Current Issues and Advances in Dissociated Cell Culturing on Nano-and Microfabricated Substrates

Advanced Semiconductor and Organic Nano-Techniques (Part III) H. Morko? (Ed.) Copyright (() 2003 Elsevier (USA). All rights reserved.

CHAPTER

5

Current Issues and Advances in Dissociated Cell Culturing on Nano- and Microfabricated Substrates H. G. Craighead, C. D. James, and A. M. P. Turner CORNELL UNIVERSITY ITHACA, NEW YORK

I. II. III.

INTRODUCTION INTRODUCTION—CHEMICAL

251 PATTERNING

252

SURFACE SCIENCE FOR CELL CULTURING

254

1. Materials 2. Cell-Surface Interface IV.

254 255

CHEMICAL PATTERNING

257

1. Historical Background 2. Methods and Results V. VI. VII.

257 259

CURRENT AND FUTURE DIRECTIONS OF CHEMICAL PATTERNING

274

INTRODUCTION: TOPOGRAPHICAL PATTERNING

275

TECHNIQUES FOR TOPOGRAPHICALLY

1. 2. 3. 4.

PATTERNING SURFACES

Materials Used for Topographical Studies Methods of Topographical Patterning Dealing with Surface Chemistry Characterizing Surface Structure

VIII. STUDIES OF CELLS ON TOPOGRAPHICALLY IX. X.

MODIFIED SURFACES

280

280 280 286 287 288

FUTURE OF TOPOGRAPHIC MODIFICATIONS OF SURFACES

301

CONCLUSIONS

306

REFERENCES

307

I.

Introduction

The exponential growth of the computer industry and the ever-present drive to produce smaller devices has led to the development of micro- and nanofabrication tools that have since been applied to other areas of research. Microfabrication technology, once limited to integrated chip production, has steadily become a significant tool in cell biology and tissue engineering in the past several decades. Advances in nanofabrication in conjunction with advances in biotechnology have led to an explosion in the interdisciplinary field that we now call nanobiotechnology (Hoch et al. 1996). The tools developed by the computer industry and applied to the field of nanobiotechnology 251

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H. G. CRAIGHEAD, C. D . JAMES, AND A. M. P. TURNER

have enabled physicists, chemists, biologists, and engineers to fabricate the devices necessary to probe biological systems down to molecular size scales. A major aspect of this merging of technologies is the construction of chemical and topographical patterns on substrates for control of cell attachment and growth. Using various lithographic techniques, surface structures can be reliably and reproducibly fabricated down to sub-cellular dimensions, on the order of tens of nanometers. Surface patterning methods offer the abihty to organize cells on surfaces, and to promote and/or inhibit cell attachment to specific locations. The three-dimensional control of substrate characteristics at the nano- and micrometer levels enables new studies in cell biology that were previously not possible. For example, selectively designed cell networks can be constructed to monitor collective behavior (Rohr et al. 1991), or microislands of cells can be isolated to evaluate single cell activity (Rao et al 2000). Topographically modified surfaces are useful for reproducing an environment more similar to the in vivo situation, and for specifically exploring topics such as contact inhibition in cultured cells (Damji et al. 1996). Surfaces with varying adhesivity can be fabricated to examine the dynamics of cell motility, and as such, chemical patterning methods have given considerable insight into the biological mechanisms responsible for cell migration and outgrowth during in vivo development. Receptor-mediated signal transduction pathways are involved in a variety of functions such as apoptosis, differentiation, glucose transport, and gene transcription regulation. Hence, cell cultures on patterned surfaces permit investigators to study not only external phenomena, for instance growth cone motility, but also internal events such as G protein activation that are triggered by extracellular chemical cues (Singhvi et al. 1994; Bailly et al. 1998; Dike et al. 1999) or topographical cues (Chou et al. 1995; den Braber et al. 1998a). Multifunctional surfaces allow researchers to examine competition between cues (Gomez and Letourneau 1994; Rajnicek et al. 1997; Esch et al. 1999) and cross-talk between connected signal cascades. In this chapter, the history and applications of chemical and topographical patterning of substrates for cell culture experiments will be reviewed, including pertinent background information on surface science, cell-substrate interactions, and fabrication techniques. In addition, recent methods and experiments performed in the authors' lab will be discussed.

II.

Introduction—Chemical Patterning

Chemically patterned surfaces are useful for many experimental research projects that involve cell-substrate or cell-cell interactions. Cell morphology and network architecture are intense areas of investigation in developmental histology, tumor metastasis, and cell differentiation. In particular, cell shape

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and cell-cell connections influence electrical impulse propagation in myocytes (Rohr et al. 1991), differentiation in endothelial cells (Dike et al. 1999), DNA synthesis and protein secretion in hepatocytes (Singhvi et al. 1994), and lamellipodia motility in carcinoma cells (Bailly et al. 1998). As previously mentioned, the ability to control cell geometry enables novel experiments to be performed that can substantiate previous work and even take research into new directions. For example, Mainen and Sejnowski (1996) simulated neocortical neurons with varying dendritic structure and found that the firing patterns change significantly, thus providing an explanation for how cells with similar distributions of ion channels can express markedly different firing activities. Chemical patterning techniques offer the possibility of constructing cells and cell networks of varying geometry to provide rigorous testing of such cell models. Surface patterning techniques not only permit the construction of surfaces with chemical/topographical features for promoting cell attachment and growth, but they also enable researchers to incorporate sensor array devices into substrates to probe cell activity. Substrates have been patterned with electrical and chemical sensors (Bratten et al. 1998; Baumann et al. 1999; Cooper 1999) in order to monitor phenomena such as neurotransmitter exocytosis, enzyme cascades, and ion fluxes that are important in metabolism, differentiation, and proliferation. This coupling of electrochemical devices to physiological activity of living cells has also sparked research that has a more practical goal: high-sensitivity cell-based detector technology. Cell-based biosensors (Gross and Rhoades 1995; Pancrazio et al. 1998,1999) offer several advantages over conventional sensor strategies such as providing physiological effects of chemical species, removing the need for expensive and timeconsuming purification of enzymes or proteins for assays, and allowing for the detection of multiple analytes with single devices. The ability to control and manipulate molecules on the surface of materials is an advance that can also be applied by the biomedical community for therapeutic implants designed to restore or enhance function in disabled patients. Macroscale implants such as hip replacements, cardiac pacemakers, and subcutaneous birth control devices have significantly improved quality of life for both handicapped and healthy individuals. However, immune responses to implants are a major concern for biomedical/bioengineering intervention, and chemical patterning methods offer the abiUty to tailor the surface properties of materials to promote improved function and histocompatibihty of such devices. For example, microfabricated neural probes (Bai et al. 2000) can not only stimulate and record brain activity, but these devices also offer reduced invasiveness due to their small size, increased functionaHty by integrating on-chip signal processing elements, and when combined with surface modification techniques, increased electrical coupling to ceUs (Cui et al. 2001) and reduced inflammation (Stelzle et al. 1997; Perizzolo et al. 2001).

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III. 1.

Surface Science for Cell Culturing

MATERIALS

The metals and semiconductors used in microfabrication are quite different from the glasses and polymers used in cell biology. But as technology in these fields has advanced, substantial overlap has occurred as the advantages of "unconventional" materials in each field have been discovered. Microfabrication researchers have long appreciated the properties of organic polymers as electron-beam and photolithography resist layers. And certain technologies common in microfabrication such as plasma processing and wet chemical etching have been used in cell culture applications for substrate preparation. Materials science efforts have progressed to the point where biological and microfabrication materials can be integrated together successfully, providing a large selection of substrates and films for experiments. The primary advantage afforded by microfabrication technology in cell culture experiments is the ability to produce various surface characteristics on different materials. Chemical/thermal pretreatment of the culture substrate is often an important factor in producing successful cell cultures, and in this respect, microfabrication provides cell biologists with a high degree of control in the production of surface chemistry and/or topography. In this respect, researchers have cultured primary neuronal cells (some of the most difficult cells to culture) on semiconductor industry materials such as thermally grown and chemical vapor deposited (CVD) silicon oxides and CVD silicon nitrides. The composition of these films is highly controlled and impurities can be kept to a minimum. Silicon oxide (SivOj;) has a surface chemistry that is, quite similar to clean glass, consisting primarily of siloxane (Si-O-Si), silanol (Si-OH), and siloxy (Si-0~) groups. Silicon nitride (Si.vN,,) is another insulating material that is used in semiconductor devices due to its superior resistance to moisture penetration and ion diffusion (Bousse and Mostarshed 1991; Lin et al. 1998). Researchers have found that the silicon nitride surface consists of silanol/siloxy and silylamine (Si-NH2, Si-NH3") groups (Harame et al. 1987), and that these silylamine groups can be oxidized to silanol groups in air and aqueous environments. Thus, the surface preparation protocols that cell biologists use to couple proteins to glass coverslips should work just as well with oxide and nitride films. Plastic is another common substrate used in cell cultures. Cultures are often performed on polystyrene dishes that have been treated with a glow discharge plasma to render the surface hydrophilic by creating polar groups such as carboxylic acid. Polymers such as poly (methyl methacrylate) are used in the semiconductor industry as etch masks, and more recently, polymers such as polystyrene and poly (ethylene glycol) (PEG) are being explored. Specifically, these polymers can be vapor deposited onto a variety of substrates (Bai et al. 2001; Bubb et al. 2001), and semiconductor films such as

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metals and oxides can be grown or deposited onto plastic substrates (Morgner et al. 1998; Kim et al. 2001); hence, materials more familiar in biological applications can easily be integrated with traditional semiconductor materials, making a diverse array of surface chemistries and sensor devices to be incorporated into cell culture experiments.

2.

CELL-SURFACE INTERFACE

The cell-substrate interface is a highly complex and dynamic environment (Fig. 1). Of important consideration are the surface properties of the substrate, which in many cases differ from the properties of the bulk material: for example, materials such as silicon and titanium readily oxidize. When surfaces are placed into cell culture media, there is substantial protein adsorption and secretion onto the designed layer (Schaffner et al 1995) as illustrated in Fig. 2. There are various proteins and factors in culture media that are participating in this mechanism and altering the surface. In addition, the physicochemical properties of the designed layer will influence not only which proteins adsorb to the surface, but also the conformation and thus the bioactivity of the adsorbed proteins (Grinnell and Feld 1982; Lewandowska et al. 1989). Cells can interact with surfaces through non-specific interactions (non-receptor mediated) with the glycocalyx and cell membrane or through specific interactions (receptor-mediated) with cell-adhesion molecules (CAMs) embedded within the cell membrane.

Cell membrane

Adsorbed molecules (1-20 nm)

Designed layer (0.5-5 nm) Surface (0.5-2 nm)

Bulk malaria!

FIG. L Cross-sectional illustration of the interface between cells cultured on designed surfaces. Dimensions not to scale.

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FIG. 2. Tapping-mode atomic force microscopy (AFM) of poly-L-lysine (PLL) structures patterned on two different glass substrates. After patterning, the sample on the right was incubated in serum-containing cell-culture medium for 2 hours: approximately 18nm of material has adsorbed onto the PLL structure.

a.

Non-Specific Interactions

Non-specific interactions between cells and surfaces include steric stabilization, London dispersion, and electrostatic forces. Steric stabilization forces are repulsive and result from the osmotic pressure that develops when extended cell-membrane molecules are compressed as a cell approaches a surface. London dispersion forces are attractive and due to charge interactions between fluctuating dipoles in neutral yet polar molecules. Finally, electrostatic forces can be attractive or repulsive, depending on the surface charges of the cell and the substrate. Important in this discussion is the glycocalyx, the shell of carbohydrates from glycoproteins, glycolipids, and proteoglycans that decorate the outer leaflet of the cell's plasma membrane. Electron microscopy and staining techniques show the glycocalyx to be from 30 to 120 nm thick on chick dorsal root ganghon (DRG) cells (James and Tresman 1972) and roughly 6nm for red blood cells (Linss et al. 1991). An important study by Schaffner et al. (1995) demonstrated that the method of cell dissociation from tissues (mechanical vs enzymatic) affects the glycocalyx, and hence cell attachment. Early work suggested that the lipid headgroups and glycocalyx of cells are involved in nonspecific interactions with polycationic polymers adsorbed onto surfaces (Yavin and Yavin 1974; McKeehan and Ham 1976), while recent studies using X-ray photoelectron spectroscopy (XPS) (Stenger et al. 1993) demonstrate that surfaces containing high ratios of protonated-to-deprotonated ([NH3^]/[NH2]) amine groups yield the best hippocampal neuron cell attachment and growth. Proteoglycans such as syndecan are found on plasma membranes, and these types of macromolecules contain long polymer chains of glycosaminoglycans which are rich in negatively charged sulfate (803") and carboxyl (CO2') groups that can interact electrostatically with positively charged substrates (Alberts et al. 1994).

5

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Specific Interactions

Specific interactions between cells and surfaces involve several classes of CAMs including cadherins, selectins, and integrins, the last of which are the primary CAMs that mediate cell-extracellular matrix (ECM) adhesion. ECM proteins contain amino acid sequences, such as arginine-glycine-aspartate (RGD) and isoleucine-lysine-valine-alanine-valine (IKVAV), which bind to integrin receptors, transmembrane proteins that protrude 20-22 nm from the cell membrane. These receptors have an intermediate affinity for their ligands ( ^ D ^ 10~^-10~^mol/l), a property that facilitates cell migration, while also allowing for strong attachment when numerous integrin/ligand bonds are formed (Alberts et al 1994; Lodish et al 1995). A recent study of the adsorption of vitronectin and fibronectin from serum onto A^-2-(aminoethyl)3 aminopropyl-trimethoxysilane (EDA) gives direct evidence of integrin receptor-mediated attachment on micropatterned substrates (McFarland et al. 2000). Fluorescence interference contrast microscopy is a technique that can be used to determine the distance between cell membranes and surfaces, possibly providing an understanding of the spatial arrangement of integrin receptors, the glycocalyx, and adsorbed molecules in cell attachment (Braun and Fromherz 1997; Sorribas et al. 2001). Surface modification with molecules that bind to integrin receptors offers the possibility of examining cellular phenomena such as the role of integrins in the structural maturation of synapses in the hippocampus (Chavis and Westbrook 2001). Future studies that examine specific and non-specific interactions between cells and surfaces may provide more information on the role of integrin and other CAM receptors in signal-transduction cascades that affect differentiation and development.

IV. 1.

Chemical Patterning

HISTORICAL BACKGROUND

A number of researchers have applied micromachining and photolithographic techniques to the study of cell attachment and migration on chemically patterned substrates (Carter 1967; Letourneau 1975; Gundersen and Park 1984; Brunette 1986; Dunn and Brown 1986; Hammerback and Letourneau 1986; Clark et al. 1987; Kleinfeld et al. 1988; Stenger et al. 1992). Neuroscience was one of the first fields in which investigators sought to exploit surface patterning techniques. Gundersen used microstructured brass blocks to create lanes of proteins to monitor the reaction of D R G cell growth cones to regions of varying adhesivity (Gundersen and Park 1984). One of the first breakthroughs in using lithography techniques was the use of transmission electron microscope (TEM) grids to shadow-mask patterns of

258

H. G. CRAIGHEAD, C. D . JAMES, AND A. M. P. TURNER

evaporated palladium onto plastic dishes (Carter 1967; Letourneau 1975). In later experiments, patterns of ECM proteins were produced with UV Hght irradiation through TEM grids to study neuronal cell attachment and process outgrowth (Hammerback and Letourneau 1986). Recent work in this area by Buettner et al. (1994) has involved producing regions of albumin and laminin to investigate the growth cone dynamics of DRG cells and rat superior cervical ganglion neurons. Time-lapse recordings enabled the development of an insightful mathematical model for growth cone extension based in part on the role of actin filament polymerization/depolymerization in filopodia (Mitchison and Kirschner 1998). Each of these surface patterning studies demonstrates the crucial role of the ECM in growth cone motility, and work such as this has provided an understanding of the biological mechanisms responsible for axonal pathfinding during in vivo nervous system development. The early work of Kleinfeld et al. (1988) was among the pioneer efforts in using conventional photohthography to construct patterns of adhesive and non-adhesive molecules for directed neuron attachment and growth. This technique used self-assembled monolayers of alkyl- and aminosilanes to produce regions of cell attachment and repulsion that yielded electrically excitable networks of cerebellar granule cells and Purkinje neurons. This work proved to be one of the seminal advancements in the evolution of surface patterning for cell culturing, and the dramatic increase in throughput, alignment capabilities, and variety of masking geometries made possible with conventional photolithography have fueled the growth of this field during the last two decades. One particular contribution of microfabrication to neuroscience has been in examining the polarization of neurons, that is, the maturation of neurites (minor processes) into ultrastructurally and electrophysiologically distinct axons and dendrites. Early work in this area studied the differentiation of minor processes after axonal transection (Dotti and Banker 1987), and other researchers focused on the role of ECM glycoproteins (Chamak and Prochiantz 1989; Lochter and Schachner 1993; Esch et al 1999). Recent research using photoHthographically patterned surfaces demonstrated the importance of substrate composition in the compartmentalization of neurites into axons and dendrites (Stenger et al. 1998). In this work, an attractive molecule and a repulsive molecule were patterned in such a way that cells were given two kinds of surfaces on which to extend neurites: continuous lanes of adhesive molecules and lanes of adhesive molecules interrupted regularly by sections of repulsive molecules. Axonal development occurred in the continuous direction, while dendrites developed in the interrupted directions, demonstrating the delicate interplay of both timing and substrate composition in the governance of cell growth and function. The culture of primary hippocampal neurons (Banker and Goslin 1998) is a strong area of research due to the implication of the hippocampus in processes such as memory and learning. Hippocampal neurons grown on

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FIG. 3. Fluorescence micrographs of hippocampal neurons grown on a uniform substrate of PLL (left) and a grid pattern of PLL (right). Images courtesy of William Shain and James N. Turner, New York State Department of Health, Wadsworth Center.

unpatterned surfaces will attach and extend processes randomly during development, while cells grown on patterned surfaces (Craighead et al. 1998; Wheeler et al. 1999; Sprossler et al 2001) open up the possibility for studies on organized cell networks with consistent and reproducible geometries (Fig. 3). Such networks would be useful in studying phenomena such as the invasion of action potentials into branched axons and dendrites, the plasticity of synaptic connections, and the effects of patterned surfaces on the electrophysiological development of neurons (Ravenscroft et al. 1998). Liu et al. (2000) have inspected the early development of synaptic activity of hippocampal neurons on patterned surfaces, indicating that micropatterned cells develop the proper pre- and post-synaptic ultrastructural specializations necessary for communication between cells. Branch et al. (2000) have used surfaces patterned covalently with poly-L-lysine (PLL) and PEG for maintaining 4-week long primary hippocampal neuron cultures, an advance that along with the continued advancement of extracellular recording technology (Jimbo et al. 1993; Craighead et al. 2001; Sprossler et al. 2001) will enable the inspection of long-term developmental changes in cell networks. Reconstructed neuronal networks are also being developed for practical applications such as cell-based biosensors of nanomolar quantities of substances such as strychnine and tetrodotoxin (Gross and Rhoades 1995; Pancrazio et al. 1998, 1999). 2.

METHODS AND RESULTS

Several strategies have been developed for using Hthographic techniques to produce chemically patterned substrates, and they can be divided into two main classes: direct and indirect Hthographic methods. Direct

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H. G. CRAIGHEAD, C. D . JAMES, AND A. M. P. TURNER

photolithographic methods involve the direct exposure of substrates with UV light, laser light, ion beams, and electron beams to cause a change in the chemical composition of the molecules on the substrate. Indirect methods use Hthography as the first step in a sequential process of microfabrication techniques such as wet and dry etching, thin film deposition, evaporation, and sputtering. a.

Direct Lithography

The first scheme we will discuss involves coating a surface with a molecule that is then selectively modified, and the second scheme involves a "hft-off' method using a patterned sacrificial layer that is removed in order to selectively immobihze molecules on a substrate. Research on patterned surfaces for cell culturing has shown that the best control of cell attachment and spreading is achieved with surfaces that both promote and repel cell attachment in complementary regions. Both of the schemes to be discussed below allow for the production of such multifunctional surfaces. The first scheme is depicted in Fig. 4. A substrate is coated uniformly with the primary molecular layer, and then irradiated in selective regions. This irradiation cleaves bonds within the molecule, producing highly reactive radicals that can then be covalently coupled to a secondary molecule. Stenger et ai (1992) have used this scheme for organizing dissociated neuronal cells

FIG. 4. Direct lithography—scheme 1. The substrate is coated with a primary molecule (a), which is then irradiated through a mask (b). The exposed regions are then coupled to a secondary molecule (c).

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(a)

(e)

(f) ijifitM!MMlMft.«MllAAlMAflAiib wAAA.«AAAAi

FIG. 5. Direct lithography—scheme 2. A mask layer is placed on the substrate (a), irradiated (b), and then developed (c). The substrate is then incubated in the primary molecule (d), and then the mask is removed (e). The final step is the incorporation of a secondary molecule to the underivatized areas of the substrate (f).

into designed networks. In their process, silica surfaces are functionalized with EDA, an aminosilane that has been shown to promote cell attachment, and has the useful property of strong absorbance at 193 nm. UV exposure of the substrate through a mask cleaves EDA molecules, leaving reactive silanol and/or short alkyl chains. These species are then covalently linked with a fluorosilane, yielding a Teflon-like surface that inhibits cell attachment. The second scheme often used for direct patterning is to cover a substrate with a masking material that can be removed with high selectivity (Fig. 5). The mask layer is patterned using photolithography and etching, and then the substrate is incubated in a solution containing the primary molecule. After allowing for the reaction of the primary molecule with the surface, the mask is removed with a solvent that leaves both the substrate and the primary layer intact. A secondary molecule with a high specificity for the bare substrate can then be incorporated. Several labs have used this style of chemical modification to produce surfaces for haptotaxis (guidance by gradients of adhesivity) studies of cells (Lom et al. 1993; Ruardij et al. 2000). Application of direct lithography—scheme 2. One specific strategy for producing chemical patterns for dissociated hippocampal neuron cultures is to use photoresist as the sacrificial layer, and PLL (a polycationic polyamino acid often used for promoting cell attachment to surfaces) as the designed layer molecule. First, silica substrates are cleaned thoroughly in nitric acid for 24 h, and rinsed thoroughly in high-grade Milli-Q filtered water

262

H. G. CRAIGHEAD, C. D . JAMES, AND A. M. P. TURNER

(18 MO-cm resistivity) for 1-3 days with several changes. Photoresist is applied to the surface, exposed with UV through a chrome mask, and then developed. A final 30-s oxygen plasma reactive-ion etch cleans up the surface for improved polypeptide adhesion to the bare silica. Solutions such as phosphate-buffered saline (PBS, pH 7.3, 0.1 M phosphate buffer, 0.15 M NaCl) or borate buffer (pH 8.5, 0.1 M) are typically used for making protein solutions. The patterned substrate is then incubated in a buffered protein solution containing 1 mg/ml of fluorescein isothiocyanate labeled PLL (PLLFITC) for 2 ^ h . After rinsing with clean buffer and water, the bulk of the photoresist mask is removed by sonicating the substrate in acetone for 30 min. A thin residue of resist may remain after the sonication, but it can be removed with the gentle rubbing of a cotton applicator soaked in acetone. The surface is then incubated at room temperature for 2-4 h in a 1-5% (v/v) solution of a block copolymer of poly (ethylene oxide) and poly(dimethylsiloxane) (PEOZ?-PDMS) in toluene (Fig. 6). This amphiphilic copolymer initially adsorbs to silica through hydrogen bonding between the siloxane backbone in the PDMS moiety and silanol groups on the surface, leaving the PEO chains extended into the solution (Fig. 7). The hydrophiHc PEO chains have flexible ether bonds, yielding a highly hydrated and mobile brush-like surface with high entropy that resists protein adsorption and cell attachment (Owen 1990; Jeon et al. 1991; Yang et al. 1999; Alcantar et ai 2000). Figure 8 shows the protein adsorption resistance of radio-frequency (RF) glow discharge and copolymer treated surfaces to several fluorescently labeled proteins. Test coverslips were subjected to a 1-min RF glow discharge air plasma followed by a 2-h PEO-/?-PDMS incubation. Surfaces were then incubated in 1-1.5 mg/ml protein solutions in PBS for 2h at 37°C. Plasma treatments alone tended to reduce protein adsorption, presumably by increasing the

Sfiaai FIG. 6. Fluorescence micrograph of PLL-FITC patterned on glass with a photoresist Hft-off method (scale bar = 50 \\m).

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CURRENT ISSUES AND ADVANCES IN DISSOCIATED CELL CULTURING -500

263

^2.0nm

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250

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FIG. 7. Tapping-mode AFM micrograph of a glass coverslip coated with PEO-/7-PDMS copolymer.

Protein adsorption resistance 0.7 0.6 0.5 0.4 §

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FIG. 8. Protein resistance of plasma and PEO-/?-PDMS treated surfaces to goat anti-mouse immunoglobulin (IgG), bovine serum albumin (BSA), Protein A, type IV collagen, and fibrinogen. Fluorescence intensity was normalized to the intensity on untreated surfaces.

hydrophilicity of the surface, and thereby curtaihng the strong hydrophobic interaction which drives the adsorption of many proteins to surfaces (Sigal et al. 1998). A cell adhesion study using cultured astrocytes on a surface coated with this polymer demonstrated drastically reduced cell attachment (averages of 15 vs 2 cells/field on a 150 x 150 |im^ field) and spreading (Fig. 9), consistent with low adhesivity. Using this scheme, grids of PLL were placed

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H. G. CRAIGHEAD, C. D . JAMES, AND A. M. P. TURNER

FIG. 9. Astrocytes grown on a silicon surface (left), and on a silicon substrate modified with PEO-/7-PDMS (right). Images courtesy of William Shain and James N. Turner, New York State Department of Health, Wadsworth Center.

FIG. 10. Phase contrast micrographs (left) and corresponding fluorescence micrographs (right) of day 6 hippocampal neurons cultured on 5-|im wide PLL line grids with spaces of 50|im. Cells were stained with rhodamine 123.

on clean coverslips, consisting of 2-5 |im wide lines, 15-|im wide nodes, and spacings of 50|im [see Fig. 6]. Hippocampal neurons were dissected from neonatal rats and plated onto the surfaces in growth medium with 10% fetal bovine serum. On the sixth day of culturing, cells were examined and videotaped under a dual phase-contrast/fluorescence inverted microscope. Cell cultures were examined for cell attachment and guidance to PLL grid patterns, and inspected for active metabolism with Rhodamine 123 a fluorescent dye that is sequestered by active mitochondria (Fig. 10). Cells have successfully adhered to the patterned PLL-FITC in grid structures and are metabolically active.

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Drawbacks and advances of direct lithography. Direct lithography methods are capable of extremely high resolution, which can be down to 200 nm for optical lithography and tens of nanometers for electron beam lithography. Some of the drawbacks for direct lithography methods include the lack of control of the location of bond cleavage in molecules, as well as the unpredictable incorporation of secondary molecules to radicals. The high dose required for photolysis of various functionalized silanes (up to 30J/cm^) (Dulcey et al. 1991) is another limiting factor for fast production of large field-size substrates, and the expensive microfabrication equipment required is available in only certain engineering facilities. One difficulty with the "liftoff" scheme is balancing the adhesion of the masking material that requires tight adhesion for well-defined patterns but loose adhesion for easy removal. In addition, primary layers must have low interaction with secondary molecules in order to produce biofunctional surfaces, and designed layers must survive harsh procedures such as solvent exposure and sonication, treatments that may destroy the functionality of bioactive molecules. Mechanical lift-off methods such as that reported by Folch et al. (2000) and Ilic and Craighead (2000) address several of these issues. In the latter case, a film of Parylene polymer is plasma deposited on a substrate and patterned using a dry reactive-ion etch. The substrate is then incubated in a protein or cell solution, rinsed, and finally the film is mechanically peeled from the substrate. This method also allows for the continuous hydration of proteins/cells, does not require solvent exposure for mask removal, and has not been demonstrated to leave a film residue on the substrate upon removal.

h.

Indirect Photolithography

Indirect photolithographic techniques use conventional photolithography to initiate the patterning process, and subsequently use other microfabrication technologies to produce a microstructured surface. One such family of techniques is the soft lithography family that includes the replica molding methods of micromolding in capillaries (MIMIC) and microcontact printing (|iCP). These two methods involve the production of PDMS elastomer replicas of microstructured surfaces that are capable of producing submicron features on substrates. The first applications developed by Whitesides and coworkers involved the deposition of self-assembled monolayers (SAMs) of alkanethiols and alkylsilanes onto surfaces to act as ultrathin resists for wet chemical etching and metal plating (Kumar et al. 1994; Hidber et al. 1996). Soft lithography techniques have evolved over the years and are now being used in an assortment of biological apphcations for producing patterns of proteins and cells on surfaces (Delamarche et al. 1991 \ Bernard et al. 1998; Craighead et al. 1998; James et al. 1998; St. John et al. 1998; Kane et al. 1999; Wheeler et al. 1999; Takayama et al. 1999). MIMIC and JLICP can be used in

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combination with more complex chemistry to produce designed layers of a variety of molecules. Ruiz-Taylor et al. (2001) have used MIMIC to produce patterns of PLL-biotin conjugates that were subsequently coupled to streptavidin molecules. Researchers have also stamped and covalently bound PLL, PEG, and synthetic bioactive peptide structures onto surfaces (Wheeler et al. 1999; SchoU et al. 2000; Sprossler et al. 2001). Several groups use the strong chemisorption of sulphur compounds onto gold and silver surfaces to produce patterned cells on metals. Mrksich et al. (1997) have used adsorbed PEG-thiols and stamped alkanethiols to selectively adsorb fibronectin to promote endothelial cell attachment, and Zhang et al. (1999) have bound thiol terminated oligopeptides and PEGs to gold surfaces to produce patterned fibroblasts and kidney cells. Application of indirect lithography. The first step in producing elastomeric stamps for MIMIC and |iCP is to create a master mold using a silicon wafer. The inverse replica of the desired stamp topography is created on the surface of the wafer using photoHthographic patterning of photoresist, polyimide, or even the bulk substrate itself (Fig. 11). The finished master can be subjected

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^*

FIG. 12.

Scanning electron microscope (SEM) micrograph of a PDMS stamp (scale bar = 20 jim).

to a surface treatment to prevent strong adhesion of the siHcone polymer to the master; detergents (such as Triton X-100 and sodium dodecyl sulfate) and fluorosilanes have proven effective. The silicone elastomer, Dow Corning Sylgard 184, consists of a mixture of one part crosslinking agent and ten parts of the silicone monomer. After vigorous mixing of the catalyst and monomer, the elastomer is poured onto the mold and placed under rough vacuum (~10~^ torr) until bubbles are removed. The elastomer can be cured at 60°C for 2 h or room temperature for roughly 24-72 h. The master and the elastomer mold are then separated, and stamps are cut into pieces with razor blades (Fig. 12). PDMS stamps are then treated to an air plasma for 10-30 s to render the surface hydrophilic. XPS, Fourier transform infraredattenuated total internal reflectance (FTIR-ATR), and static secondary ion mass spectroscopy (SSIMS) studies have shown that oxygen plasma treatments of PDMS reduce the amount of methyl groups and increase the amount of oxygen on the top surface of the siHcone, reducing contact angles from over 100° to nearly 0° (Morra et al. 1990). However, the hydrophilicity is temporary, and as silanol groups condense and polymer chains on the surface are recycled with chains diffusing from the bulk, the hydrophobicity returns. The lifetime of hydrophilicity can be extended by storing treated stamps in water, a treatment that retards the migration of alkyl chains to the surface. Researchers have also increased the hydrophilicity of native PDMS without plasma treatments by storing the stamp in deionized water for 24 h prior to use (Lauer et al. 2001). The hydrophilic stamp can now be used for MIMIC

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FIG. 13. PLL-FITC structures created by MIMIC (scale bar = 20|im).

or jiCP experiments. Substrates to be patterned are cleaned in nitric acid, and immediately before being patterned, are treated to a 1-min air plasma to increase the adhesion of proteins to the surface. MIMIC. In the case of MIMIC, the PDMS stamp is placed face down on the substrate, creating microchannels. Protein solutions are fed at the entrance of the channels, while the hydrophilic PDMS walls and the substrate surface provide a strong capillary force to pull the protein solution into the channel. After the channels have been filled and time has been allowed for protein adsorption to the surface, the channels are flushed with clean buffer and deionized water, and the mold is removed (Fig. 13). jiCP. In jiCP, protein solutions are placed on the hydrophilic stamp, allowing protein to adsorb to the surface (Fig. 11). After drying, the adsorbed protein can then be transferred to a substrate. In practice, plasma treatment of the PDMS increases the abihty of an aqueous solution of molecules to wet the stamp surface and produces more uniform stamped structures (Yan et al. 1999; personal observation). Prior to stamping, the PLL solution was filter sterilized with 0.22-iim syringe filters. To allow molecules to adsorb to the PDMS stamp, the stamps are placed face up, and covered with protein solution for 10-30 min. The stamp is then dried thoroughly with a highpurity nitrogen stream, and brought into contact with the surface. Weights can be placed on the stamp, depending on the geometry and size of the stamp and its features, with a typical pressure range of 10-500 g/cm^. After 0.55 min of contact, the stamp is removed and the substrate is rinsed in buffer and deionized water (Fig. 14). The response of hippocampal neurons to microcontact printed PLL was examined by comparing the total axon length of cells grown on substrates coated uniformly with adsorbed PLL (A-PLL) to those of cells grown on

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FIG. 14.

269

Stamped PLL-FITC on a glass substrate (scale bar = 20^m).

substrates stamped with PLL (S-PLL) consisting of 30-|im wide lines and 10|im spaces (Fig. 15). Laminin is sometimes adsorbed onto PLL coated substrates in order to promote axonal outgrowth (Lein and Higgins 1989; Lein et al. 1992), so A-PLL and S-PLL samples were prepared with adsorbed laminin (A-PLL/laminin and S-PLL/laminin). The average axon length on S-PLL and S-PLL/laminin was 77.63 and 94.97 |im, respectively, compared to 92.64 nm for A-PLL and 148.62 |im for A-PLL/laminin. The results indicate that axon extension occurs on S-PLL, and that PLL-laminin interactions still develop when using S-PLL. We attribute the reduced axon length on S-PLL to geometric constraints of the patterns. An experiment to determine the effectiveness of stamped PLL on confining cell body attachment was also performed. Grid patterns of PLL of the following dimensions were stamped onto coverslips: 2-5-|im wide Hues, 15-|im wide nodes, and 50-|im spacings between nodes. Several coverslips were cultured as is, and several coverslips were subsequently modified with PEO/}-PDMS. Cells were cultured between 1 and 4 days, and a total of 331 cells were examined (Fig. 16). The PEO-Z?-PDMS treatment increased the percentage of cell bodies that attached to PLL nodes (from 32.4 to 52.9%), while decreasing the percentage of cell bodies that attached to PLL lines (from 42.2 to 37.0%) and bare-glass spaces (from 23.9 to 10.0%). As the line width is reduced to 2 |im, the percentage of cell bodies attached to nodes increased as the percentage of cell bodies attached to lines decreased (data not shown). Issues and advances. Some of the advantages to using soft molds are that multiple replicas can yield high throughput for large-scale production, molecules can be patterned on curved or nonplanar surfaces, and the

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FIG. 15. Day 2 hippocampal neurons grown on S-PLL/laminin (scale bars= 100 and 50)im on top and bottom images, respectively). Image courtesy of Gary Banker, Oregon Health Sciences University.

dimensions of the features can be manipulated by controlled mechanical deformation of the mold (Xia and Whitesides 1998). In addition, master molds can be fabricated at engineering facilities, while the elastomeric replicas can be produced and used in any biology lab. PDMS is also a relatively inexpensive material, and the molds and elastomer replicas can be reused. The continual hydration of the surface makes MIMIC an attractive technique for producing patterned films that must be kept hydrated such as lipid bilayers (Janshoff and Kunneke 2000). MIMIC has been successful in producing highly uniform patterns of polypeptides and proteins such as PLL and laminin (Esch et al. 1999), and maintenance of biological activity is more likely since the protein can be kept fully hydrated throughout the process. However, PDMS "residue" can often be left behind on the surface after removal of the elastomer, interfering with subsequent surface modification efforts and inadvertently creating additional structural/chemical cues.

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FIG. 16. SEM micrograph of an elastomer stamp used to produce the PLL grid (blue) shown in the fluorescence micrograph on the right (scale bars= 15 and 30|im). Hippocampal neurons were stained for neurotubulin (red). Fluorescence image courtesy of William Shain and James N. Turner, New York State Department of Health, Wadsworth Center.

A more recent soft lithography technique utihzes a three-dimensional microfluidic system for direct patterning of surfaces with proteins and cells (Chiu et al. 2000). In this work, an elastomeric microfluidic system is positioned on a substrate, and protein/cell solutions are delivered selectively to surfaces in various geometries and patterns. This technique requires the fabrication of much more complex elastomeric replicas than typical MIMIC replicas, but the control over solution delivery is a substantial benefit. Both isolated (squares) and continuous (grids) features can be produced, while also allowing the continual hydration of patterned proteins and cells. |iCP is a simple technology that can effectively produce patterned substrates for various applications. Multiple chemical species can be transferred to single substrates (Fig. 17), and the time duration of molecule transfer from stamp to substrate is extremely fast, on the order of seconds (Bernard et al. 1998). Proteins such as laminin, fibronectin, bovine serum albumin (BSA), and collagen have been printed onto substrates. In terms of quantifying the transfer efficiency of jiCP, Branch et al. (2000) have stamped radiolabeled PLL and typically found a 17% transfer efficiency from stamps to the substrate, while fluorescence microscopy experiments by Bernard et al. (1998) have shown very high transfer efficiency (~90%). This discrepancy can probably be attributed to differences in stamping protocol: before stamping, Bernard et al. rinse protein-covered stamps in buffer to remove loosely bound protein, while Wheeler et al. (1999) report no rinse step before stamping. This pre-rinsing can also alleviate the smearing of protein features that result when thick protein layers are transferred to surfaces; when these stamped features are rinsed in buffer or water, weakly bound protein will be washed off and re-deposited in unintended locations. In addition, pre-rinsing can often reduce non-uniformity in stamped protein layers.

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FIG. 17. Multifunctional substrate. The substrate is patterned with 25-)im wide BSA features (red) and 2-|im wide collagen (green) features.

There are several caveats to soft lithographic techniques that are currently being addressed. As mentioned previously, PDMS replicas sometimes leave residue (probably un-crosslinked, low molecular weight PDMS chains) on surfaces after contact. However in }iCP, the molecule adsorbed to the stamp may sometimes provide a buffer zone to prevent such transfer of residue, depending on the thickness of the layer adsorbed to the stamp (unpublished observation). Also, the elasticity of the stamp contributes to the production of features with larger dimensions than intended. Of primary concern for jiCP applications is the dehydration step, a process that may damage molecules and cause an irreversible loss of secondary/ tertiary structure in proteins. The author and colleagues have conducted experiments with the direct stamping of laminin, and although laminin features are successfully transferred to substrates, cell attachment to printed laminin was minimal. Greater success at printing bioactive laminin has been achieved by covalently conjugating PLL to laminin (Kam et ai, submitted; Kam et al. 2001). However, Lauer et ai (2001) observed successful attachment and neurite outgrowth of hippocampal neurons on stamped laminin, while Wheeler et al. (1999) stamped mixtures of PLL + laminin that retained laminin's bioactivity. In this respect, different protocols (for pre-treating stamps, cleaning substrates, culturing cells, etc.) will influence the yield of bioactive stamped proteins, and a detailed study of such procedures has not been conducted. Bernard et al. (1998) have examined the bioactivity of an assortment of printed enzymes and proteins, and have observed that many retain a substantial amount of their bioactivity. The author and collaborators have printed proteins such as antibodies and Protein A and found that bioactivity was retained (Fig. 18). jiCP, while keeping the substrate and stamp in aqueous solution, seems to be a feasible task and has been encouraged by the work of Ho vis and Boxer (2001) who have used jiiCP in fluids to pattern planar lipid bilayers on substrates.

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FIG. 18. Bioactive IgG stamped onto a coverslip (scale bars = 80 and 600 iim on left and right images, respectively). Coverslips were stamped with blocks of mouse IgG, blocked with BSA, and incubated with fluorescently labeled goat anti-mouse IgG.

FIG. 19.

Custom-built ahgnment tool for |iCP on substrates.

Alignment capabilities are a particularly useful development in |iCP (Wheeler et al 1999; James et al. 2000; Lauer et al. 2001). In this scheme, the substrate to be patterned and the stamp are controlled to allow for specific placement of the chemical pattern on the surface. This enables the possibiHty of multichemical patterns for investigating an array of substances in a single culture. The custom built device shown in Fig. 19 has six degrees of

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FIG. 20. Fluorescence image of a patterned cell network grown on a planar microelectrode array (scale bar = 50|^m). Cells were stained with rhodamine 123. Inset is a recording from the electrode indicated by the arrow.

freedom for positioning and alignment of stamps to substrates (see James et al. 2000 for details). The stamp is held on a microscope stage above an objective lens, and since the stamp is transparent, the surface of the stamp and the surface of the substrate can be visualized by changing the focus. This particular instrument is capable of an alignment accuracy ~ l - 2 |im and is used for aligning chemical cues on substrates, and for placing PLL structures on the surface of planar microelectrode arrays (Fig. 20) to promote cell attachment near recording sites (James et al. 2000; Craighead

etal.imX). V.

Current and Future Directions of Chemical Patterning

Chemical patterning techniques permit novel studies in cell and molecular biology. As new growth factors and proteins are discovered, researchers need to perform experiments that not only elucidate their immediate function, but their role in signal cascades that can involve hundreds of other molecules. The techniques discussed in this section allow the production of multifunctional surfaces in an attempt to conduct such experiments. Microfluidic

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patterning methods such as those demonstrated by Chiu et al. (2000) provide the flexibihty and control that is necessary for producing compHcated surfaces with multiple proteins that remain hydrated. The abiUty of simple techniques like jiCP to produce multiple chemical patterns on substrates makes this method attractive for applications that require or benefit from simultaneous analysis of multiple analytes, such as genomic research. Photolithographic techniques have been used for years to produce microarrays for genome analysis (for a review on microarray technology, see Schena et al. 1998), and microfabrication techniques are currently being extended to the emerging field of protein chips and proteomics (the analysis of the complete protein catalog of an organism) (Bashir et al. 2001). The future work of chemical patterning for cell culture will also progress as new growth factors and adhesion molecules are identified. Researchers have already bound specific peptide sequences, such as RGD and IKVAV, to surfaces for controlling cell attachment (Ranieri et al. 1995). Past and current investigations have explored the use of cell adhesion molecules such as axonin-1 and NgCAM (Sorribas et al. 2001). In this particular work, different molecules presented to the cell produced differences in cell morphology such as neurite diameter, neurite length, and cell membrane-to-surface distance. For neuronal cell cultures, we anticipate the attempt to control the initiation of synaptogenesis using patterned chemical cues of substances such as agrin and neuroligin, proteins that have been implicated in synapse development in vivo (Brose 1999; Lesuisse et al. 2000; Rao et al. 2000; Scheiffele et al. 2000). More generally, the exploration of receptor-mediated signal cascades will be another area of interest to molecular and cell biologists, and the ability to control the spatial organization of receptor ligands on surfaces should allow for interesting experiments to be performed.

VI.

Introduction: Topographical Patterning

Although scientists for the past 100 years have been studying the effects of surface contact on the growth and development of mammalian cells, it has only been during the last two decades that various researchers around the world have been employing the tools developed by the semiconductor industry to fabricate complex surface features for use in cell culture studies. The materials conventionally used for cell culture, such as glass and polystyrene, are still highly used in these studies but more exotic materials such as silicon, titanium, epoxy resins, and silicone elastomer (PDMS) have become increasingly more popular. The types of cells being studied are just as varied as the kinds of materials being used. This group of cells has included fibroblasts, osteoblasts, endotheUal cells, cardiomyocytes, monoctyes, epitheHal

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cells, neurons, glial cells, macrophages, epitenon cells, leukocytes, hepatocytes, keratinocytes, bacteria, and fungi (see Table I). The most prevalent feature types studied have been grooves, grids, pillars, trapezoidal obstructions to flow, wells or pits, pores, ridges, spheres, cylinders, and random micrometerand nanometer-scale roughness (see Table II). These geometries have been studied for reasons ranging from the search for surface modifications that will improve tissue integration with materials used for artificial bone implants, to attempts in mimicking the topography observed in extracellular matrices and basement membranes. The results from these studies may find use in

TABLE I CELL TYPES STUDIED ON TOPOGRAPHICALLY PATTERNED SURFACES

Cell types studied Bacteria Cardiomyocytes Endothelial cells Epitenon cells Epithelial cells

Fibroblasts

Fungi Ghal cells Hepatocytes Keratinocytes Leukocytes Macrophages Monocytes Neurons

Osteoblasts

Contributors Scheuerman et al. (1998) Deutsch et al. (2000), Polonchuk et al. (2000) Goodman et al. (1996), van Kooten and von Recum (1999), Simon et al. (2000), Webster et al. (2000) Wilkinson et al. (1998), Casey et al. (1999) Brunette et al. (1983), Clark et al. (1990, 1991), Oakley and Brunette (1995b), Damji et al. (1996), Brunette and Chehroudi (1999), Evans et al. (1999), Dalton et al. (2001a,b), Brunette (1986), Clark et al. (1987, 1990, 1991), Rovensky et al. (1991, 1999), Green et al. (1994), Chou et al. (1995, 1998, 1999), Oakley and Brunette (1995a), Wojciak-Stothard (1995), Britland et al. (1996), Damji et al. (1996), den Braber et al. (1996a,b, 1997, 1998), Eisenbarth et al. (1996), Curtis and Wilkinson (1997), Oakley et al. (1997), Chen et al. (1998), den Braber (1998), Goto and Brunette (1998), van Kooten et al. (1998), Brunette and Chehroudi (1999), van Kooten and von Recum (1999), Walboomers (2000), Webster et al. (2000), Alaerts et al. (2001) Verran and Maryan (1977), Hoch et al. (1987), Terhune et al. (1993) Webb et al. (1995), Turner et al. (1997, 2000), Craighead et al. (1998, 2001) Ranucci and Moghe (1999, 2001), Ranucci et al. (2000), Semler et al. (2000) Meyle et al. (1995), Pins et al. (2000) Tan et al. (2000), Eriksson et al. (2001) Schmidt and von Recum (1991), Meyle et al. (1995), Wojciak-Stothard et al. (1996), Curtis and Wilkinson (1997), Wilkinson et al. (1998) Meyle et al. (1995) Clark et al. (1987), Clark et al. (1990, 1991), Nagata et al. (1993), Webb et al. (1995), Rajnicek and McCaig (1997), Rajnicek et al. (1997), Torimitsu (1997), Hirono et al. (1998), BayHss et al. (1999a,b), Mattson et al. (2000). Craighead et al. (2001) Lincks et al. (1998), Webster et al. (1999a,b, 2000), Anselme et al. (2000), Perizzolo et al. (2001)

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TABLE II TYPES OF SURFACE STRUCTURES FABRICATED FOR CELL-SUBSTRATUM STUDIES

Surface structures studied Cylinders Grids Grooves

Trapezoidal obstructions to flow Pillars

Pores

Random micrometer-scale texture

Random nanometer-scale texture Ridges

Spheres Wells/pits

Contributors Mattson et al. (2000) Rovensky et al. (1999) Brunette et al. (1983), Brunette (1986), Hirono et al. (1988), Clark et al. (1990, 1991), Nagata et al. (1993), Clark (1994), Oakley and Brunette (1995a), Webb et al. (1995, 1996), Wojciak-Stothard et al. (1995), Britland et al. (1996), Damji et al. (1996), den Braber et al. (1996a,b, 1997, 1998a), Chehroudi et al. (1997), Rajnicek and McCaig (1997), Rajnicek et al. (1997), Oakley et al. (1997), Chen et al. (1998), Choi et al. (1998), Goto and Brunette (1998), Scheuerman et al. (1998), van Kooten et al. (1998), Wilkinson et al. (1998), Brunette and Chehroudi (1999), van Kooten and von Recum (1999), Deutsch et al. (2000), Pins et al. (2000), Walboomers (2000), Alaerts et al. (2001), Dalton et al. (2001b), Perizzolo et al. (2001), Simon et al. (2000) Rovensky et al. (1991), Schmidt and von Recum (1991), Green et al. (1994), Craighead et al. (1998, 2001), Wilkinson et al. (1998), Casey et al. (1999), Deutsch et al. (2000), Turner et al. (2000) Bayliss et al. (1991a,b), Evans et al. (1999), Ranucci and Moghe (1999, 2001), Ranucci et al. (2000), Semler et al. (2000), Dalton et al. (2001a) Verran and Maryan (1997), Lincks et al. (1998), Chou et al. (1999), Craig and LeGeros (1999), Anselme et al. (2000), Polonchuk et al. (2000), Eriksson et al. (2001), Perizzolo et al. (2001) Eisenbarth et al. (1996), Goodman et al. (1996), Turner et al. (1997), Verran and Maryan (1997), Craighead et al. (1998), Webster et al. (1999a,b, 2000) Clark et al. (1987), Hoch et al. (1987), Hirono et al. (1988), Terhune et al. (1993), den Braber et al. (1996a), Wojciak-Stothard et al. (1996), Rajnicek and McCaig (1997), van Kooten and von Recum (1999), Pins et al. (2000) Lin and Wu (1999) Green et al. (1994), Chehroudi et al. (1997), Turner et al. (2000), Dalton et al. (2001a)

practical applications (see Table III) such as improved bone replacements and dental pieces, artificial organs, subcutaneous implants, vascular grafts and stents, advanced wound healing materials, stents for nerve regeneration, corneal implants, defined cellular networks, and optoelectronic

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TURNER

TABLE III APPLICATIONS FOR TOPOGRAPHICALLY PATTERNED SURFACES IN BIOLOGICAL SYSTEMS

Applications for topographical patterning Advanced wound healing Artificial organs Bone replacements

Corneal implants Defined cellular networks Dental pieces

Optoelectronic bio-interfaced devices Subcutaneous implants Stents for nerve regeneration

Vascular grafts and stents

Contributors

Wilkinson et al. (1998) Ranucci and Moghe (1999, 2001), Polonchuk et al. (2000), Ranucci et al. (2000), Semler et al. (2000) Chehroudi et al. (1997), Lincks et al. (1998), Chou et al. (1999), Webster et al. (1999a,b, 2000), Anselme et al. (2000), Eriksson et al. (2001), Evans et al. (1999), Dalton et al. (2001a,b) Hirono et al. (1988), Craighead et al. (2001) Brunette et al. (1983), Chehroudi et al. (1997), Verran and Maryan (1997), Goto and Brunette (1998), Chou et al. (1999), Craig and LeGeros (1999), Webster et al. (1999a,b, 2000), Perizzolo et al. (2001) Bayliss 6'/(3/. (1999a,b) den Braber et al. (1997), Walboomers (2000) Nagata et al. (1993), Gomez and Letourneau (1994), Webb et al. (1995), Rajnicek and McCaig (1997), Turner et al. (1997, 2000), Craighead et al. (1998, 2001) Goodman et al. (1996), Simon et al. (2000)

bio-interfaced devices, as well as a host of other applications that grow more apparent with each new discovery. One can draw a timeline covering the past 100 years and discover researchers in each decade who have made major contributions to the study of cell-substrate interactions. Ross G. Harrison who started his career in the early 1900s has been noted as the first to observe the behavior of attached cells to surface structure (Harrison 1911, 1914). Weiss and others in the 1930s and the 1940s pioneered the concept of "contact guidance" (Fell 1939; Weiss 1941, 1945, 1958). Weiss originally described the guiding action of oriented interfaces in the locomotion of cells in general terms as "contact action" (Weiss 1941). This basic principle still holds today and is the basis for many current studies. In the 1950s, Abercrombie studied the social behavior of cells and mutual contacts, coining the phenomena of "contact inhibition" (Abercrombie and Heaysman 1953, 1954; Abercrombie et al. 1957). He determined that cells in culture interfere with each other's movement when they make mutual contact (Abercrombie and Heaysman 1953). Curtis in the 1960s was pursuing the study of topographic factors in the control of cell behavior including contact inhibition, contact guidance, and cell spreading with fibroblasts (Curtis 1960, 1962, 1964, 1966, 1969; Curtis and Varde 1964). His findings led him to propose that the edges of cells may be

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especially adhesive and that the distance between the cell surface and the underlying substrate may be determined by the van der Waals-London and electrostatic forces between the surfaces (Curtis 1964; Curtis and Varde 1964). In the 1970s, Dunn continued with contact guidance studies by hypothesizing and proving that the shape of the substratum imposes mechanical restrictions on the formation of certain hnear bundles of microfilaments (Dunn and Heath 1976; Dunn and Ebendal 1978). Brunette, in the 1980s, was one of the first researchers to use photolithography to define surface structures for cell studies (Brunette et al. 1983; Brunette 1986). Using microelectronics fabrication techniques, he was able to observe the alignment of epithelial cells on well-defined, titanium-coated silicon grooves. Since then, scientists have realized that topographical cues play a crucial role in mediating not only biocompatibility and cell orientation, but also protein synthesis and secretion, gene expression, differentiation, and signal transduction (see Table IV). TABLE IV THE INFLUENCE OF TOPOGRAPHICAL MODIFICATIONS ON CELL PROPERTIES

Cellular responses Biocompatibility

Cell orientation

Differentiation

Gene expression Protein secretion Protein synthesis

Signal transduction

Contributors Curtis and Varde (1964), Clark (1994), Eisenbarth et al. (1996), Goodman et al. (1996), den Braber et al. (1997), Turner et al. (1997, 2000), Verran and Maryan (1997), Boeckl et al. (1998), Wilkinson et al. (1998), Bayliss et al. (1999a,b), Chou et al. (1999), Evans et al. (1999), Ratner and Shi (1999), Shi et al. (1999), Webster et al. (1999b), Anselme et al. (2000), Deutsch et al. (2000), Dalton et al. (2001a,b), Eriksson et al. (2001), Ranucci and Moghe (2001) Brunette (1986), Clark et al. (1987, 1990, 1991), Hirono et al. (1988), Rovensky et al. (1991), Nagata et al. (1993), Gomez and Letourneau (1994), Green et al. (1994), Oakley and Brunette (1995a), Webb et al. (1995), Britland et al. (1996), Damji et al. (1996), den Braber et al. (1996b), Oakley et al. (1997), Chou et al. (1998), van Kooten and von Recum (1999), Walboomers et al. (1999, 2000), Turner et al (2000), Perizzolo et al. (2001) Hoch et al. (1987), Terhune et al. (1993), Wojciak-Stothard et al. (1996), Lin and Wu (1999), Ranucci and Moghe (1999), Pins et al. (2000), Polonchuk et al. (2000), Semler et al. (2000) Chou et al. (1995, 1998), Lincks et al. (1998), van Kooten et al. (1998) Chou et al. (1995), den Braber et al. (1998a), Lincks et al. (1998), Ranucci et al. (2000) Chou et al. (1995, 1998), Oakley and Brunette (1995a), Webb et al. (1995), Britland et al. (1996), Wojciak-Stothard et al. (1996), den Braber et al. (1998a), Goto and Brunette (1998), Lincks et al. (1998), Craig and LeGeros (1999), van Kooten and von Recum (1999), Polonchuk et al. (2000), Ranucci et al. (2000), Turner et al. (2000) Rajnicek and McCaig (1997)

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VII. 1.

Techniques for Topographically Patterning Surfaces

MATERIALS USED FOR TOPOGRAPHICAL STUDIES

As a result of the continuing evolution of the computer and the technologies involved in many aspects of the manufacture of computers, the variety of materials made commercially available to the general researcher has been steadily increasing over the last few decades. Prior to this technological boom, materials researchers in industry and academia were discovering many new materials but only with time would they eventually be made available for widespread use in the private and commercial sectors. Thus, biologists were limited in the materials they could employ in cell culture for the study of cell behavior. Glass was the biologists' material of choice for years until plastics were made into viable tissue culture alternatives. Even though commercial production of polystyrene did not begin until the 1930s, it did not take long for such space-age plastics to find a permanent place in research laboratories as well as private homes. With the advent of more sophisticated analysis techniques, researchers have been able to determine some of the properties of materials that promote cell prohferation as well as cell death, opening up many doors to potential classes of biocompatible materials. Today, some of the materials being used include quartz, titanium, silicon, fused silica, polystyrene, silicone elastomer (PDMS), epoxy resins, poly(methylmethacrylate) (PMMA), polyester, polyurethane, carbon, and polycarbonate (see Table V).

2.

METHODS OF TOPOGRAPHICAL PATTERNING

If one looks at the predominant methods currently used today to fabricate substrates with topographic features, one will notice that they all involve some form of lithographic patterning. The remaining methods for structurally modifying surfaces include direct cutting or scratching with diamond or some other hard material, particle settHng, sandblasting, track etching, acid washing/etching, sintering, sanding/rubbing with an abrasive, unpatterned reactive ion etching, and unpatterned plasma deposition of materials (see top of Table VI). These non-lithographic techniques are not as commonly used as the Hthographic ones since they lack the abiUty to controllably define the geometries of the surface features being patterned. Because they allow for precision in the patterning process, photoUthography, electron beam lithography, laser holography, and laser machining (see bottom of Table VI) are the most popular methods used. What sets electron beam Uthography, X-ray Uthography, and laser holography apart from the rest of these techniques is the abiHty to controllably obtain feature sizes down to the hundreds to tens of nanometers. While these methods allow researchers to fabricate systems in which it is possible to probe sub-cellular size scale effects, they also come with

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TABLE V MATERIALS USED IN STUDIES OF CELLS AND TOPOGRAPHICALLY PATTERNED SURFACES

Materials used Carbon Epoxy resins Fused silica Polycarbonate Polyester Poly(methyl methacrylate) (PMMA) Polystyrene Polyurethane Quartz

Silicon

Silicone elastomer (PDMS)

Titanium

Contributors Lin and Wu (1999), Mattson et al. (2000) Chehroudi et al. (1997), Verran and Maryan (1997) Wojciak-Stothard et al. (1995, 1996), Britland et al. (1996) Terhune et al. (1993), Evans et al. (1999), Dalton et al. (2001a) Simon et al. (2000) Clark et al. (1987, 1990), Alaerts et al. (2001) Hoch et al. (1987), Terhune et al. (1993), Casey et al. (1999), Walboomers et al. (1999, 2000), Dalton et al. (2001b) Goodman et al. (1996) Hirono et al. (1988), Clark et al. (1991), Nagata et al. (1993), Gomez and Letourneau (1994), Webb et al. (1995), Rajnicek and McCaig (1997) Brunette et al. (1983), Rovensky et al. (1991), Chou et al. (1995), Oakley and Brunette (1995a,b), Damji et al. (1996), den Braber et al. (1996b), Oakley et al. (1997), Turner et al. (1997), Chou et al. (1998), Craighead et al. (1998, 2001), Goto and Brunette (1998), Scheuerman et al. (1998), Bayliss et al. (1999a,b), Perizzolo et al. (2001) Schmidt and von Recum (1991), Green et al. (1994), den Braber et al. (1996a, 1997, 1998a), Verran and Maryan (1997), van Kooten et al. (1998), van Kooten and von Recum (1999), Deutsch et al. (2000), Pins et al. (2000), Brunette et al. (1983), Chou et al. (1995), Oakley and Brunette (1995a,b), Damji et al. (1996), Eisenbarth et al. (1996), Chehroudi et al. (1997), Oakley et al. (1997), den Braber et al. (1998b), Chou et al. (1998), Golo and Brunette (1998), Lincks et al. (1998), Craig and LeGeros (1999), Webster et al. (1999a), Anselme et al. (2000), Polonchuk et al. (2000), Eriksson et al. (2001), Perizzolo et al. (2001)

their own drawbacks. Electron beam lithography is a very costly method of patterning and very time consuming if the patterns are very dense, complex, or cover a large area. Most X-ray Hthography faciUties are not open to outside users and, thus, less available to biologists. Laser holography is a patterning method that has been used to make very fine gratings (Clark et al. 1991; Webb et al. 1995) but requires more sophisticated techniques to create other complex patterns. It currently stands that photolithography is the most commonly used technique for patterning substrates to study cell-substrate interactions. As technology improves and chip manufacturing companies upgrade their equipment, many older machines, such as photoHthographic reduction steppers, are either donated, sold to other institutions or put up for auction.

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P.

TURNER

TABLE VI TECHNIQUES USED TO FABRICATE TOPOGRAPHICALLY PATTERNED SURFACES FOR CELL-SUBSTRATUM STUDIES

Patterning techniques used Non-lithographic methods Acid washing/etching Cutting with diamond or other materials Particle settling Rubbing/sanding with an abrasive Sandblasting Sintering of powders Track etching Unpatterned plasma deposition Unpatterned reactive-ion etching Lithographic methods Electron beam lithography

Laser holography Laser machining Photolithography

Contributors

Turner et al. (1997), Craighead (1998), BayHss et al. (1999a,b) Stepien et al. (1999), Simon et al. (2000) Craig and LeGeros (1999), Lin and Wu (1999), Mattson et al. (2000) Eisenbarth et al. (1996), Lincks et al. (1998), Anselme et al. (2000) Anselme et al. (2000) Webster et al. (1999a,b, 2000), Polonchuk et al. (2000) Evans et al. (1999), Dalton et al. (2001a) Chou et al. (1999), BayHss et al. (1999a,b) Turner et al. (1997), Casey et al. (1999) Hoch et al. (1987), Terhune et al. (1993), Gomez and Letourneau (1994), Rajnicek and McCaig (1997), Wilkinson et al. (1998), Alaerts et al. (2001) Clark et al. (1991), Webb et al. (1995) Pins et al. (2000) Brunette et al. (1983), Clark et al. (1987), Hirano et al. (1988), Clark et al. (1990), Schmidt and von Recum (1991), Nagata et al. (1993), Green et al. (1994), Chou et al. (1995, 1998), Oakley and Brunette (1995a,b), Wojciak-Stothard et al. (1995), Britland et al. (1996), Damji et al. (1996), den Braber et al. (1996a,b, 1997, 1998a,b), Wojciak-Stothard et al. (1996), Chehroudi et al. (1997), Oakley et al. (1997), Chen et al. (1998), Craighead et al. (1998), Goto and Brunette (1998), van Kooten et al. (1998), Wilkinson et al. (1998), Casey et al. (1999), van Kooten and von Recum (1999), Turner et al. (2000), Deutsch et al. (2000), Walboomers (2000), Dalton et al. (2001b), Perizzolo et al. (2001)

Thus, researchers can acquire these tools at a substantially reduced cost. Using photolithography, the smallest feature sizes one can obtain when working in the visible UV (365^36 nm) are on the order of 0.5 jim. If one moves to deep UV (248 nm), then it is possible to produce features down in the neighborhood of 0.1 jim. If it is important for a particular study to get down to feature sizes on the order of tens of nanometers, then one must make use of the higher-resolution lithography techniques mentioned earlier.

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It is assumed that the reader is knowledgeable about conventional photolithography SO the details of how patterns are transferred from a mask to a substrate will not be discussed here. A good source to reference for a review of such techniques is The Science and Engineering of Microelectronic Fabrication by Campbell (1996). Once a pattern has been transferred to a substrate, de-protected regions on the substrate are then subjected to a series of reactive or non-reactive ion and wet chemical etches that transfer the pattern into the material. If these patterned substrates are then used directly in cell surface studies, we shall refer to these substrates as having been directly patterned. If the substrate to be used in cell surface studies is fabricated by molding or imprinting with a directly patterned master, then we shall refer to these substrates as having been indirectly patterned. Methods of pattern transfer technology that include embossing and nanoimprinting, both indirect patterning methods, are very quickly growing fields of interest. These methods have been used by the data storage industry for years in the production of compact disks and are now widely used in various fields of research (Casey et al. 1997, 1999; Wilkinson et al 1998; Becker and Gartner 2000; Becker and Heim 2000; Petronis et al. 2001). Embossing involves the imprinting of a pattern from a master or die, typically one that has been patterned using some form of lithography, into a thermoplastic material which is heated to its glass transition temperature so that it flows into the die (Fig. 21) (Chou et al. 1995, 1996). The temperature of the system is then lowered below the glass transition temperature of the polymer replica such that the polymer hardens once more and can be separated from the die. Structures with small dimensions can be replicated in this manner reliably (as seen in Fig. 22). Due

t

t

FIG. 21. A cartoon of a simple embossing press. The bottom plate is raised to meet the top plate with a specified force and the temperatures of the top (Ti) and bottom {T^) plates are controlled separately. The patterned master is placed face up on the bottom plate and the piece of polymer to be embossed is placed between the master and another flat clean substrate.

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to lower costs and an increased speed in production, embossing is becoming the popular choice for pattern transfer and reproduction of expensive masters. Based on the widespread use of polymers in biotechnology, such methods of patterning plastics may open up many new areas of exploration. (a)

(b)

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(d)

jii^ii^jiillji^

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iiiiiiiiiiiiiiiiiB FIG. 22. (a) A cartoon showing the use of a master polymer chip, (b) Holes embossed in polystyrene using 10|am). (c) A cartoon showing the use of a master with chip, (d) Pillars embossed in a cyclo-olefin polymer b a r = 10)im).

with pillar structures to emboss a a master with pillars (scale bar = hole structures to emboss a polymer using a master with holes (scale

Polymers such as polystyrene and polycarbonate have been used for years in conventional cell culture; such materials are well characterized, have been made biocompatible, and are optically transparent. The issue of substrate transparency is a critical one that may determine the method of observation to be used in cell culture studies, for example, fluorescence microscopy vs phase contrast microscopy. The direct observation of living cells cultured on opaque substrates, such as silicon, is not a trivial one. Thus, the ability to fabricate patterned transparent substrates allows for various observation methods to be used. Materials such as fused siUca and quartz have been fabricated into wafers that can be used with conventional Uthography tools, but the costs associated with such materials are often non-trivial; 3-in. fused silica wafers can cost more than $100 a piece. Despite the added flexibihty in cell observation gained with the use of such materials, the transparency of the substrate makes it more difficult to correctly adjust the focus with certain hthography tools. Embossing plastics gets around this focusing problem while still maintaining the benefits of transparency. As such, the methods of embossing, injection molding and solvent casting of polymer replicas for use in cell-substratum studies are viable alternatives to silicon processing.

286 3.

H. G. CRAIGHEAD, C. D . JAMES, AND A. M. P. TURNER DEALING WITH SURFACE CHEMISTRY

When a material has been subjected to a series of process steps including reactive ion etching, wet chemical etching, and electron beam bombardment, it is possible that the surface chemistry of the substrate has been altered or affected in some way that will impact the adhesion and growth of cells on the surface. It is important that in the final steps of processing, before the cells are plated on the patterned substrate, all attempts are made to eliminate or passivate any unwanted surface charges, radicals, or chemical particulates that did not get cleared during the processing. Since we are interested, here, in the influence of topographical cues on cell adhesion and growth, we seek to eliminate any chemical cues so as to ensure that we are studying the impact of the topography alone. There are several processing steps during fabrication that might create unwanted chemical or topographical cues. Thus, it is important to use reliable cleaning methods to prevent or remove such cues. For instance, after completing a patterning step that involves resist, it is important to use a very strong chemical stripper such as Nano-Strip, a stabilized formulation of sulfuric acid and hydrogen peroxide compounds produced by Cyantek Corporation, to remove any residual resist. A weak resist stripper such as Shipley 1165 will not always remove photoresist that has been baked on as a result of an etching process. A long soak in Nano-Strip, sometimes longer than 24 h depending on the extent of baking that occurred during the etching steps, followed by an oxygen plasma will be sure to remove any scum left behind by the patterning process. Often a byproduct of reactive-ion etching with CHF3 is the formation of a Teflon-like substance over the surface of the substrate. If this Teflon-like substance is allowed to remain on the substrate, it will act as a micromask and result in the formation of grass-like structures in the next etch process. A good way to avoid this situation is to use CF4 instead of CHF3 as the etching gas for materials such as silicon dioxide. In the most severe situations involving scum or Teflon removal, it may be necessary to soak the substrate in hot Nano-Strip and employ a technique of gentle aspiration of the Nano-Strip at problem areas. After all lithography and etching steps have been completed, we typically soak all substrates in Nano-Strip, followed by an oxygen plasma to ensure that we passivate any radicals that may be residing on the surface. In the case of materials such as silicon and titanium, it is important to take into consideration that once the surface has been exposed to air a native oxide will form. Thus, when cells are cultured on these substrates, the surface they will see is titanium oxide or silicon oxide, not bare titanium or silicon. Before cells can be cultured on these substrates, the samples must be sterilized. When preparing and sterilizing samples for cell culture, patterned dice cleaved from the original substrate are either immersed in nitric acid followed by several deionized water rinses, immersed in ethanol (70% by

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volume or greater) followed by several deionized water rinses, autoclaved, or exposed to UV light. The decision to use any one method to sterilize the substrate depends on what material makes up the substrate under study and, hence, what effects each method might have on the surface chemistry. Glasslike materials such as quartz, silicon, or silicon nitride, are safe to clean with nitric acid without altering the surface chemistry or etching the material. For substrates that will be destroyed by immersion in nitric acid such as silicone elastomer, polystyrene, and polycarbonate, ethanol rinses are the method of choice. Depending on whether or not the substrate can handle steam at temperatures of 120-130°C without melting, which certain forms of polypropylene can, then autoclaving is another possibility for sterilization. Autoclaving is a particularly popular option for sterilizing glass coverslips, so it is assumed to be sufficient for patterned glass-based substrates as well. Last but not least, there is the method of sterihzation by irradiation with ultraviolet light. This is a method that is useful if the material you are using can not be subjected to acid, ethanol, or high temperatures. It is not useful if the material being used or any protein that might be applied to the surface is sensitive to UV light. It should be noted that each of these different sterilization techniques will have a different effect on the resulting surface chemistry of the substrate, and so it is important to be consistent with the methods used to achieve sterilization. Characterization of the surface can be critical in evaluating the cellular response; Auger electron spectroscopy and other spectroscopic techniques are capable of determining the atomic and chemical characteristics of the final surface. Details of such spectroscopic techniques will not be discussed here.

4.

CHARACTERIZING SURFACE STRUCTURE

For cell-substratum studies, it is important to determine not only the chemical properties of the substrate but also the topographical structures as well. Several techniques can be used to evaluate the morphology of the surface. It is most typical to use a profilometer between and after processing steps to determine the profile of the reUef structures being patterned. The probe of the profilometer is in contact with the surface as it scans and can resolve heights in the hundreds of micrometers with tens of Angstroms resolution. Scanning electron microscopy (SEM) is a "non-contact", though not always non-destructive (e.g. cleaving a wafer to image end-on), method of studying the structural characteristics of the surface and can be used to observe any unexpected sub-structures, like the ones seen in Fig. 23, that may have been produced during the processing steps. Depending on the operating voltage, the most modern scanning electron microscopes can resolve features in the tens of nanometers. In order to get even greater vertical resolution, atomic force microscopy (AFM) may be the desirable probe technique. SEM and

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H. G. CRAIGHEAD, C. D . JAMES, AND A. M. P. TURNER

FIG. 23. A SEM micrograph illustrating unwanted secondary surface structures (nanometerscale silicon grass surrounding larger micrometer-scale pillars) created during etching steps in the fabrication process (scale bar = 3|im).

AFM have very similar lateral resolutions but the differences in processing vertical changes in topography lead to one method being superior to the other depending on the topography to be imaged. An AFM is capable of resolving less than 0.5 A in the vertical direction and several nanometers in the x and y directions. While AFM is capable of resolving atomic steps on epitaxially grown thin films, it is at a disadvantage with very high aspect ratio features, structures with undercut profiles, and very rough surfaces with millimeters of vertical information (Russell et al. 2001). Thus, when depth of field is a critical issue, SEM will prove more useful in imaging over a large vertical range.

VIII.

Studies of Cells on Topographically Modified Surfaces

R. G. Harrison at Yale University was the originator of modern tissue culture (Harrison 1911, 1912). He performed experiments with tissue taken from the mesoderm and medullary tube of frog embryos in an attempt to prove that the "nerve fibers" required some form of solid support in order to carry out the growth process. The tissue samples were either suspended in frog serum without any support or suspended in frog serum and held between two layers of spider web. He observed that those pieces of tissue supported by the spider web demonstrated very active cells that extended from the main mass of tissue in long spindle conformations with many of them following along the fibers of the spider web, whereas, cells in those pieces of tissue left suspended without support showed httle to no activity and remained round in shape. It was deemed that these cells were undergoing a form of stereotropism or thigmotaxis, a movement of an organism for which the stimulus

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is contact with a solid support. Harrison went on to prove this effect in a paper he pubhshed in 1914 where he used clotted plasma, spider web, and cover glass as three types of solid supports (Harrison 1914). It is interesting to note that at the time of the first experiments in 1911, Harrison was not aware of the fact that the glass coverslips he was using to seal his test chambers might serve to support moving cells. This is something we take for granted these days, when culturing cells on glass coverslips is standard practice for the observation of cell movement. Furthermore, he went on to demonstrate that each of the three solid supports he was using produced different growth responses amongst the cells. Since that time, there have been scores of other researchers who have looked into the effects of surface structure on cell growth. Several excellent review articles have been written in the past few years covering the topic of cell response to surface topography and the impact of these studies on tissue engineering and implant design (Singhvi et al. 1994; von Recum and van Kooten 1995; Curtis and Wilkinson 1997; Flemming et al. 1999; Desai 2000; Craighead et al. 2001; Curtis and Riehle 2001; Jung et al. 2001). We will focus on those researchers who have made use of the methods of micro- and nanofabrication to create patterned surfaces. Brunette and colleagues, at the University of British Colombia, are credited as being the leaders in the application of semiconductor fabrication techniques to biological studies (Jung et al. 2001). In 1983, Brunette began to make use of methods from the microelectronics industry to fabricate micrometer scale, titanium-coated grooves in silicon for use in human gingival implants (Brunette et al. 1983). In this study and later ones, they used standard photolithographic methods to pattern silicon wafers with truncated v-shaped and flat bottom grooves and evaporatively coated these surfaces with 50 nm of titanium, based on the successes of titanium dental implants (Brunette 1986). In more recent years. Brunette and colleagues have continued to look into the alignment and migration of fibroblasts, epithelial cells, and osteoblasts cultured on micromachined surfaces and the phenomenon of contact guidance (Chou et al. 1995, 1996, 1998; Oakley and Brunette 1995a,b; Damji et al. 1996; Chehroudi et al. 1997; Oakley et al. 1997; Goto and Brunette 1998; Brunette and Chehroudi 1999; Perizzolo et al. 2001). They performed a series of particularly interesting experiments aimed at elucidating the roles of microtubules and actin microfilament bundles in cell polarization and locomotion. They cultured (on smooth and grooved titanium substrata) trypsinized fibroblasts that were resuspended in a colcemid containing solution (Oakley and Brunette 1995a). Colcemid, often used in chromosome analysis, is a mutagenic, tumorigenic, and teratogenic material that arrests mitotic cultured cells (Sigma-Aldrich). They used colcemid (A^-deacetyl-A/^-methylcolchicine) to prevent the polymerization of microtubules and, hence, determine the impact of their elimination on cell polarization, shape, and directed locomotion on grooved substrates. It was previously believed that microtubules were necessary for effecting topographic

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H. G. CRAIGHEAD, C. D . JAMES, AND A. M. P. TURNER

guidance (Vasiliev et al. 1970). This study, however, demonstrated that the colcemid-treated cells cultured on grooves were delayed but eventually did aHgn with and migrate along the grooves in a polar fashion, whereas, those cells on smooth surfaces did not polarize and migrate. In the absence of microtubules, it appears that the grooved substrate helped to overcome this deficiency by organizing and maintaining an aligned actin filament framework. They termed this effect "topographic compensation" (Oakley et al. 1995a). They later went on to study the impact of actin microfilament bundle inhibition (using cytochalasin B) on fibroblast polarization, shape and locomotion on grooved substrates (Oakley et al. 1997). Groups of cells were also treated with colcemid to inhibit microtubule formation in an attempt to determine whether actin microfilament bundles or microtubules play a more critical role in topographic guidance. All cells aligned to grooves 6-30 jim wide. The narrowest features, 0.5-|im wide grooves, caused the greatest degree of alignment in all cells containing microfilament bundles except those treated with colcemid, and thus microtubule deficient. Brunette and colleagues suggested this result implicates microtubules as pre-eminent in but not the sole contributors to the topographic guidance of fibroblasts. Besides the work discussed here, Brunette and colleagues have also studied the effects of surface topography on fibronectin mRNA (Chou et al. 1995), tenascin (Goto and Brunette 1998), and metalloproteinase-2 expression (Chou et al. 1998) in fibroblasts, as well as contact inhibition events between fibroblasts and epitheUal cells (Damji et al. 1996). Researchers at the University of Glasgow, Adam Curtis, Peter Clark (later at Imperial College), Chris Wilkinson and colleagues, were also a part of the groundbreaking movement to apply microfabrication techniques to biological studies. As early as the 1960s, Curtis was studying cell contacts and the mechanisms behind the adhesion of cells to glass (Curtis 1960, 1962, 1964; Curtis and Varde 1964). In the late 1980s, Curtis and colleagues began using photolithography to topographically patterned surfaces (Clark et al. 1987). Other research groups at the time, including Brunette and colleagues, were studying the response of cells to complex parallel groove systems. Believing such systems to be too complex, Curtis and colleagues decided to first study the response of cells to the more basic single step edge (Clark et al. 1987). They began with making simple step edges, 1-10 jam high, on pieces of plastic sheet called Perspex, a brand name for poly(methyl methacrylate) (PMMA). Four different cell types were studied: baby hamster kidney cells (BHK), chick heart fibroblasts (CHF), rabbit peritoneal neutrophils, and chick cortical nerve cells. To fabricate step edges, PMMA was etched with an O2 plasma through an aluminum hard mask that had been evaporated on and patterned using photoHthography. The oxygen plasma used to etch the PMMA created unintentional surface structures that were more noticeable on those surfaces etched for longer. In an attempt to make all surfaces equally rough, all were subjected to an O2 plasma. After culturing cells on these

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surfaces, they observed for all cells, except neutrophils, a general increase in cell alignment with an increase in step height as well as an increase in inhibition in crossing the step edges as the step height increased. From single step edges, Curtis and colleagues indeed moved on to multiple parallel groove systems, first in PMMA then in fused quartz (Clark et al. 1990,1991). In these studies they used BHK cells, Madin Darby canine kidney (MDCK) cells, and chick embryo cerebral neurons. PMMA substrates were patterned with grooves having 4-24 jim periods and depths of 0.2-1.9 jim. All cells showed an increase in alignment with an increase in groove depth and a decrease in alignment with an increase in groove spacing. Using laser holography and a fluorine-based etch (CHF3), they were able to pattern fused quartz with ultrafine grooves: 130nm wide, separated by 130nm, and 100, 210, or 400 nm deep. These ultrafine grooves were made in an attempt to mimic the topography of aligned collagen fibrils, individually 20-100 nm in diameter, found in extracellular matrix material, a stable complex of macromolecules that surrounds cells. Each cell type responded differently to the ultrafine gratings. BHK cells aligned to the substrates and demonstrated an increase in alignment with an increase in groove depth. Single MDCK cells aligned to the patterns and became increasingly elongated with an increase in grating depth but clusters or islands of MDCK cells as well as neurite outgrowth from cerebral neurons showed no discernable differences on these surfaces as opposed to smooth quartz. Based on these early observations of cell behavior on topographically modified surfaces, they proposed the "Curtis and Clark theory" that cells react to topography primarily at lines of discontinuity in the substratum by actin nucleation (Clark et al. 1980). This theory emerged from the apparent tendency of many cell types to "ridge walk" along sharp discontinuities. They suggested these sharp discontinuities in the underlying surface promoted actin condensation and, as a result, cell orientation. Further studies with fibroblasts appeared to support this hypothesis (Wojciak-Stothard et al. 1995). In more recent years, their work has combined both topographical and chemical patterning to determine any synergistic or hierarchical influences on cell guidance (Britland et al. 1996). Using methods estabhshed by Kleinfeld et al. (1988) they patterned regions with an aminosilane to create "adhesive" regions and a chlorosilane was used to create "non-adhesive" regions. Patterns containing grooves alone, silanes alone, grooves and silanes parallel to each other, and grooves and silanes perpendicular to each other were the different systems studied. The most significant degree of ahgnment was observed on the pattern with parallel grooves and chemical cues. Most recently, this collaboration has begun working with embossed polymers (Casey et al. 1997). Going back to a material they started using 10 years before, they employed electron beam lithography to pattern PMMA sheets. These pieces of patterned PMMA then served as masters for the embossing process. Some patterned polystyrene replicas contained 60 nm pillars with a

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H. G. CRAIGHEAD, C. D . JAMES, AND A. M. P. TURNER

center-to-center spacing of 300 nm. These substrates were fabricated in an effort to create controllably rough surfaces and to test the phenomena of rugophiha and rugophobia, cell loving or hating of rough surfaces (Rich and Harris 1981). Rat epitenon cells were cultured on these nanometer-scale pillars and it was observed that significantly fewer cells attached to the pillars than to the smooth polystyrene. They suggested that such nanometer-sized pillars could be used to create permanently non-adhesive surfaces for use in biomedical applications. In the past 10 years, another collaborative effort has looked at the responses of fibroblasts to patterned polymers, in this case silicone elastomer substrates. In the early 1990s, A. F. von Recum, originally at Clemson University and now at Ohio State University, and J. A. Jansen, at the University of Nijmegen, were running pilot studies and making silcone elastomer repHcas from glass masks or silicon wafers patterned lithographically with photoresist features (Schmidt and von Recum 1991). The final patterns consisted of bumps and holes 0.38-0.57 \xm deep, 2, 5, and 8 jim in diameter, with 4, 10, and 20|im center-to-center spacings. These silicone samples were either implanted subcutaneously in rabbits or placed in culture dishes and plated with macrophages. From these studies they noted increased elongation and pseudopod extension on the 2- and 5-jam textures. In addition, silicone replicas of pillars and holes were fabricated to examine the responses of abdomen fibroblasts from a continuous cell line to these textures (Green et al. 1994). The 2 and 5|im pillars increased rates of cell proHferation and cell density as compared to the 2 and 5 |im holes, while larger 10 jim pillars and wells caused a response similar to that observed on smooth silicone. Later studies looked at the interactions of rat dermal fibroblasts with 1-10 |im wide and 0.5-1.0 |im deep grooves on silicone elastomer replicas (den Braber et al. 1996a,b). Fibroblasts showed no change in rates of proliferation due to surface structures but showed a definite increase in orientation on the narrower grooves with no differences due to groove depth. The lack of an influence of groove depth on orientation differed from Curtis' published results but it is important to note that the cell lines studied were not the same. Further work with fibroblasts and endothelial cells on silicone grooves has looked at fibronectin and vitronectin deposition, vinculin and actin expression, and cell proliferation as determined by cell cycle analysis through flow cytometry (den Braber et al. 1998a; van Kooten et al. 1998; van Kooten and von Recum 1999). Most recently, Jansen and colleagues have proceeded to look at fibroblasts cultured on bulk titanium substrates as well as solvent cast polystyrene and polylactic acid replicas patterned with similar microgrooves. A summary of this work can be found in the pubUshed thesis of Frank Walboomers (2000) at the University of Nijmegen. Most researchers, such as those mentioned previously, have fabricated two-dimensional constructs for use in cell substratum studies. Others have begun to construct three-dimensional platforms for potential biomaterial

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applications. Moghe and colleagues at Rutgers University have been working with various synthetic and natural materials to create three-dimensional scaffolds for hepatocyte cell adhesion and migration. They have been looking at the response of hepatocytes to different structures and how those might regulate multicellular organization and liver-specific functions (Ranucci and Moghe 1999, 2001; Ranucci et al. 2000; Semler et al. 2000). They have made porous foams from 50/50 poly(D,L glycolic-c6>-lactic acid), PGLA, and a gel substrate derived from basement membrane (a component of extracellular matrix) that resembles Matrigel, a commercially available product made by Becton, Dickinson and Company. They observed that the mechanical compliance of the Matrigel plays a critical role in determining the role of growth factor stimulation on hepatocyte function (Semler et al. 2000). In the PGLA foams, the pore size played a significant role in the organization of the hepatocytes. The 3-|im pores induced two-dimensional cellular reorganization, while the larger 67-|im pores restricted mobility and induced threedimensional aggregations (Ranucci and Moghe 1999). Very fundamental results such as these determined for the behavior of hepatocytes in threedimensional materials could result in design parameters for building liver scaffolds for use in organ genesis. Organ genesis and tissue regeneration are areas of research with a great deal of promise for a variety of organ systems. Of particular interest to us is the area of nerve regeneration. Several potential methods for the restoration of neural function may someday be realized. Already, the use of stem cells has been able to partially repair severed spinal cords in rats (McDonald et al. 1999). Though controversy still exists over the use of stem cells in medical research the benefits that may be reaped from their use are enormous (The stem cell debate; http://www. time.com/time/2001/stemcells). Neural stents have been tested for direct placement around spinal injuries to aid in the reintegration of neurons and other CNS cells (Aebischer et al. 1990; Doolabh et al. 1996). In addition, neural probes that have the potential to circumvent the immediate wound site by making a direct connection between the command center and the unresponsive tissue are being tested in animal (including human) models (Edell et al. 1992; Rousche and Normann 1992; Schmidt et al. 1993; Hoogerwerf and Wise 1994; Anderson et al. 1997; Turner et al. 1999; Hockenberry 2001). In order for potential neural implants to function as designed, it is necessary to control the interaction of the relevant cells with the implant surface. Thus, in the design process it will be necessary to consider the topographic and chemical cues the tissue will see. The search for the response of CNS cells to topographic cues is one that started long ago and continues on today. It is interesting to note that in the early 1940s, Paul Weiss, then at the University of Chicago, was culturing CNS cells on mica surfaces patterned with scratches that look very similar in design (though not in size) to the trenches fabricated today using more sophisticated techniques (Weiss 1945). On these mica surfaces, he cultured nerve fibers obtained from spinal ganglia of chick

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embryos and Schwann cells obtained from predegenerated rat nerves. Weiss observed axons extending along the scratches and as the scratches became deeper so, too, did the degree of orientation of the nerve fibers. Work very similar to these initial studies goes on today only on a slightly smaller size scale. Clark and colleagues in the late 1980s observed the alignment of neuronal cell processes along and within single step grooves only 2|Lim deep (Clark et al. 1987). Also since the late 1980s, Kawana and colleagues in Japan have been patterning quartz gratings as simple physical models of neurite bundles to direct the outgrowth of neuronal processes (Hirono et al. 1988; Nagata et al. 1993). Not only have they looked at the guidance effects of simple gratings but also more complex hexagonal (honeycomb) patterns of oxidized metal, square wells connected by conduits, and tapered guiding grooves (Torimitsu and Kawana 1990; Jimbo and Kawana 1992; Jimbo et al. 1993). These surface structures were fabricated on planar electrode arrays to guide neuron process outgrowth specifically over embedded electrode recording sites (Hoch et al. 1996). In more recent times, McCaig and colleagues have studied the influence of surface structure on the guidance of CNS neurites (Gomez and Letourneau 1994; Rajnicek and McCaig 1997). They found that the age of the embryos from which neurons were isolated significantly influenced the frequency with which hippocampal neurites would perpendicularly align to the grooved substrate, the younger the embryo the more likely to align perpendicularly. Their results suggest that contact guidance is regulated in development (Gomez and Letourneau 1994). While several groups have looked at the influence of grooved substrates on the development of CNS cells, not many have looked at the influence of other forms of surface structure on such cells. Our research group at Cornell University in conjunction with collaborators from the New York State Department of Health, Wadsworth Center, Albany, NY and Oregon Health Sciences Univeristy, CROET, Portland, OR has looked into the influence of pillar structures on the attachment, spreading, and growth of cortical astrocytes and hippocampal neurons. This work started with the fabrication of rough nanometer-sized reactive-ion etched structures better known as silicon grass or black silicon, materials originally constructed for use in solar cells (Turner et al. 1997). The fabrication process is shown in Fig. 24. Under certain conditions, reactive-ion etching can deposit solids, either sputtered from the chamber walls or precipitated from the plasma, onto the surface of a substrate. The intentional use as a micromask of re-deposited particulates is an unconventional yet highly effective method by which to randomly pattern the surface with nanometersized structures. These particles adhere to the surface and thus form a hard micromask that can be used to transfer the random pattern into the underlying substrate using an anisotropic etch. The resulting structures, as shown in Fig. 25, averaged about 60 nm in diameter and were on the order of 230 nm in height. The substrates were then patterned on a much larger scale and

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FIG. 24. Process steps outlining the fabrication of random nanometer-scale surface structures in silicon, (a) Reactive-ion etch to deposit solids on the wafer surface forming a micromask. (b) Reactive-ion etch with plasmas of CI2, CF4, and O2 gases to create grassy surfaces, (c) Spin-coat with photoresist, (d) Expose and develop, (e) HF-nitric acid-water etch unprotected regions, (f) Strip photoresist, (g) Clean dip in weak HF-nitric acid-water.

subjected to a wet chemical etch specific for sihcon. The unprotected regions of the surface were made less rough by the wet chemical etch, thus creating substrates with two different surface textures: silicon grass and wet-etch modified grass. Astroglial cells were then cultured on these surfaces to determine the cellular response to different degrees of roughness. Primary cortical astrocytes and a continuous cell line of LRM55 cells displayed opposite preferences for the patterned surfaces (see Fig. 26). Primaries preferred the very rough surfaces whereas the continuous cell fine preferred the wet-chemical etched structures. The LRM55 cells, a transformed cell line derived from a rat CNS tumor (Martin and Shain 1979), were used to model the response of the primary cortical astrocytes but instead demonstrated

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FIG. 25. SEM micrographs of: (a) reactive-ion etching textured "silicon grass" surfaces (scale bar = 200nm) and (b) wet chemical etched reactive-ion etching textured surfaces (scale bar = 500nm).

contrasting behavior. It is not apparent why this was the case, but one theory may be a possible change in the cell Hne after many generations of passaging. As mentioned previously, Wilkinson and colleagues have also looked at the effect of nanometer-sized pillars on the attachment of cells. They observed the attachment of rat epitenon cells to nanometer-sized electron beam lithography generated polystyrene pillars and found that there was a significant decrease in the number of cells that attached as compared to the smooth control surfaces (Wilkinson et al. 1998; Casey et al. 1999). They proposed that such nano-patterned surfaces might be used as non-adhesive layers for implant devices. Based on results from the silicon grass studies and Wilkinson's observations, it is apparent that different cell lines respond

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FIG. 26. LRM55 cells growing on rough silicon grass (left side of image) and wel chemical etched surfaces (right side of image) (scale bar = 5}.im).

differently to nanometer-sized surface features. Thus, specific cell-substratum interactions should be given careful consideration when designing topographical modifications for implant devices. Given the results from the attachment studies of astroglial cells to silicon grass, it was determined that control over the dimensions of structures is critical in the attempt to understand cellular responses. Hence, we moved to using conventional photolithography to pattern pillar surfaces (Craighead et al. 1998). The first generation of structures consisted of pillars 0.5 |im in width, separated by 1.0 }im and 1.0 or 2.7 |im in height (see Fig. 27). The process steps are outlined as shown (Fig. 28). In order to gain some latitude in selectivity and sidewall profile, a thermally grown silicon oxide layer is patterned, subjected to a fluorine-based etch, and used as a hard mask for the etching of the silicon. LRM55 cells and primary cortical astrocytes were cultured on these patterned surfaces and it was observed that the cells attached to the pillar structures and spread along the tops, as shown in Fig. 29. The next generation of pillar structures were patterned in arrays with varying pillar widths (0.5, 0.75, 1.25, and 2.0 |im) and interpillar spacings (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 |im) (Fig. 30). LRM55 cells were cultured on these structures and it was determined that regardless of pillar width and interpillar spacing, the cells preferred to attach to and spread on the

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FIG. 27. SEM micrograph of the first generation of pillars averaging around 2.7 |j.m in height and 0.5 |im in width with a 1.0 |xm separation (scale bar = 2|im).

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FIG. 28. Process steps outlining the fabrication of regular arrays of pillars, (a) Silicon dioxide is thermally grown on a single crystal silicon wafer to serve as a hardmask (thickness varies depending on desired silicon etch depth), (b) Photoresist is spun on. (c) The wafer is exposed to the proper wavelength light and developed, (d) A fluorine-based plasma is used to etch through the oxide, (e) The photoresist is stripped, (f) A chlorine-based plasma is used to etch into the silicon, (g) The oxide hardmask is removed with a buffered HF etch.

FIG. 29. SEM micrograph showing LRM55 cells confined to a region of first generation pillars (scale bar = 20|im).

surfaces with pillars vs smooth silicon (Turner et al. 2000). Immunochemical methods were used to fluorescently label actin filaments and focal contacts as seen in the stained primary cortical astrocyte in Fig. 31. (Exactly the same studies were done using primary cortical astrocytes and the same results were obtained for cell preferences.) Actin filament rearrangement on the pillar structures in conjunction with focal contact placement confirmed that the cells were well attached to the pillars and making intimate contact with the pillar tops. Scanning electron microscopy confirmed these observations and the cells were seen "riding" along the tops of the pillars making Httle contact

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FIG. 31. A confocal fluorescence micrograph of a primary cortical astrocyte stained for vinculin, an attachment protein involved in the indirect binding of intracellular actin filaments to extracellular fibronectin. The underlying pillars seen in the image have an interpillar gap of 2.5 i^m and a width of 1.25 |im (scale bar = 50|im).

with the sidewalls of the structures (Fig. 32). The results from these studies may prove useful in the design of neural prostheses. Long-term biocompatibility studies have been performed on implanted neural probes and such studies have shown that performance is limited by probe rejection (Edell et al 1992; Rousche and Normann 1992; Schmidt et al 1993; Hoogerwerf and Wise 1994; Anderson et aL 1997; Turner et al. 1999). Astrocytes in the brain tissue become reactive and encapsulate the probe in a sheath of electrically insulating cells, hence rendering the probe ineffective (Fig. 33) (Anderson et al. 1997; Turner et al. 1999). By controlling surface chemistry and morphology it may be possible to mediate this effect. Thus, topographical modifications such as those presented here may prove useful in controlling the adhesion of particular CNS cells to implanted neural devices.

IX.

Future of Topographic Modifications of Surfaces

There are several future directions we can discuss for the study of the influence of surface structure on cell morphology, function, gene expression, and differentiation. One future direction will be in the area of biomimetic

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FIG. 32. A SEM micrograph showing the processes of an astrocyte cell encompassing the tops of the pillars. Very fine processes can be seen reaching down to and around the side walls of the structures (scale bar = 2 |xm).

FIG. 33. A confocal fluorescence micrograph of glial fibrillary acidic protein (GFAP) positive cells surrounding the insertion site created by a microfabricated silicon probe implanted for several weeks in the cerebral cortex of a rat (scale bar = 100 ]xm). GFAP is a specific marker for astrocytes. Image courtesy of William Shain and James N. Turner, New York State Department of Health, Wadsworth Center.

surfaces. Those researchers working in the area of biomimetics aim to recreate the natural environment a biological molecule, single cell, or region of tissue might see in vivo. This research makes use of the "lock and key" principle inherent to systems Hke proteins and enzymes. Biomimetic casting

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is similar to the jeweler's art of lost-wax casting. Materials such as proteins or tissues can be used sacrificially to mold surfaces containing structures that enzymes or cells might recognize as suitable substrates (Boeckl et al. 1998; Ratner and Shi 1999; Shi et al. 1999; Shi and Ratner 2000). Researchers at the University of Wisconsin have used polyurethane to make such replicas of sub-endothelial extracellular matrices (Goodman et al. 1996). They observed that endothelial cells cultured on these polymer castings behaved more akin to endothelial cells found in native arteries than those cells cultured on smooth polyurethane. These studies illustrate the abihty of biomimetic casting to recreate functionally intact, physical impressions of an in vivo environment. Such a task is difficult if not impossible to do using fabrication techniques that create geometrically regular features. An area of research that others have begun to look into is how microorganisms such as bacteria and fungi respond to surface structure. For example, biofilms in municipal water lines are a health concern for the general public. As such, ongoing work is looking into how chemical coatings or structural modifications on the inner walls of water lines might impact the formation and growth of these biofilms. Camper and colleagues at the Center for Biofilm Engineering have fabricated grooved silicon surfaces to determine the effect of surface topography on bacterial surface colonization under perpendicular flow conditions (Scheuerman et al. 1998). Three strains of bacteria were studied: Pseudomonas aeruginosa and motile and non-motile Pseudomonas fluorescens. P. aeruginosa is a ubiquitous bacterium that has the ability to adapt to and thrive in many ecological niches, from water and soil to plant and animal tissues, and has a remarkable abihty to cause disease in susceptible hosts (Pseudomonas genome project: Pseudomonas aeruginosa; http://www.pseudomonas.com/p_aerug.html). Motile and non-motile P. fluorescens are a group of common nonpathogenic saprophytes that colonize plant, soil, and water surface environments (Microbial genomics: Pseudomonas fluorescens genome project; http://www.jgi.doe.gov/ JGI_microbial/html/pseudomonas/pseudo_homepage.html). Researchers observed that the cells were attached greatest to the downstream edges of the grooves and only motile organisms were found in the bottoms of the grooves. In general, the rate of initial attachment of the cells to the structures was dependent on motility but independent of groove size. Perhaps, the responses of these and other bacteria to various forms of surface structure could impact the design and engineering of public water systems. A serious issue for farmers and agriculturists around the world is plant invasion by fungi. Hoch and colleagues at Cornell University started in the late 1980s fabricating artificial stomatal guard cell topographies, as found on the surface of leaves, out of solvent-cast polystyrene and polycarbonate replicas (Hoch et al. 1987, 1996; Terhune et al. 1993). They modeled the lips of the stomata using micrometer-sized ridges and observed the alignment of the bean rust fungus, Uromyces appendiculatus, to these structures. The

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Urediospore germlings were found to undergo differentiation into appressoria, the first of various infection structures necessary for stomate invasion and leaf colonization, upon contact with the ridges (Fig. 34). These experiments were interesting for reasons associated not only with the fundamental study of thigmotropic signaling in fungi, but in the potential applications of these results for use in fabricating materials or genetically engineering plants that can promote or deter fungal invasion and proliferation. Fungal invasion is not only a serious issue for the agriculturist but also for the average individual. A particularly opportunistic pathogen, yeast will invade the tissue in the mouth of denture wearers and cause a great deal of pain and discomfort. Verran and colleagues at Manchester Metropolitan University studied the adhesion of Candida albicans to both smooth and roughened substrates (Verran and Maryan 1997). Denture acrylic resin, PMMA, substrates were manually abraded with medium grade emery paper and roughened silicone elastomer substrates were formed by curing on a rough vacuum stone mold. Few yeast cells were observed on the smooth surfaces but those present were found clustered around surface defects. On the stone-molded silicone substrates, cells were found entrapped in surface depressions and, on the roughened acrylic resin, cells were found in the grooves formed by the emery paper. Thus, the researchers determined that surfaces to be used as molds for dental implants should be as smooth as possible so as not to create microstructure in the surface of the implant and hence aid in the retention of microorganisms. These studies and others like them could have a significant impact on the dental industry and human health in general. The last area of future research that we will discuss is the study of stem cell differentiation and development on topographically modified surfaces. Stem cell research is a very hot yet politically controversial area of research these days (The stem cell debate; http://www.time.com/time/2001/stemcells). Researchers have already demonstrated the potential for stem cell therapy for use in neural regeneration (McDonald et al. 1999). Neural differentiated mouse embryonic stem cells were transplanted into traumatically injured rat spinal cords nine days after injury. After several weeks, it was shown that these cells differentiated and migrated into the area surrounding the wound site, partially restoring lost function to the impaired rats. In the studies by Hoch and colleagues mentioned earlier, the influence of surface topography on the differentiation of fungi was apparent and indicative of how structural cues can impact cell behavior. Thus, if one could use nano- and microfabricated surface structures to control the differentiation of stem cells in much the same way, the applications would be limitless. These studies clearly indicate that the possibilities for stem cell research are great and there are many avenues of study that have yet to be realized, including the study of stem cell development in engineered environments such as the ones presented throughout this text. Here Hes the next generation of cell-substratum studies.

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(a)

FIG. 34. (a) Heat-embossed polystyrene replicas, prepared from corresponding silicon templates, bearing parallel ridges on which germlings of the plant pathogenic fungus Uromyces appendiculatus were grown. Germination and growth of the fungus from the spores (spiny structures) occur as elongated germtubes that are directed to grow perpendicular to the 0.1-)am high ridges (spaced 1.0 |im apart). On smooth surfaces, cell growth is random and without orientation, (b) When the fungal cells encounter and grow over the higher 0.5-|im ridges, growth ceases and the cytoplasmic contents migrate into an enlarged cell apex that becomes a specialized infection structure called an "appressorium". It is from this structure that the fungus, when grown on the surface of a host plant, gains entry into the plant tissue. Images courtesy of Harvey C. Hoch, Barton Laboratory, NYSAES, Cornell University.

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For nearly 100 years now, we have been looking into the ways we can control or influence cell growth on substrates that have been modified with structural cues. From the days of Harrison, to Abercrombie and Weiss, to Curtis, Clark and Brunette, we have been employing progressively more sophisticated methods for patterning substrates that will reveal to us new science and a greater understanding of the interplay between cells and the structural world around them. Nanobiotechnology has brought together the semicondcutor techniques of nanofabrication and the ever-changing world of biology. Armed with these fabrication techniques, researchers have a tremendous degree of control over device design and are able to probe dimensions far below the cellular size-scale. Remarkably, this field is truly still young and we have only just begun to understand the mechanisms behind cellular responses to external stimuli.

X.

Conclusions

The future for microstructuring of surfaces for cell culture is expanding, and generating considerable cross-disciplinary collaborations between molecular biology labs, microfabrication centers, and materials science groups. Investigators have found that such technology has the potential to address many physiologically relevant questions. A major area of research in the coming years will be the controlled design of three-dimensional scaffolds for tissue engineering. Understanding the effects of cell shape and orientation on cell function would aid in the advancement of tissue engineering and artificial organ research. Collagen gel matrices are being used for three-dimensional neuronal cell networks (O'Connor et al. 2000), and artificially grown tissues such as skin and cartilage are currently being produced and explored (Sacks et al. 1997; Peretti et al. 2000). Conventional scaffolds are fabricated using methods such as emulsion freeze-drying processes, and although these scaffolds can be incorporated with proteins for sustained delivery during implantation, control of the porosity is rather difficult (Whang et al. 2000). Microfabrication methods such as holographic lithography allow for the controllable production of three-dimensional porous structures over relatively large fields (Campbell et al. 2000). The ability to control the microarchitecture of tissue scaffolds would be a significant advance, in as much as the porosity influences the ability of cells to migrate into the scaffold, the connectivity between cells, and the diffusion of nutrients and wastes within the structure. Such an advance would allow for control over the intricate morphology of artificial tissue, and would significantly push current research in areas such as nerve regeneration and biochemically active artificial organs into the realm of science fiction.

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ACKNOWLEDGMENTS

We would like to thank Natalie Dowell and William Shain at the Wadsworth Center of the New York State Department of Health, and Ginger Withers and Gary Banker of the Oregon Health Sciences University, and Rachel Love of Cornell University for cell culturing and immunocytochemistry experiments.

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