The Neuro-spheroid—A novel 3D in vitro model for peripheral nerve regeneration

• A new 3D in vitro model with Schwann cell-neuron-spheroids in a collagen matrix.
• Spheroids grow interesting sprouting phenomena in this neuronal sprouting assay.
• Transferring a 2D into a 3D culture shows a significant increase of neurite length.
• It is a simple and flexible method to investigate neurite development in vitro.

BackgroundIn order to reduce in vivo animal experiments in peripheral nerve regeneration research, in vitro models are desirable. Common two dimensional (2D) co-culture models lack the complex interactions of three dimensional (3D) physiological structures. The aim of the study was to establish a neuronal 3D spheroidal sprouting assay for peripheral nerve regeneration.New methodSpheroids consisting of Schwann cells (SC, 500 cells/spheroid) and NG108-15 cells (NG, 50 cells/spheroid), a hybrid cell line, were formed in hanging drops and were embedded in a 3D collagen matrix. Spheroid sprout lengths were compared to those of the neurites of NG in a 2D co-culture with SC. Lengths were measured using phase contrast images taken every day over 10 days. Additionally we took fluorescence images to visualize the PKH26-labeled NG in both culture systems.ResultsInitially thin neurites grew out in both co-cultures, over time the sprouts’ diameter in the 3D culture increased. The direct comparison of the sprout length revealed significantly longer neurites in the 3D co-culture from day 7 until day 10 (p < 0.001).Comparison with existing methodsOther co-culture models either display processes in 2D or need complex matrices to create 3D structures. Our spheroidal model is easy to establish, highly flexible and nevertheless 3D.ConclusionsThe 3D-Schwann cell-neuron spheroid model shows that by simply transferring a 2D into a 3D co-culture with multiplication of cell-cell contacts, a significant increase of neurite length can be achieved. The model is a relatively simple method for the investigation of neurite development in vitro.