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. Author manuscript; available in PMC: 2014 Nov 25.
Published in final edited form as: J Biomed Mater Res A. 2012 Jul 30;101(2):349–357. doi: 10.1002/jbm.a.34329

Modulating patterned adhesion and repulsion of HEK 293 cells on microengineered parylene-C/SiO2 substrates

M A Hughes 1, A S Bunting 2, K Cameron 2, A F Murray 2, M J Shipston 1
PMCID: PMC4243025  EMSID: EMS61023  PMID: 22847960

Abstract

This article describes high resolution patterning of HEK 293 cells on a construct of micropatterned parylene-C and silicon dioxide. Photolithographic patterning of parylene-C on silicon dioxide is an established and consistent process. Activation of patterns by immersion in serum has previously enabled patterning of murine hippocampal neurons and glia, as well as the human hNT cell line. Adapting this protocol we now illustrate high resolution patterning of the HEK 293 cell line. We explore hypotheses that patterning is mediated by transmembrane integrin interactions with differentially absorbed serum proteins, and also by etching the surface substrate with piranha solution. Using rationalized protein activation solutions in place of serum, we show that cell patterning can be modulated or even inverted. These cell-patterning findings assist our wider goal of engineering and interfacing functional neuronal networks via a silicon semiconductor platform.

Keywords: cell adhesion, integrin, patterning, neuronal cell

INTRODUCTION

It is important to understand mechanisms that dictate cell adhesion and patterning on synthetic materials. Cell patterning platforms support diverse research goals including the generation of in vitro neuronal networks and investigation of fundamental aspects of cell physiology.1,2 It is also critical to applications such as tissue engineering, drug discovery, and the fabrication of biosensors.3-5 An armamentarium of techniques is evolving that takes advantage of biological, chemical, and physical factors that influence cell adhesion.

Photolithographic patterning of parylene-C on silicon dioxide, followed by activation via incubation in serum, has been used to pattern co-cultures of primary murine hippocampal neurons and glia effectively,6-9 as well as human teratocarcinoma cell lines (hNT).10,11 A core motivation for our work is, ultimately, to integrate cell-patterning with either optical or capacitative electrical coupling techniques. This will enable interaction between microelectronic circuits and patterned neuronal networks at a physiologically appropriate spatiotemporal scale. Towards this end, this paper describes high resolution patterning of the HEK 293 (Human Embryonal Kidney) cell line. Prior opinion considered HEK 293 cells a derivative of embryonic fibroblastic or endothelial renal cells.12 However, contemporary research points instead towards an early neuronal lineage, as suggested by the presence of mRNA and gene products typically found in neurons (neurofilament-M, neurofilament-L, α-internexin) and the endogenous expression of several voltage-gated ion currents.13,14 HEK 293 cells also have a well-described transmembrane cell adhesion molecule (CAM) profile. This profile represents an obedient phenotype for protein-dependent cell adhesion research.

The interface between a cell and adjacent foreign material is dynamic, complex, and bi-directional, mirroring aspects of the cell-to-extracellular matrix (ECM) interface in vivo.15 Proteins of the ECM are multifunctional; providing structure and strength to a tissue and also influencing cell behavior via interactions between these proteins and cell adhesion receptors. Cells interact with synthetic substrates via absorbed layers of proteins such as fibrinogen or fibronectin. Interaction with such proteins occurs via a family of heterodimeric transmembrane cell adhesion receptors called integrins.16 Integrins are expressed in most cell types and consist of non-covalently linked α and β subunits that bind to specific amino acid sequences. Twenty-four different αβ heterodimers are possible, each with its own ligand-ECM specificity.17 HEK 293 cells express at least five β1-integrin containing subunits (αVβ1, α2β1, α3β1, α5β1, α6β1). This diversity of integrin expression allows adherence to a wide variety of ECM proteins that include vitronectin, laminin, fibronectin, and collagen.18

Physico-chemical factors also determine and modulate adhesion.19 If a material is too hydrophobic, ECM molecules are absorbed in a denatured state and the resulting rigid geometric conformation impairs access to integrin binding sites. A polymer surface can be rendered more “wettable” (hydrophilic) by physical or chemical means (e.g. ions or ultraviolet light irradiation, or etching by treatment with acid or hydroxide).20 This process also increases the presence of oxygen-containing groups on the surface, increasing surface free energy and thereby enhancing the functional absorption of adhesion-promoting ECM proteins. In parallel, the binding of albumin (a cytorepulsive protein21,22 found at high concentrations in serum) decreases with hydrophilicity, as albumin molecules prefer less oxygenated and more hydrophobic surfaces. Consequently, increasing hydrophilicity can have the dual result of both enhancing cell adhesion and attenuating cell repulsion from a polymer. In the context of a patterned substrate, the capacity to selectively induce a change in polymer adhesion properties (whilst the background substrate retains baseline characteristics) could be crucial to strengthening the cell-adhesive/cell-repulsive contrast between two surfaces.

The parylene-C/SiO2 patterning mechanism may rely on differential protein absorption (quantity and/or conformation) during the serum incubation stage. X-ray Photoelectron Spectroscopy (XPS) has shown6 that chlorine and silicon peaks found on untreated parylene and SiO2, respectively, are attenuated after serum incubation. In parallel, a spectral peak at ~400eV (characteristic of nitrogen) emerges on both parylene and SiO2 surfaces. The carbon specific C1s spectra of serum-treated parylene and SiO2 both reveal characteristic contributions attributable to peptide bonds of adsorbed protein. Protein elution of serum-incubated substrates, followed by polyacrylamide electrophoresis, shows that albumin and vitronectin bind to both parylene-C and SiO2. Parylene-C substrates display a slightly higher intensity band for vitronectin than do SiO2 substrates and fibronectin does not bind to either substrate.6 The observed dominance of vitronectin ahead of other serum proteins is consistent with its known ability to bind competitively to polymer biomaterials according to a Vroman-effect hierarchy.23 These findings suggest an important pro-adhesive role for the ECM protein vitronectin. However, its presence on both parylene and SiO2 suggests that a functionally important conformational difference in protein structure is important, as opposed to a simple difference in quantity.

We hypothesize that selectively absorbed combinations of serum proteins are key to effective patterning on the parylene/SiO2 platform. Specifically, parylene-C manifests a cell-adhesive phenotype through functional absorption of vitronectin whilst background SiO2 enables ‘contrast’ by exerting a dominant cytophobic effect mediated by bound albumin. The silicon devices used were fabricated in the cleanrooms of the Scottish Microelectronics Centre. Our subsequent, established cell-growth protocol notably includes immersion in piranha solution for 10 minutes, ostensibly to clean the chip of any residual photoresist or organic material. Pirahna solution, a 5:3 mix of 30% hydrogen peroxide and 98% sulphuric acid, is a strong oxidizer which removes most organic matter and renders the treated surface extremely hydrophilic. Given the potential differential etching effect on the two patterned substances, and the latent changes in physico-chemical properties, we suggest that this step has a role beyond mere cleaning of the chip. Rather, it may enhance differences in protein binding and therefore cell adhesion.

Finally, if vitronectin binds to parylene-C ahead of other serum proteins due to its hierarchical binding status, a solution devoid of vitronectin but containing another pro-adhesive protein may then attain the ability to bind to parylene. This is important because it would afford an opportunity to define the patterned integrin-ligand, potentially allowing the parylene/SiO2 platform to be tailored according to target cell type.

To test these hypotheses, we first investigated the potential to pattern HEK 293 cells using prior established protocols on a chip consisting of a simple, reiterative parylene design. Temporal aspects of cell patterning were then evaluated by interval (daily) imaging, to account for the continued mitotic proliferation of cultured HEK293 cells. The specific impact of piranha treatment on patterning capability was measured by using amended protocols in which piranha solution was avoided. Cell patterning trials were then performed using a series of rationalized protein activation solutions. Purified solutions of only vitronectin, fibronectin, laminin, collagen, or poly-l-ornithine solutions were assessed. Solutions containing varying concentrations of bovine serum albumin (BSA) alone or in combination with either vitronectin or fibronectin were then assessed for their ability to activate the patterning process. Lastly, HEK 293 cell patterning efficacy was assessed in the context of a second chip design with more extensive geometric variation.

MATERIALS AND PROTOCOLS

Fabrication of parylene patterns on SiO2: Process flow

  1. Silicon wafers (Siltronix, Archamps, France) oxidized in an atmospheric horizontal furnace (H2 1.88 SLM and O2 1.25 SLM) at 1100°C for 40 min to produce a 500 nm SiO2 layer (measured with a Nanometrics NanoSpec 3000 reflectometer).

  2. Oxidized wafers primed with Merck Silane A174 adhesion promoter, followed by deposition of 100 nm coating of parylene-C (at 22°C at a rate of 1.298 nm/mg of dimer using a SCS Labcoter 2 deposition Unit, Model PDS2010).

  3. Hexamethyldisilazane (HMDS) adhesion promoter deposited on parylene-coated wafers in an SVG 3 inch photoresist track followed by the application of a 1 μm thick film of Rohm & Hass SPR350-1.2 positive photoresist by spinning at a speed of 4000 rpm for 30s.

  4. 60s soft bake at 90°C.

  5. Both the wafers and pre-manufactured photo mask (Compugraphics International Ltd, Glenrothes, Scotland) placed in an Suss Microtech MA/BA8 mask aligner to produce photoresist patterns on a parylene/SiO2 substrate. The primary parylene design consisted of three iterations of circular parylene nodes with a centered ‘cross-hair’ (node diameters 250 μm, 100 μm, and 50 μm, cross hairs measuring 450 μm in length for largest node size and 300 μm for smaller nodes) on chips 7.7 mm × 5.9 mm in dimension.

  6. 60s bake at 110°C and exposed photo-resist removed from the wafers after developing in Microchem MF-26A developer.

  7. Insertion into a JLS RIE80 etch system for 120s (at a 50 mTorr chamber pressure, 49 sccm O2, 100W RF power at 13.56 MHz) to etch off unprotected parylene (at an etch rate of 100 nm/min) to reveal underlying SiO2.

  8. Wafers cut with a DISCO DAD 680 Dicing Saw (spindle speed 30 000 rpm, feed speed 7 mm/s), rinsed in water, and blown dry with nitrogen.

Chip cleaning and activation: Protocol

  1. Residual photoresist was removed from all chips by brief immersion in acetone followed by rinsing in deionized distilled H2O three times.

  2. Chips were either immersed in piranha acid (5:3 ratio of 30% hydrogen peroxide and 98% sulphuric acid) for 10 min or instead left untreated.

  3. After a further diH2O wash, chips incubated for 3 hours in different activation solutions:
    • fetal bovine serum (FBS, standard protocol),
    • bovine serum albumin (BSA) alone (0.3 mg/ml, 3 mg/ml, 30 mg/ml dissolved in Hanks Balanced Salt Solution, HBSS, Invitrogen)
    • BSA with vitronectin (0.3 mg/ml, 3 mg/ml, 30 mg/ml BSA with 1 μL/ml vitronectin)
    • BSA and fibronectin (0.3 mg/ml, 3 mg/ml, 30 mg/ml BSA with 1 μL/ml fibronectin)
    • vitronectin alone (1 μg/ml)
    • fibronectin alone (1 μg/ml)
    • laminin alone (1 μg/ml)
    • collagen alone (1 μg/ml)
    • poly-L-ornithine alone (50 μg/ml)
    • diH2O (control)

Maintenance and plating of HEK 293 cells: Protocol

  1. HEK293 cells (human embryonic kidney cells; American Type Culture Collection, Virginia, USA) maintained at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Invitrogen) supplemented with 10% fetal bovine serum (FBS).

  2. For experiments using simplified protein solutions, cells passaged and transferred to Freestyle™ 293 expression medium (Gibco Invitrogen) prior to plating. This is a serum-free and protein-free growth medium chosen specifically to avoid confounding the contribution of rationalized protein activation solutions.

  3. In other patterning trials, cells plated in their standard growth medium (DMEM + 10% FBS).

  4. Cells applied in suspension at a density of 6 × 104 cells/ml for all trials. Cell viability confirmed by observed ongoing mitotic growth.

Cell imaging and analysis: Process

  1. Cells imaged alive on sequential days in vitro using a Wild Heerbrugg (Switzerland) microscope adapted for use with a Nikon Coolpix 4500 digital camera using an MDC2 relay lens. Image J (version 1.44o, National Institute for Health, USA) was used for subsequent image analysis and measurement of cell surface areas.

  2. Two indices derived to assess contrasting aspects of cell patterning:
    • Parylene Adhesion Index (PAI): calculated by dividing the surface area of cell material on parylene by the total surface area of parylene within a given Region Of Interest (ROI). Each ROI consisted of one iteration of the parylene geometric pattern (described above) surrounded by a square area of SiO2.
    • SiO2 Repulsion Index (SRI): calculated by dividing the surface area of cell material on SiO2 by the total area of SiO2 in a given ROI, and subtracting the result from 1. Hence, perfect patterning on parylene would result in a PAI of 1 (complete cell coverage of all parylene-patterned areas) and SRI of 1 (complete absence cell material from SiO2). Prism 5 for Mac OS X (GraphPad Prism Software Inc., California, USA) was used for statistical analyses.

  3. For each patterning trial, 27 ROIs were interrogated (nine for each of the three node diameters present on the chip, pooled). Charted data is illustrated as means ± SEM. Mann-Whitney U tests were used to compare patterning indices between different chip treatment and activation protocols.

RESULTS

Temporal changes of patterning indices

Figure 1 illustrates parylene adhesion and SiO2 repulsion indices from day 1 to day 7 in vitro for each of the three different node diameters, with representative images of patterned HEK 293 cultures (serum-activated chips). PAI starts low (due to the relatively low cell plating density) and increases to approach one by day seven (reflecting almost total coverage of parylene with cell matter). SRI starts and remains high for all design geometries, with a gradual decline visible at day six/seven. This growth pattern is comparable for all three pattern geometries.

FIGURE 1.

FIGURE 1

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Changes in cell patterning indices over time for the three different node diameters on chip. Piranha-treated substrates activated in FBS with cells cultured in DMEM + 10% FBS. A: 250 μm diameter node, B: 100 μm diameter, C: 50 μm diameter. FBS, fetal bovine serum.

Rationalized protein activation solutions

Figure 2(A-C) illustrates patterning indices resulting from rationalized protein activation solutions. With solutions of BSA alone, parylene becomes notably cytophobic (especially at 30 mg/mL concentration) while SiO2 becomes relatively cell-tolerant. This inverts the prior-observed patterning effect to produce a “negative” cell image. A similar result is seen with BSA codissolved with vitronectin or fibronectin, though solutions with fibronectin manifest a further significant reduction in PAI. PAI also changes according to the concentration of BSA in which vitronectin is codissolved: BSA 3 mg/mL with vitronectin and BSA 30 mg/mL with vitronectin show a significantly greater PAI compared with BSA 0.3 mg/mL with vitronectin. However, in all cases, the PAI remains far inferior to that achieved with serum-activated chips [compare Fig. 2(D,F,G)].

FIGURE 2.

FIGURE 2

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A–C: Parylene adhesion and SiO2 repulsion indices for HEK 293 cells cultured in Freestyle media on substrates activated in rationalized protein solutions. All indices measured at 4DIV. Mean ± SEM. D–G: representative images of 250 μm diameter node pattern for specific activation solutions: D: serum, E: Vn alone, F: BSA 3 mg/mL + Vn, G: BSA 3 mg/mL + Fn. BSA, bovine serum albumin, Vn, vitronectin, Fn, fibronectin, Lam, laminin, Col, collagen, and PLO, poly-l-ornithine.

The four purified solutions of only vitronectin, fibronectin, laminin, or collagen resulted in a PAI significantly greater than that for either water control, pure BSA solutions, or combinations of BSA with vitronectin or fibronectin. However, the concomitant attenuation of the SiO2 repulsion index results in a substrate that no longer manifests contrasting adhesive or repulsive qualities and therefore ceases to pattern cells. Poly-l-ornithine enabled excellent cell adhesion to parylene (PAI approaching one), comparable to that seen with serum-activated chips. However, its effect was indiscriminate with similarly confluent adhesion to SiO2 (SRI almost zero).

Effect of piranha treatment

The effect of piranha solution on patterning is illustrated in Figure 3. For water-treated chips, piranha treatment has the effect of increasing repulsion from SiO2 and, unexpectedly, of decreasing adhesion to parylene. Notably, however, cell morphology on nonpiranha treated, water-activated chips is profoundly abnormal [see Fig. 3(C)].

FIGURE 3.

FIGURE 3

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A: Cell patterning indices illustrating the impact of piranha solution treatment, and subsequent activation with FBS or water, upon HEK293 cell adhesion and patterning. B–E: Representative images of each protocol showing 250 μm node diameter pattern. Means ± SEM. Pir+ve/Pir-ve denotes treatment wth/without piranha solution.

In the context of serum activation, piranha-solution affords a dramatic rise in PAI and also pushes SRI close to the maximum of 1. The overall result of this is enhancement of effective, discriminative patterning [see Fig. 3(E)].

Morphology and parylene geometry

Figure 4 illustrates morphological changes than can be induced by patterning on serum-activated parylene. A low-density culture (2 × 104 cells/mL) on the 100 μm diameter node pattern demonstrates the ability to capture single cell bodies on parylene “cross hairs” [solid arrow, Fig. 4(B)], and the potential to dictate the direction of an extending cell process (dashed arrow). Figure 4(C) illustrates persisting high-fidelity patterning achieved using a second chip with extensive variations in parylene geometry.

FIGURE 4.

FIGURE 4

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Morphological effects of HEK293 cell patterning on serum-activated parylene. A: HEK 293 cells approaching confluence in standard polystyrene culture flask. B: Low density HEK 293 culture on parylene/SiO2 illustrating single cell capture (filled arrow) and guided process extension (dashed arrow). Parylene node diameter 100/m. C: Consistent high resolution patterning on diverse parylene geometric patterns. All images at 4DIV cultured in Freestyle media.

DISCUSSION

Temporal changes of patterning indices

HEK 293 cells will continue to proliferate in monolayer culture with a doubling time of 30–40 h, depending on culture conditions. In order to define an optimum time point for assessment of patterning trials, PAI and SRI were first measured serially in the context of piranha-treated, serum-activated chips. PAI increases towards a value of one from day 1 to day 7 in vitro, reflecting proliferation of cells adherent to parylene. SRI is high from the outset and attenuates only toward the end of the 7-day window. Cells do not migrate from SiO2 towards relatively cytophilic parylene over time - SiO2 is clearly hostile to cells from the outset. The late drop in SRI can be attributed to overgrowth of parylene-adherent cell clusters on SiO2, as illustrated by photomicrographs taken at 6 days in vitro (Fig. 1). As a result of these findings, 4 days in vitro was chosen as the primary time point for analysis when assessing rationalized activation solutions and the impact of piranha solution treatment.

Rationalized protein activation solutions

Cell patterning relies on contrasts. None of the rationalized protein solutions was able to induce the patterning behavior observed with serum activation, where SiO2 is profoundly cytophobic and parylene is cytophilic. However, under certain activation conditions, the patterning process is inverted owing to a reversal of adhesive characteristics of the two substrates.

Pure solutions of the ECM proteins vitronectin, fibronectin, laminin, or collagen enabled significantly enhanced adhesion to parylene-C compared with water controls (with vitronectin and collagen exerting the greatest effect). However, the extent of adhesion remains approximately 5 × poorer than for serum-activated chips. Importantly, the concentration of single protein used (1 μg/mL) is well below that seen in serum. It is therefore possible that solutions of higher concentrations may enable the PAI to be driven closer to that seen in serum-activated chips. In any event, patterning is undermined in these cases by attenuation of the SRI. The combination of cell tolerance on both substrates results in an absence of discriminative cell patterning.

Solutions of BSA alone cause parylene to become notably cytophobic (especially at 30 mg/mL concentration), while SiO2 becomes relatively cell-tolerant. This is an unexpected finding; our hypothesis being that albumin is responsible for imbuing SiO2 with a cyto-repulsive character. Combinations of BSA with vitronectin or BSA with fibronectin result in a similar patterning configuration. However, the presence of vitronectin in BSA solutions does somewhat mitigate the observed decrease in PAI. Nevertheless, in combination with persisting cell tolerance on SiO2, the net result is inverted, or negative, patterning.

The observed changes in PAI and SRI are modulated by the concentration of BSA in which the second protein is codissolved. The cause for this in unclear but it is, interestingly, a phenomenon that has been observed before in the context of fibronectin-binding to hydrophobic surfaces.24 One explanation is that cobound albumin alters the configuration or packing of the second protein such that its binding sites are more or less available.

Given these findings, a model of adhesion relying on pro-adhesive and prorepulsive proteins in isolation is probably an oversimplification. More likely is a combinatorial effect of different proteins in solution giving the two regions of the chip their different adhesive characteristics. Although we cannot as yet identify the key protein combinations in serum, we have developed the capacity to induce cell-tolerance to SiO2 and cell repulsion from parylene (a complete reversal of the serum-induced parylene patterning effects reported previously.6-11 This encourages the use of the parylene/SiO2 platform for effective cell patterning with a standardized and consistent activation solution and protocol, without recourse to serum (which is poorly characterized and heterogeneous between batches).

Effect of piranha treatment

For water-activated, nonpiranha-treated substrates (i.e., default material behavior), cell adhesion, and morphology is highly abnormal. A confluent sheet of cells dominates, adherent predominantly to SiO2. Piranha treatment without serum activation results in re-establishment of more normal HEK 293 morphology, with cells tolerating both surfaces similarly. This change can be attributed to globally enhanced hydrophilicity afforded by piranha. However, without the secondary impact of serum this alteration does not in itself result in patterning. Following serum-activation, both PAI and SRI are significantly increased compared with piranha-treated water controls. The key change is achieving a SRI of nearly one, enabling excellent patterning contrast. Treatment with piranha solution therefore enables differential serum-protein absorption: promoting binding of adhesive molecules to parylene and repulsive molecules to SiO2.

Morphology and parylene geometry

Whilst HEK 293 cells have some characteristics of an early neuronal lineage cell, they are far from manifesting a functional neuronal phenotype capable of generating spontaneous electrical activity, forming synapses, or releasing neurotransmitters. However, for downstream applications using functional neurons, it will be important to exert fine control over placement of the cell soma and any extending neuritic processes. As a proof of principle, the impact of varying parylene geometry upon cell morphology was evaluated. For HEK 293 cells, we have shown the capacity to reliably pattern on a wide range of different geometric patterns. In addition, single cell bodies can be isolated and cell projections can be guided along parylene (Fig. 4).

The parylene/SiO2 platform has now enabled patterning of several cell types (cocultured primary murine hippocampal neurons and glia, the hNT cell line, and now HEK 293). However, this platform is not uniformly successful. The murine N2a cell line, for instance, does not pattern accurately using our current protocols. This variation is to be expected, given the differences in CAM expression between different cell types and cell lines. A future analysis of the patterning capacity of a broader range of cell types, in combination with assessment of each cell’s proteome (with specific reference to CAM expression), may in due course allow identification of key cell membrane proteins which facilitate patterning on serum-activated parylene/SiO2 substrates.

CONCLUSIONS

We have demonstrated, and manipulated, high-resolution patterning of HEK 293 cells on microfabricated patterns of parylene-C/SiO2 of various geometries. The use of rationalized activation solutions suggests both adhesive and repulsive components in serum which, when incubated with patterns of parylene/SiO2, interact to imbue each substrate with its respective cyto-adhesive or cyto-repulsive characteristics. Etching the substrate with piranha solution results in significant surface changes that enhance both cell repulsion from SiO2 and cell adhesion to parylene, following serum-incubation. These cell-patterning insights inform our wider goal of engineering a union between functional neuronal networks and a parylene-C/SiO2 semiconductor platform.

ACKNOWLEDGMENTS

The authors thank Richard Blair and Ewan MacDonald for expertise with processing of silicon wafers.

Contract grant sponsors: Wellcome Trust and the Engineering and Physical Sciences Research Council

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