Biocompatibility Of The Polyimide Film Electrode Used For Artificial Vision And The Effects Of Electrical Stimulation On Glial Cells

ObjectiveArtificial vision applies the implanted visual prosthesis to produce the electrical signals which stimulate and activate the visual system,so that the blind or the brink of blindness in some patients can restore partial useful vision.China approved the research in 2005 through the first "973" project for visual function repair's basic theory and the key scientific questions.Earlier,we have acquired the 3-D reconstruction of microvasculature of optic nerve successfully by computer technology which determined the best window for optic nerve implantation. Biocompatibility is one of the preconditions which the visual prosthesis material can be applied in clinical.The research began with the culturing glial cells in vitro, performed manifold biological tests to evaluate the adhesion,biocompatibility between glial cell and polyimide(PI) film electrode and the best parameters of electrical pulse in vitro,providing scientific basis for the implant and the choice of stimulation parameters of artificial vision substrate material.MethodsThe research used the explant culture method to obtain the optic nerve glial cells of rat.We identified the cells by the immunofluorescent staining and selected 3-5 generation's cells to carry on the examination for biocompatibility test.Through the preparation of the polyimide film electrode extraction,we examined the cytotoxicity with the CCK-8 assay,calculated the relative cell proliferation rate and draw value-added curve.Using 6 levels of toxic staging to evaluate material toxicity, that is 0-level for＞= 100%;1 level for 75%-99%;2 level for 50%-74%;3 level for 30%-49%;4 level for 15%-29%;5 level for 0-14%.The cells were inoculated on the polyimide film electrode which was placed in the bottom of culture plate and the short-term adhesion had been observed separately in the 1st,3rd,5th,24th hour.We fixed the polyimide film electrode to the bottom of culture plate,inoculated the optic nerve tissue or the generated optic glial cells on it,observed the adhesion and growth of glial cells on the polyimide film electrode by inverted microscope and scanning electron microscope.The electrical stimulation model of glial cells of rat in vitro would be established and the biology characteristic's effect of pulse current to the glial cells would be observed.ResultsAfter 6-10 day's explant culture,cells moved out of the optic nerve tissue.Most of them were long-spindle,cord-like or fascicular arrangement.Nucleuses were round or oval.The cells cultured for 9-14 days to generate,after passing through 3-5 generations,the immunity fluorescence dyeing demonstrated that more than 95%of the cells were GFAP positive.CCK-8 assay was used to evaluate the cytotoxicity of the polyimide film electrode extraction.The OD value and cellular RGR did not have significant difference compared with the complete culture medium and displayed high Cell proliferation rate.The cytotoxicity level of extraction group was 0-1 level,namely non-cytotoxicity function,and along with the culture time's extension,the cytotoxicity tend to 0 level.The short-term adhesive test demonstrated that vaccinated at 1st＞3rd hour the adhesion of film electrode group was lower than the control group. Vaccinated for 5 hours later,there were no significant difference,indicating the adhesion of film electrode group to be increased along with the time's extension.The adhesion was fine and no obvious changes in cultured cells attached to the surface of film electrode.The growth and proliferation of the cultured cells were not significantly inhibited with the substrate materials.Under the scanning electron microscope,the glial cells were observed closely attached to the surface of film electrode,the good spreading and the connecting into a film.A regular voltage and pulse width was fixed at 2.4V,5ms respectively as well as the stimulation time.The glial cells were subjected to different frequencies of stimulation and found that the 10Hz group cells showed significant detachment and necrosis while the 1Hz group cells showed lower.The difference was significant compared to normal control group(P＜0.05).The results suggested that the frequency was lower, the impact on cell was smaller.ConclusionThe glial cells were isolated and purified after primary glial culture.It become possible to obtain higher purity and active astrocytes which meet the biocompatibility testing requirements.In-vitro experiments confirmed that the polyimide film electrode was well adhesion and biocompatibility with the glial cell of rat,which was appropriate for optic nerve implant.The established models of in-vitro pulse current stimulation could cause the film electrodes to stimulate the cell effectively and caused the cell receive continual electrical pulse stimulation under the culture conditions.As the pre-installation stimulation plan was based on the body's stimulation parameters.With these parameters,we discovered that the cells showed significant detachment and necrosis under variety of frequency,therefore it was necessary to carry on further screening under these parameters.