Article pubs.acs.org/ac
Comparison of Biocompatibility and Adsorption Properties of Different Plastics for Advanced Microfluidic Cell and Tissue Culture Models Paul M. van Midwoud,†,‡,§ Arnout Janse,† Marjolijn T. Merema,‡ Geny M. M. Groothuis,‡ and Elisabeth Verpoorte*,† †
Pharmaceutical Analysis and ‡Pharmacokinetics, Toxicology and Targeting, Department of Pharmacy, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands S Supporting Information *
ABSTRACT: Microfluidic technology is providing new routes toward advanced cell and tissue culture models to better understand human biology and disease. Many advanced devices have been made from poly(dimethylsiloxane) (PDMS) to enable experiments, for example, to study drug metabolism by use of precision-cut liver slices, that are not possible with conventional systems. However, PDMS, a silicone rubber material, is very hydrophobic and tends to exhibit significant adsorption and absorption of hydrophobic drugs and their metabolites. Although glass could be used as an alternative, thermoplastics are better from a cost and fabrication perspective. Thermoplastic polymers (plastics) allow easy surface treatment and are generally transparent and biocompatible. This study focuses on the fabrication of biocompatible microfluidic devices with low adsorption properties from the thermoplastics poly(methyl methacrylate) (PMMA), polystyrene (PS), polycarbonate (PC), and cyclic olefin copolymer (COC) as alternatives for PDMS devices. Thermoplastic surfaces were oxidized using UV-generated ozone or oxygen plasma to reduce adsorption of hydrophobic compounds. Surface hydrophilicity was assessed over 4 weeks by measuring the contact angle of water on the surface. The adsorption of 7-ethoxycoumarin, testosterone, and their metabolites was also determined after UV-ozone treatment. Biocompatibility was assessed by culturing human hepatoma (HepG2) cells on treated surfaces. Comparison of the adsorption properties and biocompatibility of devices in different plastics revealed that only UV-ozone-treated PC and COC devices satisfied both criteria. This paper lays an important foundation that will help researchers make informed decisions with respect to the materials they select for microfluidic cell-based culture experiments.
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above. Thermoplastic materials are increasingly used for the fabrication of cell culture devices;11,12 however, a lot of work is still needed to characterize the properties of thermoplastics.13 There is a broad selection of materials to choose from, so that a plastic with properties appropriate for the application at hand can generally be chosen. In conventional cell culture experiments, plastics like polycarbonate (PC) and polystyrene (PS) are used to produce culture flasks and well plates. These plastics are hydrophobic in their native form but can easily be made more hydrophilic by oxidizing the surface.14 In addition, surfaces can be chemically modified as shown by Soper et al.15 Flasks and well plates whose surfaces have been oxidized are referred to as “tissue-culture-treated”, meaning that cells can generally be successfully cultured on or in the vicinity of these surfaces.16 However, the existing literature is lacking in recent papers describing good comparative studies of different
icrofluidic technology is being increasingly utilized to develop advanced cell culture models, which are useful in vitro tools to better understand human biology and disease.1,2 By applying microfluidic technology for cell culturing, complex organ architectures can be mimicked,3 and medium flow can be applied and altered during an experiment to resemble the natural cell environment.1,2 These new in vitro chips are made primarily of poly(dimethylsiloxane) (PDMS) due to attractive physical and mechanical properties (e.g., biocompatibility, low cost, optical transparency, ease of fabrication, and gas permeability).4 PDMS is an ideal polymer to develop prototypes for the incorporation of cells or tissue on microfluidic chips, enabling experiments that are not achievable with conventional systems.5,6 Unfortunately, various studies have demonstrated the disadvantages of the use of PDMS for cell studies. PDMS can adsorb small hydrophobic molecules (like drugs),7,8 leach un-cross-linked oligomers into solution,9 and return to its hydrophobic state after surface treatment.10 As mentioned by Meyvantsson and Beebe,2 thermoplastics are the most desirable for microfluidic cell cultures and preferable to PDMS because of the disadvantages mentioned © 2012 American Chemical Society
Received: November 3, 2011 Accepted: March 23, 2012 Published: March 23, 2012 3938
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EXPERIMENTAL SECTION Information regarding chemicals, materials, detailed experimental procedures, and equipment can be found in the Supporting Information. Surface Treatment. The hydrophobicity of native plastic surfaces was altered by oxidizing the surface with UV-ozone or oxygen plasma. Samples of PMMA, PC, PS, COC, and PDMS with dimensions of 15 mm × 30 mm (L × W) were exposed to UV-ozone for 15, 30, 45, or 60 min or to oxygen plasma for 30, 60, 90, or 120 s. The effect of exposure on hydrophobicity was assessed by measuring the contact angle of water with the surface (see Supporting Information). Nine samples per plastic substrate underwent oxidation under each different set of conditions. Three pieces were characterized directly after treatment and the rest were aged in air to assess how surface characteristics changed over time. Adsorption of Hydrophobic Compounds. The adsorption of compounds onto the plastic substrates was measured by flushing medium containing 7-ethoxycoumarin (7-EC) or testosterone (TT) and their metabolites for 2 h through a microchamber formed in a sealed microfluidic chip. The chemical structures of the substrates with their metabolites are given in the Supporting Information (Figure S1). Chips were fabricated from the different plastics of interest using a hot embosser (Dr. Collin GmbH, Ebersberg, Germany). A schematic view of the chip is given in Figure 1. The total
thermoplastics for the fabrication of hydrophilic microfluidic devices for culturing cells. One paper, published by Welle and Gottwald in 2002,17 compared different plastics for cell culture applications by looking at the adhesion of HepG2 and L929 fibroblasts to UV-patterned surfaces; unfortunately, however, PDMS was not considered in this study. In addition, the paper did not discuss the adsorption of compounds and/or metabolites, an issue that has recently grown in importance, or the viability of the cells adhering to the surfaces, another critical aspect of microfluidic cell culture. Oxidation of microfluidic devices made of thermoplastics or PDMS to make surfaces more hydrophilic is easily achieved using a UV-ozone or oxygen-plasma apparatus, found in many microfluidic research laboratories. However, both UV-ozone and oxygen-plasma oxidation treatments result in temporary hydrophilicity only, with substrates returning to their hydrophobic state over time, a process known as hydrophobic recovery.10 Hydrophobic recovery has been described by several research groups for PDMS.18,19 Less information is available in the literature about the hydrophobic recovery of thermoplastics like PC and PS. This effect has been studied for a few plastics after oxygen-plasma treatment20,21 but has not been assessed for the plastics tested in this study after UVozone treatment. Hydrophilic surfaces tend not to adsorb hydrophobic compounds, which makes them suitable for metabolism and toxicity studies with hydrophobic compounds. However, an issue of concern is that surface treatment by UVozone might be toxic to cells or tissues, due to products, like peroxides, being formed on the surface during or after treatment.17,22 The aim of this study is to test several thermoplastics for their applicability as alternative materials to PDMS for the incubation and cultivation of cells and tissue without significant adsorption of hydrophobic compounds in the medium or formed by the cells (e.g., metabolites). A suitable plastic should (1) lend itself to the production of multiple chips in a highthroughput manner, (2) not adsorb hydrophobic compounds either before or after surface treatment to reduce hydrophobicity, and (3) not be toxic to cells after a surface treatment to reduce hydrophobicity. In this study, polycarbonate (PC), polystyrene (PS), poly(methyl methacrylate) (PMMA), and cyclic olefin copolymer (COC, Zeonor) were tested. All these polymers are thermoplastics and can therefore be used in injection molding or hot embossing to produce chips in bulk.23,24 The plastic surfaces were oxidized using UV-ozone and oxygenplasma treatment, and hydrophilicity was assessed by measuring the contact angle of water on the surface (angle of liquid-tosolid surface).25 Hydrophobic recoveries were also determined 1 and 4 weeks after treatment. The adsorption of 7ethoxycoumarin (7-EC) and testosterone (TT) and their liver-specific metabolites were measured after treatment. These drugs are frequently used as model compounds to test the metabolic activity of liver tissue or liver cells. Both 7-EC and TT and some of their metabolites were previously found to adsorb to PDMS.26,27 Finally, as UV-ozone appeared somewhat superior to oxygen-plasma treatment with respect to reduced adsorption, the toxicity of the plastics after UV-ozone treatment was assessed by 24-h cultivation of liver carcinoma cell line (HepG2) on the substrates. This comparative study of different thermoplastics reveals which thermoplastic is most suitable for the fabrication of microfluidic cell culture models.
Figure 1. Schematic view of the chip used for adsorption and cell culture studies. The chamber dimensions are diameter 4 mm × 200 μm (total volume ∼3.5 μL), and the flow rate of medium used was 5 μL/min.
volume of the device was approximately 3.5 μL. An extensive description of the fabrication process can be found in the Supporting Information. The experiments were performed in triplicate on three different chips made from the same material. Biocompatibility Study. Biocompatibility of the plastics and elastomer was tested by culturing HepG2 cells on the substrates. Cells were cultured in the structure shown in Figure 1 as well as on flat substrates. The substrates were coated with lyophilized rat-tail collagen (Roche, Basel, Switzerland) after UV-ozone treatment. As control, the cells were also cultivated in tissue-culture-treated polystyrene well-plates (Corning Costar, Amsterdam, The Netherlands). After a 24-h cultivation, the cells were exposed to acridine orange and propidium iodide to visualize living and dead cells simultaneously. Details regarding the cell culture and staining process can be found in the Supporting Information. The HepG2 cell experiment was 3939
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Figure 2. Contact angles of UV-ozone-treated (a) PDMS, (b) PC, (c) PS, (d) PMMA, and (e) COC as a function of treatment time. The hydrophobic recovery was measured after 1 week (white bars) and 4 weeks (gray bars). Black bars represent the contact angle of the substrates 2 h after treatment. Results are average ± standard deviation of three separate measurements on three pieces of substrate (nine measurements in total for each type of substrate, treatment, and time point after treatment).
Effect of UV-Ozone Treatment on Hydrophilicity. The results of contact-angle measurements of PDMS, PC, PS, PMMA, and COC treated with UV-ozone and stored for different lengths of time are given in Figure 2. Contact angles were measured 2 h after treatment for the first set of samples. All substrates show a decrease in contact angle after UV-ozone irradiation, indicating increased hydrophilicity. All the plastic substrates showed a tremendous decrease in contact angle, and very hydrophilic surfaces were obtained in all cases after 60 min of treatment. A contact angle of 10° for PC and PS means that many functional oxygen-containing groups are formed, resulting in a substantially charged surface that may not be suited to cell experiments for reasons discussed above. However, the contact angle of PDMS measured 2 h after treatment decreased only from 110° to approximately 70° after 60 min of treatment. The contact angle is probably low directly after treatment, as observed by others;10,18 however, it appears that hydrophobic recovery was so rapid in the first 2 h that substantial recovery had occurred before the first contact-angle measurements were made.18 It is believed that free siloxanes from the bulk migrate to the surface to return it to its native state.19 To achieve preferred contact angles of about 50° 1 week after treatment, PC and PS required only 15 min of treatment. PMMA and COC needed a somewhat longer UV-ozone oxidation of 30 min to exhibit contact angles of ∼50°, as
performed in triplicate on three different chips made from the same material.
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RESULTS AND DISCUSSION
Surface Treatment. Treatment of plastic substrates by oxidizing the surface results in a more hydrophilic surface. Oxygen-containing functional groups are formed on the surface layer during treatment, which results in higher surface free energy and lower hydrophobicity.28 This will prevent the adsorption of hydrophobic compounds. However, when the exposure time of ozone is too long, the large number of oxygencontaining groups formed on the surface can result in a substantially charged surface. This extremely hydrophilic surface will not adsorb hydrophobic compounds; however, electrostatic interaction might occur with charged compounds present in the medium.29 There is, therefore, a tipping point with respect to ozone exposure. On the other hand, however, exposures that are too short result in surfaces that exhibit significant hydrophobic interaction. The surface energy can be assessed by contact angle measurements, which give a good indication of the hydrophobicity of a substrate.25 The measured contact angle of the polystyrene tissue-cultured well plates was 45° ± 4°. Therefore the preferred contact angle is 40−50°, to prevent both adsorption of hydrophobic compounds and possible electrostatic interactions with charged surfaces. 3940
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Figure 3. Contact angles of oxygen-plasma-treated (a) PDMS, (b) PC, (c) PS, (d) PMMA, and (e) COC as a function of treatment time. The hydrophobic recovery was measured after 1 week (white bars) and 4 weeks (gray bars). Black bars represent the contact angle of the substrates 1 h after treatment. Results are average ± standard deviation of three separate measurements on three pieces of substrate (nine measurements in total for each type of substrate, treatment, and time point after treatment).
observed previously by others.29 For PC, PS, and COC, hydrophobic recovery was hardly observed between the 1- and 4-week measurement periods. In fact, PC and PS showed minimal hydrophobic recovery over this period after a treatment of 15 min with the UV-ozone cleaning system and could be used up to at least 4 weeks after treatment. COC substrates treated for 30 min do undergo more significant hydrophobic recovery, although the contact angle is still ∼50° after 4 weeks. COC could also be used up to 4 weeks after treatment. However, a relatively high recovery was observed for PMMA between 1 and 4 weeks after treatment, with measured contact angles almost back at untreated-PMMA values for samples treated for 30 min. Therefore, this substrate can be used for only 1 week after treatment. The hydrophobic recovery is expected to be due to reorientation of side groups on the molecules, as newly formed polar groups bury themselves in the subsurface, as shown for polyethylene and polypropylene by Truica-Marasescu et al.30 Effect of Oxygen-Plasma Treatment on Hydrophilicity. Oxygen-plasma treatment is harsher than UV-ozone treatment. This is because oxygen plasma contains particles with high kinetic energies, which result in a far greater production of ozone in a given time than the UV-ozone technique.19 Therefore, the exposure times required to achieve optimal contact angles will be much shorter than those used in
UV-ozone treatment, with times on the order of seconds rather than minutes. Figure 3 shows the contact angles of the different plastic substrates and PDMS after oxygen-plasma treatment. In contrast to the UV-ozone experiments, the first set of measurements was performed just one hour after treatment instead of two. As in the UV-ozone case, the contact angles of all substrates decreased upon exposure to ozone produced in the oxygen plasma. PDMS showed a very low contact angle after oxygen plasma treatment ( COC > PC > PS > PMMA).41 This gas permeability is important to maintain sufficient oxygen and carbon dioxide levels in the medium. From the perspective of gas permeability, COC and PC are preferred over PS and PMMA. Due to this low gas permeability in plastics, it is very important to introduce a flow in microfluidic cell culture devices when thermoplastic materials are used, to maintain stable and sufficient oxygen and carbon dioxide levels (important for maintaining a constant pH) over time. A flow of 5 μL/min was sufficient for our device.
Figure 5. Viability of HepG2 cells cultured on different thermoplastics and PDMS after UV-ozone treatment and collagen coating. Live cells appear green, and dead cells are red. The polymers tested were (a) PDMS, (b) PC, (c) PS, (d) PMMA, (e) COC, and (f) well plates (polystyrene). (g) The number of dead cells as a percentage of the total number of cells present. Results are average ± standard deviation of three separate devices for each substrate measured for three different HepG2 passages (nine measurements in total for each type of substrate). PC and PS were treated with UV-ozone for 15 min, and PMMA, COC, and PDMS were treated for 30 min.
to increase the hydrophilicity of the materials tested, resulting in no adsorption of 7-EC and TT and their metabolites in PS, PC, and COC. However, even after treatment, PDMS and PMMA exhibited a lower recovery of the hydrophobic compounds. An additional advantage of oxidizing the plastic surfaces was the lowered glass-transition temperature at the surface. This facilitated the bonding of substrates at temperatures below the glass transition point of the bulk of the device, preventing deformation of the hot-embossed, microfluidic structure. It was possible to fabricate all devices in the tested thermoplastics with the same instrument, including thermal bonding, without deformation of the structure.
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CONCLUSIONS The ability to culture cells in microfluidic devices is a powerful tool for studying cellular functions under conditions that mimic the in vivo situation. Thermoplastics are the most desirable for microfluidic cell cultures; however, they adsorb hydrophobic compounds due to their native surfaces being hydrophobic. It was shown that both UV-ozone and oxygen plasma can be used 3943
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UV-ozone treatment is preferred over oxygen-plasma treatment because the hydrophilicity of the surface was more stable after this treatment than after oxygen-plasma treatment. After UV-ozone treatment and coating with collagen, HepG2 cells adhered to the surface of all materials with high viability, with the exception of PMMA. This is probably due to unstable peroxides present at the PMMA surface that are formed during UV treatment. The gas permeability and chemical resistance of PC and COC to organic solvents are better than those of PS.33 When devices are flushed with organic solvents, as required for, say, liquid chromatography on-chip, the use of PC or COC is recommended. COC has the additional advantage over PC of exhibiting a lower autofluorescence, which is beneficial for optical imaging.42 In conclusion, microfluidic devices made from PC after a 15-min UV-ozone oxidation, or COC after a 30-min oxidation, are suitable for the incorporation of cells or tissue. These thermoplastics allow the low-cost production of biocompatible devices with low-adsorption profiles for both hydrophobic and hydrophilic compounds.
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ASSOCIATED CONTENT
S Supporting Information *
Additional text, one table, and one figure as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail
[email protected]; telephone +31 50 363 3337; fax +31 50 363 7582. Present Address §
Laboratory of Food & Nutrition Toxicology, ETH Zurich, Schmelzbergstrasse 9, 8092, Zurich, Switzerland. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Hella Logtenberg and Dr. Wesley R. Browne (Stratingh Institute for Chemistry, University of Groningen) for the use of their apparatus to measure contact angles.
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