Langmuir 2008, 24, 14133-14138
14133
Control of Neural Stem Cell Adhesion and Density by an Electronic Polymer Surface Switch Carmen Salto´,† Emilien Saindon,‡ Maria Bolin,‡ Anna Kanciurzewska,§ Mats Fahlman,§ Edwin W. H. Jager,‡ Pentti Tengvall,| Ernest Arenas,† and Magnus Berggren*,‡ Molecular Neurobiology, MBB, Karolinska Institutet, SE-171 77 Stockholm, Sweden, Organic Electronics, ITN, Linko¨ping UniVersity, SE-601 74 Norrko¨ping, Sweden, Organic Physics, ITN, Linko¨ping UniVersity, SE-601 74 Norrko¨ping, Sweden, and Applied Physics, IFM, Linko¨ping UniVersity, SE-581 83 Linko¨ping, Sweden ReceiVed August 29, 2008. ReVised Manuscript ReceiVed September 30, 2008 Adhesion is an essential parameter for stem cells. It regulates the overall cell density along the carrying surface, which further dictates the differentiation scheme of stem cells toward a more matured and specified population as well as tissue. Electronic control of the seeding density of neural stem cells (c17.2) is here reported. Thin electrode films of poly(3,4-ethylenedioxythiophene) (PEDOT):Tosylate were manufactured along the floor of cell growth dishes. As the oxidation state of the conjugated polymer electrodes was controlled, the seeding density could be varied by a factor of 2. Along the oxidized PEDOT:Tosylate-electrodes, a relatively lower density of, and less tightly bonded, human serum albumin (HSA) was observed as compared to reduced electrodes. We found that this favors adhesion of the specific stem cells studied. Surface analysis experiments, such as photoelectron spectroscopy, and water contact angle measurements, were carried out to investigate the mechanisms responsible for the electronic control of the seeding density of the c17.2 neural stem cells. Further, our findings may provide an opening for electronic control of stem cell differentiation.
Polymers, in vastly different forms, have extensively been explored as coatings to promote biocompatibility and to serve as the substrate in cell and biomolecule analysis. Their flexible and nonvolatile character, and the many degrees of freedom at the synthesis level, all together make polymers the natural material choice to promote specific cell-hosting and cell-adhesion characters for multiple applications. One of the most interesting applications of polymers is in the field of stem cell biology. Stem cells are capable of self-renewing, and, at the same time, they can give rise to multiple cell types. For instance, a neural stem cell can generate the cell types that characterize the nervous system: neurons, astrocytes, and oligodendrocytes. Neural stem cells are an excellent tool to study the processes that lead from one cell to a tissue. From the cell-seeding event until reaching the final state, in the form as a cell cluster or a tissue, cells pass through a cascade of intermediate states involving adhesion, proliferation, and differentiation. At each of these states, different biomolecules, including peptides, proteins, lipids, and polysaccharides, serve as the chemical cues to guide and drive the cellto-tissue evolution. In culture, the chemical and physical character of the cell-hosting surface contributes in predicting the final fate of the cell system, as much as neighbor cells regulate the fate of a new cell in a tissue during development. One important component in this process is the conformation and orientation of adhesive biomolecules. Major efforts are today devoted to understand the mechanisms by which synthetic devices and substrates interface with various biological systems, in vitro as well as in vivo. Within the area of cell growth-promoting materials and biocompatibility, one particular area aims to achieve scaffolds * Corresponding author. † Karolinska Institutet. ‡ Organic Electronics, Linko¨ping University. § Organic Physics, Linko¨ping University. | Applied Physics, Linko¨ping University.
and surfaces for nerve tissue engineering in order to improve the repair effect of traumatic injuries of the peripheral and central nervous systems.1 In this respect, stem cell therapy is today considered a very promising path, and various attempts are performed to manufacture stem cell-hosting architectures that promote a desired differentiation scheme exhibiting a specific function. Hydroxylapatite surfaces, precoated with Arg-Gly-Asp (RGD)-peptides, have been shown to interact with serum proteins to improve adhesion and spreading of mesenchymal stem cells.2 Astrocytes respond to polystyrene surfaces micropatterned with laminin by orienting parallel to the micropatterned grooves.3 Neurons also send processes, axons, and dendrites, in all three dimensions. It is therefore necessary to support and control the seeding and growth of stem cells not only along planar geometries but also in three dimensions. Biodegradable microfibers of poly(Llactide-co-glycolide) have been successfully used to improve the adhesion and to regulate the three-dimensional (3D)-growth orientation of a neural stem cell line, named c17.2.4 Similar results were obtained using braided fiber structure scaffolds.5 c17.2 neural stem cells are derived from postnatal mouse cerebellum and are known to give rise to all cell types in the nervous system, not only in vivo but also in vitro, allowing one to model development.6 Besides physical patterns and chemical cues, electrical stimulation has also proven to be useful in controlling different growth stages of cell systems. This is particularly true for neurons (1) Schmidt, C. E.; Leach, J. B. In Annual ReView of Biomedical Engineering; Annual Reviews: Palo Alto, CA, 2003; Vol. 5293-347. (2) Sawyer, A. A.; Hennessy, K. M.; Bellis, S. L. Biomaterials 2005, 26, 1467–1475. (3) Recknor, J. B.; Recknor, J. C.; Sakaguchi, D. S.; Mallapragada, S. K. Biomaterials 2004, 25, 2753–2767. (4) Bini, T. B.; Shujun, G.; Tan, T. C.; Shu, W.; Lim, A.; Hai, L. B.; Ramakrishna, S. Nanotechnology 2004, 15, 1459–1464. (5) Bini, T. B.; Shujun, G.; Shu, W.; Ramakrishna, S. J. Mater. Sci. 2006, 41, 6453–6459. (6) Snyder, E. Y.; Deitcher, D. L.; Walsh, C.; Arnoldaldea, S.; Hartwieg, E. A.; Cepko, C. L. Cell 1992, 68, 33–51.
10.1021/la8028337 CCC: $40.75 2008 American Chemical Society Published on Web 11/21/2008
14134 Langmuir, Vol. 24, No. 24, 2008
that inherently transmit electrochemical signals throughout the nervous system. Also, the combination of electrical stimuli and biochemical cues are known to enhance, for instance, neurite outgrowth. Conducting polymer electrodes have proven to be excellent transducers for signaling across the biology-technology interface.7 Recently, polypyrole (PPy) electrodes coated with neural growth factors8 were used to investigate the differentiation of PC12 cells. It is known that several different electrically conducting polymers exhibit excellent inherent biocompatibility characteristics.9 Charge conduction in polymers is not restricted to electronic current but can also include drift of ions. This allows us to construct electrochemical actuator devices that emit or receive signals other than just polarization of electric double layers. In their oxidized (reduced) state, the semiconducting polymer bulk carries and stores negatively (positively) charged counter-ions. Such doped polymer electrodes included in electrochemical devices were proven to be useful in electronic drug delivery devices10,11 and in electronic ion-pumps.12 Langer and Ingber reported already in 1994 the use of planar electrochemically controlled PPy surfaces as an active tuneable culture dish for Bovine aortic endothelial cells.13 Adhesion is also an essential event for stem cells. Neural stem cells are the source of more differentiated cells and a rich extracellular matrix that build the neural tissue. An important factor for tissue assembling is the adhesion properties of both cells and matrix. This is further regulated by the presence of specific chemoattractants and chemorepellants. Cell density is another essential parameter. While high cell densities favor the adoption of glial fates, low densities favor neuronal phenotypes. One example of secreted factors that regulate cell fate in a cell density-dependent manner is bone morphogenetic proteins, which are produced by the stem cells themselves and favor the glial differentiation of neural stem/progenitor cells. Thus cell density is a critical variable in stem cell cultures that is regulated by the adhesive properties of a substrate. Our objective in this study was to develop a surface that allows the fine-tuning of cell adhesion and density by modulation of the oxidation state of the poly(3,4-ethylenedioxythiophene) (PEDOT):Tosylate (see Figure 1) polymer. As the oxidation state of a conducting polymer film is changed, the (electro)chemical properties inside the bulk and along the outermost surface are controlled. Thus, the wetting and the adhesive properties of an electroactive polymer can be varied by changing its oxidation state. Polymer electrochemical devices are presently developed for a wide range of applications, such as electrochromic windows,14 sensors15,16 and in actuators.17 Recently, electrochemically active polymers were also studied and explored as the surface switch material to regulate surface wetting. Electrochemical switching in polymers is expected to (7) Cui, X.; Wiler, J.; Dzaman, M.; Altschuler, R. A.; Martin, D. C. Biomaterials 2003, 24, 777–787. (8) Gomez, N.; Schmidt, C. E. J. Biomed. Mater. Res., Part A 2006, 81A, 135–149. (9) Berggren, M.; Richter-Dahlfors, A. AdV. Mater. 2007, 19, 3201–3213. (10) Miller, L. L. React. Polym., Ion Exch., Sorbents 1986, 6, 341. (11) Abidian, M. R.; Kim, D.-H.; Martin, D. C. AdV. Mater. 2006, 18, 405– 409. (12) Isaksson, J.; Kja¨ll, P.; Nilsson, D.; Robinson, N.; Berggren, M.; RichterDahlfors, A. Nat. Mater. 2007, 6, 673–679. (13) Wong, J. Y.; Langer, R.; Ingber, D. E. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 3201–3204. (14) Argun, A. A.; Aubert, P.-H.; Thompson, B. C.; Schwendeman, I.; Gaupp, C. L.; Hwang, J.; Pinto, N. J.; Tanner, D. B.; MacDiarmid, A. G.; Reynolds, J. R. Chem. Mater. 2004, 16, 4401–4412. (15) Mabeck, J. T.; Defranco, J. A.; Bernards, D. A.; Malliaras, G. G.; Hocd, S.; Chase, C. J. Appl. Phys. Lett. 2005, 87, 013503. (16) Nilsson, D.; Kugler, T.; Svensson, P.-O.; Berggren, M. Sens. Actuators B: Chem. 2002, 86, 193–197. (17) Pei, Q.; Inganas, O. Synth. Met. 1993, 57(1 pt 5), 3718–3723.
Salto´ et al.
Figure 1. (a) The chemical structure of PEDOT and Tosylate. (b) PEDOT: Tosylate synthesized along the floor and walls of a 3 × 4 well plate. (c) To switch the surfaces, 1.5 V difference is applied to the two PEDOT: Tosylate electrodes. A full switch is achieved after typically 20 s. (d) The water contact angles were measured along the PEDOT:Tosylate electrode surface after they were completely switched. The water contact angles were measured for the reduced and oxidized states, respectively, and both immediately after a full switch was performed and also after an additional exposure to water.
affect the strength and the density of dipoles and the binding characteristics of doping ions, consequently offering us a simple route to achieve electronic control over the surface tension.18-21 In the present study we examined whether it is possible to modulate the attachment of neural stem cells to oxidized and reduced PEDOT surfaces. Our results show that oxidized PEDOT increased (and reduced PEDOT decreased) the adhesion of two different types of neural stem cells. Thin films of (PEDOT)22 doped with Tosylate23 were made, by vapor-phase polymerization, along the floors of polystyrene 3 × 4-well plates or standard Petri dishes (see Figure 1). As manufactured, the ground-state of PEDOT is partly oxidized, and its associated water contact angle is found to be θ ) 52.5° ( 6.8° (n ) 9). To measure the water contact angle of the switched PEDOT surfaces, a 1 mM NaCl aqueous solution was used as the electrolyte. The PEDOT:Tosylate films were patterned into two equally sized electrodes. Applying a potential difference of 1.5 V to the two electrodes induces electrochemistry to occur throughout both PEDOT films; the negatively (positively) addressed electrode reduces (oxidizes) toward the neutral (fully oxidized) state, and a complete switch takes about 20 s. Two (18) Isaksson, J.; Tengstedt, C.; Fahlman, M.; Robinson, N.; Berggren, M. AdV. Mater. 2004, 16, 316–320. (19) Causley, J.; Stitzel, S.; Brady, S.; Diamond, D.; Wallace, G. Synth. Met. 2005, 151, 60–64. (20) Sun, T.; Wang, G.; Feng, L.; Liu, B.; Ma, Y.; Jiang, L.; Zhu, D. Angew. Chem., Int. Ed. 2004, 43, 357–360. (21) Robinson, L.; Isaksson, J.; Robinson, N. D.; Berggren, M. Surf. Sci. 2006, 600, 148–152. (22) Groenendaal, L.; Jonas, F.; Freitag, D.; Pielartzik, H.; Reynolds, J. R. AdV. Mater. 2000, 12, 481–494. (23) Krebs, F. C.; Winther-Jensen, B. Sol. Energy Mater. Sol. Cells 2006, 90, 123–32.
Electronic Control of Neural Stem Cell Adhesion
Langmuir, Vol. 24, No. 24, 2008 14135
Figure 2. Differential attachment and cell density of NS cells grown on PEDOT:Tosylate polymer dishes that were electronically switched into the oxidized and reduced states. (a) c17.2 NS cells derived from the cerebellum and immortalized with c-myc were switched from serum to bFGF N2 media and examined for attachment to oxidized and reduced PEDOT polymer. High (20×) and low (10×) magnifications show nuclei of the cells stained with DAPI. Higher densities were found along the oxidized PEDOT surface. (b) Histogram showing the average cell density (number of cell attached per field after 2 h) on oxidized and reduced PEDOT polymer films. Data are mean ( standard deviation from triplicates in three independent experiments (n ) 3), *** p < 0.001. (c) Photomicrographs showing that primary, growth factor expanded, nonimmortalized, ventral midbrain-derived NS cells at the interface between the oxidized and reduced polymer. More cells attached to the oxidized polymer after 2 h in N2+bFGF.
different experiments were carried out: First, immediately after a complete electrochemical switch, the surfaces were quickly rinsed in deionized (DI) water and then dried in nitrogen. Then, the water contact angle was measured along the reduced and oxidized surfaces, respectively. The reduced polymer film exhibits a relatively lower water contact angle of θ ) 30.1° ( 5.1° (n ) 9), whereas the oxidized polymer film shows a contact angle of θ ) 58.1° ( 5.5° (n ) 9) (see Figure 1). In the second experiment, the switched PEDOT:Tosylate electrodes were left (still biased at a voltage difference of 1.5 V) in the aqueous electrolyte solution for an additional period of 20 min. The films were then rinsed in DI water and further dried with nitrogen. The corresponding water contact angles were then lowered to θ ) 18.1° ( 4.9° (n ) 9) and θ ) 19.9 ( 3.9° (n ) 9) for the reduced and oxidized PEDOT:Tosylate electrodes, respectively. The origin of the electronic control over the wettability is attributed to a change in the binding characteristics between the Tosylate ion, expressed at the surface, and the PEDOT main chains. In the oxidized state, the Tosylate ions are to a greater extent anchored to the PEDOT polymer backbone via the SO3- groups, hence directing the hydrophobic methyl group away from the surface, giving a relatively lower surface energy. In the reduced state, the Tosylate ions can easily rotate to expose the polar acid-containing end-groups toward approaching water droplets, i.e., a relatively higher surface energy. After extensive periods of times (here 20 min) of exposure to electrolyte, we suspect that ion exchange takes place along the PEDOT surface, and the Tosylate ions are either replaced by ionic species in the electrolyte or become rearranged. The consequence is that the surface tension increases for all oxidation states. Earlier studies performed on films of polyaniline doped with DBSA have shown wettability switching properties similar to those observed for the PEDOT:Tosylatebased surfaces studied here.18,24 In our experiments we used two cell lines: c17.2, a myc immortalized cell line derived from the external germinal layer of a neonatal mouse cerebellum,6 and a nonimmortalized, growth factor expanded neural stem cell (NS) derived from the ventral midbrain as described (Figure 2). Attachment experiments were performed in N2 media in the presence of basic fibroblast growth factor (bFGF), on PEDOT-coated dishes, in which half of the surface was reduced and the other half was oxidized (applying 1.5 V to the surfaces). After 2-4 h, the cells were fixed, stained
with 4′,6-diamidino-2-phenylindole (DAPI), and counted. We found that that both c17.2 and NS cells preferred to attach to the oxidized surface. The intrinsic adhesive properties of the cells were not changed by the surfaces they were presented. c17.2 cells tend to be more dispersed than NS cells from the ventral midbrain. However, the adhesion of cells to the oxidized or reduced polymer changes significantly for both cell lines. Quantification of the experiments performed with c17.2 cells revealed that the oxidized PEDOT increased cell adhesion by 2-fold compared to the reduced PEDOT. Similar results were obtained when different cell densities were examined, suggesting that this method can be applied to different cultivation protocols. Thus our data shows that it is possible to switch the adhesive properties of a PEDOT:Tosylate polymer and to achieve different cell densities in a culture, by regulating the oxidation state of this polymer. Proteins, integrins, and exposed actins can serve as the anchoring molecules for cells along artificial surfaces and also to promote binding between neighboring cells. Their adsorption, conformation upon adsorption, surface coverage, and overall interactions with humoral systems and cells may vary depending on the chemical or physical characteristics of the hosting surface.25 I125-stained human serum albumin (I125-HSA) is commonly used to study differences in protein adsorption along the surface of biomaterials, with I125 as the radioactive label to enable quantification of the amount of adsorbed protein. We have studied the differences in coverage of adsorbed I125-HSA along PEDOT: Tosylate surfaces in the pristine, oxidized, and reduced states (see Table 1). In a first experiment, aqueous I125-HSA solutions (1 mg/mL) were applied onto the active surfaces of the twoelectrode PEDOT:Tosylate surface switches. Prior to and during the exposure to the I125-HSA solutions, electrodes were biased at 0 and at ( 1.5 V to ensure that the surface switches were kept at the desired oxidation state during the entire HSA adhesion experiment. Then after 1/2 h, the surfaces were quickly dried by air, and the adsorbed I125-HSA contents were measured in a gamma counter. After this, the surface switches were either washed extensively in a 0.4% sodium dodecyl sulfate (SDS) solution or reincubated (for 30 min) in a non-I125-stained HSA solution without applying any voltage to the electrodes. From these latter experiments we achieve a quantitative measure of the protein binding strengths and associated desorption char-
(24) Isaksson, J.; Robinson, N. D.; Berggren, M. Thin Solid Films 2006, 515, 2003–8.
(25) Marchant, R. E.; Larsen, C. C.; Kligman, F.; Kottke-Marchant, K. Biomaterials 2006, 27, 4846–4855.
14136 Langmuir, Vol. 24, No. 24, 2008
Salto´ et al.
Table 1. Measured Gamma-Counts, Reflecting the Overall Quantity of I125-HSA Adsorbed on Pristine (0 V), Reduced (-1 V) and Oxidized (+1 V) PEDOT:Tosylate Surfaces
pristine (0 V) reduced (-1.5 V) oxidized (+1.5 V)
I125-HSA, 30 min seeding
I125-HSA, 30 min seeding followed by an SDS wash
I125-HSA, 30 min seeding, followed by seeding of HSA for 30 min
22700 ((1700) 40200 ((4900) 27700 ((4900)
11100 ((400) 28900 ((2300) 17400 ((1100)
11800 ((1279) 33400 ((7800) 18600 ((1000)
acteristics for the three different states, respectively. In all experiments, i.e., for the direct I125-HSA adsorption, after washing, and after HSA exchange desorption experiments (see Table 1), we measure considerably more adsorbed I125-HSA along the negatively biased (reduced) PEDOT:Tosylate electrodes as compared to what is found along the surfaces in the other states. Of particular importance for the stem cell experiments, after the washing steps and after the exchange desorption experiments, the relative loss in density of the I125-HSA is much smaller along reduced surfaces, thus indicating relatively stronger adhesion characteristics for this state. So, in comparison between the three different oxidations states, our investigations indicate that the reduced surface, first of all, to a greater extent promotes adhesion of, and second, also seems to lock the HSA matrix proteins. X-ray photoelectron spectroscopy (XPS) and ultraviolet photoelectron spectroscopy (UPS) were used to characterize the modifications on the chemical and electronic structure of the PEDOT:Tosylate surface upon oxidation/reduction. Using XPS to evaluate the surface stoichiometry, no trace elements of iron is found, and no significant variation in relative C, S, and O concentrations are seen for the different oxidation states. The elemental composition of the two materials are very similar (6C: 2O:1S vs 6C:3O:1s); however, the small variations in the PEDOT versus Tosylate concentration would fall within the error margin of the experiment. Although both PEDOT and Tosylalte contain one sulfur atom per repeated unit, the sulfur atom in PEDOT is included in the thiophene ring, whereas, in case of Tosylate, it is a part of the sulfonate moiety. Because of these different chemical environments, the S(2 p) electrons of PEDOT and Tosylate have different binding energies, and therefore changes in the composition of PEDOT:Tosylate at the surface can be probed through analysis of the S(2p) core level. The spin-split doublet originating from the PEDOT chains has its maximum located ∼164 eV.26 The spin-split doublet peak from the sulfur originating from the Tosylate groups coordinated to PEDOT has its maximum at roughly ∼168 eV. Uncoordinated Tosylate is located at slightly higher binding energy, but resides too close to the PEDOT-coordinated feature to resolve the two spin-doublets in the spectrum. The large binding energy difference (∼4 eV) between the main peaks thus make is possible to differentiate the PEDOT from the Tosylate contributions to the S(2p) core level spectrum. A further complication though is that the PEDOT sulfur doublet is asymmetric and tends toward higher binding energy as a result of the different charge environments of the sulfur atoms induced by the p-doping of the PEDOT chains.26,27 Comparing the S(2p) core level spectra of p-doped (pristinely oxidized) and reduced PEDOT:Tosylate films (see Figure 3), it is clear that the PEDOT-derived features with the main S(2p3/2) peak located at ∼164.4 eV dominate both spectra. Furthermore, because of the multitude of chemically different sulfur atoms in (26) Jo¨nsson, S. K. M.; Birgerson, J.; Crispin, X.; Greczynski, G.; Osikowicz, W.; van der Gon, A. W. D.; Salaneck, W. R.; Fahlman, M. Synth. Met. 2003, 139, 1–10. (27) Zotti, G.; Zecchin, S.; Schiavon, G.; Louwet, F.; Groenendaal, L.; Crispin, X.; Osikowicz, W.; Salaneck, W.; Fahlman, M. Macromolecules 2003, 36, 3337– 3344.
the sample and the broad photon line width of the unmonochromatized Al KR X-rays used, the individual spin-split doublets are not resolved. However, there is a significant difference between the two types of samples in terms of the Tosylate-derived features appearing as a high binding energy shoulder at ∼168 eV. For the p-doped (pristine) case, we see a stronger contribution from Tosylate than for the reduced sample, suggesting that some of the Tosylate molecules have left the surface upon reduction, as they are no longer bound as counterions to the PEDOT pluscharged segments. UPS results show no significant effect in work function upon reduction, suggesting that it is a modification of the surface chemical environment rather than modification of the surface dipole that drives the adhesion changes. Harnessing all the potential of neural stem cells is one of the key goals of stem cell biology. In this study we explored the impact of polymer surface switches to control stem cell adhesion and cell density;two basic critical variables. We use two different cell lines expanded genetically or epigenetically to explore the response of stem cells when presented to an oxidized or reduced PEDOT surface. The two stem cells explored in our study, c17.2 and midbrain NS, show a clear preference to adhere to the oxidized PEDOT-surface. We then asked: What mechanisms coupled to the oxidation state of PEDOT promote the difference in stem cell adhesion? Our surface analysis experiments gave us the following: The water contact angle is clearly different for the PEDOT:Tosylate films immediately after an electrochemical switch, and the reduced surface showed a significantly lower contact angle as compared to the pristine semioxidized or fully oxidized state. However, exposure to aqueous media over longer times yields surfaces with almost identical contact angles, independently of the oxidation state. XPS and UPS studies revealed that the oxidized surface presents a slightly higher content of the Tosylate. First, this indicates that the electrochemical switching effect occurs also along the outermost surface of the films. Second, it suggests that there might be a difference of the chemical composition along the surfaces, depending on the oxidation state. However, longer times of exposure to a cell medium or to other aqueous solutions most likely produce similar chemical composition on the PEDOT surface. The I125-HSA
Figure 3. XPS measurements. S(2p) core level spectra of p-doped PEDOT:Tosylate (solid line, circle) and reduced PEDOT:Tosylate (dashed line, triangle).
Electronic Control of Neural Stem Cell Adhesion
Langmuir, Vol. 24, No. 24, 2008 14137
of the stem cells studied. In addition, the reduced PEDOT:Tosylate surface binds proteins much stronger, which suppress attachment of the protein matrix produced by the stem cells themselves. The cell density and adhesion properties are potent parameters for regulating the differentiation of stem cells. Our findings open the door for electronic control of stem cell differentiation.
Experimental Section
Figure 4. Proposed mechanisms for the difference in adhesion and density of stem cells achieved between the reduced and oxidized PEDOT:Tosylate electrode surfaces. (a) On the oxidized PEDOT surface, a less dense layer of HSA proteins is formed; however the proteins are oriented in a favorable direction that promotes good stem cell adhesion. (b) Stem cells that are approaching a potential host surface launch proteins that form an extracellular matrix. A dense and tightly bound HSA protein layer prevents the formation of an optimal extracellular matrix along the surface.
adsorption and desorption experiments clearly show that proteins adhere to a larger extent along the reduced PEDOT surface. Also, proteins adsorbed onto the reduced surface also bind much stronger, which was evident from the HSA-desorption experiments. It is known that cell adhesion and growth is strongly dependent not only on the density of proteins along a hosting surface but also on the orientation of the protein matrix.28,29 We think that the different oxidation states of the PEDOT surface dictate the orientation of proteins, hence controlling the stem cell adhesion and density. In other words, high contents of adsorbed proteins is not favorable for optimal adhesion for the two stem cell system here studied, but rather their orientation (see Figure 4). In addition, cells coming in close proximity to a host surface start to produce their own protein matrix in order to favor good attachment and interfacing.30,31 The reduced PEDOT surface exhibits much stronger binding characteristics to serum proteins, which could suggest that it more efficiently blocks proteins produced by the stem cells. This should also suppress adhesion of stem cells along the reduced PEDOT surface. We report electronic control of the adhesion and the density of two stem cell systems using surface switch electrodes based on PEDOT:Tosylate. For both cell systems, a 2-fold increase in adhesion was found along the oxidized PEDOT:Tosylate electrode as compared to the reduced surface after 4 h of seeding. The reduced side promoted a relatively stronger and denser HSA protein layer. We propose that the reduced side provides an HSA protein layer with a relatively unfavorable orientation for adhesion (28) Barlow, S. M.; Raval, R. Surf. Sci. Rep. 2003, 50, 201–341. (29) Tengvall, P.; Lundstrom, I.; Liedberg, B. Biomaterials 1998, 19, 407– 422. (30) Shen, J.-W.; Wu, T.; Wang, Q.; Pan, H.-H. Biomaterials 2008, 29, 513– 32. (31) Fang, F.; Szleifer, I. J. Chem. Phys. 2003, 119, 1053–65. (32) Conti, L.; Cattaneo, E. Trends Neurosci. 2005, 28 (2), 57-9.
Preparation, Probing, and Characterization of PEDOT: Tosylate Thin Films. Wells and dishes were cleaned in a mixture of DI water, NH3, and H2O2 (at a volume ratio of 5:1:1) followed by extensive rinsing in DI water. PEDOT:Tosylate thin films were made via vapor phase polymerization. First, 0.29 g of pyridine was added, under nitrogen atmosphere, to 10 mL of iron(III) p-toluene sulfonate (Iron(III)-Tosylate) solution, 40% in butanol (Clevios C used as provided by H. C. Stark). Then, this solution was spin coated at 1200 rpm onto the bottom floor of dishes and wells. The samples were then immersed into a chamber containing 2,3-dihydrothieno(3,4b)-1,4-dioxin (EDOT) heated to 40 °C. The Iron(III)-Tosylate-coated well and dish samples were exposed to the EDOT vapor for 30 min. After this, the samples were rinsed in isopropanol, ethanol, and water followed by drying in nitrogen. The samples were then baked at 60 °C for 3 min. Finally, two symmetric and electronically separated electrodes were made simply by scratching the film using a scalpel. Electrical biasing, applying 1.5 V to the PEDOT:Tosylate surfaces, was performed using a Keithly 2400 sourcemeter and a homemade power supply (for cell experiments performed inside an incubator). Static water-air contact angles (θ) were measured manually using a CAM 200 (KSV). 125I-HSA Radio Labeling Procedure. The Iodobead iodination method (Pierce, USA) was used for labeling of HSA (96-99%, Sigma, Sweden) with sodium 125-iodine. The beads were rinsed in phosphate-buffered saline (PBS) at pH 6.5. The sodium iodide was mixed in PBS, and equilibrated for 5 min. Subsequently, HSA (1 mg/mL) in PBS was added, incubated, and gently stirred for 7 min. The solution with labeled HSA (without Iodobeads) was filtrated in desalting gel columns (Pierce, USA) equilibrated with PBS. From each fraction, a small volume was taken to control the activity. The two columns with the highest activity were pooled. The pooled solution was dialyzed together with 50 mM KI. Dialysis tubings of pore size 3500 D were used. Small volumes were taken from the dialysate (1000 mL, PBS), and the activity was checked. The protein solution was dialysed until the activity fell below 5000 CPM for 1 mL of the dialysate. The protein concentration was determined by spectrophotometry (Shimadzu UV- 1601PC, USA) at 280 nm. A series of solutions with known concentrations of unlabeled HSA were used for the calibration. The protein size and charge were controlled with SDS polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) (PhastSystem, Pharmacia, Sweden) before and after the labeling. This was done in order to ensure that the proteins were pure and that the labeling had minimal effect on the protein charge and stability. No changes due to the labeling procedure were noted (not shown). The labeled proteins were aliquoted and stored at -80 °C until further use. The aliquots were thawed in a water bath at room temperature (RT). Gamma Counting of Adsorbed 125I-HSA. After the adsorption process with 125I-HSA, the samples were transferred to a gamma counter (Packard Cobra II, Canberra, USA), and the activity was measured during 10 min. Samples incubated in nonlabeled HSA were used as background references. In order to correlate the gamma counter value with the amount of adsorbed protein, known volumes with known concentrations of labeled proteins were measured in the gamma counter. Control experiments with the 125I dialysate showed no, or very low, adsorption of free 125I to the test surfaces. In order to test a possible uptake of free 125I by adsorbed and nonlabeled HSA, such surfaces were incubated in the 125I-containing dialysate. No uptake of free iodine by the unlabeled HSA was then observed. UPS/XPS Measurements. The photoelectron spectroscopy was performed in an ultrahigh-vacuum system at a base pressure of 10-10 mbar. The spectrometer used for the measurements was equipped
14138 Langmuir, Vol. 24, No. 24, 2008 with a hemispherical electron energy analyzer, a monochromatized He resonance lamp (hν ) 21.2 or 40.8 eV) and a nonmonochromatized Al(Ka) X-ray source, at hν ) 1486.6 eV. The resolution of the UPS measurements were 0.1 eV, as was determined from the Fermi edge of a Au reference sample. The XPS resolution was 1.2 eV, obtained from the full width at half-maximum of the Au(4 f 7/2) core level line. The samples were switched ex-situ immediately before insertion and pump-down. Stem Cell Cultures and Cell Adhesion Experiments. C17.2 and ventral midbrain NS cells were grown and passaged for a few days, as previously described (Snyder et al., 1992 and Conti et al., 2005, respectively). Adhesion experiments were perform in triplicates, in a serum-free N2 media containing 300 mg of glucose (Sigma), 150 mg of Albumax, 25 mL of F12, 25 mL of minimum essential medium (MEM), N2 supplement, Glutamine, and HEPES (all from Invitrogen), with addition of bFGFs (20 ng/mL, from R&D). The
Salto´ et al. cells were seeded at three different concentrations of cells from 1 to 4 × 105 /ml. Briefly, the electrodes in the dish were connected to a power supply, and a voltage of 1.5 V was applied. Cells were then added to the culture media and were allowed to attach for 2-4 h at 37 °C, in a CO2 incubator. At the end of the experiment, the cells were fixed with paraformaldehide (PFA) 4% for 15 min, and washed twice with PBS. Next the cells were incubated at RT for 10 min with DAPI, to facilitate the identification of the nuclei and perform cell counts. Cells were observed on an inverted microscope, and pictures were randomly taken from non-overlapping fields at 10× and 20×. Each experiment was performed in triplicate (three wells). Cells were quantified manually using 10 pictures of each reduced and oxidized side per well at 20×. LA8028337
