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Lithography application of a novel photoresist for patterning of cells
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Biomaterials 25 (2004) 2055–2063
Lithography application of a novel photoresist for patterning of cells Wei Hea, Craig R. Halberstadtb, Kenneth E. Gonsalvesc,* a
Department of Chemistry, Institute of Material Science, University of Connecticut, Storrs, CT 06269, USA b Department of General Surgery Research, Carolinas Medical Center, Charlotte, NC 28232, USA c Department of Chemistry, University of North Carolina, 9201 University City Blvd, Charlotte, NC 28223, USA Received 19 June 2003; accepted 15 August 2003
Abstract Photolithography is the current workhorse for the microelectronic industry. It has been used extensively for the creation of patterns on two-dimensional surfaces. Various research groups have studied the use of photolithography to pattern surfaces for the alignment of cells. So far, these applications have been limited due to the use of organic solvents in the pattern developing process, which can denature biomacromolecules that would be attached to the material. To address this problem, a novel bioactive photoresist (bioresist) based on the copolymer of methyl methacrylate and 3-(t-butoxycarbonyl)-N-vinyl-2-pyrrolidone (MMA:TBNVP) was prepared and in vitro fibroblast cell growth on this resist was studied. Results demonstrated that the resist is non-adhesive to the fibroblast cells. By deprotecting the t-BOC groups into carboxyl groups (MMA:D-TBNVP), the material became cell adhesive. Furthermore, cells were able to proliferate on the MMA:D-TBNVP surface. By culturing cells on the MMA:D-TBNVP surface in serum versus serum-free medium, we reached the conclusion that the chemistry of the deprotected copolymer indirectly promoted cell attachment through its absorbance of serum proteins on the material. Patterns of 25 mm 25 mm lines were obtained by chemically manipulating the surface of the photoresist using UV lithography without any solvent development. Fibroblast cells were observed to align on the patterned surface. This resist could be a suitable candidate to improve the application of conventional lithography in direct protein patterning for the guided growth of cells. r 2003 Elsevier Ltd. All rights reserved. Keywords: Fibroblasts; Cell adhesion; Patterning; NVP; t-BOC; Chemically amplified photoresist
1. Introduction Spatial organization of mammalian cells could be valuable in applications such as cell-based sensors and diagnostic tests, interfacing regenerating neurons with solid-state devices in vivo, construction of neural networks in vitro, medical and dental implants, cocultures of different cell types in vitro, and microfabrication of devices and tissues in vitro [1–5]. Such spatial organization could be achieved by micropatterning substrates in order to specifically align proteins and cells in defined patterns. Various approaches have been studied for micropatterning which include conventional lithography that has been widely used in the semiconductor industry, and soft lithography that uses a poly(dimethyl siloxane) (PDMS) elastomeric stamp for pattern transfer. There are, however, several drawbacks *Corresponding author. E-mail address: [email protected] (K.E. Gonsalves). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.08.055
related to such methods. The patternable area in soft lithography is limited by the size of the PDMS stamps, usually around 1 cm2, therefore affecting its application in large-scale pattern production. Furthermore, pairing and sagging [6] of the stamp is unavoidable due to the elastomeric properties of PDMS. In conventional lithography, organic solvents are normally utilized to dissolve the photoresist in order to form the desired pattern. Such processes can lead to the denaturation of the biomolecules, and thus, it is not suitable for application in biomolecule and cell patterning. Recently, an alternative approach for patterning of biomolecular layers was introduced based on the design of a modified chemically amplified photoresist material that is derived from poly(t-butyl acrylate) [7]. It allows positive imaging with very dilute basic solutions that are tolerated by selected biomolecules. In this paper, a novel chemically amplified photoresist material (bioresist) is presented and its behavior evaluated with fibroblast cells. A unique feature of this
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bioresist is that, unlike in the conventional lithographic approach, no further process of development is needed to remove the remaining resist after the photolithography patterning. The patterned resists can be used directly for the coupling of specific cell-adhesion peptides or proteins for the alignment of cells.
CH3 CH2
C m C
CH2
O
CH n N
O
O
2. Materials and methods
CH3
t-BOC
2.1. Materials
t-BOC: -COOC(CH3 )3 All the chemical reagents were obtained from Aldrich and/or Acros. N,N0 -azobisisobutyronitrile (AIBN) was purified by recrystallization in methanol. Tetrahydrofuran (THF) was distilled over sodium using benzophenone as an indicator. Methyl methacrylate (MMA) was vacuum distilled under nitrogen prior to use to remove inhibitors. Other chemicals were used as received. Triphenyl sulfonium hexafluoroantimonate (TPSHA), used as a photoacid generator (PAG) for resist formulation, was purchased from Polysciences. It was a 50 wt% solution in propylene glycol methyl ether (PGME). Monomer 3-(t-butoxycarbonyl)-N-vinyl-2pyrrolidone (TBNVP) was synthesized as described elsewhere [8]. 2.2. Bioresist synthesis MMA/TBNVP copolymer was prepared by AIBNinitiated free radical polymerization. A solution of 2.1 g of TBNVP and 0.7 g MMA was dissolved in 16 ml of THF (monomer concentration 1.0 mol/l) along with 0.0398 g of AIBN (1.5 102 mol/l) and the mixture was heated to 65–75 C under nitrogen for 24 h. To isolate the polymer, the reaction mixture was added dropwise into sufficient petroleum ether. The polymer was filtered and washed thoroughly with petroleum ether. The polymer was then redissolved in THF and reprecipitated to minimize the presence of residual unreacted monomer. Finally, the polymer was dried in vacuum at 40 C to a constant weight (2.38 g, 85% yield). The copolymer will be referred to as MMA:TBNVP (64:36), indicating that MMA comprises 64 mol% of the polymer composition. The t-BOC protection groups from the copolymer were converted into COOH groups using trifluoroacetic acid (TFA) similar to that in the homopolymer [8]. The copolymer and deprotected copolymer (shown as MMA:D-TBNVP) were deposited onto glass coverslips from solution for the cell culture studies, and their chemical structures are illustrated in Scheme 1. 2.3. Bioresist characterization Fourier transform infrared (FTIR) spectra of the polymers were recorded on a Nicolet spectrometer. For
CH3 CH2
C m C
CH2
O
CH n N
O
O CH3
COOH
Scheme 1. Chemical structures of MMA:TBNVP and MMA: D-TBNVP.
FTIR spectra, the solid polymers and KBr (spectroscopic grade) were thoroughly mixed and this mixture was pressed to form a pellet. Solution 1H NMR spectra were recorded with a Varian UNITY 300 spectrometer. Gel permeation chromatography (GPC) measurements for molecular weights and molecular weight distributions were carried out on a Waters 2410 (eluent THF, narrow molecular weight polystyrene standards, refractive index detection). Thermal analyses of the polymers were conducted using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) measurements. TGA was performed on a TA instruments TGA 2950 at a heating rate of 20 C/min in a nitrogen atmosphere. DSC was conducted on a TA instruments DSC 2920 in N2 at a heating rate of 10 C/ min for glass transition determination. Glass transition temperature (Tg ) of the polymers was obtained from the second run of DSC thermograms. 2.4. Patterning with bioresist Circular glass coverslips (VWR) of 25 mm diameter were patterned photolithographically to generate lines of 25 mm width with a 25 mm spacing with the copolymer. Patterning of the copolymer is similar to that described for the homopolymer patterning [8]. Briefly, 10 wt% of MMA:TBNVP (64:36)/PGME solution with 5 wt% of TPSHA was prepared and filtered. The copolymer film was spin-cast onto the circular glass
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and the pattern was achieved via UV lithography. The pattern was revealed by soft bake for 1 min at 120 C immediately after the exposure. No further development was performed. 2.5. Cell culture and assays All cell culture reagents were obtained from SIGMA unless otherwise noted. This study was performed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AALAC International)-approved vivarium with the approval of, and strict adherence to, the guidelines of the Institutional Animal Care and Use Committee (IACUC). Primary Wistar furth rat fibroblast (RFB) cells were isolated from rat dermis using a modified collagenase digestion procedure [9]. The isolated cells were cultured in medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20% heat-inactivated fetal bovine serum (FBS), 1.25% l-glutamine (200 nm, GIBCO), 1% fungizone (GIBCO), 1% penicillin (10,000 U/ml)-streptomycin (10 mg/ml), and 2.5% HEPES buffer solution (1 m, GIBCO). The cultures were incubated at 37 C in an 8% carbon dioxide, humidified atmosphere. 2.5.1. Cell attachment assay Following exposure to sterile phosphate buffered saline (PBS) for 5 min at 25 C, the glass coverslips coated with MMA:TBNVP and MMA:D-TBNVP were positioned on the bottom of sterile 24-well culture plates. The wells were precoated with a thin film of 12% poly(hydroxylethylmethacrylate) (PHEMA) (Polysciences) to guarantee that cells adhered only to the substrates. RFB cells were grown near confluence in T75 flasks. Cells were released from the culture flasks by incubation with 0.1% (w/v) trypsin/0.02% ethylenediaminetetraacetic acid (EDTA) in PBS at 37 C for approximately 1 min, suspended by tapping the flask, then collected by centrifugation at 1200 rpm for 5 min, and resuspended in fresh cell culture medium. Cell concentration was determined using a hemacytometer. A plating concentration of 2.5 104 cells per well in a total volume of 0.5 ml was applied to each sample. After seeding, the cultures were incubated for 2 and 6 h, respectively, at 37 C in 8% CO2 air. At the end of each period, the substrates were washed twice with PBS to remove any non-attached cells. The number of viable cells attached to the different substrates was evaluated by the CellTiter 96s AQueous One Solution Cell Proliferation Assay (PROMEGA). The reagent contains a novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (inner salt, MTS) and an electron coupling reagent phenazine ethosulfate (PES). In this assay, the MTS
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tetrazolium compound is bioreduced by cells into a colored formazan product. The quantity of formazan product as measured by the amount of absorbance at 490 nm is directly proportional to the number of living cells in culture. Each experimental time point was performed in triplicate. As a reference material, cells seeded on plain glass were included in each run as positive controls. Significant differences between experimental values were determined based on a 95% confidence interval (Student’s t-test). 2.5.2. Cell proliferation assay Additionally, fibroblast cell proliferation on MMA:D-TBNVP was also studied. Fibroblast cells were seeded onto the surface of polymer coated glasses at a plating density of 1 105 cells/well and cultured at 37 C in 8% CO2/air atmosphere for 7 days. Twelve-well tissue culture plates were used and the plates were coated with PHEMA. Cell growth was quantified at 1, 2, 3, 5 and 7 days by measuring the cell metabolic activity with the MTS method mentioned above. The nonattached cells were removed by rinsing with PBS prior to the assay to ensure accuracy. In each experiment, the materials were performed in triplicate, and untreated polystyrene dishes were used as negative control. 2.5.3. Study of serum effect on cell attachment Glass coverslips coated with MMA:D-TBNVP were rinsed several times with sterile PBS and placed in sixwell tissue culture plates which were pretreated with PHEMA. Plain glasses were used as positive controls. Fibroblasts were lifted from the culture flask after reaching confluence, centrifuged and resuspended. The cells were then washed three times with serum-free medium, consisting of DMEM supplemented with 1.25% l-glutamine (200 nm), 1% fungizone, 1% penicillin (10,000 U/ml)-streptomycin (10 mg/ml), 2.5% HEPES buffer solution (1 m) and 0.1% insulin-transferrin-selenium-A (GIBCO). Cells were seeded onto the samples at a plating density of 1.5 105 cells/well. For each set of polymer, cells were cultured both in serum and serum-free medium in triplicate. After 2, 6 and 24 h, respectively, the non-adherent cells were removed by washing with PBS. Adherent cells were quantified by MTS assays. Student’s t-test and one-way ANOVA were used for statistical analysis. 2.5.4. Cell culture on the patterned surfaces Fibroblast cells were also cultured on micropatterned surfaces of MMA:TBNVP. Glass cover slides with patterns were rinsed several times with sterile PBS. The patterned slides were then placed in six-well tissue culture plates precoated with PHEMA. Cells were washed with serum-free medium before seeded onto the samples at a density of 1.5 105 cells/well. Nonpatterned regular glass slides were used as a control. Cell
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cultures were carried out both in serum and serum-free medium. After seeding, fibroblast cells were allowed to stay in contact with the surfaces in an incubator at 37 C with 8% CO2 for various periods of time. At the end of each incubation period, the samples were rinsed with PBS to remove non-attached cells and cell morphology was observed using an inverted Olympus phase-contrast microscope. 2.5.5. Large-scale patterning and cell culture A 2-inch glass substrate was patterned with the bioresist MMA:TBNVP using similar techniques as described above and RFB cells were cultured on it under physiological conditions. The cells were cultured on the material for a total time of 2 weeks. The culture media were collected every 3 days and lactate production was measured by using the YellowSprings glucose/lactate analyzer (YellowSprings, Inc., YellowSprings, Ohio). A plain non-tissue culture treated polystyrene dish was used as a control. Cell morphology was examined frequently during the culture period. At the end of the 2-week incubation period, both the patterned 2-in sample and the plain polystyrene dish were fixed in formalin and stained with Masson’s Trichrome stain. Aniline blue is a component of Masson’s Trichrome stain and it stains collagen blue. Therefore, it allows for the in situ detection of total collagen matrix deposition on these samples.
3. Results and discussion This study was initiated by the fact that polymers bearing t-BOC groups can be used as photoresist materials. Polymers derived from NVP and MMA are generally biocompatible. Therefore, the copolymer of MMA and TBNVP was prepared which would combine both properties to make a bioactive photoresist which we have termed as a bioresist. In the present study, in vitro methods to evaluate the material properties were performed. In vitro investigations have previously been shown to be suitable in examining cell interactions with polymeric materials because in vitro conditions provide a relatively simplistic environment in which information on the effect of material properties on cellular adhesion and growth could be obtained without the influence of other systemic events [10,11].
protection groups were removed and the molecular weight decreased to 17,500 with a polydispersity of 1.40. In the FTIR spectra (Fig. 1), a broad peak in the region of 3400–2500 cm1 was observed for MMA:D-TBNVP, due to the O–H stretching from the carboxyl groups. In order to study the composition of the copolymer, NMR was applied. The 1H NMR spectra of the copolymers before and after deprotection are shown in Fig. 2. The peak at d1.4 is due to the tert-butyl protons, while the peak appearing at d3.5–d3.7 was assigned to the methyl protons in MMA. Before deprotection, the molar ratio of MMA:TBNVP was obtained by the ratio of integrated area per unit proton of each characteristic peak (CH3 of MMA at d3.5–d3.7 and C(CH3)3 of TBNVP at d1.4), which belonged to different compositions. From the integration, the composition of the copolymer is 64:36 molar ratio of MMA:TBNVP. In addition, the intensity of the signal for the tert-butyl protons was greatly decreased after the deprotection with TFA.
C=O
Absorbance
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(b)
(a)
4000
3000 Wavenumbers
2000
1000
(cm-1)
Fig. 1. FTIR spectra of (a) MMA:TBNVP and (b) MMA:D-TBNVP.
(b)
3.1. Bioresist—synthesis and characterization Copolymerization reactions provide an excellent way of preparing macromolecules with controlled properties such as hydrophilic/hydrophobic balances, polarity and film formation [12,13]. GPC showed that the molecular weight of MMA:TBNVP was 24,000 and the polydispersity was 1.80. After reacting with TFA, the t-BOC
(a)
Fig. 2. 1H NMR spectra of (a) MMA:TBNVP (in CDCl3) and (b) MMA:D-TBNVP (in CD2Cl2).
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The thermal behaviors of MMA:TBNVP and MMA:D-TBNVP were examined by TGA, the plot of which is shown in Fig. 3. The thermal stability of the copolymer can be used to provide an assessment of the tBOC content in the copolymer. The TGA curve of MMA:TBNVP (Fig. 3a) shows that the polymer is thermally stable until B180 C. Due to the t-BOC groups the polymer starts to lose weight at 180 C by giving off carbon dioxide and isoproprene and continues until 240 C in a nitrogen environment at a heating rate of 20 C/min. The weight loss is 25.6%, which is in very good agreement with the expected value (25.7%). After reacting with TFA (Fig. 3b), the polymer starts decomposing at B150 C, due to the t-BOC groups and the newly formed carboxyl groups. Comparing 3a and 3b, the decrease in the weight change between 140 and 240 C further indicated the removal of the t-BOC protecting groups. In the DSC thermograms, a glass transition (Tg ) at 118 C was observed for MMA:TBNVP, while a very subtle transition was noticed at 73 C for MMA:D-TBNVP. Because of the bulky t-BOC protecting groups, the mobility of polymer chains is decreased, which leads to a higher glass transition temperature. After the deprotection, the bulky t-BOC group is replaced by a much smaller carboxyl group, which also has the great possibility of forming intramolecular H-bonding between the OH and the carbonyl group on the two-position of the lactam ring. It enables polymer chain motion occurring at a lower temperature, i.e. a lower Tg [14]. 3.2. Cell culture on bioresist For anchorage-dependent cells, such as fibroblasts, adhesion is the critical prerequisite for subsequent cell functions such as cell proliferation and deposition of extracellular matrix (ECM). Therefore, initial attachment is a very critical process for cell growth on
materials. To study how fibroblast cells would interact with the copolymer before and after deprotection, cells were cultured on both MMA:TBNVP and MMA: D-TBNVP surfaces for 2 and 6 h, respectively, and the number of cells attached was measured by the absorbance at 490 nm obtained by MTS assay. As shown in Fig. 4, cells respond differently to various surfaces. Two hours after seeding cells onto the materials, there was a minimal cell attachment on the MMA:TBNVP and MMA:D-TBNVP surfaces as compared to the plain glass surface. When the incubation time was increased to 6 h, the absorbance was significantly (po0:05) increased on MMA:D-TBNVP samples compared to that of 2 h. The increment is much more obvious than that of the MMA:TBNVP samples. It indicates that cells adhered better on the MMA:D-TBNVP surfaces where COOH groups were present. The difference in cell adhesion on these surfaces could be due to different surface chemistries of the materials. The exposure of the carboxyl groups by deprotection of the t-BOC groups made MMA:D-TBNVP more hydrophilic than MMA:TBNVP and also changed the surface electric charge. In addition, these carboxyl groups can be used for the further incorporation of drugs or other bioactive agents [15–19]. For example, Hyun et al. [20] have used microstamping to pattern a cell-adhesive Arg-Gly-Asp (RGD) peptide onto the surface of a COOH-derivatized comb polymer. Cell–material interactions are mainly due to the interactions of cell-adhesion proteins (ligands) bound to a material surface and cell surface receptors (integrins). The tripeptide RGD is known as the active sequence of adhesive proteins of the ECM that binds to the integrin receptors [21]. Therefore, RGD-containing peptides could be covalently coupled to the polymeric material through the COOH to promote cellular adhesion. The growth behavior of fibroblasts on MMA: D-TBNVP is shown in Fig. 5. As shown the cells not only attached on the surface, but also proliferated over a 7-day culture period indicating that the material does not hinder cell growth. There was a 3-day lag period on Attachment 0.3 Absorbance at 490 nm
Fig. 3. TGA thermograms of (a) MMA:TBNVP and (b) MMA: D-TBNVP.
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0.25 0.2
MMA:TBNVP MMA:D-TBNVP
0.15
Control
0.1 0.05 0 2
6
Incubation Time (hours)
Fig. 4. Results for cell attachment assay. Averages and standard deviations are plotted based on a sample size of three.
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the MMA:D-TBNVP prior to the cells reaching the exponential phase of growth. Generally, fibroblast cell growth on tissue culture treated polystyrene (TCPS) enters exponential growth phase by day 1 post-seeding (data not shown). 3.3. Study of serum effect on cell attachment A further study was conducted in order to determine whether surface chemistry directly affected cell adhesion or indirectly by influencing serum protein adsorption. As mentioned above, cell adhesion on MMA:TBNVP and MMA:D-TBNVP was different. Various hypotheses may be proposed to explain such a difference. It is well known that surface properties of polymers affect cell adhesion, spreading and proliferation via their adsorptive properties with respect to serum proteins. In order to determine whether surface chemistry of the copolymer has any direct influence on cell attachment, fibroblast cells were cultured on these different polymer surfaces both in serum and serum-free medium. Results shown in Fig. 6 indicated that better cell adhesion on MMA:D-TBNVP correlates with serum protein adsorption. At each selected time point, the MTS absorbance obtained in the presence of serum is significantly higher than serum-free conditions. This observation may be due to the physical properties of the material such as its Proliferation 300000
Cell Number
250000 200000 150000
MMA:D-TBNVP
100000
Control
50000 0 1
-50000
2
3
5
7
Incubation Time (days)
Fig. 5. Fibroblast proliferation (mean7SD, n ¼ 3) in direct contact with MMA:D-TBNVP and control (a plain polystyrene dish) evaluated by MTS assay up to 7 days.
Absorbance at 490 nm
Serum vs. Serum-free 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0
I I
II
I
II
II
Control MMA:DTBNVP
2
6
24
Incubation Time (hours)
Fig. 6. Cell culture in both serum and serum-free condition. Values represent the mean7SD of three samples (I: serum; II: serum-free).
hydrophilic nature and its chemical composition. These components are important in promoting and presenting serum proteins to the cells in the appropriate configuration. For most synthetic polymer surfaces, adhesion of cells requires the presence of serum and, therefore, this optimum is probably related to the ability of proteins, such as fibronectin [22,23] and vitronectin [22,24], to absorb to the surface. Many recent studies have demonstrated the involvement of serum proteins in the attachment of various cells to the substrate. For example, the attachment of endothelial cells is dependent upon vitronectin [25]. Studies have also shown that fibronectin and vitronectin adsorption and conformation on surfaces are related to the characteristics of the surfaces such as wettability [26]. 3.4. Cell culture on patterned bioresist surfaces Because of the t-BOC groups, MMA:TBNVP can be used as a chemically amplified photoresist in the presence of a PAG. The process of patterning is controlled by the successful creation of differences between the exposed and unexposed areas in terms of the hydrophobic/hydrophilic nature of the material and its chemistry. Micron-sized patterns were obtained with MMA:TBNVP using deep UV lithography. The exposed areas are rich in carboxylic groups due to the photolysis of the t-BOC groups. The features were revealed without any treatment of development, which means the patterns are actually composed of alternating MMA:TBNVP and MMA:D-TBNVP. The surface of the unexposed MMA:TBNVP resist is hydrophobic, while the hydrophilic areas in the exposed region are created due to the formation of carboxylic groups. Fibroblast cells were cultured on these patterned surfaces both in serum and serum-free medium for various periods of times. The cell morphology is shown in Figs. 7 and 8. Fig. 7 shows the optical micrographs of cells cultured in serum containing medium. After 24 h, cells were randomly attached on the plain glass with spindle shapes (Fig. 7a), while on the patterned surface, cells were aligned along the patterns in a longitudinal fashion (Fig. 7b), i.e. they were exclusively attached in the exposed area, where MMA:D-TBNVP was produced. We did see cells reaching out to their adjacent neighbors occasionally, due to the short distance between the patterned lines (25 mm). Such contact could be avoided with increased distance between the patterns. This finding is consistent with the data shown above, where cells adhered preferentially to the deprotected copolymer. After 2 days of incubation, compared to the random proliferation on the plain surface, cells were growing in a more organized way. Because of proliferation, cells bridged across the patterns and formed a confluent layer on top of the pattern: the main vector of growth is along the direction of the pattern.
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(a)
(c)
(b)
(d)
unexposed
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exposed
Fig. 7. Optical micrographs of fibroblast cells cultured on the plain glass surface (a, c) and the patterned photoresist surface (b, d) after 24 h (a, b) and 48 h (c, d) incubation in serum containing medium, respectively (20 magnification, bar=50 mm).
(a)
(c)
(b)
(d)
Fig. 8. Optical micrographs of fibroblast cells cultured on the plain glass surface (a, c) and the patterned photoresist surface (b, d) after 24 h (a, b) and 48 h (c, d) incubation in serum-free medium, respectively (10 magnification, bar=100 mm).
To assess the effect of serum components on the alignment phenomenon, we seeded cells on the patterned sample in the absence of serum proteins. Under such condition, cells did attach on both plain and patterned surfaces, but they remained rounded which indicated non-specific cell adhesion. In addition, the number of cells attached was less compared to those that were seeded in the presence of serum. As shown in Fig. 8, after 2 days of incubation, cells began to spread on the plain surface, while on the patterned surface, they
remained rounded. It is worth noting that on the patterned surface, the majority of the cells attached to the exposed area (MMA:D-TBNVP), which implies that in the absence of serum, cells were attracted to the exposed area more than the unexposed area (MMA:TBNVP). By comparing serum versus serum-free conditions, we reached a conclusion that cell alignment on the patterned surface is mostly related to serum protein adsorption. Serum proteins preferentially adsorbed onto
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the exposed area, which is more hydrophilic, and led to cell attachment in these areas. In this study, the surface is essentially coplanar, thereby eliminating topographical cues such as contact guidance. An important advantage of this bioresist is that it could be combined with the well-developed lithographic process to generate patterns as large as 8 in. To demonstrate this scalability, we used the bioresist on a 2-inch glass substrate and cultured fibroblast cells on it for a period of 2 weeks. Cell metabolism was tracked during the incubation. Fig. 9 shows the results of the lactate production rate of cells cultured on the patterned surface as well as the control. As seen there is an increase in the rate of lactate production for cells growing on the patterned surface, which indicates that cells were more metabolically active. Upon visual inspection, cells on the patterned sample appeared to have a higher density than that on the control. Although a direct cell count to correlate a per cell production of lactate was not preformed, it appears that the increase in the rate of lactate production may be due to an increase in cell number on the patterned surface. The morphology of the cells cultured on the patterned 2-in sample was similar to the 1-in sample, where cell alignment was observed. In addition, the cell-seeded disks were fixed and stained with Masson’s Trichrome stain for the detection of collagen deposition on the samples. The result is shown in Fig. 10. There was only a small area of the control sample that remained adhered after going through the fixation and staining process. This may be
due to the low deposition of ECM proteins and/or the non-specific binding of cells on the surface of polystyrene material. The cells had a random appearance of cell growth with a disorganized pattern of collagen deposition. In comparison, the patterned surface had a welldefined matrix deposition pattern that appears to be oriented along the patterned surface. This was observed throughout the sampled area. Similar formation of adhesive and non-adhesive domains via photolithography process on the classical DNQ/novolak resist was previously reported for neuronal cell culture [27]. In the study diazo-naphthoquinone/novolak photoresist was used. Since the main component in the DNQ/novolak system is the aromatic rings, there is a concern that this polymer could be toxic. Our system is non-toxic, and additionally, the COOH groups formed on the surface of the polymer can be used for further surface modifications. It also implies a potential for expanding conventional lithography application in large-scale direct cell patterning based on the bioresist. Cell alignment observed in this study implied a potential application of using this technique together with 3-D structures to generate an oriented tissue-like construct from fibroblasts, which will be mechanically strong yet have certain flexibility similar to the normal tissue. The ability to control cell orientation in tissue engineering has been shown to be critical for the development of advanced forms of tissue repair and cell engineering therapies, such as peripheral nerve repair, production of tendon and ligament substitutes in vitro, and the control of microvascular repair [28].
Lactate Production Rate (∆Lactate/∆Day)
Lactate Production of Fibroblast Cells 3.5 3 2.5 2
4. Conclusions control pattern
1.5 1 0.5 0 1
4
7
12
Days
Fig. 9. Lactate production rate of fibroblast cells cultured on the control surface (a plain polystyrene dish) and the patterned 2-in substrate.
(a)
A novel photoresist material has been assessed in terms of its biocompatibility and potential for patterning of fibroblast cells. Photo-induced chemical transformation of the surface of the resist thin film resulted in changes in the surface hydrophobic/hydrophilic properties and surface chemistry. These changes affected protein adsorption that led to cell seeding and alignment on the patterned surfaces. This development-free,
(b)
Fig. 10. Masson’s Trichrome stain (blue dye) for collagen deposition on (a) control (a plain polystyrene dish) and (b) patterned surfaces after 2 weeks of culture (200 magnification).
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patternable, and ‘‘cell-friendly’’ resist presents the potential of solving the solvent problem in applying conventional lithographic processes for cell and protein patterning. Our ongoing studies are directed to coupling the RGD peptide to the surface through the COOH groups and study its effect on cell adhesion. Additionally, more patterns will be processed with various shapes and dimensions, and different cell types will be tested on these cell-friendly resist surfaces. Acknowledgements KEG acknowledges partial funding from the North Carolina Biotechnology Center, Durham, NC. References [1] Healy KE, Thomas CH, Rezania A, Kim JE, Mckeown PJ, Lom B, Hockberger PE. Kinetics of bone cell organization and mineralization on materials with patterned surface Chemistry. Biomaterials 1996;17:195–208. [2] Patel N, Padera R, Sanders GH, Cannizzaro SM, Davies MC, Langer R, Roberts CJ, Tendler SJ, Williams PM, Shakesheff KM. Spatially controlled cell engineering on biodegradable polymer surfaces. FASEB J 1998;12:1446–54. [3] Bhatia SN, Balis UJ, Yarmush ML, Toner M. Probing heterotypic cell interactions: hepatocyte function in microfabricated cocultures. J Biomater Sci Polym Ed 1998;9:1137–60. [4] Rowe CA, Scruggs SB, Feldstein MJ, Golden JP, Ligler FS. An array immunosensor for simultaneous detection of clinical analytes. Anal Chem 1999;71:433–9. [5] Kane RS, Takayama S, Ostuni E, Ingber DE, Whitesides GM. Patterning proteins and cells using soft lithography. Biomaterials 1999;20:2363–76. [6] Xia Y, Whitesides GM. Soft lithography. Angew Chem Int Ed 1998;37:550–75. [7] Douvas A, Argitis P, Misiakos K, Dimotikali D, Petrou PS, Kakabakos SE. Biocompatible photolithographic process for the patterning of biomolecules. Biosens Bioelectron 2002;17:269–78. [8] He W, Gonsalves KE, Pickett JH, Halberstadt CR. Synthesis, characterization, and preliminary biological study of poly(3-(tbutoxycarbonyl)-N-vinyl-2-pyrrolidone). Biomacromolecules 2003;4:75–9. [9] Doyle A, Griffiths JB, Newell DG. Dissociation of dermal fibroblasts in cell and tissue culture: laboratory procedures. New York: Wiley; 1996. [10] Peppas NA, Langer R. New challenges in biomaterials. Science 1994;263:1715–20. [11] Morrison C, Macnair R, MacDonald C, Wykman A, Goldie I, Grant MH. In vitro biocompatibility testing of polymers for orthopedic implants using cultured fibroblasts and osteoblasts. Biomaterials 1995;16:987–92.
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[12] Gallardo A, Lemus AR, San Roman J, Cifuentes A, Diez-Masa JC. Micellar electrokinetic chromatography applied to copolymer systems with heterogeneous distribution. Macromolecules 1999; 32:610–7. [13] Brar AS, Kumar R. One- and two-dimensional NMR characterization of N-vinyl-2-pyrrolidone/methyl acrylate copolymers. J Polym Sci A: Polym Chem 2002;40:2225–36. [14] Odian G. Principles of polymerization. New York: Wiley; 1991. [15] Na K, Park KH. Conjugation of Arg-Gly-Asp sequence into thermo-reversible gel as an extracellular matrix for 3T3-L1 fibroblast cell culture. Biotechnol Lett 2000;22:1553–6. [16] Irvine DJ, Mayes AM, Griffith LG. Nanoscale clustering of RGD peptides at surfaces using comb polymers. 1. Synthesis and characterization of comb thin films. Biomacromolecules 2001; 2:85–94. [17] Jo S, Engel PS, Mikos AG. Synthesis of poly(ethylene glycol)tethered poly(propylene fumarate) and its modification with GRGD peptide. Polymer 2000;41:7595–604. [18] Drumheller PD, Hubbell JA. Polymer networks with grafted cell adhesion peptides for highly biospecific cell adhesive substrates. Anal Biochem 1994;222:380–8. . usderelioglu . . [19] Gum M, Turkoglu H. Biomodification of non-woven polyester fabrics by insulin and RGD for use in serum-free cultivation of tissue cells. Biomaterials 2002;23:3927–35. [20] Hyun J, Ma HW, Banerjee P, Cole J, Gonsalve KE, Chikoti A. Micropatterns of a cell-adhesive peptide on an amphiphilic comb polymer film. Langmuir 2002;18:2975–9. [21] Ruoslahti E. RGD and other recognition sequences for integrins. Ann Rev Cell Dev Biol 1996;12:697–723. [22] Saltzman WM. Cell interactions with polymers. In: Lanza PP, Langer R, Vacanti J, editors. Principles of tissue engineering. New York: Academic Press; 2000. p. 221–35. [23] Horbett T, Schway M. Correlations between mouse 3T3 cell spreading and serum fibronectin adsorption on glass and hydroxyethylmethacrlate-ethylmethacrylate copolymers. J Biomed Mater Res 1988;22:763–93. [24] McFarland CD, Thomas CH, DeFilippis C, Steele JG, Healy KE. Protein adsorption and cell attachment to patterned surfaces. J Biomed Mater Res 2000;49:200–10. [25] Steele JG, Johnson G, McFarland C, et al. Roles of serum vitronectin and fibronectin in initial attachment of human vein endothelial cells and dermal fibroblasts on oxygen- and nitrogencontaining surfaces made by radiofrequency plasmas. J Biomater Sci Polym Ed 1994;6:511–32. [26] Bergkvist M, Carlsson J, Oscarsson S. Surface-dependent conformations of human plasma fibronectin adsorbed to silica, mica, and hydrophobic surfaces, studied with use of Atomic Force Microscopy. J Biomed Mater Res 2003;64A: 349–56. [27] Nicolau DV, Taguchi T, Tanigawa H, Yoshikawa S. Control of the neuronal cell attachment by functionality manipulation of diazo-naphtho-quinone/novolak photoresist surface. Biosens Bioelectron 1996;11:1237–52. [28] Mudera VC, Pleass R, Eastwood M, Tarnuzzer R, Schultz G, Khaw P, McGrouther DA, Brown RA. Molecular response of human dermal fibroblasts to dual cues: contact guidance and mechanical load. Cell Motil Cytoskel 2000;45:1–9.
Biomaterials 25 (2004) 2055–2063
Lithography application of a novel photoresist for patterning of cells Wei Hea, Craig R. Halberstadtb, Kenneth E. Gonsalvesc,* a
Department of Chemistry, Institute of Material Science, University of Connecticut, Storrs, CT 06269, USA b Department of General Surgery Research, Carolinas Medical Center, Charlotte, NC 28232, USA c Department of Chemistry, University of North Carolina, 9201 University City Blvd, Charlotte, NC 28223, USA Received 19 June 2003; accepted 15 August 2003
Abstract Photolithography is the current workhorse for the microelectronic industry. It has been used extensively for the creation of patterns on two-dimensional surfaces. Various research groups have studied the use of photolithography to pattern surfaces for the alignment of cells. So far, these applications have been limited due to the use of organic solvents in the pattern developing process, which can denature biomacromolecules that would be attached to the material. To address this problem, a novel bioactive photoresist (bioresist) based on the copolymer of methyl methacrylate and 3-(t-butoxycarbonyl)-N-vinyl-2-pyrrolidone (MMA:TBNVP) was prepared and in vitro fibroblast cell growth on this resist was studied. Results demonstrated that the resist is non-adhesive to the fibroblast cells. By deprotecting the t-BOC groups into carboxyl groups (MMA:D-TBNVP), the material became cell adhesive. Furthermore, cells were able to proliferate on the MMA:D-TBNVP surface. By culturing cells on the MMA:D-TBNVP surface in serum versus serum-free medium, we reached the conclusion that the chemistry of the deprotected copolymer indirectly promoted cell attachment through its absorbance of serum proteins on the material. Patterns of 25 mm 25 mm lines were obtained by chemically manipulating the surface of the photoresist using UV lithography without any solvent development. Fibroblast cells were observed to align on the patterned surface. This resist could be a suitable candidate to improve the application of conventional lithography in direct protein patterning for the guided growth of cells. r 2003 Elsevier Ltd. All rights reserved. Keywords: Fibroblasts; Cell adhesion; Patterning; NVP; t-BOC; Chemically amplified photoresist
1. Introduction Spatial organization of mammalian cells could be valuable in applications such as cell-based sensors and diagnostic tests, interfacing regenerating neurons with solid-state devices in vivo, construction of neural networks in vitro, medical and dental implants, cocultures of different cell types in vitro, and microfabrication of devices and tissues in vitro [1–5]. Such spatial organization could be achieved by micropatterning substrates in order to specifically align proteins and cells in defined patterns. Various approaches have been studied for micropatterning which include conventional lithography that has been widely used in the semiconductor industry, and soft lithography that uses a poly(dimethyl siloxane) (PDMS) elastomeric stamp for pattern transfer. There are, however, several drawbacks *Corresponding author. E-mail address: [email protected] (K.E. Gonsalves). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.08.055
related to such methods. The patternable area in soft lithography is limited by the size of the PDMS stamps, usually around 1 cm2, therefore affecting its application in large-scale pattern production. Furthermore, pairing and sagging [6] of the stamp is unavoidable due to the elastomeric properties of PDMS. In conventional lithography, organic solvents are normally utilized to dissolve the photoresist in order to form the desired pattern. Such processes can lead to the denaturation of the biomolecules, and thus, it is not suitable for application in biomolecule and cell patterning. Recently, an alternative approach for patterning of biomolecular layers was introduced based on the design of a modified chemically amplified photoresist material that is derived from poly(t-butyl acrylate) [7]. It allows positive imaging with very dilute basic solutions that are tolerated by selected biomolecules. In this paper, a novel chemically amplified photoresist material (bioresist) is presented and its behavior evaluated with fibroblast cells. A unique feature of this
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bioresist is that, unlike in the conventional lithographic approach, no further process of development is needed to remove the remaining resist after the photolithography patterning. The patterned resists can be used directly for the coupling of specific cell-adhesion peptides or proteins for the alignment of cells.
CH3 CH2
C m C
CH2
O
CH n N
O
O
2. Materials and methods
CH3
t-BOC
2.1. Materials
t-BOC: -COOC(CH3 )3 All the chemical reagents were obtained from Aldrich and/or Acros. N,N0 -azobisisobutyronitrile (AIBN) was purified by recrystallization in methanol. Tetrahydrofuran (THF) was distilled over sodium using benzophenone as an indicator. Methyl methacrylate (MMA) was vacuum distilled under nitrogen prior to use to remove inhibitors. Other chemicals were used as received. Triphenyl sulfonium hexafluoroantimonate (TPSHA), used as a photoacid generator (PAG) for resist formulation, was purchased from Polysciences. It was a 50 wt% solution in propylene glycol methyl ether (PGME). Monomer 3-(t-butoxycarbonyl)-N-vinyl-2pyrrolidone (TBNVP) was synthesized as described elsewhere [8]. 2.2. Bioresist synthesis MMA/TBNVP copolymer was prepared by AIBNinitiated free radical polymerization. A solution of 2.1 g of TBNVP and 0.7 g MMA was dissolved in 16 ml of THF (monomer concentration 1.0 mol/l) along with 0.0398 g of AIBN (1.5 102 mol/l) and the mixture was heated to 65–75 C under nitrogen for 24 h. To isolate the polymer, the reaction mixture was added dropwise into sufficient petroleum ether. The polymer was filtered and washed thoroughly with petroleum ether. The polymer was then redissolved in THF and reprecipitated to minimize the presence of residual unreacted monomer. Finally, the polymer was dried in vacuum at 40 C to a constant weight (2.38 g, 85% yield). The copolymer will be referred to as MMA:TBNVP (64:36), indicating that MMA comprises 64 mol% of the polymer composition. The t-BOC protection groups from the copolymer were converted into COOH groups using trifluoroacetic acid (TFA) similar to that in the homopolymer [8]. The copolymer and deprotected copolymer (shown as MMA:D-TBNVP) were deposited onto glass coverslips from solution for the cell culture studies, and their chemical structures are illustrated in Scheme 1. 2.3. Bioresist characterization Fourier transform infrared (FTIR) spectra of the polymers were recorded on a Nicolet spectrometer. For
CH3 CH2
C m C
CH2
O
CH n N
O
O CH3
COOH
Scheme 1. Chemical structures of MMA:TBNVP and MMA: D-TBNVP.
FTIR spectra, the solid polymers and KBr (spectroscopic grade) were thoroughly mixed and this mixture was pressed to form a pellet. Solution 1H NMR spectra were recorded with a Varian UNITY 300 spectrometer. Gel permeation chromatography (GPC) measurements for molecular weights and molecular weight distributions were carried out on a Waters 2410 (eluent THF, narrow molecular weight polystyrene standards, refractive index detection). Thermal analyses of the polymers were conducted using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) measurements. TGA was performed on a TA instruments TGA 2950 at a heating rate of 20 C/min in a nitrogen atmosphere. DSC was conducted on a TA instruments DSC 2920 in N2 at a heating rate of 10 C/ min for glass transition determination. Glass transition temperature (Tg ) of the polymers was obtained from the second run of DSC thermograms. 2.4. Patterning with bioresist Circular glass coverslips (VWR) of 25 mm diameter were patterned photolithographically to generate lines of 25 mm width with a 25 mm spacing with the copolymer. Patterning of the copolymer is similar to that described for the homopolymer patterning [8]. Briefly, 10 wt% of MMA:TBNVP (64:36)/PGME solution with 5 wt% of TPSHA was prepared and filtered. The copolymer film was spin-cast onto the circular glass
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and the pattern was achieved via UV lithography. The pattern was revealed by soft bake for 1 min at 120 C immediately after the exposure. No further development was performed. 2.5. Cell culture and assays All cell culture reagents were obtained from SIGMA unless otherwise noted. This study was performed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AALAC International)-approved vivarium with the approval of, and strict adherence to, the guidelines of the Institutional Animal Care and Use Committee (IACUC). Primary Wistar furth rat fibroblast (RFB) cells were isolated from rat dermis using a modified collagenase digestion procedure [9]. The isolated cells were cultured in medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20% heat-inactivated fetal bovine serum (FBS), 1.25% l-glutamine (200 nm, GIBCO), 1% fungizone (GIBCO), 1% penicillin (10,000 U/ml)-streptomycin (10 mg/ml), and 2.5% HEPES buffer solution (1 m, GIBCO). The cultures were incubated at 37 C in an 8% carbon dioxide, humidified atmosphere. 2.5.1. Cell attachment assay Following exposure to sterile phosphate buffered saline (PBS) for 5 min at 25 C, the glass coverslips coated with MMA:TBNVP and MMA:D-TBNVP were positioned on the bottom of sterile 24-well culture plates. The wells were precoated with a thin film of 12% poly(hydroxylethylmethacrylate) (PHEMA) (Polysciences) to guarantee that cells adhered only to the substrates. RFB cells were grown near confluence in T75 flasks. Cells were released from the culture flasks by incubation with 0.1% (w/v) trypsin/0.02% ethylenediaminetetraacetic acid (EDTA) in PBS at 37 C for approximately 1 min, suspended by tapping the flask, then collected by centrifugation at 1200 rpm for 5 min, and resuspended in fresh cell culture medium. Cell concentration was determined using a hemacytometer. A plating concentration of 2.5 104 cells per well in a total volume of 0.5 ml was applied to each sample. After seeding, the cultures were incubated for 2 and 6 h, respectively, at 37 C in 8% CO2 air. At the end of each period, the substrates were washed twice with PBS to remove any non-attached cells. The number of viable cells attached to the different substrates was evaluated by the CellTiter 96s AQueous One Solution Cell Proliferation Assay (PROMEGA). The reagent contains a novel tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (inner salt, MTS) and an electron coupling reagent phenazine ethosulfate (PES). In this assay, the MTS
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tetrazolium compound is bioreduced by cells into a colored formazan product. The quantity of formazan product as measured by the amount of absorbance at 490 nm is directly proportional to the number of living cells in culture. Each experimental time point was performed in triplicate. As a reference material, cells seeded on plain glass were included in each run as positive controls. Significant differences between experimental values were determined based on a 95% confidence interval (Student’s t-test). 2.5.2. Cell proliferation assay Additionally, fibroblast cell proliferation on MMA:D-TBNVP was also studied. Fibroblast cells were seeded onto the surface of polymer coated glasses at a plating density of 1 105 cells/well and cultured at 37 C in 8% CO2/air atmosphere for 7 days. Twelve-well tissue culture plates were used and the plates were coated with PHEMA. Cell growth was quantified at 1, 2, 3, 5 and 7 days by measuring the cell metabolic activity with the MTS method mentioned above. The nonattached cells were removed by rinsing with PBS prior to the assay to ensure accuracy. In each experiment, the materials were performed in triplicate, and untreated polystyrene dishes were used as negative control. 2.5.3. Study of serum effect on cell attachment Glass coverslips coated with MMA:D-TBNVP were rinsed several times with sterile PBS and placed in sixwell tissue culture plates which were pretreated with PHEMA. Plain glasses were used as positive controls. Fibroblasts were lifted from the culture flask after reaching confluence, centrifuged and resuspended. The cells were then washed three times with serum-free medium, consisting of DMEM supplemented with 1.25% l-glutamine (200 nm), 1% fungizone, 1% penicillin (10,000 U/ml)-streptomycin (10 mg/ml), 2.5% HEPES buffer solution (1 m) and 0.1% insulin-transferrin-selenium-A (GIBCO). Cells were seeded onto the samples at a plating density of 1.5 105 cells/well. For each set of polymer, cells were cultured both in serum and serum-free medium in triplicate. After 2, 6 and 24 h, respectively, the non-adherent cells were removed by washing with PBS. Adherent cells were quantified by MTS assays. Student’s t-test and one-way ANOVA were used for statistical analysis. 2.5.4. Cell culture on the patterned surfaces Fibroblast cells were also cultured on micropatterned surfaces of MMA:TBNVP. Glass cover slides with patterns were rinsed several times with sterile PBS. The patterned slides were then placed in six-well tissue culture plates precoated with PHEMA. Cells were washed with serum-free medium before seeded onto the samples at a density of 1.5 105 cells/well. Nonpatterned regular glass slides were used as a control. Cell
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cultures were carried out both in serum and serum-free medium. After seeding, fibroblast cells were allowed to stay in contact with the surfaces in an incubator at 37 C with 8% CO2 for various periods of time. At the end of each incubation period, the samples were rinsed with PBS to remove non-attached cells and cell morphology was observed using an inverted Olympus phase-contrast microscope. 2.5.5. Large-scale patterning and cell culture A 2-inch glass substrate was patterned with the bioresist MMA:TBNVP using similar techniques as described above and RFB cells were cultured on it under physiological conditions. The cells were cultured on the material for a total time of 2 weeks. The culture media were collected every 3 days and lactate production was measured by using the YellowSprings glucose/lactate analyzer (YellowSprings, Inc., YellowSprings, Ohio). A plain non-tissue culture treated polystyrene dish was used as a control. Cell morphology was examined frequently during the culture period. At the end of the 2-week incubation period, both the patterned 2-in sample and the plain polystyrene dish were fixed in formalin and stained with Masson’s Trichrome stain. Aniline blue is a component of Masson’s Trichrome stain and it stains collagen blue. Therefore, it allows for the in situ detection of total collagen matrix deposition on these samples.
3. Results and discussion This study was initiated by the fact that polymers bearing t-BOC groups can be used as photoresist materials. Polymers derived from NVP and MMA are generally biocompatible. Therefore, the copolymer of MMA and TBNVP was prepared which would combine both properties to make a bioactive photoresist which we have termed as a bioresist. In the present study, in vitro methods to evaluate the material properties were performed. In vitro investigations have previously been shown to be suitable in examining cell interactions with polymeric materials because in vitro conditions provide a relatively simplistic environment in which information on the effect of material properties on cellular adhesion and growth could be obtained without the influence of other systemic events [10,11].
protection groups were removed and the molecular weight decreased to 17,500 with a polydispersity of 1.40. In the FTIR spectra (Fig. 1), a broad peak in the region of 3400–2500 cm1 was observed for MMA:D-TBNVP, due to the O–H stretching from the carboxyl groups. In order to study the composition of the copolymer, NMR was applied. The 1H NMR spectra of the copolymers before and after deprotection are shown in Fig. 2. The peak at d1.4 is due to the tert-butyl protons, while the peak appearing at d3.5–d3.7 was assigned to the methyl protons in MMA. Before deprotection, the molar ratio of MMA:TBNVP was obtained by the ratio of integrated area per unit proton of each characteristic peak (CH3 of MMA at d3.5–d3.7 and C(CH3)3 of TBNVP at d1.4), which belonged to different compositions. From the integration, the composition of the copolymer is 64:36 molar ratio of MMA:TBNVP. In addition, the intensity of the signal for the tert-butyl protons was greatly decreased after the deprotection with TFA.
C=O
Absorbance
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(b)
(a)
4000
3000 Wavenumbers
2000
1000
(cm-1)
Fig. 1. FTIR spectra of (a) MMA:TBNVP and (b) MMA:D-TBNVP.
(b)
3.1. Bioresist—synthesis and characterization Copolymerization reactions provide an excellent way of preparing macromolecules with controlled properties such as hydrophilic/hydrophobic balances, polarity and film formation [12,13]. GPC showed that the molecular weight of MMA:TBNVP was 24,000 and the polydispersity was 1.80. After reacting with TFA, the t-BOC
(a)
Fig. 2. 1H NMR spectra of (a) MMA:TBNVP (in CDCl3) and (b) MMA:D-TBNVP (in CD2Cl2).
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The thermal behaviors of MMA:TBNVP and MMA:D-TBNVP were examined by TGA, the plot of which is shown in Fig. 3. The thermal stability of the copolymer can be used to provide an assessment of the tBOC content in the copolymer. The TGA curve of MMA:TBNVP (Fig. 3a) shows that the polymer is thermally stable until B180 C. Due to the t-BOC groups the polymer starts to lose weight at 180 C by giving off carbon dioxide and isoproprene and continues until 240 C in a nitrogen environment at a heating rate of 20 C/min. The weight loss is 25.6%, which is in very good agreement with the expected value (25.7%). After reacting with TFA (Fig. 3b), the polymer starts decomposing at B150 C, due to the t-BOC groups and the newly formed carboxyl groups. Comparing 3a and 3b, the decrease in the weight change between 140 and 240 C further indicated the removal of the t-BOC protecting groups. In the DSC thermograms, a glass transition (Tg ) at 118 C was observed for MMA:TBNVP, while a very subtle transition was noticed at 73 C for MMA:D-TBNVP. Because of the bulky t-BOC protecting groups, the mobility of polymer chains is decreased, which leads to a higher glass transition temperature. After the deprotection, the bulky t-BOC group is replaced by a much smaller carboxyl group, which also has the great possibility of forming intramolecular H-bonding between the OH and the carbonyl group on the two-position of the lactam ring. It enables polymer chain motion occurring at a lower temperature, i.e. a lower Tg [14]. 3.2. Cell culture on bioresist For anchorage-dependent cells, such as fibroblasts, adhesion is the critical prerequisite for subsequent cell functions such as cell proliferation and deposition of extracellular matrix (ECM). Therefore, initial attachment is a very critical process for cell growth on
materials. To study how fibroblast cells would interact with the copolymer before and after deprotection, cells were cultured on both MMA:TBNVP and MMA: D-TBNVP surfaces for 2 and 6 h, respectively, and the number of cells attached was measured by the absorbance at 490 nm obtained by MTS assay. As shown in Fig. 4, cells respond differently to various surfaces. Two hours after seeding cells onto the materials, there was a minimal cell attachment on the MMA:TBNVP and MMA:D-TBNVP surfaces as compared to the plain glass surface. When the incubation time was increased to 6 h, the absorbance was significantly (po0:05) increased on MMA:D-TBNVP samples compared to that of 2 h. The increment is much more obvious than that of the MMA:TBNVP samples. It indicates that cells adhered better on the MMA:D-TBNVP surfaces where COOH groups were present. The difference in cell adhesion on these surfaces could be due to different surface chemistries of the materials. The exposure of the carboxyl groups by deprotection of the t-BOC groups made MMA:D-TBNVP more hydrophilic than MMA:TBNVP and also changed the surface electric charge. In addition, these carboxyl groups can be used for the further incorporation of drugs or other bioactive agents [15–19]. For example, Hyun et al. [20] have used microstamping to pattern a cell-adhesive Arg-Gly-Asp (RGD) peptide onto the surface of a COOH-derivatized comb polymer. Cell–material interactions are mainly due to the interactions of cell-adhesion proteins (ligands) bound to a material surface and cell surface receptors (integrins). The tripeptide RGD is known as the active sequence of adhesive proteins of the ECM that binds to the integrin receptors [21]. Therefore, RGD-containing peptides could be covalently coupled to the polymeric material through the COOH to promote cellular adhesion. The growth behavior of fibroblasts on MMA: D-TBNVP is shown in Fig. 5. As shown the cells not only attached on the surface, but also proliferated over a 7-day culture period indicating that the material does not hinder cell growth. There was a 3-day lag period on Attachment 0.3 Absorbance at 490 nm
Fig. 3. TGA thermograms of (a) MMA:TBNVP and (b) MMA: D-TBNVP.
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0.25 0.2
MMA:TBNVP MMA:D-TBNVP
0.15
Control
0.1 0.05 0 2
6
Incubation Time (hours)
Fig. 4. Results for cell attachment assay. Averages and standard deviations are plotted based on a sample size of three.
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the MMA:D-TBNVP prior to the cells reaching the exponential phase of growth. Generally, fibroblast cell growth on tissue culture treated polystyrene (TCPS) enters exponential growth phase by day 1 post-seeding (data not shown). 3.3. Study of serum effect on cell attachment A further study was conducted in order to determine whether surface chemistry directly affected cell adhesion or indirectly by influencing serum protein adsorption. As mentioned above, cell adhesion on MMA:TBNVP and MMA:D-TBNVP was different. Various hypotheses may be proposed to explain such a difference. It is well known that surface properties of polymers affect cell adhesion, spreading and proliferation via their adsorptive properties with respect to serum proteins. In order to determine whether surface chemistry of the copolymer has any direct influence on cell attachment, fibroblast cells were cultured on these different polymer surfaces both in serum and serum-free medium. Results shown in Fig. 6 indicated that better cell adhesion on MMA:D-TBNVP correlates with serum protein adsorption. At each selected time point, the MTS absorbance obtained in the presence of serum is significantly higher than serum-free conditions. This observation may be due to the physical properties of the material such as its Proliferation 300000
Cell Number
250000 200000 150000
MMA:D-TBNVP
100000
Control
50000 0 1
-50000
2
3
5
7
Incubation Time (days)
Fig. 5. Fibroblast proliferation (mean7SD, n ¼ 3) in direct contact with MMA:D-TBNVP and control (a plain polystyrene dish) evaluated by MTS assay up to 7 days.
Absorbance at 490 nm
Serum vs. Serum-free 0.5 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0
I I
II
I
II
II
Control MMA:DTBNVP
2
6
24
Incubation Time (hours)
Fig. 6. Cell culture in both serum and serum-free condition. Values represent the mean7SD of three samples (I: serum; II: serum-free).
hydrophilic nature and its chemical composition. These components are important in promoting and presenting serum proteins to the cells in the appropriate configuration. For most synthetic polymer surfaces, adhesion of cells requires the presence of serum and, therefore, this optimum is probably related to the ability of proteins, such as fibronectin [22,23] and vitronectin [22,24], to absorb to the surface. Many recent studies have demonstrated the involvement of serum proteins in the attachment of various cells to the substrate. For example, the attachment of endothelial cells is dependent upon vitronectin [25]. Studies have also shown that fibronectin and vitronectin adsorption and conformation on surfaces are related to the characteristics of the surfaces such as wettability [26]. 3.4. Cell culture on patterned bioresist surfaces Because of the t-BOC groups, MMA:TBNVP can be used as a chemically amplified photoresist in the presence of a PAG. The process of patterning is controlled by the successful creation of differences between the exposed and unexposed areas in terms of the hydrophobic/hydrophilic nature of the material and its chemistry. Micron-sized patterns were obtained with MMA:TBNVP using deep UV lithography. The exposed areas are rich in carboxylic groups due to the photolysis of the t-BOC groups. The features were revealed without any treatment of development, which means the patterns are actually composed of alternating MMA:TBNVP and MMA:D-TBNVP. The surface of the unexposed MMA:TBNVP resist is hydrophobic, while the hydrophilic areas in the exposed region are created due to the formation of carboxylic groups. Fibroblast cells were cultured on these patterned surfaces both in serum and serum-free medium for various periods of times. The cell morphology is shown in Figs. 7 and 8. Fig. 7 shows the optical micrographs of cells cultured in serum containing medium. After 24 h, cells were randomly attached on the plain glass with spindle shapes (Fig. 7a), while on the patterned surface, cells were aligned along the patterns in a longitudinal fashion (Fig. 7b), i.e. they were exclusively attached in the exposed area, where MMA:D-TBNVP was produced. We did see cells reaching out to their adjacent neighbors occasionally, due to the short distance between the patterned lines (25 mm). Such contact could be avoided with increased distance between the patterns. This finding is consistent with the data shown above, where cells adhered preferentially to the deprotected copolymer. After 2 days of incubation, compared to the random proliferation on the plain surface, cells were growing in a more organized way. Because of proliferation, cells bridged across the patterns and formed a confluent layer on top of the pattern: the main vector of growth is along the direction of the pattern.
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(a)
(c)
(b)
(d)
unexposed
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exposed
Fig. 7. Optical micrographs of fibroblast cells cultured on the plain glass surface (a, c) and the patterned photoresist surface (b, d) after 24 h (a, b) and 48 h (c, d) incubation in serum containing medium, respectively (20 magnification, bar=50 mm).
(a)
(c)
(b)
(d)
Fig. 8. Optical micrographs of fibroblast cells cultured on the plain glass surface (a, c) and the patterned photoresist surface (b, d) after 24 h (a, b) and 48 h (c, d) incubation in serum-free medium, respectively (10 magnification, bar=100 mm).
To assess the effect of serum components on the alignment phenomenon, we seeded cells on the patterned sample in the absence of serum proteins. Under such condition, cells did attach on both plain and patterned surfaces, but they remained rounded which indicated non-specific cell adhesion. In addition, the number of cells attached was less compared to those that were seeded in the presence of serum. As shown in Fig. 8, after 2 days of incubation, cells began to spread on the plain surface, while on the patterned surface, they
remained rounded. It is worth noting that on the patterned surface, the majority of the cells attached to the exposed area (MMA:D-TBNVP), which implies that in the absence of serum, cells were attracted to the exposed area more than the unexposed area (MMA:TBNVP). By comparing serum versus serum-free conditions, we reached a conclusion that cell alignment on the patterned surface is mostly related to serum protein adsorption. Serum proteins preferentially adsorbed onto
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the exposed area, which is more hydrophilic, and led to cell attachment in these areas. In this study, the surface is essentially coplanar, thereby eliminating topographical cues such as contact guidance. An important advantage of this bioresist is that it could be combined with the well-developed lithographic process to generate patterns as large as 8 in. To demonstrate this scalability, we used the bioresist on a 2-inch glass substrate and cultured fibroblast cells on it for a period of 2 weeks. Cell metabolism was tracked during the incubation. Fig. 9 shows the results of the lactate production rate of cells cultured on the patterned surface as well as the control. As seen there is an increase in the rate of lactate production for cells growing on the patterned surface, which indicates that cells were more metabolically active. Upon visual inspection, cells on the patterned sample appeared to have a higher density than that on the control. Although a direct cell count to correlate a per cell production of lactate was not preformed, it appears that the increase in the rate of lactate production may be due to an increase in cell number on the patterned surface. The morphology of the cells cultured on the patterned 2-in sample was similar to the 1-in sample, where cell alignment was observed. In addition, the cell-seeded disks were fixed and stained with Masson’s Trichrome stain for the detection of collagen deposition on the samples. The result is shown in Fig. 10. There was only a small area of the control sample that remained adhered after going through the fixation and staining process. This may be
due to the low deposition of ECM proteins and/or the non-specific binding of cells on the surface of polystyrene material. The cells had a random appearance of cell growth with a disorganized pattern of collagen deposition. In comparison, the patterned surface had a welldefined matrix deposition pattern that appears to be oriented along the patterned surface. This was observed throughout the sampled area. Similar formation of adhesive and non-adhesive domains via photolithography process on the classical DNQ/novolak resist was previously reported for neuronal cell culture [27]. In the study diazo-naphthoquinone/novolak photoresist was used. Since the main component in the DNQ/novolak system is the aromatic rings, there is a concern that this polymer could be toxic. Our system is non-toxic, and additionally, the COOH groups formed on the surface of the polymer can be used for further surface modifications. It also implies a potential for expanding conventional lithography application in large-scale direct cell patterning based on the bioresist. Cell alignment observed in this study implied a potential application of using this technique together with 3-D structures to generate an oriented tissue-like construct from fibroblasts, which will be mechanically strong yet have certain flexibility similar to the normal tissue. The ability to control cell orientation in tissue engineering has been shown to be critical for the development of advanced forms of tissue repair and cell engineering therapies, such as peripheral nerve repair, production of tendon and ligament substitutes in vitro, and the control of microvascular repair [28].
Lactate Production Rate (∆Lactate/∆Day)
Lactate Production of Fibroblast Cells 3.5 3 2.5 2
4. Conclusions control pattern
1.5 1 0.5 0 1
4
7
12
Days
Fig. 9. Lactate production rate of fibroblast cells cultured on the control surface (a plain polystyrene dish) and the patterned 2-in substrate.
(a)
A novel photoresist material has been assessed in terms of its biocompatibility and potential for patterning of fibroblast cells. Photo-induced chemical transformation of the surface of the resist thin film resulted in changes in the surface hydrophobic/hydrophilic properties and surface chemistry. These changes affected protein adsorption that led to cell seeding and alignment on the patterned surfaces. This development-free,
(b)
Fig. 10. Masson’s Trichrome stain (blue dye) for collagen deposition on (a) control (a plain polystyrene dish) and (b) patterned surfaces after 2 weeks of culture (200 magnification).
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patternable, and ‘‘cell-friendly’’ resist presents the potential of solving the solvent problem in applying conventional lithographic processes for cell and protein patterning. Our ongoing studies are directed to coupling the RGD peptide to the surface through the COOH groups and study its effect on cell adhesion. Additionally, more patterns will be processed with various shapes and dimensions, and different cell types will be tested on these cell-friendly resist surfaces. Acknowledgements KEG acknowledges partial funding from the North Carolina Biotechnology Center, Durham, NC. References [1] Healy KE, Thomas CH, Rezania A, Kim JE, Mckeown PJ, Lom B, Hockberger PE. Kinetics of bone cell organization and mineralization on materials with patterned surface Chemistry. Biomaterials 1996;17:195–208. [2] Patel N, Padera R, Sanders GH, Cannizzaro SM, Davies MC, Langer R, Roberts CJ, Tendler SJ, Williams PM, Shakesheff KM. Spatially controlled cell engineering on biodegradable polymer surfaces. FASEB J 1998;12:1446–54. [3] Bhatia SN, Balis UJ, Yarmush ML, Toner M. Probing heterotypic cell interactions: hepatocyte function in microfabricated cocultures. J Biomater Sci Polym Ed 1998;9:1137–60. [4] Rowe CA, Scruggs SB, Feldstein MJ, Golden JP, Ligler FS. An array immunosensor for simultaneous detection of clinical analytes. Anal Chem 1999;71:433–9. [5] Kane RS, Takayama S, Ostuni E, Ingber DE, Whitesides GM. Patterning proteins and cells using soft lithography. Biomaterials 1999;20:2363–76. [6] Xia Y, Whitesides GM. Soft lithography. Angew Chem Int Ed 1998;37:550–75. [7] Douvas A, Argitis P, Misiakos K, Dimotikali D, Petrou PS, Kakabakos SE. Biocompatible photolithographic process for the patterning of biomolecules. Biosens Bioelectron 2002;17:269–78. [8] He W, Gonsalves KE, Pickett JH, Halberstadt CR. Synthesis, characterization, and preliminary biological study of poly(3-(tbutoxycarbonyl)-N-vinyl-2-pyrrolidone). Biomacromolecules 2003;4:75–9. [9] Doyle A, Griffiths JB, Newell DG. Dissociation of dermal fibroblasts in cell and tissue culture: laboratory procedures. New York: Wiley; 1996. [10] Peppas NA, Langer R. New challenges in biomaterials. Science 1994;263:1715–20. [11] Morrison C, Macnair R, MacDonald C, Wykman A, Goldie I, Grant MH. In vitro biocompatibility testing of polymers for orthopedic implants using cultured fibroblasts and osteoblasts. Biomaterials 1995;16:987–92.
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