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Surfaces, Interfaces, and Applications

3D microcontact printing for combined chemical and topographical patterning on porous cell culture membrane. Justyna Borowiec, Joerg Hampl, Sukhdeep Singh, Sebastian Haefner, Karin Friedel, Patrick Mai, Dana Brauer, Florian Ruther, Liliana Liverani, Aldo Roberto Boccaccini, and Andreas Schober ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06585 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 11, 2018

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ACS Applied Materials & Interfaces

3D microcontact printing for combined chemical and topographical patterning on porous cell culture membrane. Justyna Borowiec1*, Joerg Hampl1, Sukhdeep Singh1, Sebastian Haefner1, Karin Friedel1, Patrick Mai1, Dana Brauer1, Florian Ruther2, Liliana Liverani2, Aldo R. Boccaccini2, Andreas Schober1

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Department of Nanobiosystem Technology, Institute of Micro- and Nanotechnologies MacroNano, Institute of Chemistry and Biotechnology, Ilmenau University of Technology, 98693 Ilmenau, Germany

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Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, 91058 Erlangen, Germany Corresponding Author * Tel.: +49 3677 69 1965; e-Mail: [email protected]; Postal address: Department of Nanobiosystem Technology, Institute of Micro- and Nanotechnologies MacroNano, Ilmenau University of Technology, Gustav-Kirchhoff-Strasse 7, Feynmanbuilding Room 205, 98693 Ilmenau, Germany

Keywords: 3D micropattern technique; microcontact printing; thermoforming; cell adhesion; extracellular matrix

Abstract Micrometer-scale biochemical or topographical patterning is commonly used to guide the cell attachment and growth, but the ability to combine these patterns into an integrated surface with defined chemical and geometrical characteristics still remains a technical challenge. Here, we present a technical solution for simultaneous construction of 3D morphologies, in the form of channels, on porous membranes along with precise transfer of extracellular matrix proteins into the channels to create patterns with geometrically restricting features. By combining the advantages of microthermoforming and microcontact printing this technique offers a unique patterning process that provides spatiotemporal control over morphological and chemical feature in a single step. Using our 3D-microcontact printing (3DµCP), determined microstructures like 1

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channels with different depths and widths even with more complex patterns can be fabricated. Collagen, fibronectin and laminin were successfully transferred inside the pre-designed geometries and the validity of the process was confirmed by antibody staining. Cells cultivated on 3DµCP patterned polycarbonate membrane have shown selective adhesion and growth. This technique offers a novel tool for creating freeform combinatorial patterning on the thermoformable surface.

1. Introduction Emerging concepts of mimicking cellular hierarchy using principles of 3D cell cultivation1, biotechnical multiscale engineering2 and organ-on-a-chip devices3 have attracted various engineering disciplines to work together with life sciences. Creating a user-defined vital multicellular morphology on artificial substrate is a key factor for understanding the cellular bases of organ function4. Therefore, techniques to control 3D arrangement of cells at the microscale level are of high importance in modern biotechnology. Recent progress in

3D

bioprinting5-7 hybrid hydrogels8-11, surface modification12-13 or click chemistry14 techniques has enabled the direct immobilization of cells and biomolecules in 3D matrices. However, the complexity and material dependent nature of those methods make them less general. In contrast, microfabrication techniques like photolithography15-16, soft lithography17-18 , stimuli responsive polymers19, or micro molding20 are able to create patterned surface on various solid materials, but obtaining simultaneous control on chemical composition and surface topology are challenging with these techniques. In particular, soft lithographic techniques like microcontact printing (µCP) have emerged as simple and efficient manually operated methods to pattern different planar substrates with proteins or polymers21 even in an automated way22. Typically µCP technique is applied only to 2


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the planar substrates. Kehr et al. have extended the scope of µCP by demonstrating the use of µCP for non-planar materials of nano- and micrometer sizes by printing zeolites on SAMs of zeolite L crystals23. Similarly, Fisher et al. explored the use of micro contacting to pattern RGD functionalized zeolites on melanic hydrogel thin films24. These studies have shown the organized spatial controlled cell adhesion onto nano or micro patterned surfaces. It has been shown that there is a synergistic effect of integrated chemical and topographical surface patterning on controlling the 3D-shape of single cells

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, noninvasive control of

endothelial cells elongation 27 and cytoarchitectural polarization in primary neurons28. Therefore, developing methods to obtain an integrated patterning are of high interest in this field. In one attempt, Waterkotte et al. have demonstrated the possibility of using microthermoforming of a chemically pre-patterned surfaces, to obtain randomly distributed chemical cues on microstructured surface29. Another approach used “PoT” printing technique enables the transfer of bioactive molecules inside of geometrically restricted sites by using specially designed soft stamps30. Moraes et al. have used a crack-based approach to produce fiber-like patterns on microfabricated substrates31. However, to generate combinatorial patterns for these studies, multistep process and wet-printing methods were used, which limit the efficiency in terms of high throughput. Despite a step forward, these methods are limited in terms of defining chemical patterns specifically in relation to geometrical patterning. With present microcontact printing techniques, there is no provision to create microscopic geometrical barriers along with chemical patterning. In other words, technical means of transferring biochemical molecules to the pre-structured micro-geometries in precise, reproducible and high throughput manner are limited. Therefore, the applicability of conventional microcontact printing remains restricted when complex 3D

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surfaces e.g. channels or tubular structures with defined chemical and topographical micropatterns are desirable. In this study, we report a 3-dimensional microcontact printing (3D µCP) technique, which is capable of forming microtopographies on the surface and precise transfer of the extracellular matrix (ECM) proteins into the obtained geometries simultaneously. We combined the advantages of microcontact printing with microthermoforming to synchronize the chemical and topological patterning in one step, thereby extending the scope of 2D surface patterning to 3D. For this purpose we used conventional stamps made from polydimethylsiloxane (PDMS)32. In brief, a stamp with a target pattern was selectively inked with biomolecules and subsequently used as mold for microthermoforming. Therefore, microstructuring and chemical patterning were performed at the same time. During thermoforming a polymer film is heated up to the thermoplastic state and formed under gas pressure onto a mold to replicate its topography33. Microthermoforming is highly reproducible, efficient for mass production, and works for most thermoplastics polymers, including biodegradable polymers used in tissue engineering as well as porous and permeable polymer films34. We chose polycarbonate membranes as substrate because they are commonly used for cell culture applications, highly transparent and are available in different thickness and pore sizes35. The validity of our process has been demonstrated by microscopic measurements, fluorescence staining and finally, by testing the substrates to cell adhesion response.

2. Methods 2.1 Fabrication of PDMS stamp. Stamp masters were produced with standard photolithographic methods36. A photomask with a number of different features (lines ranging from 30 to 400 µm in widths, 30 to 75 µm in depth and 400 µm in spacing) was used. PDMS replicas were produced by pouring the uncured mixture over the whole wafer, which was previously vigorously stirred and degassed in vacuum (Sylgard 184, Dow Corning, US, 1:10 ratio of curing agent to prepolymer). The mixture was then cured on a hot plate (70 °C, 30 min). After 24 h at room temperature, PDMS was peeled off from the 4


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master mold and the required areas were extracted by cutting. The thickness of the stamps was about 2 mm. Before using, the stamps were heat treated for 1 h at 200 °C to outgas the moisture and remaining monomers and reduce shrinkage during the thermoforming process. 2.2 Inking of PDMS Stamp. For the subsequent patterning of proteins, collagen was applied only to the top features of PDMS stamp by inverted µCP. A glass coverslip was covered with collagen type I (C3867, Sigma) diluted to a concentration of 200 µg/ml in distilled water, incubated for 1h and dried. Laminin (L2020, Sigma) and fibronectin (F1141, Sigma) were applied similarly except laminin was diluted to working concentration of 100 µg/ml and fibronectin was diluted to 50 µg/ml in distilled water. Meantime, PDMS stamp was treated (150W, 120 s) with oxygen plasma (PVA TePla 200), to achieve hydrophilicity. An oxidized stamp was then turned upside down such that the features were facing down and gently placed onto the collagen-coated glass. To ensure a good contact of the stamp with the glass surface, the stamp was shortly pressed down using tweezers. After 30 min the stamp was carefully removed and finally transferred to microthermoforming process. 2.3 Topographical and chemical patterning - 3DµCP A microstructuring process such as microthermoforming was applied to produce the topographical patterns on the heavy ion etched polycarbonate (PC) membrane (it4ip, Louvain-laNeuve, Belgium) with a thickness of 50 µm. In order to simultaneously perform chemical modifications inside the formed features, a PDMS stamp previously inked with ECM proteins was used as mold for thermoforming. Therefore, microstructuring and microcontact printing were performed in one step. First the patterned face of the PDMS stamp was placed in contact with the porous PC membrane. For closing the pores during processing, a 50 µm thick poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP) foil was placed underneath PC foil as protection and force transducing layer (Fig. 1a). The assembled stack was then inserted into the chamber of the microthermoforming machine (WLP 1600S, WICKERT Presstech, Landau). The assembled stack was heated to 100°C; the process chamber was completely closed and subsequently evacuated. At 158° C the foil was tempered for 30 s and a pressure of 40 bars was applied to stretch the PC membrane over the PDMS mold. Afterwards, the machine was cooled down and vented to atmospheric pressure. Finally, the newly patterned PC membrane was carefully released from the PDMS mold and FEP layer to obtain patterned PC membrane (Fig. 1b). 2.4 Characterization of the patterned surface. For the characterization of topological patterning after thermoforming, samples were sputtercoated with platinum and observed using laser scanning digital microscope (Olympus LEXT OLS4100, Fig. 1c). The LSM images were analyzed to measure the widths and depths of thermoformed channels. All obtained data were expressed as mean ± standard deviation of measurements from 3 independent samples and at least 3 channels in each sample. Nanoindentation measurements were performed to assess local mechanical properties, by using an optical nanoindenter (PIUMA, Optics 11, The Netherlands) at room temperature. Two different types of probe were used, with cantilever stiffness of 0.49 N/m and 142 N/m. The 5



measurements were performed on dried samples in air and on samples immersed in Hank´s Balanced Salt Solution (HBSS, Sigma Aldrich, Germany). Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analysis was used to investigate the presence of collagen on the PC substrate and eventual interaction between them, by using a spectrometer (Nicolet 6700, Thermo Scientific, Germany) with 32 scans at resolution of 4 cm-1 in the wavenumber range between 4000 and 550 cm-1. Patterning of ECM molecules was characterized by fluorescent staining using specific antibodies. Samples were fixed for 30 min with 4% paraformaldehyde and subsequently washed with PBS, followed by 15 min of blocking with 5% BSA/PBS and again PBS washing. After this pretreatment, samples patterned with collagen were incubated for 1h with a conformation dependent anti-collagen I monoclonal antibody (ab6306, Abcam, UK), diluted 1:50 in 1% BSA/PBS, washed with PBS and incubated with a purified Alexa Fluor_ 555 conjugate goat antimouse secondary antibody (A-21422, Thermo Fisher Scientific) diluted 1:200 in 1% BSA/PBS for 1 h. Samples printed with fibronectin were stained according to the same protocol using monoclonal anti-fibronectin antibody produced in mouse (F0791, Sigma) followed by staining with Alexa Fluor® 405 - conjugated goat anti-mouse secondary antibody (ab175661, Abcam). Samples printed with laminin were labeled using anti-laminin antibody produced in rabbit (L9393, Sigma) followed by staining with Alexa Fluor® 488-conjugated goat anti-rabbit secondary antibody (A11034, Thermo Fisher Scientific). All samples were then mounted using Mowiol (Sigma) and examined using a laser scanning microscope FV1000 (Olympus, Hamburg, Germany). 2.5 Cell response to patterned surface. The 3DµCP patterned PC samples were placed in 24 well microtiter plates and sterilized with ultraviolet (UV) treatment in a cell culture hood for 15 min. To demonstrate the principal biological applicability of patterned samples, the human endothelial-like EA.hy926 cell line (ATCC, Rockville, USA), the mouse fibroblast cell line L929 (ATCC, LGC Promochem, Wesel, Germany) and the hepatoma cell line HepG2 (ATCC, LGC Promochem, Wesel, Germany) were used. EA.hy926 cells were cultivated in Dulbecco's Modified Eagle's Medium (Sigma), supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 2% L-glutamine and 1% sodium pyruvate. 50 µl of cell suspension containing 1 × 104 cells was seeded per substrate. After 2 h 1 ml medium was added. The cells were cultured for 24 h or longer. L929 cells were cultivated in RPMI 1640Medium (Sigma) and supplemented with 10% fetal calf serum (FCS, Biochrom), 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine. 50 µl of cell suspension containing 4 × 104 cells was seeded per substrate. After 2 h 1 ml medium was added. The cells were cultured for 24 h. HepG2 cells were cultivated in Minimum Essential Medium (Sigma), supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine. 50 µl of cell suspension containing 2 × 104 cells was seeded per substrate. After 2 h 1 ml medium was added. The cells were cultured for 24 h. All tests were repeated in triplicate.

2.6 Actin cytoskeleton labeling Different cell species were fixed with 4 % paraformaldehyde for 15 min. For fluorescent staining of actin filaments, samples were incubated in 0.25 % Triton X-100 for 15 min, blocked with 5% 6


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BSA for 30 min and incubated with 100 nM Alexa Fluor 488-labeled Phalloidin (Thermo Fisher Scientific) for 30 min at room temperature. Cell nuclei were stained with 4´,6-diamidino-2phenylindole (DAPI; D8417; Sigma–Aldrich). Cells were 2 times rinsed with PBS and then mounted with Mowiol. Imaging takes place via laser scanning microscope FV1000 (Olympus, Hamburg, Germany).

2.7 Live/Dead staining For Live–Dead staining cells were incubated with the Live/Dead cellular staining kit II (PromoCell GmbH, Heidelberg, Germany) according to the manufacturer's instructions. Samples were imaged using laser scanning microscope FV1000 (Olympus, Hamburg, Germany). 2.8. SDS-PAGE and Coomassie staining 50 µg of collagen, laminin and fibronectin were thermoformed and subsequently dissolved in 70 µl of Laemmli buffer without (collagen) or with mercaptoethanol (laminin, fibronectin) for 2h at RT with gentle shaking. The corresponding native ECM molecules were treated in exactly the same way. All samples were heat-denaturated for 5 min at 95°C. 30 µl of the thermoformed and 15 µg of the native ECM molecules in Laemmli-buffer were separated on 6 % polyacrylamide gels. Gels were stained with Coomassie brilliant blue according to a common protocol. 2.9. SEM analysis Cells were fixed using 4% paraformaldehyde at 4° C for 15 min. Samples were dried, coated with platinum and examined by scanning electron microscopy (SEM Hitachi S 4800).

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Fig.1 3DµCP patterned scaffolds fabrication and characterization. Schematic of inverted printing of PDMS stamp and one-step microstructuring and microcontact printing of PC foils (a), surface topography of thermoformed scaffolds visualized by SEM (b), 3DuCP patterned channel visualized and measured using laser scanning digital microscope (c) and pattern size fidelity according to designed size and PDMS master (d). 8


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3. Result and discussion. 3.1 Surface fabrication and characterization. The fabrication and characterization process for creating film-based porous substrates with chemical and topographical guidance cues is shown in Fig. 1. The initial step was inking of the PDMS stamp with ECM proteins just to the parts where the molecules were needed for the subsequent thermoforming process. For different proteins we used the protein concentrations recommended in protocols of µCP described in literature30, 37-38. However, to prevent the inking of the entire surface, the stamp was not exposed to the protein solution used in conventional microcontact printing, but only to the protein-coated glass slice that mediates the transfer of the biomolecules on PDMS stamp features. Consequently, stamp features were not subjected to the capillary forces or swelling39. To promote good transfer of proteins from a glass surface to the PDMS stamp features, it was activated with oxygen plasma. After inking with ECM proteins the stamp was used as a mold in the microstructuring process. Thermoforming of PC foil with an elastomeric master turned out to be a reliable technique to form up to 6 micropatterned PC scaffolds during one thermoforming cycle. Elasticity of the PDMS mold enabled simple removal of the master after forming process, preventing damage to the sample or PDMS features. Microchannel arrays were produced with widths ranging from 30 µm to 400 µm and depths of 30 µm to 75 µm. By using the confocal microscope, the surface profile of the thermoformed foils was measured and compared to designed size and to the PDMS master (Fig.1d). For the samples with larger channel dimensions, the foil fully replicated both the height and width of the PDMS features. Smaller structures showed less accurate retention of height, probably due to high ratio of foil thickness to mold structure height. High shape fidelity was observed for all measured samples, indicating no deformation or displacement of the stamp during thermoforming. It was,

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however, not possible to form features with respect to a ratio (high/width) higher than 2. Exceeding this ratio the forming pressure exerted too much stress on the elastomeric structure and caused them to collapse and generate defects in the patterns. During the thermoforming process, different ECM proteins, including collagen, laminin and fibronectin, were transferred from the PDMS stamp features onto the surface of PC. In order to assess the local mechanical properties and possible variations due to the presence of the collagen biomolecules, nanoindentation measurements were performed in air and in wet conditions (by immersing samples in HBSS). No significant differences were detected among the samples with biomolecular micropatterns and the PC substrate without pattern, used as control. These results indicate that the biomolecular layer thickness is in sub-micrometer range and with this analysis is not possible to assess the local mechanical properties. From the analysis of ATR-FTIR spectra (SI, Fig. S1), it is possible to notice the prevalence of all the main bands ascribable to PC substrate in all samples (with and without collagen pattern). The presence of collagen is shown by two weak bands at 1650 and 1555 cm-1, ascribable to amides I and amides II vibrations, respectively. Alternatively, the presence of printed biomolecules was successfully visualized by immunofluorescence staining (Fig. 2). Immuno-labeled proteins were observed on the bottom of the thermoformed channels.

Fig.2. Examination of ECM protein coating after 3DµCP process. Micropatterned PC samples with 45µm bright channels were stained using protein specific antibody. Immuno-labeled 10


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collagen (a), laminin (b) and fibronectin (c) are observed on the bottom of the thermoformed channels (top view). Scale bar indicates 100 µm. To further investigate whether thermoforming affects the structure of ECM molecules, SDS gel electrophoresis and Coomassie staining were used. Collagen remained intact after thermoforming; α1-, α2-, β- and γ-chains were still detectable (SI, Fig.S2a). In addition, visualization of the collagen pattern by a conformational-dependent antibody that binds exclusively to non-denatured native collagen molecules confirmed this assumption. These data suggest that after 3DµCP process native collagen structure was present on the thermoformed surface. Although denaturation of native hydrated collagen in physiological conditions occurs at 65°C (±10°C), in a dry environment the conformational change of the collagen molecules from a triple helix to a random coil was detected first at 220°C (±10°C). This temperature has been referred as the denaturation temperature of the dehydrated collagen40. Moreover, collagen fibrils in dry conditions had been shown to maintain their native structure even after 90 min of heating41. Thermoforming of the heterotrimer laminin and the dimer fibronectin resulted in a partial decomposition of the respective protein chains (SI, Fig.S2, b and c). This decomposition was detected in SDS-PAGE experiments only under reducing conditions, which does not represent the three-dimensional state of the proteins in dry and non-reducing state. In both cases a fragment pattern in addition to the intact protein chains could be observed, which can be attributed to a breakdown of the protein after dissolution of disulfide bridges with the reducing agent mercaptoethanol. We conclude that despite the partial decomposition of fibronectin and laminin in a non-dry state, essential cell-binding motifs have been preserved after the thermoforming process, since cells (SI, Fig.S3) and antibodies (Fig.2) bind to the ECM-coated channels. Because, only certain areas of the protein structure are necessary for the binding of the cells, but

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not the entire protein, it can be assumed that a moderate decomposition of the molecules by thermoforming does not adversely affect cell adhesion. 3.2 Cells adhesion on surface patterns

Fig3. Cell adhesion preference. Representative phase contrast images of L929 (a), HepG2 (b) and EA.hy926 (c) cells attached to the patterned regions of the PC foils printed with collagen after 24h in cell culture. Microphotographs show 100 µm width channels at lower and higher magnification. Scale bars indicate 100 µm. To study the potential of the patterned surface for guided adhesion of different cell types, EA.hy926, HepG2 and L929 cells were seeded on the 3DµCP patterned PC foils with 100 µm wide channels printed with collagen and examined for preferential adhesion and pattern recognition (Fig.3). After 24 h all three cell types were observed preferentially attached to patterned regions of the microthermoformed surface. Due to the variety of the cell types that were used, a different cell distribution in the channel was observed. Endothelial and fibroblast cells attached more uniformly to the printed channels and formed single layer of cells, while the hepatoma cells presented more irregular morphology with some aggregates. However, for all cell types cell attachment was restricted to the channels and only few cells were observed on non12


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printed surfaces. Similar results were observed for the samples patterned with laminin and fibronectin (SI, Fig. S3).

Fig4. Cell adhesion preference. Representative phase contrast images of Ea.Hy926 cells attached to the patterned regions of the PC foils with 30 µm (a), 45 µm (b), 60 µm (c), 200 µm (d) and 400 µm (e) width channels printed with collagen after 24 h in cell culture. Microphotographs show channels at lower and higher magnification. Scale bars indicate 100 µm.

To ensure that pattern recognition was independent of pattern geometry, EA.hy926 cells were seeded on the thermoformed PC foils with channels of different dimensions. As shown in Fig. 4, for all thermoformed surfaces, cell adhesion occurred preferentially on the patterned regions. On the surfaces with wide channels (200 µm and 400 µm), cell attachment was restricted to the channels and no cells were observed between the patterns. Cells adhered uniformly within the channels and showed an elongated and frequently flattened morphology. Seeding cells on the surface with narrow channels (30 µm, 45 µm and 60 µm) resulted in more irregular cell

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attachment. For all patterned structures, the intervals between the channels were identical, thus scaffolds with narrow channels results in much smaller surfaces available for cell adhesion. Therefore, the substrates with 100 µm channel width were considered as suitable for experimentation with these cell types. Since unprinted polycarbonate has low surface free energy and shows relatively low cell attachment42, the cells between the patterns remained mostly unattached and were removed by medium exchange. To better observe how the patterned scaffolds were inducing cellular organization, EA.hy926 cells cultured on the scaffolds with different dimensions of channels were stained for actin and nuclei and imaged using confocal microscopy (Fig.5). After 24 h cells were individually distributed within the channels. Subsequently, cells contracted into multi-cellular structures, and after 72 h an extended chain of cells occurred along the formed channels.

Fig.5 Cells adhesion and growth in 3DµCP-patterned channels printed with collagen. Images show cells in channels with different dimensions after culture for 24 and 72h. Samples were stained for actin fibers (green) and nuclei (blue). Scale bare indicates 100µm.

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The microchannel pattern utilized was chosen for its illustrative potential; however, in principle microstructuring can be achieved in arbitrary configurations (Fig.6a). To test how the cells attach to the patterned surface with irregular shape, surfaces with 100 µm width channels with rounded and sharp angles were fabricated. As shown in Fig.6b, cell binding was restricted to the patterned regions, and when the shape of the channel was changing, the cells were also aligning parallel to the direction of the channel.

Fig.6 PC scaffolds with 100 µm width channels patterned with collagen with irregular shape. (a) SEM images of the thermoformed surface and (b) DAPI staining, F-actin staining and merged images of Ea.Hy926 cells cultured into scaffolds for 24h. Scale bars indicate 200 µm.

3.3 Long-term cell culture. To test the feasibility of long-term culture on the patterned surface with 100 µm width channels, in serum containing media, cells were examined for 10 days. Previously, conventional µCP methods had been shown as a powerful approach to control the cell adhesion, but cells failed to remain on the patterns over time43. In the present study, the surface chemistry provides chemical attraction and repulsion for controlling cell attachment, while the topography provides additional geometrical restrictions for controlling cell placement and growth. Culturing cells in 3D cues provides another dimension for external mechanical inputs and could induce cells to polarize and interact with neighboring cells44. Chosen dimensions of the channels had enough space to hold seeded cells for a longer time. Cells do not overfill the limited space of the channels and overgrow out of the scaffold under these conditions. Fig.7a shows cell distribution in channel 15



over time. After 2 days in culture scaffolds enabled formation of single layer of cells with random orientation. After 4 days, when the surface for cell attachment within the channels was limited, cells began to interact with other cells and populated, forming cell bundles and some aggregates. Some cells were attaching or migrating onto the unstructured regions, but typically cells grown on non-collagen coated surfaces had a rounded morphology. The maximum aggregation was observed after 8 days in culture, and the cells started to overfill the channels. After 10 days cell bundles were partially detached from the channel surface as a continuous chain of cells (Fig.7a; for further images see SI, Fig.S4).

Fig.7. 3DµCP enhance 3D cell culture formation. Ea.Hy926 cells were cultured on 3DµCP patterned PC surface with 100 um width channels printed with collagen. Cell growth was observed for 10 days. (a) SEM images of representative cell structures observed after 2, 4, 6, 8 and 10 days.(b) Stack LSM images (XZ, XY and YZ planes) of cell distribution. Samples were 16


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stained for nuclei. Yellow dashed curves represent the channels that included the cells. (c) Live/dead staining of cells. Green: live cells, red: dead cells. Cells were examined after 2, 6 and 10 days of culture. Scale bars indicate 100µm.

These cell culture data were further confirmed by scanning electron microscopy. Cells were stained for nuclei and imaged using LSM and 3D reconstruction software. The LSM images of cross-section of channels with cells (XZ and YZ) (Fig.7b) confirmed that cells structures changed from flat layer of the cells at the bottom of the channel at day 2 to chain of cells overgrowing from the channels at day 10, indicating cellular aggregation and 3D growth. The live-dead staining of the 3D cell structures showed high viability of the cultured cells (Fig.7c). Porous structure of the PC foils could provide better oxygen diffusion and nutrient transfer to the bottom parts of the channels. The 3DµCP process obviates the use of organic solvents and does not lead to any cytotoxicity. In spite of height temperature used during thermoforming, patterned ECM was recognized by cells. Moreover, in some cases cell attachment to thermally denaturated ECM was previously shown to be as good as, or better then to native collagen45-46.

4. Conclusion. This study proposed a direct and simple method for dual patterning of microporous polymer substrates for directing cell adhesion onto a microtopographically complex surface. 3DµCP approach was able to reproducibly create arrays of bioactive microchannels with variety of different dimensions on PC microporous foils. During the microstructuring process, different ECM proteins were successfully transferred into the bottom of the channels. Cell culture results revealed that the patterned surface has promising capability to manipulate cell adhesion and multicellular organizations. This technique has the potential to be used on other thermoplastic biopolymers, and additionally more complex geometries could be implemented. This opens promising perspectives for mimicking and biofabrication of free-form complex morphologies 17



with applications in biotechnology e.g. more native tissue–like microarchitectures2 or bio fuel cells47.

Supporting Information ATR-FTIR of polycarbonate with thermoformed collagen on surface, Images of SDS-PAGE and Coomassie staining of ECM molecules, SEM images of cell binding on 3D patterned surface with different ECM proteins, SEM images of time dependent 3D cell culture on 3D patterned surface

Acknowledgements This work was supported by Federal Ministry of Education and Research and the Thuringian Ministry of Culture (BioMepSens: FKZ03ZIK062, MKF: FKZ03ZIK465, Nanozellkulturen: FKZB714-09064, Optimi II: FKZ16SV5473, WK-Basis: FKZ 03WKCB010, Biolithomorphie: FKZ03Z1M511 and FKZ03Z1M512) within the Initiative “Centre for Innovation Competence”, MacroNano®, In vitroTox: AIF/BMWi KF 27311209AJ4 and by the Carl Zeiss Foundation (3D Felxible Sensorik: FKZ 0563-2.8/399/1).

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3DµCP patterned scaffolds fabrication and characterization. Schematic of inverted printing of PDMS stamp and one-step microstructuring and microcontact printing of PC foils (a), surface topography of thermoformed scaffolds visualized by SEM (b), 3DuCP patterned channel visualized and measured using laser scanning digital microscope (c) and pattern size fidelity according to designed size and PDMS master (d). 174x265mm (300 x 300 DPI)



Examination of ECM protein coating after 3DµCP process. Micropatterned PC samples with 45µm bright channels were stained using protein specific antibody. Immuno-labeled collagen (a), laminin (b) and fibronectin (c) are observed on the bottom of the thermoformed channels (top view). Scale bar indicates 100 µm. 178x44mm (300 x 300 DPI)


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Cell adhesion preference. Representative phase contrast images of L929 (a), HepG2 (b) and EA.hy926 (c) cells attached to the patterned regions of the PC foils printed with collagen after 24h in cell culture. Microphotographs show 100 µm width channels at lower and higher magnification. Scale bars indicate 100 µm. 185x83mm (300 x 300 DPI)



Cell adhesion preference. Representative phase contrast images of Ea.Hy926 cells attached to the patterned regions of the PC foils with 30 µm (a), 45 µm (b), 60 µm (c), 200 µm (d) and 400 µm (e) width channels printed with collagen after 24 h in cell culture. Microphotographs show channels at lower and higher magnification. Scale bars indicate 100 µm. 135x96mm (300 x 300 DPI)


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Cells adhesion and growth in 3DµCP-patterned channels printed with collagen. Images show cells in channels with different dimensions after culture for 24 and 72h. Samples were stained for actin fibers (green) and nuclei (blue). Scale bare indicates 100µm. 173x113mm (300 x 300 DPI)



PC scaffolds with 100 µm width channels patterned with collagen with irregular shape. (a) SEM images of the thermoformed surface and (b) DAPI staining, F-actin staining and merged images of Ea.Hy926 cells cultured into scaffolds for 24h. Scale bars indicate 200 µm. 231x46mm (300 x 300 DPI)


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3DµCP enhance 3D cell culture formation. Ea.Hy926 cells were cultured on 3DµCP patterned PC surface with 100 um width channels printed with collagen. Cell growth was observed for 10 days. (a) SEM images of representative cell structures observed after 2, 4, 6, 8 and 10 days.(b) Stack LSM images (XZ, XY and YZ planes) of cell distribution. Samples were stained for nuclei. Yellow dashed curves represent the channels that included the cells. (c) Live/dead staining of cells. Green: live cells, red: dead cells. Cells were examined after 2, 6 and 10 days of culture. Scale bars indicate 100µm. 332x220mm (300 x 300 DPI)



82x53mm (300 x 300 DPI)


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3D Microcontact Printing for Combined Chemical ... - ACS Publications

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