Microfluidic co-culture system model to study the interplay between sensory neurons and mesenchymal stem cells: new clues towards osteogenesis

Abstract

Event Abstract Back to Event Microfluidic co-culture system model to study the interplay between sensory neurons and mesenchymal stem cells: new clues towards osteogenesis Hugo Oliveira1*, Bruno P. Dos Santos1*, Diana I. Silva1*, Jacques Leng2* and Joelle Amedee1* 1 Inserm U1026, Université Bordeaux Segalen, BioIngénierie Tissulaire, France 2 UMR 5258 CNRS/SOLVAY/Univ. BORDEAUX, LaboratoireduFutur, France Introduction: The existence of nerve fibers in bone and the expression of neural factors in close vicinity to bone cells have been reported. Neuron-derived molecules have demonstrated to play a role on bone turnover [1] and accumulating evidence has revealed the importance of the sensory nervous system in the orchestration of bone repair [2]. The aim of this work is to establish a co-culture system between sensorial neurons and mesenchymal stem cells (MSC) and to study the cellular interplay, between these cells, in view of osteogenesis. Materials and Methods: In order to allow the establishment of co-culture between rat dorsal root ganglia (DRG) sensory neurons and rat bone marrow MSCs we fabricated PDMS microfluidic chambers, using standard photolithography techniques. This approach allows to physically separate cellular bodies from the distinct cell types, to use two different cell culture medias and to some extent to mimic the in vivo physiological environment. The primary cultures were co-cultured during 7 and 14 days in microfluidics system using DMEM (high glucose) with 2% B27 medium or DMEM with 10%FBS medium for DRGs and MSCs, respectively. At designated time points (i.e. 7 and 14 days), metabolic activity, immunostaining and gene expression profiles (qPCR) were performed. Results: DRGs were able to spread neurites through microchannels toward MSCs (Figure 1). Figure 1. Co-culture microfluidic system. DRG neurons and MSCs are cultured separately but cells can interact through channels. Immunocytochemistry of DRG neurons (anti-beta III tubulin, green) and MSCs (actin, red) co-cultures at 7 and 14 days of culture. MSCs metabolic activity was not influenced by the co-culture with DRGs. MSCs in co-culture conditions shown an upregulation of the osteogenic markers Runx2, Col1 and Sp7 (Osx). Additionally, in view of further evaluate the mechanism involved in this regulation we assessed the gene expression of SMAD1 and Ctnnb1 (beta-catenin) (Figure 2). Figure 2. Quantitative gene expression of Runx2, collagen type I (Col1), osterix (osx), beta cathenin (Bcath) and Smad 1 of MSCs in mono and co-culture with sensory neurons, at 7 and 14 days of culture (Aver±SD, n=3, * denotes p <0.05). Discussion: We show that that sensorial neurons can closely interact with MSCs and modulate their phenotype toward the osteogenic lineage. Further studies are underway in order to further understand the mechanism underlying this cellular communication. Conclusion: These co-culture systems will allow the broadening of current knowledge regarding the impact of the sensorial nervous system in bone remodeling and repair, and can open new avenues for the establishment of new strategies for bone tissue regeneration.