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.

Three-dimensional extracellular matrix engineering in the nervous system

“…19 The results are also comparable to the data obtained by Ranieri et al, who showed that in the case of a 2D substrate, cell attachment of NG 108-15 cells was significantly reduced when the surface had been derivatized with a CDPGYIGSK instead of a CDPGYIGSR peptide sequence. 23 Ostheimer et al showed by conducting a 2D 1 H-1 H-NMR study of the CDPGYIGSR peptide that the exchange of only one amino acid had an effect on the ''conformation'' of the peptide, possibly interfering with its receptor binding activity. 24 The specificity of cell-matrix interaction was further validated through competitive binding assays.…”

Stable Non‐Covalent Large Area Patterning of Inert Teflon‐AF Surface: A New Approach to Multiscale Cell Guidance

“…Few reports addressed the use of Teflon as substrate for patterning and controlling cell adhesion. [21][22][23] Teflon could become an interesting material for cell guidance, else as cell carrier, or scaffold upon a selective pattern with cell-adhesion biomolecules. A stable functionalization of the Teflon surface by non-covalent attachment of molecules and avoiding the chemical modification of the Teflon surface is challenging.…”

Biology on a Chip: Microfabrication for Studying the Behavior of Cultured Cells

“…Since then many studies in neuronal patterning have been performed in a variety of labs, using various methods, including surface modification by silane chemistry 113 137 Ti, 140 polystyrene, 130,131 Teflon (fluorinated ethylene propylene), [132][133][134][141][142][143][144] and biodegradable polymers. 145 These studies mainly yielded insights into the adhesion and guidance of neuronal growth by insoluble factors and could prove valuable for selective neuronal immobilization on future microsensors.…”