cell types to study structures like the blood–brain barrier. Future adaptations might allow use with adult brain or human surgical tissue, bringing research even closer to real disease conditions. The approach could help refine brain research methods while reducing reliance on animal experiments.