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Cell patterning using molecular vapor deposition of self-assembled monolayers and lift-off technique
Acta Biomaterialia 7 (2011) 1094–1103
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Cell patterning using molecular vapor deposition of self-assembled monolayers and lift-off technique Gaoshan Jing a, Yu Wang a, Tianyi Zhou a, Susan F. Perry b, Michael T. Grimes c, Svetlana Tatic-Lucic a,⇑ a
Sherman Fairchild Center, Department of Electrical & Computer Engineering, Lehigh University, Bethlehem, PA, USA Department of Chemical Engineering, Lehigh University, Bethlehem USA c Applied Microstructures, Inc., San Jose, CA, USA b
a r t i c l e
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Article history: Received 14 January 2010 Received in revised form 19 September 2010 Accepted 29 September 2010 Available online 8 October 2010 Keywords: Single cell patterning Self-assembled monolayers (SAMs) Multi-electrode arrays (MEAs) Molecular vapor deposition (MVD) Biosensors
a b s t r a c t This paper reports a precise, live cell-patterning method by means of patterning a silicon or glass substrate with alternating cytophilic and cytophobic self-assembled monolayers (SAMs) deposited via molecular vapor deposition. Specifically, a stack of hydrophobic heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane SAMs and a silicon oxide adhesion layer were patterned on the substrate surface, and a hydrophilic SAM derived from 3-trimethoxysilyl propyldiethylenetriamine was coated on the remaining non-treated areas on the substrate surface to promote cell growth. The primary characteristics of the reported method include: (i) single-cell resolution; (ii) easy alignment of the patterns with the pre-existing patterns on the substrate; (iii) easy formation of nanoscale patterns (depending on the exposure equipment); (iv) long shelf life of the substrate pattern prior to cell culturing; (v) compatibility with conventional, inverted, optical microscopes for simple visualization of patterns formed on a glass wafer; and (vi) the ability to support patterned cell (osteoblast) networks for at least 2 weeks. Here, we describe the deposition technique and the characterization of the deposited layers, as well as the application of this method in the fabrication of multielectrode arrays supporting patterned neuronal networks. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Patterned growth of cultured cells, pioneered by Kleinfeld et al. at AT&T Bell Laboratories [1], is a technique gaining importance in a variety of applications and fields, such as cell-based sensors, neurobiology and tissue engineering [2,3]. The critical step in cell patterning is the formation of alternating patterns of permissive (cytophilic) or non-permissive (cytophobic) surface regions for respectively promoting and suppressing cell growth. It is well known that, in vivo, most mammalian cells require extracellular matrices (ECM), containing cytophilic proteins such as fibronectin and collagen [2], and chemical growth factors for physical attachment, survival and growth. The ECM plays a critical role in multiple cellular functions, ranging from migration to proliferation to apoptosis [2–4]. Thus, patterning of the ECM or chemicals which can promote cell attachment on a substrate is a prerequisite for the growth of patterned cells, in vitro. A number of research groups have developed various methods to pattern cytophilic and cytophobic chemicals on a substrate [5– 7]. Such methods usually combine microfabrication, chemical sur⇑ Corresponding author. Address: Sherman Fairchild Center, Department of Electrical & Computer Engineering, Lehigh University, 16A Memorial Dr. East, Bethlehem, PA 18015, USA. Tel.: +1 610 758 4552; fax: +1 610 758 6279. E-mail address: [email protected] (S. Tatic-Lucic).
face modification and material processing. Among them, microcontact printing (lCP) is the most frequently used [7–9]. This simple and cost-effective technique first requires the fabrication of desired topographical features on a silicon wafer using well-developed semiconductor processing technology. Polydimethylsiloxane is then molded onto the patterned silicon surface structure and peeled off as a stamp. Finally, the stamp, coated with a specific chemical which is favorable or non-favorable for cell growth, is brought into contact with a substrate, transferring the corresponding cytophilic or cytophobic molecules to the substrate, similar to traditional ink printing. Other patterning techniques include dip-pen nanolithography [10,11], the parylene-based dry liftoff technique [12], inkjet printing for cell patterning [13], laser scanning lithography [14] and microfluidic patterning [15]. Recently, the patterning of self-assembled monolayers (SAMs) has been intensely explored as an alternative to protein patterning. Patterning SAMs is an attractive alternative because the SAM molecules are highly orderly and exhibit controllable properties for a variety of desired applications, depending on the functionality of the terminal group (such as hydrophobic or hydrophilic control) or the chain length [16,17]. Most frequently, self-assembled monolayers have been deposited using micro-contact printing [18] or adsorption [19,20]. They have been used, among other applications, for biosensors [16,21] and making arrays of proteins and cells on chips [19,22].
1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.09.040
G. Jing et al. / Acta Biomaterialia 7 (2011) 1094–1103
However, most patterning techniques, whether based on patterning proteins or patterning self-assembled monolayers, have some limitations, such as an inability to combine single-cell resolution and simple alignment of the pattern to features and/or structures already existing on the substrate. For example, our target application is the fabrication of multielectrode arrays (MEAs) capable of supporting patterned neuronal networks with single neurons positioned on top of underlying electrodes, and defined narrow pathways connecting the electrodes, capable of supporting the outgrowth of neurites but not the attachment of neuronal cell bodies. For that application, the desired cell patterning technique must be capable of providing single-cell resolution and must be easily aligned with the underlying electrodes. Additionally, it is desirable for this technique to generate patterns which are both suitable for visualization by inverted microscopy (both prior to and during cell culture) and able to support patterned neuronal networks for extended time periods (2 weeks or more) to allow longer-term experiments. The surface patterns should also have a relatively long shelf life, making them more convenient for utilization. We sought to develop a technique that satisfied all of the requirements mentioned above at the same time. To accomplish this, we built upon several significant and relevant studies. For example, several patterning techniques have achieved single-cell patterning, such as lCP [23,24], using magnetic microposts [25] and dielectrophoresis [26]. Two related, and particularly relevant, publications describe techniques developed by Stenger et al. [27] and Ravenscroft et al. [28], whereby high-resolution substrates for cell patterning were created, based on self-assembled monolayers; our choice of surface treatment was based on these important studies. However, these previously developed cell patterning methodologies required deep UV lithography, which requires specialized equipment that was not available in our laboratory. It should also be mentioned that some earlier cell patterning techniques have demonstrated a good alignment of lCP-created patterns with respect to the underlying features, such as microelectrodes in MEAs [29,30], where modified optical aligners were utilized. Finally, extended-time experiments have been performed successfully using certain patterning methods [22,31,32]. Indeed, Branch et al. [32] have demonstrated long-term monitoring of electrical signals on patterned substrates via microelectrode arrays for four weeks. In doing so, two important goals were achieved: the formation of relatively coarse neuronal networks patterned on top of microelectrode structures, and the maintenance of the survival and activity of these networks for longer time periods. This previous study provided important groundwork; however, our envisioned networks have a finer resolution (single cell level) than that demonstrated by the previous work. In the current study, we implement, for the first time in cell patterning efforts, a recently developed SAM deposition technique from the gaseous phase (rather than the traditional method of soaking) via molecular vapor deposition (MVDÒ). Vapor-phase deposition has a potential advantage of small feature formation that would be more difficult to achieve from the traditional liquid-phase deposition. Additionally, because of its repeatability, it might have an advantage in mass manufacturing conditions. Here, we report a new, simple and effective cell patterning technique that accomplishes all of our desired goals. Using this technique, predefined patterns for cell growth can be visualized prior to cell culturing, single-cell patterning resolution is achieved and the patterns can be used to form a complicated biosensor system with accurate alignment using a conventional aligner. This method uses a patterned bilayer of a hydrophobic fluorine-based SAM, heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane (FDTS), on a silicon oxide (SiO2) adhesion layer, using the MVD process to create a cytophobic surface that prevents cell growth. A hydrophilic
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SAM, 3-trimethoxysilyl propyldiethylenetriamine (DETA), which has been shown to be cytophilic and encourages cell adhesion [27,28,33], is then backfilled on the open silicon oxide surface to promote cell growth in alternating regions. The predefined patterns we obtain can be conveniently visualized prior to cell culturing. As this method is photolithography-based, it also achieves high resolution and single-cell positioning and patterning, which can be maintained for extended culture periods. Additionally, the deposited materials display long-term reliability. Using this method, we have successfully patterned immortalized mouse hypothalamic (GT1–7) neurons and mouse (MC3T3) osteoblast cells, with osteoblast patterns being maintained for at least 14 days in vitro.
2. Materials and methods 2.1. Microfabrication of substrate The microfabrication process, illustrated in Fig. 1, starts from a single crystal silicon wafer, three inches in diameter. First, a silicon oxide layer of 100 nm thickness was deposited at 130 °C using a plasma-enhanced chemical vapor deposition (PECVD) method (Fig. 1a). Then positive photoresist (Shipley 1818, Shipley Company Inc, MA) was spin-coated at 3000 rpm for 30 s to obtain a 2.2 lm thick thin film (Fig. 1b). After prebaking at 90 °C for 60 s, the photoresist was exposed to UV light (wavelength k = 405 nm, power density = 12 mW cm–2) for 6.4 s using an EV620 mask aligner (EV Group Inc., NY), developed in Shipley MF321 developer for 90 s, then flood-exposed for 60 s to assist photoresist stripping later (Fig. 1c). Once photolithography was completed, the substrate with patterned photoresist was coated successively with a 10 nm thick silicon oxide adhesion layer followed by a self-assembled organosilane functional monolayer of FDTS (chemical structure illustrated in Fig. 1) using an MVD system that was initially developed for applying anti-stiction coatings to micromechanical systems (MEMS) devices. The MVD system (schematically shown in Fig. 2) and the deposition procedure have been described in detail elsewhere [34] and are also presented in the ‘‘Supplementary Material” to this paper. Following the MVD processing (Fig. 1d), the whole 3 inch wafer was cut into dies, 1.5 1.5 cm in size, using a K&S 7100 dicing saw (Kulicke & Soffa, PA). This die size was chosen so that the die would fit in a well of a six-well culture plate (BD Biosciences, NJ) during subsequent cell culturing. Next, the photoresist was stripped in acetone for 5 min, and the wafers were cleaned by an ultrasonic cleaner (VWR, PA; Fig. 1e), immersed in a 9 mM solution of DETA (Gelest, Inc., PA) for 1 h and withdrawn. The DETA has been shown to function in a manner similar to poly-D-lysine, and promotes cell attachment and growth [33]. Due to the high DETA/FDTS inter-surface energy, DETA does not rest on the FDTS SAM film. The postMVD immersion and withdrawal resulted in a cytophilic DETA SAM pattern complementary to the cytophobic pattern of the MVD-produced FDTS SAM/SiO2 bilayer. The substrates were rinsed with ethanol and deionized (DI) water and dried with a stream of nitrogen to remove any residual solvent (Fig. 1f) before cell culturing (Fig. 1g). While the EV620 contact aligner can reach line widths of approximately 2 lm, projection optics in an Autostep stepper (AS200, GCA Corporation, MA) allowed us to achieve a 0.5 lm line width of patterned photoresist. In this case, we used OiR 620-7i iline photoresist (Fuji films, Japan), 0.45 lm thick, soft-baked at 90 °C for 1 h, exposed for 0.168 s and baked at 115 °C for 1 min, followed by development in AZ 300 MIF developer (AZ Electronics Materials US Corporation, NJ) for 1 min. Then FDTS SAM/SiO2 bilayer was deposited using the MVD process and subsequently
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Fig. 1. (a–g) Schematic illustration of the cell patterning procedure based on molecular vapor deposition of self-assembled monolayers and lift-off technique. (h) Chemical structure of FDTS.
beam current of 300 pA. The electron high voltage was 2 kV, the aperture 30 lm and the working distance 5 mm. Developing was performed in a 3:1 mixture of IPA:methyl isobutyl ketone (Sigma Alrdich, MO, US) for 70 s with gentle agitation. 2.2. Characterization of patterned culturing substrate
Fig. 2. Schematic of the MVD vapor deposition system [34].
patterned using the lift-off technique. The above-mentioned microfabrication process was executed on both silicon wafers coated with PECVD oxide and on bare glass wafers. Finally, nanoscale features in our FDTS layer were demonstrated, using E-beam photolithography and the lift-off method. First, 950 PMMA resist (Microchem Inc) was mixed with A – thinner (Microchem Inc) in a 3:1 ratio. Then, the photoresist was spin coated at 3000 rpm and baked in a convection oven at 150 °C for 1 h. It was exposed using E-beam write Leo 1550DVP (Leo Electron Microscopy Inc, UK). The writing dose was 250 lC cm–2, with a
The oxide thicknesses were measured using an ellipsometer (LSE Stokes, Gaertner Scientific Corporation, IL) using a wavelength of 632.8 nm (HeNe laser source) at a 70° angle of incidence. Films were modeled using a refractive index of 1.46 at the above wavelength for silicon oxide. The uncertainty in the thickness measurements is ±0.1 nm. The topography of the patterned substrate was characterized using a scanning electron microscope (SEM; Hitachi S-4300, CA), operated at an accelerating voltage of 3 kV, and a Dimension 3100 scanning probe microscope (SPM; Veeco, CA), operated in the tapping mode. Typical SPM height and amplitude images of the patterned substrate surface were recorded as data files, and processed using the offline image processing software WSxM (Nanotec, IN) to obtain three-dimension topography images and average cross-section profiles. To confirm the hydrophilic and hydrophobic nature of different areas on the patterned substrate, the substrate surface was sprayed with DI water and observed under an optical microscope (Nikon 4500, NY). Its contact angle with water was measured by a contact angle meter (VCA Optima Contact Angle Meter, MA).
G. Jing et al. / Acta Biomaterialia 7 (2011) 1094–1103
Whereas PECVD silicon oxide is free of it, FDTS contains fluorine, which has a characteristic KLL Auger electron energy of 659 eV [35]. Therefore a peak at 659 eV in an Auger spectrum will definitely confirm the presence of FDTS on our substrate surface. Taking such a characteristic into consideration, the chemical nature of our patterned surface was analyzed using a PHI 670 scanning Auger microscope (Physical Electronics, MN), which is also equipped with a secondary electron detector and can obtain conventional secondary electron images. During the operation, the sample chamber was in ultrahigh vacuum (under 10 10 torr) and the primary electron beam was accelerated by 10 kV. First, the substrate pattern was imaged in the conventional secondary electron mode, then the primary electron beam was focused on typical regions revealed by the secondary electron micrograph and Auger spectra were collected with energy scanned from 50 to 1700 eV. Finally, the intensity of the fluorine KLL Auger electron was used to construct micrographs which clearly illustrate the distribution of fluorine, or FDTS, over the surface.
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graphs using the imaging software, SPOT (Diagnostic Instruments, MI). 3.3. Patterned cell imaging by scanning electron microscopy Cell patterns were further analyzed using an SEM (Hitachi S4300, CA) operated at an accelerating voltage of 3 kV, following a standard procedure of sample preparation [37]. Briefly, the patterned cells were rinsed with PBS (pH 7.4) solution for 5 min and fixed in 10% neutral buffered formalin solution for 4 h. After the fixation, the substrate (with patterned cells) was rinsed in DI water, dehydrated in a series of ethanol solutions (from 35% to 100%), soaked in 100% hexamethyldisilazane (HMDS) (VWR, PA) for 5 min, and left in air for drying for 30 min, and finally coated with a 5.5 nm thick gold layer by sputtering. 4. Results 4.1. Culturing substrate
3. Culturing and imaging of patterned cells 3.1. Cell dissociation and culturing Immortalized mouse hypothalamic neurons (GT1–7 cell line) were chosen to assess the effect of the SAM patterns on cell positioning and growth (Fig. 1g). GT1–7 cells were maintained in 25 cm2 flasks (Fisher Scientific, GA) in a humidified environment at 37 °C in the presence of 8% carbon dioxide in a culture medium consisting of Dulbecco’s modified Eagle’s medium (Gibco, NY) supplemented with 1 mM sodium pyruvate, 10 mM sodium bicarbonate, 2 mM L-glutamine, 10 mM Hepes buffer and 10% fetal bovine serum (Invitrogen, CA) [36]. Immortalized mouse osteoblast (MC3T3) cells were also patterned using our technique, for the purpose of investigating the long-term maintenance of cell patterns. The MC3T3 cells were grown in minimum essential medium, a modification (Invitrogen, CA), supplemented with 2 mM L-glutamine, 1% penicillin and streptomycin, and 10% fetal bovine serum (Invitrogen, CA). The MC3T3 cells were maintained in 25 cm2 flasks (Fisher Scientific, GA) in a humidified environment at 37 °C in the presence of 5% carbon dioxide. Silicon dies, with complementary cytophobic (FDTS) and cytophilic (DETA) SAM patterns, were placed into wells of six-well culture plates (BD Biosciences, CA). GT1–7 and MC3T3 cells were harvested by incubating in 0.125% (w/v) trypsin solution at 37 °C for 8 min, centrifuging at 750 rpm for 5 min, resuspending in the culture medium and transplanting into wells containing silicon dies at a density of 2 104 cells ml 1. The cells were maintained under the above-mentioned standard conditions until their patterns became observable under an optical microscope (typically 48 h later). We also maintained patterned M3T3 cells for up to 2 weeks (as documented in Section 4.2), and the same cell protocol was implemented for extended culturing times, with additional medium changes every 3–4 days. 3.2. Patterned cell imaging by epifluorescent microscopy The patterned cells were rinsed with phosphate-buffered saline (PBS) solution, incubated with a fluorescent live cell stain, calcein AM (2 lM in cell-specific, serum-free medium; Invitrogen, CA), at room temperature for 20 min, and observed using an IX-70 inverted microscope (Olympus, NY). Phase-contrast and fluorescence images were captured through a SPOT, RTKE/SE digital camera (Diagnostic Instruments, MI). The magnification of captured micrographs was calibrated using an OB-M 1/100 stage micrometer (Olympus Inc, NY) and an accurate scale bar was added to micro-
The average thickness of an oxide adhesion layer is 10 nm by ellipsometric measurement and the average thickness of a patterned FDTS SAM/silicon oxide bilayer is 11 nm as determined by SPM measurement. Fig. 3a shows a conventional (or secondary electron) SEM micrograph of patterned regions analyzed by Auger electron microscopy. The Auger spectra presented in Fig. 3b were collected from the rectangular bright and dark areas, the top surfaces of which are presumed to be an FDTS SAM/silicon oxide bilayer and PECVD silicon oxide, respectively. Whereas there is no peak around 659 eV in the Auger spectrum (dark line) from the dark area, the spectrum from the bright area (red line) has an obvious peak at 659 eV (Fig. 3b), which is attributed to fluorine KLL Auger electrons [35]. This indicates that the dark area is free of FDTS, whereas the bright area is covered by FDTS. The scanning Auger electron micrograph shown in Fig. 3c further indicates the distribution of FDTS and PECVD silicon oxide on the top surface of the prepatterned substrate for cell patterning, over a larger area. The micrograph matches very well with the secondary electron micrograph (Fig. 3d), except that the former has larger image pixels (or a lower resolution) than the latter because of the resolution limitation of the instrument. All these results clearly confirm that the top surface chemical patterning was as expected. Additionally, we have demonstrated FDTS SAM/oxide bilayer features 200 nm in diameter using E-beam lithography and a liftoff technique, as shown in Fig. 4. 4.2. Cell patterns on silicon substrate Typical intermediate and final cell patterns on silicon substrates are presented in Fig. 5. The photoresist pattern was transferred to the cytophobic FDTS SAM/silicon oxide patterns (as shown in Fig. 5a) following the MVD and lift-off processes. After the entire wafer is immersed and withdrawn from DETA solution, the DETA SAM is coated on regions complementary to FDTS areas, not on top of the FDTS SAM, as discussed and concluded in the previous subsection. The patterns of cytophilic DETA and cytophobic FDTS SAM/silicon oxide were replicated by the GT1–7 cells, as shown by the cell patterns displayed in Fig. 5b. Comparison of Fig. 5a with Fig. 5b indicates that the final cell patterns are faithful replicas of pre-existing patterns on the culturing substrates. To pattern a neural network, we designed a pre-culturing pattern containing islands connected by lines of different widths, where the islands and their connecting lines, respectively, were coated with cytophilic DETA SAM for neuronal cell bodies and neurites, and the surrounding area was coated with a cytophobic FDTS
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Fig. 3. (a) SEM micrograph of the regions analyzed by Auger electron microscopy. (b) Auger spectra collected from the rectangular areas 1 (bright, red line) and 2 (dark, black line), shown in (a), are typical of designed FDTS SAM/SiO2 bilayer and PECVD SiO2 regions, respectively. The peak around 659 eV is attributed to the fluorine KLL Auger electron. (c) Fluorine Auger electron and (d) secondary electron SEM micrograph of the same patterned area. In both micrographs, the bright and dark areas are FDTS SAM/ SiO2 bilayer and PECVD SiO2, respectively. Since the F Auger electron was very weak, a pixel region in (c) is larger than that in (d) so as to collect enough Auger electron intensity for a reasonable amount of time.
SAM/silicon oxide bilayer to prevent cell anchoring and growth. Our scheme succeeded, as is shown in Figs. 5b and 6b. From observations of a wide variety of cell patterns, it was determined that the island size and line width of the connecting line play important roles in the neural network patterning. An island size of 20 lm is recommended for a single neural cell body to anchor, and a line width of 5 lm is sufficient for neurites to grow from one island to another (Fig. 5). Whereas neurites cannot grow without the connecting line (Fig. 5), lines wider than 10 lm are occasionally populated by cell bodies (Fig. 5). With an optimal combination of island shape, size and connecting line width, a complex neural network can be patterned. Fig. 6b shows an example of cells positioned onto circular islands, 12 lm in diameter, connected by 6 lm wide pathways. Because our technique is a passive way to pattern cells, which means cells find appropriate sites to survive by themselves, vacant islands can be observed occasionally, as shown in Fig. 6b. Accurate cell patterns can also be observed using an SEM. Fig. 7 demonstrates the manner in which a GT1–7 cell body can be defined by and attached to a cytophobic/cytophilic pattern on a
culturing substrate. The circular island, 20 lm in diameter, was coated with cytophilic DETA SAM and surrounded by an area coated with the cytophobic FDTS/silicon oxide bilayer. The cell body is attached to the island so accurately that the island actually defines the cell’s body shape. It is interesting to note that both GT1–7 cells and MC3T3 osteoblasts can adapt their bodies to anchor on FDTS SAM/silicon oxide bilayer lines only 0.5 lm in width (in the rare cases when they manage to anchor on them), even though the line width was much smaller than the size of both of these cells in suspension, which is approximately 10 lm [38]. Fig. 8 shows two MC3T3 cells anchored on a line that is only half a micron wide. It is obvious that cells anchored on thin cytophilic lines (0.5 lm) are more elongated than those anchored on wider lines. Additionally, we have performed long-term monitoring of cell patterns (specifically M3T3 cells) on silicon culturing substrates with MVD patterns. Our results are illustrated in Fig. 9. It can be seen that MC3T3 cell patterns are clear and cells are viable, as determined by calcein-AM staining, even after 14 days in culture, indicating the long-term capability of our novel patterning
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Fig. 4. FDTS SAM/silicon oxide bilayer circles with a diameter of 200 nm were fabricated by E-beam lithography and a lift-off technique. The artifact in the bottom right corner is believed to be due to the lift-off process imperfection.
Fig. 6. (a) A complex pre-culturing network of circular islands, 12 lm in diameter, connected by lines 6 lm in width after photoresist lift-off; (b) complex neural network of GT1–7 cells obtained after 48 h. Cytophobic FDTS/oxide bilayer regions are greenish-yellow whereas PECVD oxide regions are dark green in (a). Some islands are not occupied by any cells in (b).
onto islands and lines on transparent glass substrates, as well as predefined FDTS SAM/silicon oxide bilayer patterns. 5. Discussion
Fig. 5. (a) Pre-culturing patterns with greenish-yellow FDTS/oxide bilayer and dark green PECVD oxide patterns. The island size is 20 20 lm, and the line widths are 5 and 10 lm. (b) Final GT1–7 neural cell pattern after 48 h. The right part of the top figure shows a well-developed neural network, where cell bodies occupy most of the islands and networking neurites grow neatly along 5 lm wide cytophilic pathways.
method. Longer patterning times are essential for many studies, particularly for experiments on differentiation. 4.3. Cell patterns on transparent glass substrates For applications in tissue engineering, it is desirable to pattern cells on a transparent (rather than opaque) glass substrate, so that their growth can be monitored by a conventional inverted microscope [6]. Although the FDTS SAM/silicon oxide bilayer patterns on transparent glass are still invisible under a conventional optical microscope in the bright-field, dark-field or phase-contrast mode, they can be visualized by an inverted microscope in the differential interference contrast (DIC) mode. In the DIC mode, the step height of patterned FDTS SAM/silicon oxide layers (11 nm) was utilized to derive contrast between different topographic regions. Fig. 10 shows DIC micrographs of GT1–7 neurons successfully patterned
In our investigations of the FDTS SAM/silicon oxide bilayer deposited using molecular vapor deposition, we first focused on the necessity of depositing a silicon oxide layer below the hydrophobic SAM. It was determined that the MVD silicon oxide adhesion layer plays several critical roles. Firstly, it enhances the attachment of FDTS SAM to the substrate. On the one hand, it has a strong adhesion to the underlying PECVD oxide or to the transparent glass substrate. On the other hand, during the MVD FDTS deposition step, the freshly in situ deposited silicon oxide layer provides an ideal surface on which to covalently bond the subsequent FDTS SAM layer. Secondly, the MVD silicon oxide adhesion layer slightly increases the hydrophobicity of the FDTS coating and significantly enhances the stability of the coating. Enhancing the stability of hydrophobic films MVD silicon oxide adhesion layers has been previously demonstrated for FDTS [39]. An FDTS, or other similar organosilane SAM film, such as tridecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane (FOTS), when coated on an in situ MVD deposited silicon oxide adhesion layer, retains its high water contact angle (>100°) for at least 14 days after deposition. In contrast, the water contact angle of an FDTS or FOTS film without the MVD adhesion layer decreases significantly after only 8 days [39,40]. Even more importantly, we have discovered that our MVD FDTS SAM keeps its strong hydrophobicity for at least 2 years, as
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Fig. 7. SEM micrographs of a circular island 20 lm in diameter covered by DETA SAM and surrounded by an area coated with the cytophobic FDTS SAM/oxide bilayer (a) with and (b) without an attached GT1–7 neuronal cell. The circular halo around the perimeter of the island is due to an imperfect lift-off process implemented here. (Note: The process applied here was contact photolithography.).
Fig. 9. (a) Pre-culturing patterns with SAM-treated cytophilic PECVD oxide islands, 30 30 lm square, with 20 lm wide interconnecting lines, and FDTS/oxide bilayer in the surrounding area. (b) Final MC3T3 cell patterns after 14 divisions. Cells were observed using an upright fluorescent microscope.
Fig. 8. SEM micrograph of two MC3T3 cells anchored along an FDTS SAM/oxide bilayer line of 0.5 lm width (fabricated using projection lithography and a lift-off technique).
demonstrated by the fact that the water contact angle did not change significantly during that period of time. Immediately after deposition, the contact angle was 118°; 2 years later, during which
period the sample was not stored in a clean-room environment, the contact angle was 110°, as measured by a contact angle meter (VCA Optima Contact Angle Meter, MA). This compares favorably with the other cytophobic materials commonly used in cell patterning, such as polyethylene glycol [18], which has a shorter shelf life in terms of effectively preventing cell growth, as it is easily oxidized in the ambient environment [7]. Thirdly, the MVD silicon oxide adhesion layer enhances the contrast of FDTS SAM patterns, making them visible under a conventional optical microscope. A single FDTS SAM is optically invisible because its thickness is only about 1 nm. With the introduction of the 10 nm silicon oxide adhesion layer, the combined bilayer has a thickness of 11 nm and its patterns on silicon wafers and transparent glass can be visualized using an optical microscope in reflection and DIC modes, respectively (as shown in Fig. 10).
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combination of these attributes (if not the specific characteristics by themselves) makes this technique unique. Whereas single-cell resolution of cell-patterning methods has been demonstrated before [25,26], it has rarely been combined with the alignment capability (without requiring specialized or custom-modified equipment) [29,30], patternability in the nanoscale regime or the ability to visualize the cytophobic/cytophilic pattern prior to cell growth. Moreover, the long shelf life (at least 2 years) and the ability to support longer-term experiments make this technique even more attractive. The ultimate application of this technique in our laboratory is the creation of MEA chips able to support patterned cell networks, as shown Fig. 11. The benefits of combining such performance
Fig. 10. Typical DIC micrographs of GT1–7 neurons patterned onto (a) lines of width varying from 20 to 50 lm and (b) 50 50 lm square islands on transparent glass substrates. Note that the FDTS SAM/silicon dioxide bilayer has been stored for 2 years in an ambient environment and DETA SAM was freshly prepared just before cell culturing.
We should also note that profilometry has indicated that the bilayer region formed using contact lithography has a sharp edge due to a suboptimal lift-off process (as shown in Fig. 7b). We do not believe that it had a major role in our cell patterning efforts, however, due to the fact that we have observed that cells do not have trouble traversing heights as large as 22 nm unless the cytophobic/cytophilic pattern is present on a wafer [41]. Having determined that the silicon oxide adhesion layer greatly enhances the formation, stability and visibility of hydrophobic FDTS-coated regions, we then evaluated the performance characteristics of the described patterning technique. We have determined that our technique has a single-cell resolution (in fact, submicron features were successfully patterned, Fig. 8), and that, due to the photolithographic nature of the process, the cytophilic patterns can be defined so that they are easily aligned with the features already in existence on the substrate (e.g. microelectrodes in this article). Moreover, these patterns can be visualized prior to cell growth, so that there is no ambiguity about whether a compromised cell pattern is due to an ill-defined substrate or to the cell culturing process itself. Finally, we have demonstrated that osteoblast cell patterns, generated as a result of our technique, can be maintained for at least 2 weeks, which makes this technique appropriate for longer-term experiments with cultured cells. The
Fig. 11. (a) An MEA supporting patterned cell growth which utilizes MVDdeposited SAMs for defining of cell locations and pathways between electrodes photographed prior to lift-off. (b) Mouse osteoblast cells (MC3T3) cultured on an MVD MEA chip after 7 divisions.
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characteristics are easily recognizable for other applications as well. For instance, utilization of this patterning process could improve the assembly of complex biosensors or aid in the creation of arrays for interrogating multiple cells at the same time, while fulfilling the need for those cells to be positioned at predefined locations. One important question that needs to be addressed is whether it is the MVD technique itself or the molecules used for SAMs that are responsible for the obtained results. In this work, we did not directly compare the cell-patterning performance of the MVD-deposited SAMs with respect to identical SAMs using other methods (such as soaking). It could be said that the MVD deposition is highly compatible with the standard types of semiconductor processing, such as photolithographic definition of the desired patterns and the lift-off process, so the high-resolution feature definition and alignment capability could be attributed to the SAM deposition from the gaseous phase (MVD) rather than soaking (liquid phase). Additionally, MVD deposition enables deposition of a silicon oxide layer directly beneath the FDTS using the same equipment, which (as previously discussed) enhances the adhesion of this layer, slightly increases the hydrophobicity of the FDTS coating, significantly enhances the stability of the coating and enhances the contrast of FDTS SAM patterns, making them visible under a conventional optical microscope. The other beneficial characteristics, such as long shelf life and capability to maintain patterns of some cell types (for example, osteoblasts) for at least 14 days in vitro, stem from the SAM molecules, themselves. Additionally, it is well accepted that both cell-dependent processes, such as the secretion of proteolytic or oxidative enzymes, and cell-independent processes, e.g. hydration of nonadhesive layers or the presence of serum proteins in the culture medium, play a role in the stability of micropatterned surfaces [42,43], and thus in the fidelity of generated cell patterns. Desorption and/or exchange processes of the passivating FDTS SAM/silicon oxide bilayer layer by components in the culture medium or byproducts of cellular processes may ultimately lead to degradation of the surface molecules; consequently, pattern degradation could occur over time. However, the stability of the cell patterns for up to 2 weeks suggests a relatively strong interaction between the passivating layer and the substrate surface, especially for the MVD FDTS SAM, which keeps its strong hydrophobicity for at least 2 years.
6. Conclusion We have developed a simple and effective cell-patterning method based on a combination of photolithography and MVD of an FDTS SAM/silicon oxide bilayer. The culturing substrate was prepatterned with alternating cytophilic DETA SAM areas and cytophobic FDTS SAM/silicon oxide bilayer regions deposited by MVD. While photolithography patterning of culturing substrate guarantees precise pattern alignment, the introduction of the MVD silicon oxide layer enhances the adhesion of the FDTS SAM to the substrate, increases the stability of the hydrophobic FDTS SAM, and greatly facilitates the visualization of cytophobic/cytophilic patterns prior to and during patterned cell culturing. This method has the following characteristics: (i) single-cell resolution; (ii) easy alignment of the patterns with the pre-existing patterns on the substrate; (iii) easy formation of nanoscale patterns (depending on the exposure equipment); (iv) long shelf life of the substrate pattern prior to cell culturing; (v) compatibility with conventional, inverted, optical microscopes for simple visualization of patterns formed on a glass wafer; and (vi) the ability to support patterned cell (osteoblast) networks for at least 2 weeks. These performance traits make our method suitable for making complex, mass-manufacturable devices supporting cultured cell patterns.
Acknowledgements This work was supported by National Science Foundation (CAREER grant ECS-0448886 and NER grant BES-0608742), as well as Pennsylvania Infrastructure Technology Alliance (Grant PA-DCED C000016682). The fabrication part of this work was performed at the Cornell NanoScale Facility (CNF), a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation (Grant ECS 03-35765). We are grateful to Kanlun Li, of our laboratory, for her work related to cell culturing experiments. We also would like to thank Prof. Gregory Ferguson with the Department of Chemical Engineering at Lehigh University for his constructive advice, and thank Mr. Vince Bojan with the Materials Characterization Lab at Penn State University for his advice and help with the Auger electron spectroscopy experiments. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1, 2, 3, 5, 6, 9 and 11 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio. 2010.09.040. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2010.09.040. References [1] Kleinfeld D, Kahler KH, Hockberger PE. Controlled outgrowth of dissociated neurons on patterned substrates. J Neurosci 1988;8:4098–120. [2] Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular biology of the cell. fourth ed. New York: Garland; 2002. [3] Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Geometric control of cell life and death. Science 1997;276:1425–8. [4] Dike LE, Chen CS, Mrksich M, Tien J, Whitesides GM, Ingber DE. Geometric control of switching between growth, apoptosis, and differentiation during angiogenesis using micropatterned substrates. In Vitro Cell Dev Biol Anim 1999;35:441–8. [5] Yarmush M, King K. Living-cell microarrays. Annu Rev Biomed Eng 2009;11:235–57. [6] Falconnet D, Csucs G, Michelle Grandin H, Textor M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006;27:3044–63. [7] Whitesides GM, Ostuni E, Takayama S, Jiang X, Ingber DE. Soft lithography in biology and biochemistry. Annu Rev Biomed Eng 2001;3:335–73. [8] Ruiz SA, Chen CS. Microcontact printing; a tool to pattern. Soft Matter 2007;3:168–77. [9] Thibault C, Le Berre V, Casimirius S, Trevisiol E, Francois J, Vieu C. Direct microcontact printing of oligonucleotides for biochip applications. J Nanobiotechnol 2005;3:7. [10] Piner RD, Zhu J, Xu F, Hong S, Mirkin CA. Dip-pen nanolithography. Science 1999;283:661–3. [11] Salaita K, Wang Y, Mirkin CA. Applications of dip-pen nanolithography. Nat Nanotechnol 2007;2:145–55. [12] Ilic B, Craighead HG. Topographical patterning of chemically sensitive biological materials using a polymer-based dry lift off. Biomed Microdevices 2000;2:317–22. [13] Roth EA, Xu T, Das M, Gregory C, Hickman JJ, Boland T. Inkjet printing for highthroughput cell patterning. Biomaterials 2004;25:3707–15. [14] Miller JS, Bethencourt MI, Hahn M, Lee TR, West JL. Laser-scanning lithography (LSL) for the soft lithographic patterning of cell-adhesive self-assembled monolayers. Biotechnol Bioeng 2006;93:1060. [15] Chiu DT, Jeon NL, Huang S, Kane RS, Wargo CJ, Choi IS, et al. Patterned deposition of cells and proteins onto surfaces by using three-dimensional microfluidic systems. PNAS 2000;97:2408–13. [16] Franks W, Tosatti S, Heer F, Seif P, Textor M, Hierlemann A. Patterned cell adhesion by self-assembled structures for use with a CMOS cell-based biosensor. Biosens Bioelectron 2007;22:1426–33. [17] Arya S, Solanki P, Datta M, Malhotra B. Recent advances in self-assembled monolayers based biomolecular electronic devices. Biosens Bioelectron 2009;24:2810–7. [18] Smith RK, Lewis PA, Weiss PS. Patterning self-assembled monolayers. Prog Surf Sci 2004;75:1–68.
G. Jing et al. / Acta Biomaterialia 7 (2011) 1094–1103 [19] Yamauchi F, Kato K, Iwata H. Micropatterned, self-assembled monolayers for fabrication of transfected cell microarrays. Biochim Biophys Acta, Gen Subj 2004;1672:138–47. [20] Jing G, Yao Y, Gnerlich M, Perry S, Tatic-Lucic S. Towards a multi-electrode array (MEA) system for patterned neural networks. Procedia Chem 2009;1:329–32. [21] Chaki N, Vijayamohanan K. Self-assembled monolayers as a tunable platform for biosensor applications. Biosens Bioelectron 2002;17:1–12. [22] Kira A, Okano K, Hosokawa Y, Naito A, Fuwa K, Yuyama J, et al. Micropatterning of perfluoroalkyl self-assembled monolayers for arraying proteins and cells on chips. Appl Surf Sci 2009;255:7647–51. [23] Wheeler BC, Corey JM, Brewer GJ, Branch DW. Microcontact printing for precise control of nerve cell growth in culture. J Biomech Eng 1999;121:73–8. [24] McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS. Cell shape, cytoskeletal tension, and rhoa regulate stem cell lineage commitment. Dev Cell 2004;6:483–95. [25] Sniadecki N, Anguelouch A, Yang M, Lamb C, Liu Z, Kirschner S, et al. Magnetic microposts as an approach to apply forces to living cells. Proc Nat Acad Sci 2007;104:4553. [26] Mittal N, Rosenthal A, Voldman J. NDEP microwells for single-cell patterning in physiological media. Lab Chip 2007;7:1146–53. [27] Stenger D, Hickman J, Bateman K, Ravenscroft M, Ma W, Pancrazio J, et al. Microlithographic determination of axonal/dendritic polarity in cultured hippocampal neurons. J Neurosci Meth 1998;82:167–73. [28] Ravenscroft M, Bateman K, Shaffer K, Schessler H, Jung D, Schneider T, et al. Developmental neurobiology implications from fabrication and analysis of hippocampal neuronal networks on patterned silane-modified surfaces. J Am Chem Soc 1998;120:12169–77. [29] James CD, Spence AJH, Dowell-Mesfin NM, Hussain RJ, Smith KL, Craighead HG, et al. Extracellular recordings from patterned neuronal networks using planar microelectrode arrays. IEEE Trans Biomed Eng 2004;51:1640–8. [30] Chang JC, Brewer GJ, Wheeler BC. A modified microstamping technique enhances polylysine transfer and neuronal cell patterning. Biomaterials 2003;24:2863–70. [31] Naka Y, Eda A, Takei H, Shimizu N. Neurite outgrowths of neurons on patterned self-assembled monolayers. J Biosci Bioeng 2002;94:434–9.
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[32] Branch DW, Wheeler BC, Brewer GJ, Leckband DE. Long-term maintenance of patterns of hippocampal pyramidal cells on substrates of polyethylene glycol and microstamped polylysine. IEEE Trans Biomed Eng 2000;47: 290–300. [33] Hickman JJ, Stenger DA. Interactions of cultured neurons with defined surfaces. In: Stenger DA, McKenna TM, editors. Enabling technologies for cultured neural networks. San Diego, CA: Academic Press; 1994. p. 51–76. [34] Kobrin B, Fuentes V, Dasaradhi S, Yi R, Nowak R, Chinn J, et al. Molecular vapor deposition–an improved vapor-phase deposition technique for molecular coatings for MEMS devices. San Francisco, CA: Semicon West; 2004. [35] Albridge R, Hamrin K, Johansson G, Fahlman A. The KLL Auger spectrum of fluorine. Zeitschrift für Physik A Hadrons and Nuclei 1968;209:419–27. [36] Liposits Z, Merchenthaler I, Wetsel WC, Reid JJ, Mellon PL, Weiner RI, et al. Morphological characterization of immortalized hypothalamic neurons synthesizing luteinizing hormone-releasing hormone. Endocrinology 1991;129:1575–83. [37] See http://www.bdbiosciences.com/external_files/dl/doc/tech_bulletin/live/ web_enabled/tb405.pdf#search=%28Technical%20Bulletin%20405%29. [38] Jing G, Labukas JP, Iqbal A, Perry SF, Ferguson GS, Tatic-Lucic S. A novel method for accurate patterning and positioning of biological cells. Proc SPIE 2007;6592:65920Y. [39] Kobrin B, Zhang T, Grimes MT, Chong K, Wanebo M, Chinn J, et al. An improved chemical resistance and mechanical durability of hydrophobic FDTS coatings. J Phys Conf Ser 2006:454. [40] Kobrin B, Chinn J, Ashurst R. Vapor deposition of composite organic inorganic films. Washington, DC: World Tribology Congress; 2005. [41] Jing G, Perry S, Tatic-Lucic S. Precise cell patterning using cytophobic selfassembled monolayer deposited on top of semi-transparent gold. Biomed Microdevices 2010;12:935–48. [42] Nelson C, Raghavan S, Tan J, Chen C. Degradation of micropatterned surfaces by cell-dependent and independent processes. Langmuir 2003;19:1493–9. [43] Lussi J, Falconnet D, Hubbell J, Textor M, Csucs G. Pattern stability under cell culture conditions–a comparative study of patterning methods based on PLLg-PEG background passivation. Biomaterials 2006;27:2534–41.
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Cell patterning using molecular vapor deposition of self-assembled monolayers and lift-off technique Gaoshan Jing a, Yu Wang a, Tianyi Zhou a, Susan F. Perry b, Michael T. Grimes c, Svetlana Tatic-Lucic a,⇑ a
Sherman Fairchild Center, Department of Electrical & Computer Engineering, Lehigh University, Bethlehem, PA, USA Department of Chemical Engineering, Lehigh University, Bethlehem USA c Applied Microstructures, Inc., San Jose, CA, USA b
a r t i c l e
i n f o
Article history: Received 14 January 2010 Received in revised form 19 September 2010 Accepted 29 September 2010 Available online 8 October 2010 Keywords: Single cell patterning Self-assembled monolayers (SAMs) Multi-electrode arrays (MEAs) Molecular vapor deposition (MVD) Biosensors
a b s t r a c t This paper reports a precise, live cell-patterning method by means of patterning a silicon or glass substrate with alternating cytophilic and cytophobic self-assembled monolayers (SAMs) deposited via molecular vapor deposition. Specifically, a stack of hydrophobic heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane SAMs and a silicon oxide adhesion layer were patterned on the substrate surface, and a hydrophilic SAM derived from 3-trimethoxysilyl propyldiethylenetriamine was coated on the remaining non-treated areas on the substrate surface to promote cell growth. The primary characteristics of the reported method include: (i) single-cell resolution; (ii) easy alignment of the patterns with the pre-existing patterns on the substrate; (iii) easy formation of nanoscale patterns (depending on the exposure equipment); (iv) long shelf life of the substrate pattern prior to cell culturing; (v) compatibility with conventional, inverted, optical microscopes for simple visualization of patterns formed on a glass wafer; and (vi) the ability to support patterned cell (osteoblast) networks for at least 2 weeks. Here, we describe the deposition technique and the characterization of the deposited layers, as well as the application of this method in the fabrication of multielectrode arrays supporting patterned neuronal networks. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Patterned growth of cultured cells, pioneered by Kleinfeld et al. at AT&T Bell Laboratories [1], is a technique gaining importance in a variety of applications and fields, such as cell-based sensors, neurobiology and tissue engineering [2,3]. The critical step in cell patterning is the formation of alternating patterns of permissive (cytophilic) or non-permissive (cytophobic) surface regions for respectively promoting and suppressing cell growth. It is well known that, in vivo, most mammalian cells require extracellular matrices (ECM), containing cytophilic proteins such as fibronectin and collagen [2], and chemical growth factors for physical attachment, survival and growth. The ECM plays a critical role in multiple cellular functions, ranging from migration to proliferation to apoptosis [2–4]. Thus, patterning of the ECM or chemicals which can promote cell attachment on a substrate is a prerequisite for the growth of patterned cells, in vitro. A number of research groups have developed various methods to pattern cytophilic and cytophobic chemicals on a substrate [5– 7]. Such methods usually combine microfabrication, chemical sur⇑ Corresponding author. Address: Sherman Fairchild Center, Department of Electrical & Computer Engineering, Lehigh University, 16A Memorial Dr. East, Bethlehem, PA 18015, USA. Tel.: +1 610 758 4552; fax: +1 610 758 6279. E-mail address: [email protected] (S. Tatic-Lucic).
face modification and material processing. Among them, microcontact printing (lCP) is the most frequently used [7–9]. This simple and cost-effective technique first requires the fabrication of desired topographical features on a silicon wafer using well-developed semiconductor processing technology. Polydimethylsiloxane is then molded onto the patterned silicon surface structure and peeled off as a stamp. Finally, the stamp, coated with a specific chemical which is favorable or non-favorable for cell growth, is brought into contact with a substrate, transferring the corresponding cytophilic or cytophobic molecules to the substrate, similar to traditional ink printing. Other patterning techniques include dip-pen nanolithography [10,11], the parylene-based dry liftoff technique [12], inkjet printing for cell patterning [13], laser scanning lithography [14] and microfluidic patterning [15]. Recently, the patterning of self-assembled monolayers (SAMs) has been intensely explored as an alternative to protein patterning. Patterning SAMs is an attractive alternative because the SAM molecules are highly orderly and exhibit controllable properties for a variety of desired applications, depending on the functionality of the terminal group (such as hydrophobic or hydrophilic control) or the chain length [16,17]. Most frequently, self-assembled monolayers have been deposited using micro-contact printing [18] or adsorption [19,20]. They have been used, among other applications, for biosensors [16,21] and making arrays of proteins and cells on chips [19,22].
1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.09.040
G. Jing et al. / Acta Biomaterialia 7 (2011) 1094–1103
However, most patterning techniques, whether based on patterning proteins or patterning self-assembled monolayers, have some limitations, such as an inability to combine single-cell resolution and simple alignment of the pattern to features and/or structures already existing on the substrate. For example, our target application is the fabrication of multielectrode arrays (MEAs) capable of supporting patterned neuronal networks with single neurons positioned on top of underlying electrodes, and defined narrow pathways connecting the electrodes, capable of supporting the outgrowth of neurites but not the attachment of neuronal cell bodies. For that application, the desired cell patterning technique must be capable of providing single-cell resolution and must be easily aligned with the underlying electrodes. Additionally, it is desirable for this technique to generate patterns which are both suitable for visualization by inverted microscopy (both prior to and during cell culture) and able to support patterned neuronal networks for extended time periods (2 weeks or more) to allow longer-term experiments. The surface patterns should also have a relatively long shelf life, making them more convenient for utilization. We sought to develop a technique that satisfied all of the requirements mentioned above at the same time. To accomplish this, we built upon several significant and relevant studies. For example, several patterning techniques have achieved single-cell patterning, such as lCP [23,24], using magnetic microposts [25] and dielectrophoresis [26]. Two related, and particularly relevant, publications describe techniques developed by Stenger et al. [27] and Ravenscroft et al. [28], whereby high-resolution substrates for cell patterning were created, based on self-assembled monolayers; our choice of surface treatment was based on these important studies. However, these previously developed cell patterning methodologies required deep UV lithography, which requires specialized equipment that was not available in our laboratory. It should also be mentioned that some earlier cell patterning techniques have demonstrated a good alignment of lCP-created patterns with respect to the underlying features, such as microelectrodes in MEAs [29,30], where modified optical aligners were utilized. Finally, extended-time experiments have been performed successfully using certain patterning methods [22,31,32]. Indeed, Branch et al. [32] have demonstrated long-term monitoring of electrical signals on patterned substrates via microelectrode arrays for four weeks. In doing so, two important goals were achieved: the formation of relatively coarse neuronal networks patterned on top of microelectrode structures, and the maintenance of the survival and activity of these networks for longer time periods. This previous study provided important groundwork; however, our envisioned networks have a finer resolution (single cell level) than that demonstrated by the previous work. In the current study, we implement, for the first time in cell patterning efforts, a recently developed SAM deposition technique from the gaseous phase (rather than the traditional method of soaking) via molecular vapor deposition (MVDÒ). Vapor-phase deposition has a potential advantage of small feature formation that would be more difficult to achieve from the traditional liquid-phase deposition. Additionally, because of its repeatability, it might have an advantage in mass manufacturing conditions. Here, we report a new, simple and effective cell patterning technique that accomplishes all of our desired goals. Using this technique, predefined patterns for cell growth can be visualized prior to cell culturing, single-cell patterning resolution is achieved and the patterns can be used to form a complicated biosensor system with accurate alignment using a conventional aligner. This method uses a patterned bilayer of a hydrophobic fluorine-based SAM, heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane (FDTS), on a silicon oxide (SiO2) adhesion layer, using the MVD process to create a cytophobic surface that prevents cell growth. A hydrophilic
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SAM, 3-trimethoxysilyl propyldiethylenetriamine (DETA), which has been shown to be cytophilic and encourages cell adhesion [27,28,33], is then backfilled on the open silicon oxide surface to promote cell growth in alternating regions. The predefined patterns we obtain can be conveniently visualized prior to cell culturing. As this method is photolithography-based, it also achieves high resolution and single-cell positioning and patterning, which can be maintained for extended culture periods. Additionally, the deposited materials display long-term reliability. Using this method, we have successfully patterned immortalized mouse hypothalamic (GT1–7) neurons and mouse (MC3T3) osteoblast cells, with osteoblast patterns being maintained for at least 14 days in vitro.
2. Materials and methods 2.1. Microfabrication of substrate The microfabrication process, illustrated in Fig. 1, starts from a single crystal silicon wafer, three inches in diameter. First, a silicon oxide layer of 100 nm thickness was deposited at 130 °C using a plasma-enhanced chemical vapor deposition (PECVD) method (Fig. 1a). Then positive photoresist (Shipley 1818, Shipley Company Inc, MA) was spin-coated at 3000 rpm for 30 s to obtain a 2.2 lm thick thin film (Fig. 1b). After prebaking at 90 °C for 60 s, the photoresist was exposed to UV light (wavelength k = 405 nm, power density = 12 mW cm–2) for 6.4 s using an EV620 mask aligner (EV Group Inc., NY), developed in Shipley MF321 developer for 90 s, then flood-exposed for 60 s to assist photoresist stripping later (Fig. 1c). Once photolithography was completed, the substrate with patterned photoresist was coated successively with a 10 nm thick silicon oxide adhesion layer followed by a self-assembled organosilane functional monolayer of FDTS (chemical structure illustrated in Fig. 1) using an MVD system that was initially developed for applying anti-stiction coatings to micromechanical systems (MEMS) devices. The MVD system (schematically shown in Fig. 2) and the deposition procedure have been described in detail elsewhere [34] and are also presented in the ‘‘Supplementary Material” to this paper. Following the MVD processing (Fig. 1d), the whole 3 inch wafer was cut into dies, 1.5 1.5 cm in size, using a K&S 7100 dicing saw (Kulicke & Soffa, PA). This die size was chosen so that the die would fit in a well of a six-well culture plate (BD Biosciences, NJ) during subsequent cell culturing. Next, the photoresist was stripped in acetone for 5 min, and the wafers were cleaned by an ultrasonic cleaner (VWR, PA; Fig. 1e), immersed in a 9 mM solution of DETA (Gelest, Inc., PA) for 1 h and withdrawn. The DETA has been shown to function in a manner similar to poly-D-lysine, and promotes cell attachment and growth [33]. Due to the high DETA/FDTS inter-surface energy, DETA does not rest on the FDTS SAM film. The postMVD immersion and withdrawal resulted in a cytophilic DETA SAM pattern complementary to the cytophobic pattern of the MVD-produced FDTS SAM/SiO2 bilayer. The substrates were rinsed with ethanol and deionized (DI) water and dried with a stream of nitrogen to remove any residual solvent (Fig. 1f) before cell culturing (Fig. 1g). While the EV620 contact aligner can reach line widths of approximately 2 lm, projection optics in an Autostep stepper (AS200, GCA Corporation, MA) allowed us to achieve a 0.5 lm line width of patterned photoresist. In this case, we used OiR 620-7i iline photoresist (Fuji films, Japan), 0.45 lm thick, soft-baked at 90 °C for 1 h, exposed for 0.168 s and baked at 115 °C for 1 min, followed by development in AZ 300 MIF developer (AZ Electronics Materials US Corporation, NJ) for 1 min. Then FDTS SAM/SiO2 bilayer was deposited using the MVD process and subsequently
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Fig. 1. (a–g) Schematic illustration of the cell patterning procedure based on molecular vapor deposition of self-assembled monolayers and lift-off technique. (h) Chemical structure of FDTS.
beam current of 300 pA. The electron high voltage was 2 kV, the aperture 30 lm and the working distance 5 mm. Developing was performed in a 3:1 mixture of IPA:methyl isobutyl ketone (Sigma Alrdich, MO, US) for 70 s with gentle agitation. 2.2. Characterization of patterned culturing substrate
Fig. 2. Schematic of the MVD vapor deposition system [34].
patterned using the lift-off technique. The above-mentioned microfabrication process was executed on both silicon wafers coated with PECVD oxide and on bare glass wafers. Finally, nanoscale features in our FDTS layer were demonstrated, using E-beam photolithography and the lift-off method. First, 950 PMMA resist (Microchem Inc) was mixed with A – thinner (Microchem Inc) in a 3:1 ratio. Then, the photoresist was spin coated at 3000 rpm and baked in a convection oven at 150 °C for 1 h. It was exposed using E-beam write Leo 1550DVP (Leo Electron Microscopy Inc, UK). The writing dose was 250 lC cm–2, with a
The oxide thicknesses were measured using an ellipsometer (LSE Stokes, Gaertner Scientific Corporation, IL) using a wavelength of 632.8 nm (HeNe laser source) at a 70° angle of incidence. Films were modeled using a refractive index of 1.46 at the above wavelength for silicon oxide. The uncertainty in the thickness measurements is ±0.1 nm. The topography of the patterned substrate was characterized using a scanning electron microscope (SEM; Hitachi S-4300, CA), operated at an accelerating voltage of 3 kV, and a Dimension 3100 scanning probe microscope (SPM; Veeco, CA), operated in the tapping mode. Typical SPM height and amplitude images of the patterned substrate surface were recorded as data files, and processed using the offline image processing software WSxM (Nanotec, IN) to obtain three-dimension topography images and average cross-section profiles. To confirm the hydrophilic and hydrophobic nature of different areas on the patterned substrate, the substrate surface was sprayed with DI water and observed under an optical microscope (Nikon 4500, NY). Its contact angle with water was measured by a contact angle meter (VCA Optima Contact Angle Meter, MA).
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Whereas PECVD silicon oxide is free of it, FDTS contains fluorine, which has a characteristic KLL Auger electron energy of 659 eV [35]. Therefore a peak at 659 eV in an Auger spectrum will definitely confirm the presence of FDTS on our substrate surface. Taking such a characteristic into consideration, the chemical nature of our patterned surface was analyzed using a PHI 670 scanning Auger microscope (Physical Electronics, MN), which is also equipped with a secondary electron detector and can obtain conventional secondary electron images. During the operation, the sample chamber was in ultrahigh vacuum (under 10 10 torr) and the primary electron beam was accelerated by 10 kV. First, the substrate pattern was imaged in the conventional secondary electron mode, then the primary electron beam was focused on typical regions revealed by the secondary electron micrograph and Auger spectra were collected with energy scanned from 50 to 1700 eV. Finally, the intensity of the fluorine KLL Auger electron was used to construct micrographs which clearly illustrate the distribution of fluorine, or FDTS, over the surface.
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graphs using the imaging software, SPOT (Diagnostic Instruments, MI). 3.3. Patterned cell imaging by scanning electron microscopy Cell patterns were further analyzed using an SEM (Hitachi S4300, CA) operated at an accelerating voltage of 3 kV, following a standard procedure of sample preparation [37]. Briefly, the patterned cells were rinsed with PBS (pH 7.4) solution for 5 min and fixed in 10% neutral buffered formalin solution for 4 h. After the fixation, the substrate (with patterned cells) was rinsed in DI water, dehydrated in a series of ethanol solutions (from 35% to 100%), soaked in 100% hexamethyldisilazane (HMDS) (VWR, PA) for 5 min, and left in air for drying for 30 min, and finally coated with a 5.5 nm thick gold layer by sputtering. 4. Results 4.1. Culturing substrate
3. Culturing and imaging of patterned cells 3.1. Cell dissociation and culturing Immortalized mouse hypothalamic neurons (GT1–7 cell line) were chosen to assess the effect of the SAM patterns on cell positioning and growth (Fig. 1g). GT1–7 cells were maintained in 25 cm2 flasks (Fisher Scientific, GA) in a humidified environment at 37 °C in the presence of 8% carbon dioxide in a culture medium consisting of Dulbecco’s modified Eagle’s medium (Gibco, NY) supplemented with 1 mM sodium pyruvate, 10 mM sodium bicarbonate, 2 mM L-glutamine, 10 mM Hepes buffer and 10% fetal bovine serum (Invitrogen, CA) [36]. Immortalized mouse osteoblast (MC3T3) cells were also patterned using our technique, for the purpose of investigating the long-term maintenance of cell patterns. The MC3T3 cells were grown in minimum essential medium, a modification (Invitrogen, CA), supplemented with 2 mM L-glutamine, 1% penicillin and streptomycin, and 10% fetal bovine serum (Invitrogen, CA). The MC3T3 cells were maintained in 25 cm2 flasks (Fisher Scientific, GA) in a humidified environment at 37 °C in the presence of 5% carbon dioxide. Silicon dies, with complementary cytophobic (FDTS) and cytophilic (DETA) SAM patterns, were placed into wells of six-well culture plates (BD Biosciences, CA). GT1–7 and MC3T3 cells were harvested by incubating in 0.125% (w/v) trypsin solution at 37 °C for 8 min, centrifuging at 750 rpm for 5 min, resuspending in the culture medium and transplanting into wells containing silicon dies at a density of 2 104 cells ml 1. The cells were maintained under the above-mentioned standard conditions until their patterns became observable under an optical microscope (typically 48 h later). We also maintained patterned M3T3 cells for up to 2 weeks (as documented in Section 4.2), and the same cell protocol was implemented for extended culturing times, with additional medium changes every 3–4 days. 3.2. Patterned cell imaging by epifluorescent microscopy The patterned cells were rinsed with phosphate-buffered saline (PBS) solution, incubated with a fluorescent live cell stain, calcein AM (2 lM in cell-specific, serum-free medium; Invitrogen, CA), at room temperature for 20 min, and observed using an IX-70 inverted microscope (Olympus, NY). Phase-contrast and fluorescence images were captured through a SPOT, RTKE/SE digital camera (Diagnostic Instruments, MI). The magnification of captured micrographs was calibrated using an OB-M 1/100 stage micrometer (Olympus Inc, NY) and an accurate scale bar was added to micro-
The average thickness of an oxide adhesion layer is 10 nm by ellipsometric measurement and the average thickness of a patterned FDTS SAM/silicon oxide bilayer is 11 nm as determined by SPM measurement. Fig. 3a shows a conventional (or secondary electron) SEM micrograph of patterned regions analyzed by Auger electron microscopy. The Auger spectra presented in Fig. 3b were collected from the rectangular bright and dark areas, the top surfaces of which are presumed to be an FDTS SAM/silicon oxide bilayer and PECVD silicon oxide, respectively. Whereas there is no peak around 659 eV in the Auger spectrum (dark line) from the dark area, the spectrum from the bright area (red line) has an obvious peak at 659 eV (Fig. 3b), which is attributed to fluorine KLL Auger electrons [35]. This indicates that the dark area is free of FDTS, whereas the bright area is covered by FDTS. The scanning Auger electron micrograph shown in Fig. 3c further indicates the distribution of FDTS and PECVD silicon oxide on the top surface of the prepatterned substrate for cell patterning, over a larger area. The micrograph matches very well with the secondary electron micrograph (Fig. 3d), except that the former has larger image pixels (or a lower resolution) than the latter because of the resolution limitation of the instrument. All these results clearly confirm that the top surface chemical patterning was as expected. Additionally, we have demonstrated FDTS SAM/oxide bilayer features 200 nm in diameter using E-beam lithography and a liftoff technique, as shown in Fig. 4. 4.2. Cell patterns on silicon substrate Typical intermediate and final cell patterns on silicon substrates are presented in Fig. 5. The photoresist pattern was transferred to the cytophobic FDTS SAM/silicon oxide patterns (as shown in Fig. 5a) following the MVD and lift-off processes. After the entire wafer is immersed and withdrawn from DETA solution, the DETA SAM is coated on regions complementary to FDTS areas, not on top of the FDTS SAM, as discussed and concluded in the previous subsection. The patterns of cytophilic DETA and cytophobic FDTS SAM/silicon oxide were replicated by the GT1–7 cells, as shown by the cell patterns displayed in Fig. 5b. Comparison of Fig. 5a with Fig. 5b indicates that the final cell patterns are faithful replicas of pre-existing patterns on the culturing substrates. To pattern a neural network, we designed a pre-culturing pattern containing islands connected by lines of different widths, where the islands and their connecting lines, respectively, were coated with cytophilic DETA SAM for neuronal cell bodies and neurites, and the surrounding area was coated with a cytophobic FDTS
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Fig. 3. (a) SEM micrograph of the regions analyzed by Auger electron microscopy. (b) Auger spectra collected from the rectangular areas 1 (bright, red line) and 2 (dark, black line), shown in (a), are typical of designed FDTS SAM/SiO2 bilayer and PECVD SiO2 regions, respectively. The peak around 659 eV is attributed to the fluorine KLL Auger electron. (c) Fluorine Auger electron and (d) secondary electron SEM micrograph of the same patterned area. In both micrographs, the bright and dark areas are FDTS SAM/ SiO2 bilayer and PECVD SiO2, respectively. Since the F Auger electron was very weak, a pixel region in (c) is larger than that in (d) so as to collect enough Auger electron intensity for a reasonable amount of time.
SAM/silicon oxide bilayer to prevent cell anchoring and growth. Our scheme succeeded, as is shown in Figs. 5b and 6b. From observations of a wide variety of cell patterns, it was determined that the island size and line width of the connecting line play important roles in the neural network patterning. An island size of 20 lm is recommended for a single neural cell body to anchor, and a line width of 5 lm is sufficient for neurites to grow from one island to another (Fig. 5). Whereas neurites cannot grow without the connecting line (Fig. 5), lines wider than 10 lm are occasionally populated by cell bodies (Fig. 5). With an optimal combination of island shape, size and connecting line width, a complex neural network can be patterned. Fig. 6b shows an example of cells positioned onto circular islands, 12 lm in diameter, connected by 6 lm wide pathways. Because our technique is a passive way to pattern cells, which means cells find appropriate sites to survive by themselves, vacant islands can be observed occasionally, as shown in Fig. 6b. Accurate cell patterns can also be observed using an SEM. Fig. 7 demonstrates the manner in which a GT1–7 cell body can be defined by and attached to a cytophobic/cytophilic pattern on a
culturing substrate. The circular island, 20 lm in diameter, was coated with cytophilic DETA SAM and surrounded by an area coated with the cytophobic FDTS/silicon oxide bilayer. The cell body is attached to the island so accurately that the island actually defines the cell’s body shape. It is interesting to note that both GT1–7 cells and MC3T3 osteoblasts can adapt their bodies to anchor on FDTS SAM/silicon oxide bilayer lines only 0.5 lm in width (in the rare cases when they manage to anchor on them), even though the line width was much smaller than the size of both of these cells in suspension, which is approximately 10 lm [38]. Fig. 8 shows two MC3T3 cells anchored on a line that is only half a micron wide. It is obvious that cells anchored on thin cytophilic lines (0.5 lm) are more elongated than those anchored on wider lines. Additionally, we have performed long-term monitoring of cell patterns (specifically M3T3 cells) on silicon culturing substrates with MVD patterns. Our results are illustrated in Fig. 9. It can be seen that MC3T3 cell patterns are clear and cells are viable, as determined by calcein-AM staining, even after 14 days in culture, indicating the long-term capability of our novel patterning
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Fig. 4. FDTS SAM/silicon oxide bilayer circles with a diameter of 200 nm were fabricated by E-beam lithography and a lift-off technique. The artifact in the bottom right corner is believed to be due to the lift-off process imperfection.
Fig. 6. (a) A complex pre-culturing network of circular islands, 12 lm in diameter, connected by lines 6 lm in width after photoresist lift-off; (b) complex neural network of GT1–7 cells obtained after 48 h. Cytophobic FDTS/oxide bilayer regions are greenish-yellow whereas PECVD oxide regions are dark green in (a). Some islands are not occupied by any cells in (b).
onto islands and lines on transparent glass substrates, as well as predefined FDTS SAM/silicon oxide bilayer patterns. 5. Discussion
Fig. 5. (a) Pre-culturing patterns with greenish-yellow FDTS/oxide bilayer and dark green PECVD oxide patterns. The island size is 20 20 lm, and the line widths are 5 and 10 lm. (b) Final GT1–7 neural cell pattern after 48 h. The right part of the top figure shows a well-developed neural network, where cell bodies occupy most of the islands and networking neurites grow neatly along 5 lm wide cytophilic pathways.
method. Longer patterning times are essential for many studies, particularly for experiments on differentiation. 4.3. Cell patterns on transparent glass substrates For applications in tissue engineering, it is desirable to pattern cells on a transparent (rather than opaque) glass substrate, so that their growth can be monitored by a conventional inverted microscope [6]. Although the FDTS SAM/silicon oxide bilayer patterns on transparent glass are still invisible under a conventional optical microscope in the bright-field, dark-field or phase-contrast mode, they can be visualized by an inverted microscope in the differential interference contrast (DIC) mode. In the DIC mode, the step height of patterned FDTS SAM/silicon oxide layers (11 nm) was utilized to derive contrast between different topographic regions. Fig. 10 shows DIC micrographs of GT1–7 neurons successfully patterned
In our investigations of the FDTS SAM/silicon oxide bilayer deposited using molecular vapor deposition, we first focused on the necessity of depositing a silicon oxide layer below the hydrophobic SAM. It was determined that the MVD silicon oxide adhesion layer plays several critical roles. Firstly, it enhances the attachment of FDTS SAM to the substrate. On the one hand, it has a strong adhesion to the underlying PECVD oxide or to the transparent glass substrate. On the other hand, during the MVD FDTS deposition step, the freshly in situ deposited silicon oxide layer provides an ideal surface on which to covalently bond the subsequent FDTS SAM layer. Secondly, the MVD silicon oxide adhesion layer slightly increases the hydrophobicity of the FDTS coating and significantly enhances the stability of the coating. Enhancing the stability of hydrophobic films MVD silicon oxide adhesion layers has been previously demonstrated for FDTS [39]. An FDTS, or other similar organosilane SAM film, such as tridecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane (FOTS), when coated on an in situ MVD deposited silicon oxide adhesion layer, retains its high water contact angle (>100°) for at least 14 days after deposition. In contrast, the water contact angle of an FDTS or FOTS film without the MVD adhesion layer decreases significantly after only 8 days [39,40]. Even more importantly, we have discovered that our MVD FDTS SAM keeps its strong hydrophobicity for at least 2 years, as
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Fig. 7. SEM micrographs of a circular island 20 lm in diameter covered by DETA SAM and surrounded by an area coated with the cytophobic FDTS SAM/oxide bilayer (a) with and (b) without an attached GT1–7 neuronal cell. The circular halo around the perimeter of the island is due to an imperfect lift-off process implemented here. (Note: The process applied here was contact photolithography.).
Fig. 9. (a) Pre-culturing patterns with SAM-treated cytophilic PECVD oxide islands, 30 30 lm square, with 20 lm wide interconnecting lines, and FDTS/oxide bilayer in the surrounding area. (b) Final MC3T3 cell patterns after 14 divisions. Cells were observed using an upright fluorescent microscope.
Fig. 8. SEM micrograph of two MC3T3 cells anchored along an FDTS SAM/oxide bilayer line of 0.5 lm width (fabricated using projection lithography and a lift-off technique).
demonstrated by the fact that the water contact angle did not change significantly during that period of time. Immediately after deposition, the contact angle was 118°; 2 years later, during which
period the sample was not stored in a clean-room environment, the contact angle was 110°, as measured by a contact angle meter (VCA Optima Contact Angle Meter, MA). This compares favorably with the other cytophobic materials commonly used in cell patterning, such as polyethylene glycol [18], which has a shorter shelf life in terms of effectively preventing cell growth, as it is easily oxidized in the ambient environment [7]. Thirdly, the MVD silicon oxide adhesion layer enhances the contrast of FDTS SAM patterns, making them visible under a conventional optical microscope. A single FDTS SAM is optically invisible because its thickness is only about 1 nm. With the introduction of the 10 nm silicon oxide adhesion layer, the combined bilayer has a thickness of 11 nm and its patterns on silicon wafers and transparent glass can be visualized using an optical microscope in reflection and DIC modes, respectively (as shown in Fig. 10).
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combination of these attributes (if not the specific characteristics by themselves) makes this technique unique. Whereas single-cell resolution of cell-patterning methods has been demonstrated before [25,26], it has rarely been combined with the alignment capability (without requiring specialized or custom-modified equipment) [29,30], patternability in the nanoscale regime or the ability to visualize the cytophobic/cytophilic pattern prior to cell growth. Moreover, the long shelf life (at least 2 years) and the ability to support longer-term experiments make this technique even more attractive. The ultimate application of this technique in our laboratory is the creation of MEA chips able to support patterned cell networks, as shown Fig. 11. The benefits of combining such performance
Fig. 10. Typical DIC micrographs of GT1–7 neurons patterned onto (a) lines of width varying from 20 to 50 lm and (b) 50 50 lm square islands on transparent glass substrates. Note that the FDTS SAM/silicon dioxide bilayer has been stored for 2 years in an ambient environment and DETA SAM was freshly prepared just before cell culturing.
We should also note that profilometry has indicated that the bilayer region formed using contact lithography has a sharp edge due to a suboptimal lift-off process (as shown in Fig. 7b). We do not believe that it had a major role in our cell patterning efforts, however, due to the fact that we have observed that cells do not have trouble traversing heights as large as 22 nm unless the cytophobic/cytophilic pattern is present on a wafer [41]. Having determined that the silicon oxide adhesion layer greatly enhances the formation, stability and visibility of hydrophobic FDTS-coated regions, we then evaluated the performance characteristics of the described patterning technique. We have determined that our technique has a single-cell resolution (in fact, submicron features were successfully patterned, Fig. 8), and that, due to the photolithographic nature of the process, the cytophilic patterns can be defined so that they are easily aligned with the features already in existence on the substrate (e.g. microelectrodes in this article). Moreover, these patterns can be visualized prior to cell growth, so that there is no ambiguity about whether a compromised cell pattern is due to an ill-defined substrate or to the cell culturing process itself. Finally, we have demonstrated that osteoblast cell patterns, generated as a result of our technique, can be maintained for at least 2 weeks, which makes this technique appropriate for longer-term experiments with cultured cells. The
Fig. 11. (a) An MEA supporting patterned cell growth which utilizes MVDdeposited SAMs for defining of cell locations and pathways between electrodes photographed prior to lift-off. (b) Mouse osteoblast cells (MC3T3) cultured on an MVD MEA chip after 7 divisions.
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characteristics are easily recognizable for other applications as well. For instance, utilization of this patterning process could improve the assembly of complex biosensors or aid in the creation of arrays for interrogating multiple cells at the same time, while fulfilling the need for those cells to be positioned at predefined locations. One important question that needs to be addressed is whether it is the MVD technique itself or the molecules used for SAMs that are responsible for the obtained results. In this work, we did not directly compare the cell-patterning performance of the MVD-deposited SAMs with respect to identical SAMs using other methods (such as soaking). It could be said that the MVD deposition is highly compatible with the standard types of semiconductor processing, such as photolithographic definition of the desired patterns and the lift-off process, so the high-resolution feature definition and alignment capability could be attributed to the SAM deposition from the gaseous phase (MVD) rather than soaking (liquid phase). Additionally, MVD deposition enables deposition of a silicon oxide layer directly beneath the FDTS using the same equipment, which (as previously discussed) enhances the adhesion of this layer, slightly increases the hydrophobicity of the FDTS coating, significantly enhances the stability of the coating and enhances the contrast of FDTS SAM patterns, making them visible under a conventional optical microscope. The other beneficial characteristics, such as long shelf life and capability to maintain patterns of some cell types (for example, osteoblasts) for at least 14 days in vitro, stem from the SAM molecules, themselves. Additionally, it is well accepted that both cell-dependent processes, such as the secretion of proteolytic or oxidative enzymes, and cell-independent processes, e.g. hydration of nonadhesive layers or the presence of serum proteins in the culture medium, play a role in the stability of micropatterned surfaces [42,43], and thus in the fidelity of generated cell patterns. Desorption and/or exchange processes of the passivating FDTS SAM/silicon oxide bilayer layer by components in the culture medium or byproducts of cellular processes may ultimately lead to degradation of the surface molecules; consequently, pattern degradation could occur over time. However, the stability of the cell patterns for up to 2 weeks suggests a relatively strong interaction between the passivating layer and the substrate surface, especially for the MVD FDTS SAM, which keeps its strong hydrophobicity for at least 2 years.
6. Conclusion We have developed a simple and effective cell-patterning method based on a combination of photolithography and MVD of an FDTS SAM/silicon oxide bilayer. The culturing substrate was prepatterned with alternating cytophilic DETA SAM areas and cytophobic FDTS SAM/silicon oxide bilayer regions deposited by MVD. While photolithography patterning of culturing substrate guarantees precise pattern alignment, the introduction of the MVD silicon oxide layer enhances the adhesion of the FDTS SAM to the substrate, increases the stability of the hydrophobic FDTS SAM, and greatly facilitates the visualization of cytophobic/cytophilic patterns prior to and during patterned cell culturing. This method has the following characteristics: (i) single-cell resolution; (ii) easy alignment of the patterns with the pre-existing patterns on the substrate; (iii) easy formation of nanoscale patterns (depending on the exposure equipment); (iv) long shelf life of the substrate pattern prior to cell culturing; (v) compatibility with conventional, inverted, optical microscopes for simple visualization of patterns formed on a glass wafer; and (vi) the ability to support patterned cell (osteoblast) networks for at least 2 weeks. These performance traits make our method suitable for making complex, mass-manufacturable devices supporting cultured cell patterns.
Acknowledgements This work was supported by National Science Foundation (CAREER grant ECS-0448886 and NER grant BES-0608742), as well as Pennsylvania Infrastructure Technology Alliance (Grant PA-DCED C000016682). The fabrication part of this work was performed at the Cornell NanoScale Facility (CNF), a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation (Grant ECS 03-35765). We are grateful to Kanlun Li, of our laboratory, for her work related to cell culturing experiments. We also would like to thank Prof. Gregory Ferguson with the Department of Chemical Engineering at Lehigh University for his constructive advice, and thank Mr. Vince Bojan with the Materials Characterization Lab at Penn State University for his advice and help with the Auger electron spectroscopy experiments. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1, 2, 3, 5, 6, 9 and 11 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio. 2010.09.040. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2010.09.040. References [1] Kleinfeld D, Kahler KH, Hockberger PE. Controlled outgrowth of dissociated neurons on patterned substrates. J Neurosci 1988;8:4098–120. [2] Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular biology of the cell. fourth ed. New York: Garland; 2002. [3] Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE. Geometric control of cell life and death. Science 1997;276:1425–8. [4] Dike LE, Chen CS, Mrksich M, Tien J, Whitesides GM, Ingber DE. Geometric control of switching between growth, apoptosis, and differentiation during angiogenesis using micropatterned substrates. In Vitro Cell Dev Biol Anim 1999;35:441–8. [5] Yarmush M, King K. Living-cell microarrays. Annu Rev Biomed Eng 2009;11:235–57. [6] Falconnet D, Csucs G, Michelle Grandin H, Textor M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006;27:3044–63. [7] Whitesides GM, Ostuni E, Takayama S, Jiang X, Ingber DE. Soft lithography in biology and biochemistry. Annu Rev Biomed Eng 2001;3:335–73. [8] Ruiz SA, Chen CS. Microcontact printing; a tool to pattern. Soft Matter 2007;3:168–77. [9] Thibault C, Le Berre V, Casimirius S, Trevisiol E, Francois J, Vieu C. Direct microcontact printing of oligonucleotides for biochip applications. J Nanobiotechnol 2005;3:7. [10] Piner RD, Zhu J, Xu F, Hong S, Mirkin CA. Dip-pen nanolithography. Science 1999;283:661–3. [11] Salaita K, Wang Y, Mirkin CA. Applications of dip-pen nanolithography. Nat Nanotechnol 2007;2:145–55. [12] Ilic B, Craighead HG. Topographical patterning of chemically sensitive biological materials using a polymer-based dry lift off. Biomed Microdevices 2000;2:317–22. [13] Roth EA, Xu T, Das M, Gregory C, Hickman JJ, Boland T. Inkjet printing for highthroughput cell patterning. Biomaterials 2004;25:3707–15. [14] Miller JS, Bethencourt MI, Hahn M, Lee TR, West JL. Laser-scanning lithography (LSL) for the soft lithographic patterning of cell-adhesive self-assembled monolayers. Biotechnol Bioeng 2006;93:1060. [15] Chiu DT, Jeon NL, Huang S, Kane RS, Wargo CJ, Choi IS, et al. Patterned deposition of cells and proteins onto surfaces by using three-dimensional microfluidic systems. PNAS 2000;97:2408–13. [16] Franks W, Tosatti S, Heer F, Seif P, Textor M, Hierlemann A. Patterned cell adhesion by self-assembled structures for use with a CMOS cell-based biosensor. Biosens Bioelectron 2007;22:1426–33. [17] Arya S, Solanki P, Datta M, Malhotra B. Recent advances in self-assembled monolayers based biomolecular electronic devices. Biosens Bioelectron 2009;24:2810–7. [18] Smith RK, Lewis PA, Weiss PS. Patterning self-assembled monolayers. Prog Surf Sci 2004;75:1–68.
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