ARTICLE pubs.acs.org/Langmuir
Photopatterning of Cell-Adhesive-Modified Poly(ethyleneimine) for Guided Neuronal Growth Nam Seob Baek, Ji-Hyun Lee, Yong Hee Kim, Bong Joon Lee, Gook Hwa Kim, Ik-Hyun Kim, Myung-Ae Chung, and Sang-Don Jung* IT Convergence Technology Research Laboratory, Electronics and Telecommunications Research Institute, 138 Gajeongno, Yuseong-gu, Daejeon 305-700, Republic of Korea ABSTRACT: We describe photopatterning technique that employs the photodegradation of cell-adhesive-modified poly(ethyleneimine) (m-PEI) to fabricate precise micropatterns on the indium tin oxide (ITO) substrate for guided neuronal growth. The photodegradation of m-PEI coated on hydroxyl group-terminated ITO substrate created micropatterns over a large area through deep UV irradiation. The photopatterned m-PEI layer can effectively guide neurite outgrowth and control neurite extensions from individual neurons.
’ INTRODUCTION Cell-adhesive polymers have attracted considerable interest in a wide variety of neuroelectronics,1-3 neural engineering,4,5 and basic neuroscience.6-8 In neuroelectronics, based on their celladhesive capability, they play an important role in forming welldefined networks of neuronal cells on the multielectrode array (MEA)9-12 and field-effect transistor.13-16 A well-defined network of neuronal cells can provide a useful test vehicle for examining the communication between neuronal cells and semiconductor devices under controlled conditions. They also have been employed in adjusting adhesion property of the neural prosthetic electrodes, by reducing the immune response, to achieve long-term chronic recording of a neural signal.8,17-19 Poly(ethyleneimine) (PEI), owing to its biocompatibility and positively charged surface, can interact with negatively charged neuronal cells, resulting in the strong adhesion of neuronal cells on PEI-coated substrates. Several techniques have been used to obtain PEI patterns for guiding neuronal cells including soft lithography like microcontact printing with a polydimethylsiloxane stamp,20-23 photoresist process,24,25 and deep UV lithography.26 It is generally accepted that adhesion molecules physically bound to a substrate are prone to dissolve from the substrate in the cell culture medium so that long-term adhesion is hardly expected. To check this, we have preliminarily cultured neuronal cells on the PEI physically bound to the ITO (indium tin oxide) surface. As expected, neuronal cells were easily detached from the substrate and spread toward the rim of the substrate, resulting in no adhesion (see Figure 1a). This preliminary result is attributed to the weak physical interaction between PEI and the ITO surface, leading to the dissolution of PEI during the culture period. In order to cope with the dissolution problem, we have selected PEI modified with the trimethoxylsilane moiety which can form covalent bonds with the hydroxyl groups formed on the ITO surface. In this paper we propose the patterned photodegradation of r 2011 American Chemical Society
modified PEI (m-PEI) as a new efficient method to fabricate micropatterns for guided neuronal growth.
’ EXPERIMENTAL SECTION Materials and Instruments. Poly(ethyleneimine) (Mn ∼ 60 000, 50% (w/v) in water) and trimethoxysilylpropyl-modified poly(ethyleneimine) (Mw 1500-1800, 50% in isopropanol) were purchased from Sigma and Gelest Inc., respectively. Anhydrous ethanol, hydrogen peroxide, and ammonia were purchased from Aldrich Co. and used as received. The attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrum was taken on a Nicolet iN10 spectrometer with Ge crystal, and 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 kV, and 250 W. The base pressure of the system was 5 10-10 Torr. The XPS spectra were acquired at a 90° takeoff angle relative to the surface. Tapping mode atomic force microscopy (AFM) images were taken on a Veeco multimode AFM using a nanoscope III controller. Surface Modification and Photopatterning. Before surface modification, the ITO substrates were cut into 1 cm 1 cm sized pieces. They were sonicated sequentially in a bath of methanol, acetone, and pure water for 10 min each followed by immersion into a NH3:H2O2: H2O mixture solution (1:4:20 v/v) at 60 °C for 1 h and rinsed with ultrapure water. Subsequently, hydroxyl group-terminated ITO surface was silanized using a standard self-assembly method by immersing them into a 2% modified poly(ethyleneimine) in anhydrous ethanol for 12 h at room temperature followed by baking at 120 °C for 10 min to enhance the covalent bonding of silanol moieties. The m-PEI-coated substrate was sonicated in an ethanol solution for 5 min to remove the nonspecifically bound m-PEI. The covalently bound m-PEI was exposed to a 150 W Xe light (Muller Elektronik-Optik Lampenversorgung type Received: August 24, 2010 Revised: December 22, 2010 Published: February 03, 2011 2717
dx.doi.org/10.1021/la103372v | Langmuir 2011, 27, 2717–2722
Langmuir
ARTICLE
Figure 1. Cells grown on (a) PEI-coated ITO substrate and (b) modified-PEI-coated ITO substrate. The phase-contrast micrograph was taken at 4 days after cell seeding. All scale bars are 50 μm. SVX1530, Germany, P254 nm = 300 mW/cm2) through a Cr photomask for 90 min. After UV exposure, the substrate was then immersed in ethanol solution for 10 min followed by sonication in ethanol solution for 5 min. Neuronal Cell Culture. Primary cultures were obtained from the cerebral cortex of fetal ICR mice (15-16 days gestation). Cortical neuron cell cultures were prepared from embryonic day 15 ICR mice, as described previously with minor modifications.27 Briefly, the cerebral cortical regions from embryonic day 15 mouse fetuses were dissected in ice cold Ca2þ/Mg2þfree Hanks’ salt solution, pH = 7.4. After removing the meninges, the cells were dissociated by mild trypsinization (0.25%) (Invitrogen, Carlsbad, CA) followed by gently blowing through an autoclaved pipet. The cell suspension was then plated at a density of 5.0 104 cells on modified PEI-coated and photopatterned substrate in a 12-well culture plate. The culture medium of neurobasal medium (Invitrogen) was supplemented with a 2% B27 supplement and 1% N2 supplement, 2.0 mM glutamine with antibiotics. To prevent the proliferation of non-neuronal cells, cytosine arabinoside (3 μM) was added to the cultures 24 h after plating. The culture medium was replaced with fresh medium without cytosine arabinoside every 3 days. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. Immunofluorescent Staining. Neurons were identified by immunofluorescent staining of anti-β-tubulin (neuron marker; Covance, Emeryville, CA). Neurons comprised ∼98% of the total cells. The experiments were performed after 4 days of neuron culture. For immunofluorescent imaging, the cultured cells were fixed with 4% formaldehyde for 10 min. After washing the cells with phosphate buffer solution (PBS) and permeabilized with 0.2% Triton X-100, they were incubated in 2% bovine serum albumin and 0.1% Triton X-100 in PBS for 30 min at room temperature. The primary antibody was added and incubated at 37 °C for 2 h. Mouse anti-β-tubulin monoclonal antibody (Tul1) was diluted to 4.0 μg/mL in blocking solution. The ITO substrates were washed three times with PBS and incubated for 30 min at room temperature with the secondary antibodies diluted with a blocking solution. Secondary antibodies includes Alexa Fluor 488 goat antimouse IgG (2.0 mg/mL, Invitrogen), diluted to 20 μg/mL. After washing with PBS three times, 40 ,6-diamidino-2phenylindole dihydrochloride (DAPI 1:1000 in blocking solution; Invitrogen, Carlsbad, CA) was added and incubated at room temperature for 5 min followed by washing with PBS for three times. Micrographs were captured on a Nikon fluorescence microscope (DS-Ri1 CCD camera).
’ RESULTS AND DISCUSSION Figure 1 shows neuronal cells grown on physically bound PEI and covalently bound m-PEI. It is clear from the Figure 1b that neuronal cells adhere not to physically bound PEI but to covalently bound m-PEI. This preliminary result confirms that covalent binding of m-PEI is quite effective in achieving long-term adhesion. Figure 2 shows a schematic diagram of the photopatterning procedure. Details of the photopattering procedure, surface characterization, and guided neuronal growth are described below. The hydrophilic hydroxyl group-terminated ITO substrate was immersed in a 2% m-PEI solution under a N2 atmosphere. The positively charged m-PEIs were
Figure 2. Schematic diagram of the micropatterning procedure of m-PEI on hydroxyl group-terminated ITO surface.
Figure 3. ATR-FTIR spectra of hydroxyl group-terminated ITO, before and after the photoexposure to the m-PEI layer.
linked covalently to the hydroxyl group-terminated ITO surface through a sol-gel process in an anhydrous EtOH solution.28 The m-PEI-coated ITO was then placed in a UV exposure box equipped with a constant power supply. The UV exposure and ethanol treatment resulted in the photodegradation of m-PEI and the removal of photodegraded m-PEI, respectively, yielding micropatterns over a large area (Figure 2). In our previous report PEI degrades under deep UV irradiation (
