Enhancement of neuronal cell adhesion by covalent binding of poly-d-lysine

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Abstract

We have prepared the poly-d-lysine (PDL) bound surfaces for neuron cell culture by covalent binding between the poly-d-Lysine and substrates and investigated neuronal cell adhesion properties and cell growth morphology. The number of neuronal cell and the number of neurite per neuronal cell on PDL bound surfaces was much more than those on PDL coated surfaces and also the neuronal cells on PDL bounded surfaces survived a longer time. On the pattern of covalently bound PDL, neuronal cells and their neurites are confined within the grid line leading to patterned neuronal networks with the long-term survival.

Highlights

► Surface modification through covalent binding between poly-d-lysine and substrates for neuron cell culture. ► The number of neuronal cell and neurite per neuronal cell on PDL bounded surfaces was much more than those on PDL coated surfaces. ► Neuronal cells and their neurites on the pattern of covalently bound PDL are confined within the grid line leading to patterned neuronal networks.

Introduction

Long-term adhesion of neuronal cells with the artificial solid surface is one of the key demands in the field of tissue regeneration, neuronal cell-based biosensor, and measurement of neuronal signal in vivo and in vitro (Widge et al., 2007, Koh et al., 2008). Although there were reports that the surface roughness, stiffness, and electrical properties play an important role in neuronal adhesion and neurite outgrowth, funtionalization of the solid surface with cell adhesion molecule (CAM) has constituted the main stream of neuronal cell adhesion enhancement. CAM had been used in combination with various surface modification methods including physical adhesion (Koh et al., 2008, Haile et al., 2008), blending (Mahoney and Anseth, 2006), electrostatic attachment (Tang et al., 2006), electrochemical polymerization (Guimarda et al., 2007), and covalent bonding (Tong and Shoichet, 1998).

Poly-d-lysine (PDL) is the most widely used CAM due to its excellent neural adhesion capability which is originated from the positively charged nature (James et al., 2000, Harnett et al., 2007). It attracts neuron by electrostatic interaction with the negatively charged cell membrane and promotes neurite outgrowth. However, curiously enough, physical adhesion had been involved in most of the previous reports on PDL-based neuronal cell adhesion through simple dropping and/or dipping. It has been well-established that covalent bonding of CAM is superior to physical and electrostatic attachment and blending in that it can produce much more stable CAM layers on which neuronal cells can be cultured for a long time (Rao and Winter, 2009). Even though PDL is widely used as a reference or control, there are very few reports on the attachment of PDL by covalent bonding.

In this study we have bound PDL chemically onto the surface of sputtered silicon dioxide (SiO2) and indium-tin oxide (ITO), compared the neuronal cell adhesion property of chemically bound PDL with that of physically bound PDL, and examined the difference in cell growth morphology of cultures on both surfaces. The SiO2 and ITO substrate were selected because they are biocompatible and the most widely used materials in the fabrication of multi-electrode array. We have also fabricated grid type patterns of chemically bound PDL and physically bound PDL and then checked the fidelity of primarily cultured neuronal cells to the patterned PDL grid.

Section snippets

Materials and instruments

Poly-d-lysine and PLL-FITC was purchased from Sigma Chemical and 3-aminopropyltriethoxysilane (APTES), glutaraldehyde, anhydrous ethanol, hydrogenperoxide and ammonia were purchased from Aldrich Co. and used as received. Positive photoresist (PR, AZ GXR 601) and developer (AZ 300 MIF) were purchased from AZ Electronic Materials. X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB 200R instrument (VG scientific) with monochromized Al KR X-ray radiation operated at 1486.6 eV, 12.5

Characterization of PDL binding by contact angle measurements

One of the most effective ways to check the surface property is the contact angle measurement which can characterize the hydrophilicity and hydrophobicity of a given surface. The change in surface chemical composition induced by covalent binding processes of PDL was monitored by contact angle measurement and the result is listed in Table 1. The contact angle of bare ITO was 36.3° which decreased to <10° after the treatment with RCA solution, indicating that the ITO surface is covered with

Acknowledgments

This study was supported by the Ministry of Education, Science and Technology through Global Partnership Program (2009-00503) and Electronics and Telecommunications Research Institute (Project No. 11ZC1130).

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