Development of a functional silicon chamber for in vitro spinal cord tissue regeneration with support for cell migration and neurite outgrowth.

The mammalian central nervous system doesn't have the ability to regenerate due to the presence of inhibitory molecules, such as those derived from the breakdown of myelin. Strategies to overcome this limitation have received increasing attention over the years, mainly in the development and application of biomaterials. The aim of this work was to evaluate the growth of motor neuron axons from the spinal cord of embryonic rats (CEUA 092/20) from a tissue culture model using modified chambers and surfaces.  Poly-ε-caprolactone (PLC) filaments and silicone chambers (Sylgard 184) were prepared in three steps: surface hydration with "plasma-O₂⁺ ", washing and sterilization with ultraviolet radiation. For the preparation of the culture medium, the chambers were placed in Petri dishes and the filaments were cut to uniform thickness and length before being placed in the chambers. The spinal cords were dissected and a precision loupe was used to access the ventral region of the H-medulla to obtain small fragments to place on the filaments. One set of chambers was filled with 100 µL of neurobasal medium and another set with 100 µL of motor neuron selective medium (Cold Spring Harbor protocol). After 6 days of incubation, fixation, immunostaining (Tuj-1, Olig-2, GFAP) and histological staining (alfa-bugarotoxin and DAPI), the filaments were visualized by fluorescence microscopy (Colibri Zeiss). A consistent adherence of the explants to the filaments was observed. With DAPI, we observed intense cell migration along the filaments, while immunostaining for Tuj-1 showed that these neurons survived and projected neurites along the filaments. The culture system using Cold Spring Harbor medium showed longer neurite outgrowth and a higher density of migrated cells compared to the Neurobasal culture medium. Given the success of this model and its viability, our future aim is to characterize the population of migrated cells, believing that the majority are motor neurons.