Patch method for culture of primary hippocampal neurons

Abstract

Culture of primary neurons, and especially hippocampal neurons, is important for understanding cellular mechanisms in neurobiology. Actually, this is achieved by using culture dish or glass slide with surface coated proteins. Here, we proposed a patch method for culture of primary neurons on a monolayer of gelatin nanofibers electrospun and crosslinked on a honeycomb microframe of poly (ethylene glycol) diacrylate (PEGDA). This method allows us to minimize exogenous material contact of cells and largely increase the exposure area of cells to the culture medium. We found that neurons, and especially astrocytes, have a more in vivo like morphology comparing to that on culture dish or on glass slide. We also found that neurons were preferentially located in the suspended areas of the monolayer nanofibers. Finally, calcium imaging revealed that primary neurons have a higher degree of neural activity on the patch than on glass. These results suggest that crosslinked and monolayer gelatin nanofibers closely mimic the extracellular matrix structure and allow more effective culture of primary neurons than conventional methods, thus facilitating advanced studies of neural functions as well as cell-based assays.