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Thin Polymer Layers as Supports for Hippocampal Cell Cultures Dierk Beyer,*,† Mieko Matsuzawa, Aiko Nakao, and Wolfgang Knoll Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama, 351-01 Japan Received December 22, 1997. In Final Form: March 6, 1998 Layers from poly[(1-methylvinyl isocyanate)-alt-(maleic anhydride)] on aminosilanized glass substrates were assembled and reacted in a two-step procedure, first with different mixtures of a sequence from the protein laminin, the peptide (PA22-2), and the aminopoly(ethylene oxide) (APEG) and second with the R,ω-diamine JA, to yield functionalized hydrophilic polymer films which were examined using X-ray photoelectron spectroscopy. Cell-growth experiments of embryonic hippocampal cells were performed on these surfaces, showing only cell adhesion to polymer films which were modified with the sequence from the protein laminin and the peptide (PA22-2). The cell cultures were investigated using phase contrast microscopy and immunostaining techniques together with fluorescence microscopy after 1 and 3 days, showing neuritic outgrowth of the cells after a period of 3 days.
Introduction The cultivation of fetal rat hippocampal cells on solid substrates is, in analogy to polymer-supported lipid bilayers,1,2 of scientific interest because a large number of analytical methods which were originally developed for surface analysis can be applied to such a setup. This opens up the opportunity to use surface-attached biological interfaces as a whole (cells) or in part (tethered supported cell membranes) as analytical devices which ultimately may lead to a new generation of biosensors. As one example, the concept of investigating the signal transduction of neuronal cells on a silicon-based field effect transistor (FET) can be seen as a particularly intriguing application for the investigation of signal transduction in neuronal cells.3 One of the problems which are frequently encountered in using silicon or silicon oxide substrates for cell cultivation is the behavior of the part of the cell membrane in direct contact with the substrate.4 This contact can lead to differences in the lateral diffusion of the lipids in contact with the substrate, to complete lysis of the membrane, and/or to the denaturation of incorporated membrane proteins. In order to eliminate such effects, ideally a polymer “cushion layer” is introduced which insulates the cell membrane from the influence of the substrate and avoids unspecific interaction between cells and cushion layer but, on the other hand, still allows cell attachment to the surface via a specific cell-polymer layer interaction. As one example for substrates which greatly reduce unspecific attachment of neuronal cells, it could be shown that thin polymer films of N-isopropylacrylamide on glass substrates lead to a strong reduction of cell attachment to the surface, indicating a low interaction between the †
Current address: Clariant GmbH, Division CP, F+E PM I, D-65926 Frankfurt am Main, Germany. (1) Sackmann, E. Science 1996, 271, 43-48. (2) Beyer, D.; Elender, G.; Knoll, W.; Ku¨hner, M.; Maus, S.; Ringsdorf, H.; Sackmann, E. Angew. Chem. 1996, 108, 1791-1794. (3) Fromhertz, P.; Offenha¨user, A.; Vetter, T.; Weis, J. Science 1991, 252, 1290. (4) Thompson, N.; Poglitsch, C.; Timbs, M.; Pisarchick, M. Acc. Chem. Res. 1993, 26, 567.
cells and the polymer film.5 In another approach Matsuzawa et al.6 could show that hippocampal cells on patterned silicon oxide surfaces modified with a synthetic peptide derived from an extracellular matrix protein laminin show a directed neurite outgrowth on these substrates along this preformed peptide-functionalized pattern through specific recognition of the peptide sequence by the cells.7 Since laminin is a large basement membrane protein with many biologically active sites, we have used in this study a synthetic peptide consisting of 19 amino acids (PA22-2) which contains a IKVAV sequence derived from the laminin R-chain. This sequence has been identified as one of the cell adhesive and neurite-promoting sites of laminin. Affinity chromatography of neonate mouse brain has identified a 110 kDa cell surface protein as a receptor for PA22-2.7 In order to achieve a functionalizable polymer cushion which ideally reduces the nonspecific binding of the cells but also promotes a specific attachment through recognition sites for cell adhesion and neurite outgrowth, such as the laminin sequence PA22-2, a thin polymer layer can be attached to cover the substrate using the concept of reactive polymer adsorption with subsequent derivatization8,9 of the resulting reactive layer. This concept allows tuning of the essential layer properties concerning cell attachment and cell-polymer interaction. Experimental Section 1. Polymer Adsorption and Functionalization. Polymer adsorption was carried through on cleaned and aminosilanized glass substrates prepared according to the procedure described elsewhere.2 Polymer IAP (poly[(1-methylvinyl isocyanate)-alt(maleic anhydride)]) synthesis was performed according to the procedure described elsewhere.2 The synthetic peptide PA22-2 (5) Bohanon, T. M.; Elender, G.; Knoll, W.; Koeberle, P.; Lee, J.-S.; Offenha¨usser, A.; Ringsdorf, H.; Sackmann, E.; Tovar, J.; Winnik, F. J. Biomater. Sci. Polym. Ed. 1996, 8, 19. (6) Matsuzawa, M.; Umemura, K.; Beyer, D.; Sugioka, K.; Knoll, W. Thin Solid Films 1997, 305, 74. (7) Kibbey, M. C.; Johnson, B.; Petryshyn, R.; Jucker, M.; Kleinman, H. K. J. Neurosci. Res. 1995, 42, 314. (8) Beyer, D.; Bohanon, T. M.; Knoll, W.; Ringsdorf, H.; Elender, G.; Sackmann, E. Langmuir 1996, 12, 2514. (9) Zhou, Y.; Bruening, M. L.; Bergbreiter, D. E.; Crooks, R. M.; Wells, M. J. Am. Chem. Soc. 1996, 15, 3773.
S0743-7463(97)01400-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 05/02/1998
Supports for Hippocampal Cell Cultures Table 1. Survey over Surface Modification and Cell Adsorption to Polymer Films Functionalized with Various Concentrations of the Peptide PA22-2 and APEG sample 1 2 3 4 5 6
c(PA22-2) [µM]
1 2 5 10
c(APEG) [µM]
c(JA) [M]
cell growth after 3 days
10 5 5 5
0.5 0.5 0.5 0.5 0.5 0.5
no cells attached no cells attached few cells attached cells attached cells attached cells attached, signs of intoxication
(CSRARKQAASIKVAVSADR), derived from the R-chain of mouse laminin, was obtained from RBI (Research Biochemicals International), Natick, MA. The aminosilanized substrate is immersed for 4 h in a 2.2 × 10-3 M solution of poly[(1-methylvinyl isocyanate)-alt-(maleic anhydride)] (IAP) in DMF/CH2Cl2 (1:9). After the adsorption of the reactive polymer IAP onto the aminosilanized glass substrate, the slides are rinsed several times with DMF/CH2Cl2 (1:9) and pure CH2Cl2. In order to introduce the synthetic peptide sequence PA22-2, 0.5 mg of this peptide were dissolved in 1 mL of DMSO and diluted with CH2Cl2. A solution of (aminopropyl)poly(ethylene glycol) monomethyl ether, APEG (Mw ) 1050), in CH2Cl2 was prepared and mixed with the peptide (PA22-2) solution in different ratios. Several reaction solutions with different concentrations of peptide (PA22-2) and APEG were prepared according to this procedure and allowed to react with the polymer layer on the glass substrate for 12 h (see Table 1). In order to react the remaining functional groups of the polymer, the modified substrates are subsequently immersed for 2 h in a 0.5 M solution of bis(aminoethyl)diethylene glycol ether (Jeffamine, JA) in CH2Cl2. The slides were rinsed with CH2Cl2, ethanol, and water (MilliQ), dried in a culture hood, and directly used for cell cultivation. 2. Cell Culture. Hippocampal tissues were dissected from 18-days-old rat fetuses and were dissociated into single cells as described elsewhere.10,11 The dissociated hippocampal cells were placed on the substrates at low density (5000 cells/cm2) in chemically defined media supplemented with B27.12,13 These culture conditions (low-density cells and serum-free media) were used to observe the direct effects of growth surfaces on cell attachment and outgrowth. The cells were incubated in an atmosphere of 95% air and 5% CO2 at 36.5 °C. After a predetermined period (1 or 3 days), the attachment and outgrowth of living hippocampal cells were examined using an Olympus microscope with phase-contrast optics. The neuronal morphology was then evaluated by an immunostaining technique using a mouse monoclonal antibody against R-tubulin protein (Cappel) as a primary antibody. Details of the immunostaining procedure are desribed elsewhere.15 In brief the cells were fixed using a 4% formaldehyde solution in phosphate-buffered saline (PBS) and were then permeabilized in -20 °C methanol for 5 min. After rehydrating the cells in PBS for 5 min, the primary antibody was applied to the cells for 2 h at 30 °C, which was then followed by the application of a secondary antibody, goat anti-mouse IgG labeled with fluorescein (Cappel). The immunostained cells were mounted on glass slides using MOWIOL (Aldrich), and the neuronal morphology was indirectly visualized by the detection of the distribution of tubulin protein using an Olympus fluorescent microscope with an appropriate filter combination. 3. X-ray Photoelectron Spectroscopy Measurements. X-ray photoelectron spectroscopy (XPS) measurements were performed using a PHI model 5600 MultiTechnique system (Perkin-Elmer) equipped with a Mg KR source (1253.6 eV). The substrates were analyzed at an electron take-off angle of 30°, measured with respect to the surface plane. (10) Trencner, E. Culturing nerve cells; MIT Press: Cambridge, MA, 1991; p 283. (11) Banker, B.; Goslin, K. Culturing nerve cells; MIT Press: Cambridge, MA, 1991; p 252. (12) Bottenstein, J. E.; Sato, G. H. Proc. Natl. Acad. Sci. 1979, 76, 514. (13) Brewer, G. J.; Torricelli, J. R.; Evege, E. K.; Price, P. J. J. Neurosci. Res. 1993, 35, 567.
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Results and Discussion A number of slides were prepared by applying three different reaction paths for the reactive polymer layer, as is presented in Scheme 1. In Figure 1B the XPS spectra of the polymer layer after direct reaction with JA with all reactive polymer functionalities transformed into amide, carboxylic acid, and urea moieties are presented. In the XPS spectra in Figure 1C, the reactive polymer layer was first reacted using a 10 µM solution of APEG and then the remaining reactive groups were passivated using JA. In Figure 1D XPS spectra of the polymer after a first reaction with a 10 µM solution of the peptide (PA22-2) and subsequent passivation using JA are displayed. Additional samples were prepared using different ratios of the peptide (PA22-2) and APEG in the first reaction solution (see Table 1), and subsequently the remaining reactive groups on these slides were passivated using Jeffamine JA. The attachment and growth of the hippocampal cells on these substrates after 1 and 3 days was investigated. In order to verify the attachment of PA22-2, XPS spectra were taken from samples with and without modification of the polymer layer with the Jeffamine JA, (aminopropyl)poly(ethylene glycol) monomethyl ether, APEG (Mw ) 1050), and the peptide PA22-2. The high-resolution C 1s and O 1s XPS spectra of such modified glass slides are displayed in Figure 1. In Figure 1A the XPS C 1s and O 1s high-resolution spectra of the reactive polymer layer, which has been extensively characterized in ref 8 using FTIR, are displayed. In the C 1s spectrum the band with a binding energy of 289.6 eV can be assigned to the anhydride functionality of the polymer, whereas the band at 287.3 eV may be assigned to the isocyanate functionality. The relative silicon content of this sample (13.3% Si) indicates that the thickness of this reactive polymer layer either is below the penetration depth of the XPS analysis and therefore the substrate is detected together with the polymer layer or the coverage of the polymer layer on the substrate is incomplete. The same argument is obvious for the oxygen content (26.0% O) and the O 1s spectra of the reactive polymer layer. In the latter case two bands, corresponding to Si-O (533.2 eV) and CdO (531.9 eV) functionalities, are observed. In Figure 1B the XPS data of the polymer layer reacted with a 0.5 M solution of bis(aminoethyl)diethylene glycol ether (JA) in CH2Cl2 are presented. As could be shown in a previous investigation, the thickness of this polymer layer on a silicon oxide subtrate is 81 ( 5 Å.2 The relative silicon and oxygen contents of this layer have decreased toward the sample displayed in Figure 1A, indicating a thicker layer than that in the unreacted polymer film or a more complete coverage of the substrate with the polymer film reacted with JA. As can be observed easily in the C 1s spectrum, the band at 289.6 eV, which is attributed to the anhydride moiety of the original polymer layer, has disappeared entirely. Furthermore, the band at 288.1 eV can be attributed to the formation of carboxylic acid or amide groups, which are reaction products from the derivatization of the reactive polymer layer with JA. The band at 286.2 eV exhibits an increased intensity as compared to Figure 1A, which is attributed to the number of C-O functionalities introduced by the reaction with bis(aminoethyl)diethylene glycol ether (JA), whereas the presence of a band at 284.7 eV accounts for the aliphatic groups in this system. In the O 1s spectrum in Figure 1B, the increase in the band at 532.6 eV can be attributed to ether-oxygen atoms due to the reaction with JA and the
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Scheme 1. Overview of the Different Modification Paths of the Reactive Polymer Filma
aTo each structure is assigned the corresponding XPS spectrum in Figure 1. Please note that these modifications present the subsequently functionalized repeating unit of the polymer and therefore do not represent those repeating units where the polymer has reacted with the aminosilanized surface.
band at 531.2 eV can be attributed to the presence of carbonyl-oxygen in the reacted layer. These observations are in good accordance with the results derived from the grazing incidence-FTIR measurements stated in refs 2 and 8, where it could be shown that the entire reactive polymer layer (Figure 1C) may be derivatized using JA, yielding a layer with carboxylic acid, amide urea, and ethylene oxide moieties. In Figure 1C the data from the reaction of the polymer layer first with a 10 µM solution of (aminopropyl)poly(ethylene glycol) monomethyl ether, APEG (Mw ) 1050), and then with a 0.5 M solution of bis(aminoethyl)diethylene glycol ether (Jeffamine, JA) in CH2Cl2 are displayed. The C 1s spectra of parts B and C of Figure 1 do not show any significant differences, although the oxygen content of the layer in Figure 1C has slightly increased toward the polymer layer reacted with JA only (Figure 1B). In the O 1s spectrum of Figure 1C the band at 532.3 eV has increased in direct comparison with the O 1s spectrum in Figure 1B, indicating the additional presence of ether-oxygen and therefore the reaction of APEG with the reactive polymer layer. The C 1s and O 1s spectra in Figure 1D (reactive polymer layer after reaction with PA22-2 and JA) are significantly different from parts B and C of Figure 1. The increase in the bands at 287.9 and 284.7 eV and the decrease in the band at 285.9 eV clearly show the attachment of the peptide PA22-2. The increase of the band at 287.9 eV in the C 1s spectrum and the increase of the band at 531.3 eV in the O 1s spectrum can be attributed to the increased number of carbonyl functionalities which are introduced
by the functionalization of the reactive polymer layer with the peptide PA22-2. The band at 285.9 eV in the C 1s spectrum and the band at 532.6 eV in the O 1s spectrum may be attributed to the subsequent reaction of the remaining reactive groups with JA. Table 1 represents a survey over the slides for cell adsorption which were prepared in analogy to the samples for the XPS analysis, using different ratios of the peptide (PA22-2) and APEG in the reaction solution and subsequently reacting the remaining functional groups with JA. As can be seen from Table 1 and as is shown in Figure 2, after 1 day no cell attachment was observed on the polymer layer which was not modified with the peptide (PA22-2). The hippocampal cells are still mobile in the culturing medium and show intercellular aggregation effects on the substrate. No difference in cell adsorption could be observed if the reactive polymer layer was prepared by exposure to a reaction solution containing 10 µM APEG in CH2Cl2 for 24 h before passivating the remaining reactive groups using a 0.5 M solution of JA (Figure 2b) or by direct reaction of the polymer layer with the JA solution (Figure 2a). This effect leads to the conclusion that the hydrophilic polymer layer (contact angle θa ) 35 ( 5°, and θr ) 22 ( 5°) without a specific peptide that has been known to interact with a cell surface receptor14 attached does not provide sufficient interaction with the cell surface in order (14) Kleinman, H. K.; Weeks, B. S.; Cannon, F. B.; Sweeney, T. M.; Sephel, G. C.; Clement, B.; Zain, M.; Olson, M. O. J.; Jucker, M.; Burrous, B. A. Arch. Biochem. Biophys. 1991, 290, 320.
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Figure 1. High-resolution C 1s and O 1s XPS spectra of the polymer layer: (A) prior to any reaction; (B) after reaction with JA only; (C) after the reaction with a 10 µM solution of APEG; (D) after reaction with a 10 µM solution of PA22-2. Both c and d are after passivating the remaining reactive groups with JA.
to enable the cell to attach directly to the substrate. This observation is in agreement with results obtained for cells interacting with hydrophilic poly(N-isopropylacrylamide) polymer layers.5 In contrast to this observation, hippocampal cells do adhere readily to clean glass substrates modified with an aminosilane within 1 day, developing morphologies with poor neuritic outgrowth after 3 days.15 Depending on the concentration of peptide in the first
reaction solution, polymer layers which are modified with mixtures of the peptide (PA22-2) and APEG (contact angle θa ) 38 ( 5° and θr ) 23 ( 5°) supported the cell attachment after 1 day and promoted the outgrowth of neuritic processes. Within 3 days in culture, multiple short and single neuritic processes were extended from individual (15) Matsuzawa, M. J. Neurosci. Methods 1996, 69, 189.
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Figure 2. (a) Phase-contrast microscopy image of hippocampal cells on a polymer film (sample 1) not functionalized with PA22-2 or APEG after 3 days. The cells are not attached to the substrate but are mobile in the nurturing solution. (b) Phase-contrast microscopy image of hippocampal cells on a polymer film (sample 2) functionalized with APEG but not functionalized with PA22-2 after 3 days. Showing essentially the same effect as in sample 1 but without APEG functionalization.
cell bodies (Figure 3). The long processes usually exhibited an arborized morphology which is characteristic of hippocampal neurons in vivo.11 Hippocampal neurons grown on samples 4 and 5 (Table 1) usually exhibited such an arborized morphology with multiple short and single neuritic processes (Figure 3). This effect clearly shows the specific attachment of hippocampal cells to the modified substrate due to the presence of the peptide (PA22-2) in the thin polymer layer and the neurite outgrowth promoting effect of the peptide upon the cells. Furthermore, it can be observed that apparently a critical concentration of peptide on the surface is necessary in order to allow cell attachment. On the other hand, increasing peptide concentration in the reaction solution apparently leads to a higher degree of functionalization of the polymer layer. In this case the highly functionalized polymer substrates caused degenerative effects on the hippocampal neurons (sample 6, Table 1). The neurons appeared flat and exhibited a phasedark appearance in the soma with some extended fragmented neuritic processes. Additionally, a pronounced
Beyer et al.
Figure 3. (a) Phase-contrast microscopy image of hippocampal cells on a polymer film (sample 5) functionalized with PA22-2 and APEG after 3 days. The cells extend long neuritic processes from phase-bright cell bodies. (b) Fluorescence micrograph showing that a hippocampal cell grown on a polymer film (sample 5), functionalized with PA22-2 and APEG, develops typical morphological characteristics of hippocampal neurons in vivo. The cells were immunocytochmically stained using an anti-R-tubulin antibody and visualized using fluorescence microscopy.
damage like nucleus extrusion on these highly functionalized surfaces was frequently observed. Considering that similar degenerative effects were observed in a culture of hippocampal neurons in the presence of a high concentration of the neurotransmitter glutamic acid,16 the effects caused by an excess amount of PA22-2 may be due to an overstimulation of cellular activities through surface receptor proteins, one of which is likely a 110 kDa membrane protein.7 Following the interaction of PA22-2 with the receptor, cell behaviors such as adhesion and neurite outgrowth can be modulated through the cytoskeletal machinery. One suggested mechanism underlying such a signaling system is the involvement of the protein (16) Mattson, M. P.; Dou, P.; Kater, S. B. J. Neurosci. 1988, 8, 2087.
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kinase C, which phosphorylates signaling molecules and affects the cytoskeletal protein production.17 Conclusions It could be shown that embryonic hippocampal cells do attach to thin hydrophilic polymer films modified with a laminin sequence, the peptide (PA22-2). On polymer layers which were not modified with this peptide, no cell attachment could be observed. Depending upon the concentration of peptide used in the solution for derivatization of the polymer film, the cells exhibited either (17) Luckenbill-Edds, L. Brain Res. Rev. 1997, 23, 1.
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clustering effects due to a low surface concentration of peptide, neuritic outgrowth or slightly toxic effects due to high surface concentration. These results lead to a better understanding of the mechanism of cell attachment and neuritic outgrowth on a surface and may be useful in optimizing cell cultures for biosensor applications. Of particular interest will be the functionalization of a reactive polymer layer with photocleavable groups in order to introduce a lateral structure in the cell attachment and their neuritic outgrowth in order to grow defined cell networks. LA971400B
