D Kraus1, V Boyle2, N Leibig3, G B Stark4, V Penna4. 1. Department of Plastic and Hand Surgery, University Medical Center Freiburg, Freiburg, Germany. Electronic address: daniel.kraus@uniklinik-freiburg.de. 2. Clinic for Neurology, Ortenau Klinikum Lahr-Ettenheim, Lahr, Germany. 3. Department of Hand, Plastic and Reconstructive Surgery, Burn Center, BG Trauma Center Ludwigshafen, University of Heidelberg, Ludwigshafen, Germany. 4. Department of Plastic and Hand Surgery, University Medical Center Freiburg, Freiburg, Germany.
Abstract
BACKGROUND: In 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 METHOD: Spheroids 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. RESULTS: Initially 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 METHODS: Other 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. CONCLUSIONS: The 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.
BACKGROUND: In 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 METHOD: Spheroids 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. RESULTS: Initially 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 METHODS: Other 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. CONCLUSIONS: The 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.