Covalent bonding of lysine to EVAL membrane surface to improve survival of cultured cerebellar granule neurons

Abstract

Polylysine is a commonly used coating material in the preparation of neuronal cultures. In the present study, poly (ethylene-co-vinyl alcohol) (EVAL) membranes were modified by the covalent bonding of lysine via isocyanation of surface hydroxyl groups to improve cell behavior in cultured cerebellar granule neurons from 7-day-old Wistar rats. Cell survival and death ratio were assayed by MTT reduction activity and lactate dehydrogenase release, respectively. Cell morphology and neurite growth were observed by a scanning electron microscope. It was found that immobilizing lysine onto the EVAL membranes could improve neuronal MTT reduction activity and delay the death rate of neurons. In addition, neurons seeded on the lysine-immobilized EVAL membrane were able to regenerate with the formation of an extensive neuritic network, indicating that the surface modification on the EVAL membrane should be useful for culturing of neurons. Furthermore, the influence exerted by the modification of different amino acids (glycine, arginine and ornithine) onto the EVAL membranes on the neuronal behavior were investigated. We found that the effect of the terminal primary amine of the side chain of the basic amino acids on the neuronal behavior should be taken into account because the immobilized glycine molecules did not improve neuronal cultures. In order to interpret the phenomenon further, in the surface modification process, both the two amino groups of lysine were designed to react with the EVAL membrane surface to form a cyclic structure. On such a surface, neuronal MTT reduction activity was decreased and neuronal death was not delayed. The result shows that the lysine molecules immobilized on the EVAL membrane surface can mediate cellular response to promote neuronal cell survival and guidance of neuritic processes, indicating a delicate interaction of neuron with lysine molecules on the EVAL membrane surface.