Spatial control of neuronal cell attachment and differentiation on covalently patterned laminin oligopeptide substrates

Abstract

The spatial control of neuronal cell attachment and differentiation via specific receptor mediated interactions, may provide an effective means for the in vitro reconstruction of neuronal cell architecture. In this study, receptor-specific oligopeptide sequences derived from the extracellular matrix (ECM) molecule laminin, a potent neural cell attachment and differentiation promoter were covalently bound on fluorinated ethylene propylene (FEP) films. The degree of receptor-specific cell attachment and the ability to spatially control neurite outgrowth by covalently patterning the oligopeptide sequences on the FEP film surface were assessed.FEP films were first chemically activated with a Radio Frequency Glow Discharge (RFGD) process that covalently replaces the surface fluorine atoms with reactive hydroxyl groups. Oligopeptides containing the YIGSR sequence from the B1 chain of laminin and the water soluble oligopeptide containing the IKVAV sequence (CSRARKQAASIKVAVSADR) from the A chain were covalently bound to the hydroxylated FEP films. Electron Spectroscopy for Chemical Analysis (ESCA) verified the covalent attachment of the oligopeptides to the material surface.The degree of receptor mediated NG108-15 cell attachment on immobilized CDPGYIGSR films was determined using competitive binding media. A 78% reduction in cell attachment was observed on films containing CDPGYIGSR in the cell plating medium. Only a 23% reduction in cell attachment was noted on films plated in medium containing a mock CDPGYIGSK sequence. FEP films immobilized with the IKVAV oligopeptide sequence were shown to mediate PC12 cell attachment and a competitive binding medium also significantly attenuated cell attachment on the immobilized films.The spatial patterning of these oligopeptide sequences to the FEP surface was shown to localize cell attachment and neurite extension on the patterned pathways. The surrounding unmodified FEP surface was inhibitory in serum containing medium and prevented cellular interactions outside the oligopeptide modifications. The spatial immobilization of laminin oligopeptides on FEP films provides a means to organize the attachment and differentiation of neuronal cells in a receptor-specific manner.