Ormocomp-Modified Glass Increases Collagen Binding and Promotes

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Ormocomp-Modified Glass Increases Collagen Binding and Promotes the Adherence and Maturation of Human Embryonic Stem CellDerived Retinal Pigment Epithelial Cells Elli Kap̈ yla,̈ †,§,⊥ Anni Sorkio,†,‡,⊥ Shokoufeh Teymouri,†,§ Kimmo Lahtonen,∥ Leena Vuori,∥ Mika Valden,∥ Heli Skottman,†,‡ Minna Kellomak̈ i,†,§ and Kati Juuti-Uusitalo*,†,‡ †

BioMediTech, Tampere, Finland BioMediTech, University of Tampere, Biokatu 12, 33014 Tampere, Finland § Department of Electronics and Communications Engineering, Tampere University of Technology, P.O. Box 692, 33101 Tampere, Finland ∥ Surface Science Laboratory, Tampere University of Technology, P.O. Box 692, 33101 Tampere, Finland ‡

ABSTRACT: In in vitro live-cell imaging, it would be beneficial to grow and assess human embryonic stem cellderived retinal pigment epithelial (hESC-RPE) cells on thin, transparent, rigid surfaces such as cover glasses. In this study, we assessed how the silanization of glass with 3-aminopropyltriethoxysilane (APTES), 3-(trimethoxysilyl)propyl methacrylate (MAPTMS), or polymer−ceramic material Ormocomp affects the surface properties, protein binding, and maturation of hESC-RPE cells. The surface properties were studied by contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and a protein binding assay. The cell adherence and proliferation were evaluated by culturing hESCRPE cells on collagen IV-coated untreated or silanized surfaces for 42 days. The Ormocomp treatment significantly increased the hydrophobicity and roughness of glass surfaces compared to the APTES and MAPTMS treatments. The XPS results indicated that the Ormocomp treatment changes the chemical composition of the glass surface by increasing the carbon content and the number of C−O/O bonds. The protein-binding test confirmed that the Ormocomp-treated surfaces bound more collagen IV than did APTES- or MAPTMS-treated surfaces. All of the silane treatments increased the number of cells: after 42 days of culture, Ormocomp had 0.38, APTES had 0.16, MAPTMS had 0.19, and untreated glass had only 0.062, all presented as million cells cm−2. There were no differences in cell numbers compared to smoother to rougher Ormocomp surfaces, suggesting that the surface chemistry and, more specifically, the collagen binding in combination with Ormocomp are beneficial to hESC-RPE cell culture. This study clearly demonstrates that Ormocomp treatment combined with collagen coating significantly increases hESC-RPE cell attachment compared to commonly used silanizing agents APTES and MAPTMS. Ormocomp silanization could thus enable the use of microscopic live cell imaging methods for hESC-RPE cells.

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INTRODUCTION An immense body of convergent data has shown that human pluripotent stem cells (hPSC), i.e., human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC), can be differentiated to retinal pigment epithelial cells (RPE) that express RPE specific marker genes and exhibit RPE specific morphology.1 The generated hESC-RPE cells are proposed to be valuable in in vitro model systems to elucidate the mechanisms of RPE-derived diseases, drug testing, and targeted drug therapy.2 Cell behavior, such as proliferation, maturation, and motility, is directly affected by the culture substrata, and the response differs from cell type to cell type.3−5 This has recently been demonstrated for hESC-RPE cells by Subrizi and Sorkio, who showed that some substrata that have supported the growth of other cell types were not beneficial to hESC-RPE cells.6,7 © XXXX American Chemical Society

Glass is a standard cell culture substrate that benefits from transparency to wavelengths employed in optical microscopy.8 Unmodified glass substrates, however, have only a limited capability to support cell adhesion.9 One of the most widely used methods to increase protein binding is to functionalize glass surfaces by silanization, in which organosilane molecules are covalently bound to the hydroxyl groups on the glass surface via stable Si−O−Si links.10,11 Depending on the type of organofunctional group, silanization can either promote or inhibit cell adhesion on glass surfaces.8 Aminosilanes, such as 3aminopropyltriethoxysilane (APTES), are one of the most commonly used adhesion promoters.12,13 The amine functionReceived: February 13, 2014 Revised: October 30, 2014

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dx.doi.org/10.1021/la5023642 | Langmuir XXXX, XXX, XXX−XXX

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water followed by air drying. The MAPTMS samples were also prepared in three steps: coverslips were cleaned with a strong soap solution, rinsed thoroughly with ion-exchanged water and 99.5% (v/v) ethanol and air dried, then immersed in a solution of 1 mL of MAPTMS in 200 mL of 99.5% (v/v) ethanol and 6 mL of dilute acetic acid (1:10 glacial acetic acid/water) for approximately 3 min, and finally rinsed thoroughly with 99.5% (v/v) ethanol and air dried. Both the Ormodev and HMDS samples were first rinsed with 99.5% (v/v) ethanol and then either immersed in Ormodev for approximately 5 min or immersed in HMDS for approximately 1 min. As a final step, all of the samples were treated with 70% (v/v) and 99.5% (v/v) ethanol for 15 min each. The ethanol treatment was also used to disinfect the samples before cell culturing. Samples treated only with ethanol served as untreated controls. To prepare smoother surfaces, thin films of Ormocomp-I127 were prepared by UV polymerization. A drop of Ormocomp-I127 was sandwiched between a glass slide and a cover glass separated by a 150μm-thick stainless steel spacer. The films were exposed to UV light (300−450 nm, ∼3000 mW/cm2 maximum intensity, BlueWave 50 UV curing spot lamp, DYMAX Corporation, USA) for 10 s, immersed in Ormodev for 5 min, and rinsed with HMDS. The films were then immersed in Ormocomp for 2 h, again immersed in Ormodev for 5 min, and rinsed with HMDS. The films were disinfected in the same manner as silanized cover glasses. Contact Angle Measurements. The static water contact angles were analyzed with the sessile drop method at room temperature with a Theta Lite optical tensiometer (Attension, Biolin Scientific AB, Sweden). The contact angles were measured with ion-exchanged water, and a total of 10 samples were examined with each surface treatment. Protein Coating and Binding on Surface-Treated Materials. The protein-binding capacity was assessed from untreated APTES, MAPTMS, and Ormocomp-I127 samples. An extracellular matrix protein, human collagen IV (Col IV), was chosen because it is the major component of the native Bruch membrane.32 Furthermore, we have previously used Col IV for the enrichment and maturation of hESC-RPE.7 Col IV from human placenta (Sigma-Aldrich, St. Louis, MO, USA) was used at a concentration of 5 μg/cm2. The samples were rinsed with Dulbecco’s phosphate-buffered saline (DPBS) (Lonza Group Ltd., Basel, Switzerland). Thereafter, the samples were incubated in a protein coating solution for 3 h at 37 °C and rinsed twice with DPBS to remove any remaining unbound protein. The binding of Col IV was determined with indirect immunofluorescence staining. Uncoated samples were used as negative controls. First, the samples were blocked with 3% BSA (SigmaAldrich) at RT for 1 h, followed by incubation with primary antibody mouse anticollagen IV 1:100 (Neomarkers) for 1 h at RT. Thereafter, samples were washed several times with DPBS and labeled with secondary antibody Alexa Fluor 488-conjugated donkey antirabbit IgG (Molecular Probes, Life Technologies) diluted in ratio of 1:800 with 0.5% BSA-DPBS for 1 h at RT. After repeated washes with DPBS, samples were included with mounting media (Vector Laboratories Inc., Burlingame, CA) to prevent the samples from drying. 8-bit images were taken with an Olympus IX 51 fluorescence microscope (Olympus, Tokyo, Japan). The protein binding was quantified with ImageJ image processing and analysis software (http://imagej.nih.gov/ ij/index.html) through pixel intensity normalization. The pixel intensities of negative controls were subtracted from the pixel intensities of the Col IV-coated samples in order to remove the background caused by primary and secondary antibodies. Normalization was carried out against an ethanol control. Atomic Force Microscopy. The morphologies of APTES, MAPTMS, Ormocomp-I127 and ethanol-treated glass surfaces, and Ormocomp-I127 thin films and their Col IV-coated duplicates were characterized using an atomic force microscope (AFM, XE-100, Park Systems Corp, USA). Before being imaged, the Col IV-coated samples were soaked in PBS for 20 min and allowed to air dry. For one to two samples, a total of six 5 μm2 areas were scanned in noncontact mode, under air and at room temperature. The probe was supported on an APPNANO AFM cantilever (type ACTA, L = 125 μm, tip radius < 10

ality of APTES enables simple coupling to proteins and other biomolecules.14 For example, APTES has been shown to support the growth and maturation of cardiomyocytes extracted from rat embryos.15 Within ophthalmology, APTES has been used to bind histological sections of retina16 but not to grow retinal cells. Another common silanization agent is 3(trimethoxysilyl)propyl methacrylate (MAPTMS), which is often used to bind (meth)acrylate polymers to inorganic substrates such as glass.17,18 MAPTMS has also been used to increase the surface adherence of pluripotent stem cells cultured on different extracellular matrix components.19 Even though APTES and MAPTES have been used to enhance the adherence of pluripotent hES and cardiomyocytes,15,19 to our knowledge neither APTES nor MAPTMS has been previously used to functionalize glass surfaces for hESC-RPE cells. Ormocers (organically modified ceramics) are hybrid polymer-ceramic materials that are based on organically crosslinked heteropolysiloxanes. Ormocers are commonly prepared by sol−gel processing, which enables the sequential synthesis of inorganic and organic networks.20 Because of the interaction of the alkoxysilanes with hydroxyl groups, Ormocers adhere well to inorganic substrates, such as glasses, which makes them ideal for coating applications.21,22 An Ormocer by the trade name of Ormocomp is transparent over the 400−1600 nm wavelength range23 and has been shown to bind proteins24 and to be biocompatible with various cell types.25−27 Ormocomp has previously been used as micropatterned 3D cell culture substrates26,28−30 and as tissue-engineering scaffolds,5,24,25,31 but to the best of our knowledge, it has not been used in 2D as a cell culture substratum with nanoscale structure. To gain a better understanding of the function of hESC-RPE cells, it would be beneficial to have a means to assess dynamic changes in living cells. This necessitates that cells can be grown on a thin, transparent, rigid surface such as cover glass. To our knowledge, the culturing of hESC-RPE cells on silanefunctionalized glass surfaces has not been studied until now. In this work, we show how silanization with Ormocomp, APTES, or MAPTMS affects the chemical and physical surface properties of glass, the protein binding, and the hESC-RPE survival.

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MATERIALS AND METHODS

Surface Treatments. The silanized glass substrates were prepared by treating round borosilicate glass coverslips (9 mm diameter, 0.13 mm thickness, VWR Collection, VWR, Finland) with Ormocomp, 3aminopropyltriethoxysilane (APTES), or 3-(trimethoxysilyl)propyl methacrylate (MAPTMS) (both from Sigma-Aldrich, St. Louis, MO, USA). Additional samples were prepared by treatment with Ormodev solvent (50:50 4-methyl-2-pentanone/2-propanol, Micro Resist Technology GmbH, Germany), hexamethyldisilazane (HMDS), and ethanol. The Ormocomp samples were prepared in four steps: coverslips were cleaned by rinsing with 99.5% (v/v) ethanol, immersed in Ormocomp (Micro Resist Technology GmbH, Germany) for approximately 2 h, immersed in Ormodev solvent (50:50 4-methyl-2pentanone and 2-propanol, Micro Resist Technology GmbH, Germany) for approximately 5 min, and finally rinsed thoroughly with HMDS. Two different types of Ormocomp samples were prepared, Ormocomp and Ormocomp-I127, with Ormocomp used either as received or in combination with 2 wt % (w/w) photoinitiator Irgacure 127 (Ciba Specialty Chemicals, Switzerland, hereafter called Ormocomp-I127). Pure Ormocomp contains 1% photoinitiator DAROCUR TPO. The APTES samples were prepared in three steps: coverslips were soaked in methanol for 2 min followed by air drying, then dipped in a 2% (v/v) solution of APTES in acetone for approximately 2 min, and finally washed two times with ion-exchanged B


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nm, f = 200−400 kHz, spring constant = 25−75 N m−1, coating aluminum). The scan rate was 0.5 Hz. The arithmetic mean of the surface roughness (Ra) and the surface area ratio were calculated from the roughness profiles determined by AFM with XEI image-processing software (Park Systems). The surface area ratio was calculated by dividing the difference between the surface area and the geometric area by the geometric area and multiplying this ratio by 100%. X-ray Photoelectron Spectroscopy. The Ormocomp-I127 samples were examined with an X-ray photoelectron spectroscope (XPS) and compared to the ethanol-treated control samples. The spectrs of two Ormocomp-I127 samples and one control sample were obtained. By identifying the chemical composition of the outermost surface layers of the samples, the effect of the Ormocomp treatment on the composition of the glass surface and the reproducibility of the Ormocomp treatments were studied. The freshly prepared samples were introduced into the UHV system,33 and the loadlock was evacuated from atmospheric pressure to 5 × 10−8 mbar in 1 h before transferring the sample to the analysis chamber with a base pressure below 1 × 10−10 mbar. In XPS, nonmonochromatized Al Kα X-rays (1486.6 eV) were utilized for excitation, and the measurements were carried out at normal (0°) and grazing (60°) emission angles with a detection area of ∼600 μm in diameter. The surface elemental concentrations and chemical states of compounds were identified by analyzing the high-resolution spectra of C 1s, O 1s, Si 2p, B 1s, K 2p, Na 1s, Ti 2p, and Zn 2p transitions. After subtracting a Shirley- or linear-type background, the spectral components were fitted with a combination of Gaussian (70%) and Lorentzian (30%) line shapes. The sampling depths of C 1s, O 1s, Si 2p, B 1s, K 2p, Na 1s, Ti 2p, and Zn 2p signals, for example, in SiO2 are 10.1, 8.4, 11.2, 10.7, 10.0, 4.6, 8.9, and 5.0 nm, respectively. Ninety-five percent of the XPS signal is acquired from the surface layer with the thickness defined by the sampling depth. Human Embryonic Stem Cells. Previously derived hESC line Regea 08/017 (46, XX) was used in this study.34 The undifferentiated hESC was cultured on γ-irradiated (40 Gy) human foreskin fibroblast feeder cells (CRL-2429TM, ATCC, Manassas, VA, USA) under serum-free conditions described previously.7 The culture medium was changed five times a week, and undifferentiated colonies were manually passaged onto new feeder cells once a week. Cell culturing was done as previously described in.7 Briefly, undifferentiated hESC colonies were manually cut onto low cell binding six-well plates (Nalgene NUNC, Tokyo, Japan), where the cells formed floating aggregates. These aggregates were differentiated in medium consisting of the same reagents as the medium used to maintain hESCs with the modifications of 15% KnockOut Serum Replacement (KO-SR) and no basic fibroblast growth factor (bFGF).7 During differentiation, the medium was changed three times a week. After 90 to 150 days of differentiation in suspension culture, the pigmented areas of the floating aggregates were manually cut under a light microscope with a lancet into small pieces. The pigmented cell clusters were dissociated with 1× Trypsin-EDTA (Lonza, Walkersville, MD). The cells were filtered through a BD Falcon cell strainer (BD Biosciences) and seeded on Col IV-coated surface-treated materials at a density of 5.5 × 105 cells/cm2. Col IV coating of the unsilanized, ethanol, APTES, MAPTMS, or Ormocomp-treated substrata was carried out as described above. The cells were cultured on investigated materials for 7 or 42 days. Immunofluorescence. The protein expression and localization were investigated with immunofluorescence (IF) staining after 42 days of culture on the investigated materials. First, cells were washed twice with DPBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature (RT). Fixing was followed by three washes with DPBS and permeabilization of the cells with 0.1% Triton X-100 in DPBS (Sigma-Aldrich) at RT for 10 min. Next, unspecific binding sites were blocked with 3% BSA (Sigma-Aldrich) at RT for 1 h. Thereafter, samples were incubated with primary antibodies for 1 h at RT and rabbit antibestrophin 1:200, mouse anticellular retinaldehyde-binding protein (CRALBP) 1:500, mouse anti-Na+/K +ATPase 1:200 (all from Abcam, Cambridge, U.K.), and rabbit antizonula occludens 1 (ZO-1) 1:250 (Invitrogen). Cells were washed

several times with DPBS and labeled with secondary antibodies diluted in ratio of 1:800 with 0.5% BSA-DPBS/Alexa Fluor 568-conjugated goat antimouse IgG and goat antirabbit Ig G, and Alexa Fluor 488conjugated donkey antirabbit IgG and donkey antimouse IgG (all from Molecular Probes, Life Technologies). In addition, phalloidintetramethylrhodamine B isothiocyanate 1:300 (Sigma-Aldrich) was used for labeling filamentous actin. Samples were incubated in secondary antibody dilutions for 1 h at RT following repeated washes in DPBS. 4′,6′Diamidino-2-phenylidole (DAPI) included in the mounting media was used to stain the nuclei (Vector Laboratories Inc., Burlingame, CA). Images were taken with an AxioScope A1 fluorescence microscope (Carl Zeiss, Jena, Germany) using a 63× oilimmersion objective. Images were edited using ZEN 2011 Light Edition (Carl Zeiss) and Adobe Photoshop CS4. Analysis of Cell Number. The attachment and proliferation of hESC-RPE cells on surface-treated glass slides were determined with cell counts after either 7 or 42 days of culture. Six randomly chosen areas on each sample with DAPI-stained nuclei were captured with an AxioScope A1 fluorescence microscope and a 10× objective. Two parallel samples were analyzed in each experiment, and the data was collected from two individual cell culture experiments. The cell number on each image was counted with with the aid of the ImageJ image processing and analysis software cell counter plugin. Statistical Analyses. The contact-angle data were analyzed with the two-tailed student’s t test comparing each surface treatment with the ethanol control. The surface roughness and the surface area ratio data were analyzed with one-way ANOVA (analysis of variance) followed by Tukey’s multiple comparison test. A Mann−Whitney Utest and IBM SPSS statistics software were used to determine the statistical significance of cell number counts and Col IV absorption. Average (median) values of cell number and pixel intensity obtained from the Col IV coating on each sample were compared to average (median) values obtained on ethanol (reference) with the Mann− Whitney U-test. p values of ≤0.05 were considered to be statistically significant. Ethical Issues. The National Authority for Medicolegal Affairs Finland has approved our research on human embryos (Dnro 1426/ 32/300/05). We also have a supportive statement from the local ethics committee of the Pirkanmaa hospital district Finland to derive and expand hESC lines from surplus embryos not used in the treatment of infertility by donating couples and to use these cell lines for research purposes (R05116). No new cell lines were derived in this study.

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RESULTS Contact Angles. To gain better insight into the wettability of surface-treated samples, samples without a Col IV coating were assessed by contact-angle measurements (Figure 1). All of the samples were found to be hydrophilic, with average contact angles smaller than 90°. There was no significant difference between the contact angles of the ethanol controls and the APTES, MAPTMS, and Ormodev samples, which all had contact angles close to 60°. However, the Ormocomp and HMDS treatments increased the contact angle of the glass surface by approximately 20° compared to the ethanol control, making these surfaces significantly more hydrophobic (p < 0.001). Surface Roughness and Surface Area Ratio. The surface roughness (Ra) and the surface area ratio of the APTES, MAPTMS, and Ormocomp-I127 samples with and without the Col IV coating were studied with atomic force microscopy. There were no statistically significant differences in the surface roughness of the noncoated and Col IV-coated samples, although a slight increase was found for the control, MAPTMS, and Ormocomp-I127 samples (Figure 2a). The OrmocompI127-treated surfaces without the Col IV coating were significantly rougher (p < 0.001) than the surface of the untreated control, APTES, and MAPTMS samples. The C


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APTES-treated surface was also rougher than the untreated control. Among the Col IV-coated samples, the OrmocompI127-treated surfaces were significantly rougher (p < 0.001) than the APTES, MAPTMS, and control surfaces, which were all equally smooth. As shown by Figure 2b,c, the noncoated Ormocomp-I127 had a significantly higher surface area ratio than the Col IV-coated Ormocomp-I127 (p = 0.034), control, or MAPTMS samples (p < 0.001). This was also evident by the large number of surface features on the noncoated OrmocompI127 surface (Figure 2c). The Col IV coating decreased the number of surface features and the surface area ratio of the Ormocomp-I127 samples to comparable values with the control, APTES, and MAPTMS samples. According to the surface roughness data (Figure 3, left side), UV-polymerized Ormocomp-I127 thin films were significantly smoother (p < 0.001) than the Ormocomp-I127-treated glass surface. The noncoated Ormocomp-I127 thin films also had a significantly lower (p < 0.005) surface area ratio (Figure 3, right side) than did the Ormocomp-I127-treated glass surface. The Col IV coating significantly increased the surface area ratio and the roughness of the Ormocomp-I127 thin films to a level comparable to that of the Ormocomp-I127-treated glass surface.

Figure 1. (a) Representative photographs of water droplets on each of the treated surfaces. (b) Static water contact angles of samples with different surface treatments. The data points represent mean ± standard deviation (n = 10). *** (p < 0.001) indicates significance.

Figure 2. (a) Surface roughness (Ra) and (b) surface area ratio of the ethanol control, APTES, MAPTMS, and Ormocomp-I127 samples with and without the Col IV coating. The data points represent the mean ± standard deviation (n = 6). * (p < 0.05) indicates the significance between the noncoated and Col IV-coated samples, + (p < 0.05) indicates the significance among the noncoated samples, and − (p < 0.05) indicates the significance among the Col IV-coated samples. (c) Representative AFM images of the ethanol control, APTES, MAPTMS, and Ormocomp-I127 samples with and without the Col IV coating. D


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immunofluorescence staining and analyzed the the results with ImageJ software. The highest absorption of human Col IV was detected on Ormocomp-I127 samples (1.32-fold compared to the ethanol control), whereas lower values were seen with APTES and MAPTMS (both 0.90-fold against the ethanol control) (Figure 5). The absorption of human Col IV was significantly higher on Ormocomp-I127 than on the ethanol control (p = 0.014), APTES (p = 0.027), and MAPTMS (p = 0.014). Furthermore, the protein binding on Ormocomp-I127 samples was rather uniform throughout the samples, whereas more heterogeneous absorption was detected on APTES, as can be seen with a higher standard deviation (Figure 5). Analysis of Cell Number. The hESC-RPE cells are commonly grown on top of the extracellular matrix coating.1,7 Thus, the cell attachment and proliferation of hESC-RPE cells was evaluated after 42 days of culture on untreated, APTES, MAPTMS, Ormocomp-I127, and Ormocomp-treated samples all having an additional Col IV coating (Figure 6). All silane treatments increased the cell number on glass substrates compared to the ethanol control. The highest cell numbers of 401 534 ± 125 408 and 383 563 ± 113 316 cells cm−2 were detected on Ormocomp-I127 and Ormocomp, respectively. Cell numbers reached 162 200 ± 216 441 cells cm−2 for APTES and 191 236 ± 166 244 cells cm−2 for MAPTMS. The lowest cell number was obtained on the ethanol control with 62 483 ± 122 540 cells cm−2. The average cell number on surfacemodified substrates, except that for APTES, significantly (p < 0.05) differed from the average cell number of the ethanol control. Furthermore, the cell numbers on Ormocomp-I127 and Ormocomp were significantly higher (p < 0.005) than for APTES and MAPTMS. To investigate whether the increased number of cells on Ormocomp-treated surfaces is due to enhanced protein binding on the material surface, we evaluated the cell attachment of hESC-RPE cells on uncoated Ormocomp-I127-treated glass and on Ormocomp-I127-treated glass that had an additional Col IV coating. After 7 days of culture, the cell number was significantly higher (p < 0.001) on Ormocomp-I127-treated glass with a Col IV coating compared to the uncoated sample. The cell numbers reached 167 798 ± 31 066 cells cm−2 for Col IV-coated Ormocomp-I127 samples and 40 956 ± 15 113 cells cm−2 for uncoated Ormcomp-I127 samples. The number of cells on uncoated Ormocomp-I127 samples was slightly higher than the number of cells of 17313 ± 9751 cells cm−2 on the ethanol control, but the difference was not statistically significant. Cell number analysis was also done for UV-polymerized Ormocomp-I127 thin films and Ormocomp-I127 glass surfaces that were coated with Col IV. The hESC-RPE cells were cultured for 7 days (Figure 8a−d) after which cell number analysis was done in the same manner as for other samples. The cell number analysis (Figure 8e) showed that the OrmocompI127-treated glass surfaces had slightly more cells (255 952 ± 30 573 cells cm−2) than Ormocomp-I127 thin films (222 065 ± 34 332 cells cm−2), but the difference was not statistically significant. Analysis of hESC-RPE Cell Maturation. Cell adhesion and maturation were examined with phase-contrast imaging and immunofluorescence staining after 42 days of culture of hESC-RPE cells on untreated, APTES, MAPTMS, OrmocompI127, and Ormocomp-treated coverslips that were coated with Col IV (Figure 9). The hESC-RPE cells on Ormocomp-I127 and Ormocomp samples had formed a confluent and uniform

Figure 3. (a) Surface roughness Ra (left side) and surface area ratio (right side) of UV-polymerized Ormocomp-I127 thin films and Ormocomp-I127-treated glass surfaces with and without the Col IV coating. The data points represent the mean ± standard deviation (n = 6). * (p < 0.05) indicates the significance between the noncoated and Col IV-coated samples, + (p < 0.05) indicates the significance among the noncoated samples, and − (p < 0.05) indicates the significance among the Col IV-coated samples. Representative AFM images of Ormocomp-I127 thin films (b) without and (c) with Col IV coating.

XPS Analysis. The changes in the elemental composition of the glass surface after Ormocomp treatment were evaluated by XPS. The Ormocomp treatment doubled the relative surface concentration of carbon (C) on the glass surface from 8.1 atom % to the average of 17.5 atom % (Table 1). The thicker carbonTable 1. Elemental Surface Concentrations of the Ethanol Control Sample and Ormocomp-I127 Sample Surfaces relative elemental surface concentratios (atom%) sample ethanol control OrmocompI127-1 OrmocompI127-2

C

O

Si

B

K

Na

Ti

Zn

8.1

54.8

27.3

2.9

2.5

2.1

1.2

1.1

15.8

51.4

25.2

2.9

1.7

1.5

1.0

0.5

18.5

49.9

24.1

2.9

1.6

1.3

1.1

0.6

containing layer on the glass surface attenuated the photoelectron signals from the elements present only in the borosilicate glass. The C 1s transition was broadened toward higher binding energies because of the Ormocomp treatment, suggesting an increased number of C−O compounds (Figure4). Protein Binding on Surface-Treated Materials. The protein-binding properties of untreated, APTES, MAPTMS, and Ormocomp-I127 samples were investigated with indirect E


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Figure 4. XP spectra measured on the surface of the ethanol control and Ormocomp-I127 sample. The inset shows high-resolution spectra of K 2p and C 1s. Σ1 (285.0 eV): C−C, C−H. Σ2 (286.0 eV): C−O. Σ3 (289.0 eV): CO, O−CO, O−CO−O.

Figure 5. Protein binding of human Col IV on surface-treated materials. The data is represented as normalized values against the ethanol control (protein binding equals 1), * (p < 0.05) indicates significance compared to the ethanol control.

Figure 6. (a) Representative images of nuclear stain DAPI used for cell number analysis. (b) Cell number analysis after 42 days of culture on surface-treated materials that had an additional Col IV coating (n = 2), with ** (p < 0.005) and *** (p < 0.001) indicating the significance compared to the ethanol control.

RPE monolayer with abundant pigmentation and typical hexagonal RPE cell morphology (Figure 9a−c). In contrast, the hESC-RPE cells grew as raftlike heterogeneous structures on APTES and MAPTMS surfaces, and confluent cultures were not reached during the 42-day culture period. Moreover, on APTES and MAPTMS, the cell morphology was irregular with abundant fibroblast-like elongated cells; fewer cells with hexagonal RPE morphology were seen. On the ethanol controls, poor cell attachment was detected; cells grew as spherical clumps. In immunofluorescence staining, the hESC-RPE cells on Ormocomp-I127 and Ormocomp samples were strongly positive for bestrophin and CRALBP proteins that are specific to mature RPE-cells (Figure 9d,e). Bestrophin and CRALBP positive cells were also seen on the APTES and MAPTMS

samples and ethanol controls. Uniform expression of tight junction protein ZO-1 and polarization marker Na+/K+ATPase was detected on Ormocomp-I127 and Ormocomp, whereas on APTES, MAPTMS, and the ethanol control the expression of these proteins was discontinuous.

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DISCUSSION Glass is thin, transparent, and easy to clean, which makes it a widely used substrate for studying cell functions with microscopic methods.8 However, glass can either induce or inhibit the adherence of cells, and special methods are often F


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Figure 7. Low-magnification phase contrast and immunofluorescence images showing the cell attachment after 7 days of culture on the ethanol control (a and d), uncoated Ormocomp-I127-treated glass (b and e), and Ormocomp-I127-treated glass that had an additional Col IV coating (c and f). Filamentous actin staining (red) with phalloidin that illustrates the cell morphology of hESC-RPE cells on surface-treated materials. The nuclei were counterstained with DAPI (blue). The scale bar is 200 μm. (g) The cell number analysis of the 7-day-cultured hESC-RPE cells on OrmocompI127-treated glass with and without an additional Col IV coating. *** (p < 0.001).

Figure 8. Low-magnification phase-contrast images elucidating the cell attachment after 7 days of culture on Ormocomp-I127 thin films (b and d) and Ormocomp-I127-treated glass (a and c) that had an additional Col IV coating. Filamentous actin staining (red) with phalloidin that illustrates the cell morphology of hESC-RPE cells on surface-treated materials. The nuclei were counterstained with DAPI (blue). The scale bar is 200 μm. The cell number analysis of the 7-day-cultured hESC-RPE cells on both Ormocomp-I127 thin films and Ormocomp-I127 glass (e).

needed to enhance the adherence of cells to glass.8,15,35 HESCRPE cells are shown to have a closer resemblance to native RPE cells than several immortalized RPE cell lines.36 Although the clinical transplantation trials with these have already started,37 there are still many aspects of signal propagation that would be preferably studied by live cell microscopy methods. The lack of published data implies and our own empirical tests have shown that hESC-RPE cells do not adhere well to glass. A good adhesion promoter on glass should have high transparency and preferably a low processing temperature together with sufficient thermal stability.21 We used the two most common adhesion promoters APTES13 and MAPTMS, both of which have been previously applied to enhance the adherence of several cell types.19 We also tested the polymer− ceramic material Ormocomp, which has been used to fabricate 3D cell culture surfaces by direct laser writing.25,28,29 All of the above-mentioned materials share the optimal features of a good adhesion promoter.21,26 The wettability of the surface is influenced by the surface chemistry but also by the surface roughness.5 Wettability is an important feature when estimating the suitability of a surface treatment for cell culture5 as values that are too low inhibit cell spreading and values that are too high inhibit adherence.38

Different cell types prefer different water contact angles. For several different cell types, the optimal contact-angle range has been shown to be between 65 and 80°.39 However, for an immortalized human RPE cell line (D407), a contact angle of 43−53° has been shown to be optimal.38,39 According to our knowledge, the effect of the surface contact angle on the culturing of hESC-RPE cells has not yet been evaluated. The previously reported water contact-angle values of APTES coatings on glass range from approximately 45°15,40 to 65°41 and 75°.42 The great variation is explained by the multiple possible orientations of APTES molecules, which depend on reaction conditions such as the concentration, time, temperature, amount of water, and measurement technique.15,40,42 In our study, the contact angle of APTES-treated glass was 59 ± 2°, which was essentially the same as the value for untreated glass. The relatively high contact angle indicates hydrophilic amine groups directed toward the glass surface due to hydrogen bonding.43,44 The MAPTMS coating increased the average contact angle of glass by 10° to almost 70°; however, there was variation between the samples, suggesting a nonuniform coating. In previous studies, MAPTMS treatment has been shown to decrease the contact angle from 99 to 46°,45 G


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Figure 9. (a) Low-magnification phase-contrast images elucidating the cell attachment after 42 days of culture on surface-treated materials that had an additional Col IV coating. The scale bar is 200 μm. (b) Representative higher-magnification phase contrast images showing the cell morphology and degree of pigmentation of hESC-RPE cells. The scale bar is 100 μm. (c) Filamentous actin staining with phalloidin illustrates the cell morphology of hESC-RPE cells on surface-treated materials. IF staining with RPE-specific proteins (d) bestrophin and (e) CRALBP. Localization of tight junction protein ZO-1 (f) and polarization marker Na+/K+ATPase (g). The nuclei were counterstained with DAPI (blue). The scale bars for IF images is 50 μm.

but in these cases, MAPTMS was in mixtures45−47 and not in its pure form as in this study. Ormocers are known to bind covalently on glass surfaces because of their polysiloxane network.21 This was also indicated in our study by the increased hydrophobicity of the glass surfaces after Ormocomp treatment. Among the samples studied here, the Ormocomp treatment resulted in the most hydrophobic surface with a contact angle of approximately 80°. This value is slightly higher than the previously reported values of 70° for Ormocomp on glass48 and 72° on silicon,49 which is likely due to differences in the coating procedures. Treatment with organosilicon compound HMDS also increased the hydrophobicity to a value close to that of the Ormocomptreated glass. This is not unexpected because HMDS is also

known to bind covalently to the surface hydroxyl groups with the methyl groups increasing the surface hydrophobicity.50 The binding of Ormocomp to the borosilicate glass surface was further confirmed by the XPS results, which indicated that the Ormocomp treatment altered the chemical state of the glass surface by increasing the number of molecules containing C−O and CO bonds. This is also consistent with previously reported ATR-FTIR results showing that Ormocomp contains polar O−H and CO bonds, which increases the polarity of the surface and may also facilitate cell adhesion.29 Surface roughness affects the type and strength of interactions in the biomaterial−biological environment and therefore it is an important feature of the cell culture substratum.4,38,39 In several previous studies, APTES treatment H


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RPE cells. This is in accordance with the previously reported noncytotoxicity of I127 with hESC-derived neuronal cells55 and adipose stem cells.27 Our results show that Ormocomp as a 2D substrate permits not only the adherence but also the migration of hESC-RPE cells and supports the generation of a homogeneous and mature cell culture. This notably improved adhesion and maturation of hESC-RPE cells on the Ormocomp-I127 substrata was most likely due to enhanced Col IV binding, which was verified by an immunofluorescence protein-binding assay. This enhanced protein-binding property of the Ormocomp-I127 surfaces could be due to the changes in the glass hydrophobicity, surface roughness, or surface chemistry. Because the HMDS-treated surfaces with a comparable contact angle did not support hESC-RPE growth (data not shown), it is unlikely that increased hydrophobicity alone attributed to the enhanced Col IV binding. The Ormocomp-I127-treated glass was significantly rougher than untreated glass, APTES, and MAPTMS, and the effect of surface roughness was therefore studied further. Significantly smoother Ormocomp-I127-treated surfaces, i.e., UV-polymerized Ormocomp-I127 thin films, were prepared and cultured with hESC-RPE cells. The hESC-RPE cells adhered and matured equally well on smoother Col IVcoated Ormocomp-I127 thin films and on rougher Col IVcoated Ormocomp-I127-treated glass. On the contrary, Ormocomp-I127-treated glass without the additional collagen coating resulted in an inadequate cellular response. This was a clear indication that it was likely not the surface roughness but the surface chemistry and, more specifically, the enhanced collagen binding on Ormocomp, which is beneficial to hESCRPE cell culture. This suggests that Ormocomp positively affects cell maturation, which might be beneficial in live cell imaging studies.

has been shown to increase the surface roughness in the 5−15 nm range,44 and the surface roughness was further increased by protein binding.51,52 Our results of APTES surface roughness both with and without protein coating were in agreement with the earlier studies. MAPTMS coatings have been previously shown to be rather smooth when studied by AFM,53 which is also consistent with our findings. In this study, the Ormocomp-treated surface was 1.2−7.1 times rougher than the untreated or APTES- or MAPTMStreated surfaces and the difference in roughness became even more evident after the Col IV coating. According to the supplier, Ormocomp coatings are typically very smooth with a root-mean-square (rms) surface roughness of 2−4 nm.28 An even lower rms roughness value of 0.97 nm has recently been reported by Malainou at el.48 Without the Col IV coating, we measured an average rms surface roughness of 4.6 ±1.0 nm. Thus, our coating procedure resulted in a rougher Ormocomp surface, which also supported protein binding. The enhanced protein binding property of Ormocomp compared to the untreated, APTES-, or MAPTMS-treated surfaces was further verified by an immunofluorescence protein binding assay. Although APTES is a widely used cell-adhesion promoter,13 the growth of many cell types on APTES has not been homogeneous.15 This was also evident in our experiments wherein the hESC-RPE cells failed to form a homogeneous cell monolayer on APTES. This could be linked to the typically nonuniform nature of the APTES coatings, which instead of a monolayer consists of islands with a gradient cross-linking density.10 MAPTMS has also been used in various cell culture and coating experiments and has been showed to support the growth of several cell lines.19 However, according to our knowledge, this is the first study in which hESC-RPE cells were cultured on MAPTMS. On the basis of our experiments, MAPTMS does not support hESC-RPE cell adherence under serum-free conditions. Ormocomp has previously been shown to support the attachment and growth of various cell types in 3D scaffolds.25,26,29 In this work, we used Ormocomp in an unconventional way as a 2D adhesion promoter. The cell count after 42 days of culture showed that Ormocomp noticeably supported hESC-RPE cell adhesion: on Ormocomp and Ormocomp-I127 samples there were four times more cells than on untreated glass and two times more cells than on APTES or MAPTMS. The high cell count on the Ormocomptreated surfaces was most likely due to the homogeneity of the culture. Cell attachment, cell density, and homogeneity are important features for cell culture because they affect cell maturation and thus the function of the entire culture.35 The results clearly showed that Ormocomp enabled the formation of a more homogeneous and pigmented cell layer than the untreated, APTES-, or MAPTMS-treated surfaces. The hESCRPE cells matured well on the Ormocomp surfaces, evincing the homogeneous expression of tight junction marker ZO-1, RPE maturation marker CRALBP and bestrophin, and polarization marker Na+/K+ATPase localization. Several photoinitiators are shown to reduce the viability of cells.54 According to the above-mentioned protein markers, the maturation status of hESC-RPEs was equally good on Ormocomp substrata with and without photoinitiator I127. Also according to a cell number analysis, the viability of the hESC-RPE cells was even better on the I127-containing substrata than on the substrata without I127. Thus, we conclude that the Ormocomp-I127 combination does not adversely affect the viability of hESC-

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CONCLUSIONS In this study, we showed that a simple 2D Ormocomp treatment of glass increased the surface hydrophobicity, surface roughness, and Col IV binding compared to commonly used commercial silanizing agents APTES and MAPTMS. Ormocomp treatment also significantly increased the adherence of the hESC-RPE cells, thus enabling the formation of a homogeneous cell monolayer. There were no differences in cell numbers between Ormocomp-treated surfaces with different roughnesses, suggesting that the surface chemistry and, more specifically, the enhanced collagen binding after the Ormocomp treatment support hESC-RPE cell adherence and maturation. Our results propose that Ormocomp silanization could in the future be utilized to culture hESC-RPE cells for microscopic live cell imaging methods.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: kati.juuti-uusitalo@uta.fi. Author Contributions ⊥

E.K. and A.S. made equal contributions to this work.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. I


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ACKNOWLEDGMENTS Part of the data was presented at ARVO 2014 meeting in Orlando, USA. We thank Elina Pajula, Outi Heikkilä, Outi Melin, Hanna Pekkanen, Elina Konsén, and Alexandra Mikhailova for culturing the cells and Samu Hemmilä for technical assistance with the contact-angle measurements. This study was financially supported by the Academy of Finland (grant numbers 218050, 137801, 253134, and 310059), the Finnish Cultural Foundation, the Finnish Funding Agency for Technology and Innovation, and the Doctoral Program of Tampere University of Technology’s President.

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Ormocomp-Modified Glass Increases Collagen Binding and Promotes

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