Cell culture system

Abstract

The present disclosure relates to a system (200) for cell culture, in particular a photoreconfigurable system comprising a support substrate (201), an azobenzene-containing layer (202), and a protective coating layer (203). The disclosure also relates to reversibly inscribing a topography on the surface of the azobenzene-containing material of the system, a method for culturing cells on the system, and the cell culture obtained by the method. A method for manufacturing the system is also disclosed.

Description

Technical Field

[0001] The present disclosure relates to a system for cell culture, particularly to a photo-reconfigurable system comprising a two-layer structure including an azobenzene-containing layer and a protective coating layer.

Background Art

[0002] In pharmaceutical development, more than 90% of the newly developed molecules fail in clinical-stage trials. The main cause of the low success rate is the cell models used in in vitro research. These model systems cannot reproduce the natural state of the human body. In vivo, the microenvironment of cells regulates various cell functions. Therefore, there is clearly a need for better cell-based models. For this purpose, it is important to mimic as faithfully as possible the dynamic in vivo conditions in in vitro cell culture, including the microenvironment of cells.

[0003] Currently, cells are mainly cultured in cell culture disks, flasks, and plates without proper simulation of the dynamic in vivo microenvironment, the cell niche, of the cells. One of the major drawbacks of these conditions is the lack of surface features, i.e., surface topography, which does not sufficiently reflect the environment that cells encounter in the human body. Furthermore, the adherent cells in our body are constantly changing and interacting with their surroundings. This behavior creates a dynamic extracellular environment, but it is difficult to reproduce this in vitro.

[0004] To construct micro-patterned cell culture substrates, various micro-engineering techniques have been used, but they usually feature a square topography rather than a smooth structure and are not similar to the cell environment. Furthermore, they still suffer from a lack of reconfigurability or dynamic modification of the surface and thus provide only a static environment for the cells.

[0005] Therefore, a reconfigurable surface for cell culture is still needed.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The present invention is based on the observation that by creating photoinduced surface features using a two-layer structure including an azobenzene-containing layer and a protective coating layer, the surface topography can be repeatedly changed, erased, or entirely reconfigured.

[0007] Accordingly, an object of the present invention is The patterned according to claim 1 to provide a cell culture system.

Means for Solving the Problems

[0009] Also, an object of the present invention is to provide a method for reversibly engraving topography on the surface of the azobenzene-containing material of the system, the method including focusing a light beam on the material or projecting an interference pattern of laser light onto the material.

[0012] Yet another object of the present invention is to provide a method for erasing the topographical features of a patterned system, the method including exposing the topographical features to light in the range of nm to 600 nm, preferably 460 nm to 530 nm, generated by a laser, preferably a continuous-wave laser, or by a fluorescent lamp or an LED. 430 nm to 600 nm of light, preferably 460 nm to 530 nm of light.

[0013] Yet another object of the present invention is to provide the use of a system or a patterned system as a cell culture platform.

[0015] Yet another object of the present invention is to provide a cell culture obtained by a method of culturing cells on a patterned system.

[0016] Further objects of the present invention are described in the appended dependent claims.

[0017] Exemplary and non - limiting embodiments of the present invention, both as to structure and method of operation, together with additional objects and advantages thereof, will be best understood from the following description of specific exemplary embodiments when read in conjunction with the accompanying drawings.

[0018] In this document, the verbs “comprise” and “include” are used as open limitations that do not exclude or require the presence of functions not recited. The features recited in the appended dependent claims can be freely combined with each other unless specifically stated otherwise. Further, it should be understood that the use of “a” or “an” throughout this document, i.e., in the singular, does not exclude the plural.

Brief Description of the Drawings

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[0032] According to one aspect, the present disclosure relates to a system for cell culture. An exemplary system 100 is shown in FIG. 1. This system comprises a support structure 101, an azobenzene-containing intermediate layer 102, and a top layer 103.

[0033] The support structure can be any support structure used for cell culture. Exemplary support structures are cell culture disks such as Petri dishes, microscope cover slips, and well plates. The support structure is typically made of plastic or glass. Examples of Petri dish formats are poly(styrene) and glass-bottom Petri dishes.

[0034] The material containing azobenzene is photoreconfigurable. As defined herein, a photoreconfigurable material is a material whose shape is reconfigurable when exposed to light. The azobenzene molecules may be substituted or unsubstituted. An exemplary photoinduced conversion of azobenzene units is shown in FIG. 2. Here, R and R‘ refer to different para substituents. Different substituents can also be added at the meta and ortho positions. An example of azobenzene suitable for this technology is ethyl-N-(2-hydroxyethyl)-4-(4-nitrophenylazo)aniline.

[0035] The top layer is composed of a protective polymer such as an elastomer or a hydrogel. Exemplary elastomers are siloxane-containing polymers such as polydimethylsiloxane, PDMS.

[0036] According to another embodiment, the protective polymer comprises parylene, preferably parylene C, i.e., poly(chloro-para-xylylene). Various substituted [2.2] para-cyclophanes exist, which may introduce functional groups into the phenyl ring. These functional groups enable the deposition of functionalized parylene films or further functionalization for the immobilization of bioactive molecules.

[0037] PDMS and parylene C are preferred coating agents because they have excellent mechanical properties, barrier properties, hydrophobicity, chemical resistance, and biocompatibility. A further advantage of parylene C is that a parylene C vapor deposition can produce an ultrathin film without pinholes. Furthermore, despite being semi-crystalline, parylene C is highly transparent within the thickness range of interest.

[0038] According to one embodiment, the thickness d1 of the azobenzene-containing layer is 50 nm to 5 μm, and the thickness d2 of the top layer is 20 nm to 100 μm, preferably 20 nm to 200 nm.

[0039] According to another embodiment, the thickness d1 of the azobenzene-containing layer is 50 nm to 5 μm, and the thickness d2 of the top layer is 50 nm to 100 μm. An example of the thickness of the azobenzene-containing layer is 500 nm.

[0040] When the protective polymer is PDMS or parylene C, the thickness of the top layer is preferably less than 90 nm.

[0041] When the protective polymer is a hydrogel, the thickness of the top layer is preferably less than 50 μm.

[0042] The present disclosure also relates to a method of manufacturing a system 100 for cell culture, the method including the following steps. a) providing a support structure 101, b) coating the support structure with an azobenzene-containing layer 102, and c) coating the azobenzene-containing layer with a top layer 103 containing a protective polymer.

[0043] The support structure is preferably selected from a Petri dish, a microscope cover slip, and a well plate. According to one embodiment, the protective polymer includes a hydrogel or an elastomer. An example of the elastomer is a siloxane. A specific siloxane is PDMS.

[0044] According to another embodiment, the protective polymer includes parylene. A specific parylene is parylene C.

[0045] According to one embodiment, the coating in step b) includes spin coating.

[0046] According to another embodiment, the coating in step c) includes spin coating. When the protective polymer is a siloxane such as PDMS, spin coating is preferred.

[0047] According to another embodiment, the coating in step c) includes chemical vapor deposition polymerization. This is a preferred method when the protective polymer includes a parylene such as parylene C.

[0048] Micro and sub-micrometer scale topography can be reversibly etched onto the surface of the azobenzene-containing material by optical interference lithography, digital micromirror devices, microlens arrays, or simply by scanning a laser beam (e.g., from a laser scanning microscope) over the film surface. In the presence of a focused light beam, a thin azobenzene-based coating tends to accumulate within the focal volume of the light beam or leak from the focal volume of the light ray. Therefore, by the scanning operation of the light beam, any shape can be written in the same way as a drawing tool.

[0049] A schematic diagram of the generation and erasure of topographical features for an exemplary system of the present invention is shown in FIG. 3.

[0050] Therefore, it is also an aspect of the present disclosure to provide a method for reversibly etching topography onto the surface of the azobenzene-containing material of the system. According to one embodiment, this method includes the step of focusing a light beam on the material. According to a specific embodiment, this method includes the step of scanning a laser beam over the azobenzene-containing layer.

[0051] According to another embodiment, a method for reversibly engraving topography on the surface of an azobenzene-containing material of a system utilizes interference lithography. According to this embodiment, the method includes projecting an interference pattern of laser light onto the material.

[0052] Since the intermediate layer is coated with the top layer, topography is also formed on the top layer.

[0053] Reversibly engraving includes patterning and erasing. The wavelength of the light used for patterning and erasing is typically 400 nm to 600 nm, preferably 430 nm to 530 nm. The appropriate intensity range varies depending on the technique used. For example, for interference lithography, 100 mW / cm -2 ~ 600 mW / cm -2 is sufficient, but for patterning / erasing with a laser scanning confocal microscope, about 1 W / cm -2 ~ 5 W / cm -2 is used.

[0054] According to one embodiment, for patterning, the method includes exposing one or more regions of the azobenzene-containing layer to light of 400 nm to 600 nm, preferably 430 nm to 530 nm, generated by a laser, preferably a continuous wave laser, thereby generating topographical features in the system. An exemplary intensity of the light generated by the laser is preferably 1 W / cm -2 ~ 5 W / cm -2 is.

[0055] According to an embodiment, for erasing, the topographical features of the patterned system are exposed to light of 400 nm to 600 nm, preferably 430 nm to 530 nm, generated by a laser, preferably a continuous wave laser. An example of the intensity of the light generated by the laser is 1 W / cm -2 ~ 5 W / cm -2 is.

[0056] An exemplary system 300 of the present disclosure obtainable by the method disclosed above is shown in FIG. 4. This figure also shows a sinusoidal surface relief grating etched by interference lithography, a 3D projection of an atomic force microscope, an average cross-sectional profile and topography etched by laser scanning of the system.

[0057] According to another aspect, the present disclosure relates to a method of patterning a system by exposing one or more regions of an azobenzene-containing layer of the system to light in the range of 400 nm to 600 nm, preferably 430 nm to 530 nm, generated by a laser, preferably a continuous wave laser, thereby generating topographical features in the system. The intensity of the light is typically 1 W / cm -2 ~5 W / cm -2 . An exemplary wavelength is 488 nm, which is a preferred wavelength when the azobenzene-containing substance is ethyl-N-(2-hydroxyethyl)-4-(4-nitrophenylazo)-aniline. An exemplary intensity is 1 W / cm -2 .

[0058] The present disclosure also relates to a method of erasing topographical features from a system by exposing the topographical features to light in the range of 460 nm to 530 nm generated by a laser, preferably a continuous wave laser. Exemplary wavelengths are 470 nm, 488 nm, and 530 nm. According to another embodiment, the erasure is performed by using light generated by a fluorescent lamp filtered in the range of 430 nm to 530 nm. The exemplary intensity generated by the laser is 1 W / cm -2 . The intensity of the light generated by the fluorescent lamp is typically 1 W / cm -2 ~5 W / cm -2 . The erasure can also be performed using an LED in addition to the above-mentioned laser and fluorescent lamp.

[0059] The present invention enables the creation of free-form topographic patterns on a cell culture substrate. Further, even when cells are already growing on the substrate, the topography can be erased using a uniform light source (such as a fluorescent lamp, LED light, laser, etc.) and rewritten to create a new pattern in the culture dish, thereby enabling better mimicry of the dynamic state of the human body and the creation of dynamic topography.

[0060] Accordingly, a further aspect of the present disclosure is to provide a method for culturing cells on a system having topographical features on its upper surface. The system having topographical features is obtainable as disclosed above.

[0061] According to an exemplary embodiment, the method includes the following steps: a) providing a patterned system; b) coating the topmost layer of the patterned system with a cell adhesion protein; and c) seeding cells on the cell adhesion protein.

[0062] According to a preferred embodiment, the method includes subjecting the system to oxygen plasma treatment before step b).

[0063] Exemplary cells are selected from the group consisting of epithelial cells, fibroblasts, endothelial cells, neurons, mesenchymal stem cells, astrocytes, cardiomyocytes, and cancer cells.

[0064] Examples of cell adhesion proteins suitable for this method are selected from the group consisting of collagen, fibronectin, and laminin. The selection of the cell adhesion protein varies depending on the type of cells to be seeded.

[0065] The system of the present invention enables robust and reconfigurable control of surface topography based on generating photoinduced surface features using an azobenzene-containing bilayer. According to one embodiment, the cell culture disk is coated with an azobenzene-containing polymer film that provides a protective siloxane layer that enables easy chemical modification of the surface, ensures biocompatibility, and fully supports existing protein deposition techniques. According to another embodiment, the cell culture disk is coated with an azobenzene-containing polymer film that provides a protective parylene C layer that enables easy chemical modification of the surface, ensures biocompatibility, and fully supports existing protein deposition techniques.

[0066] In vivo, the dynamic interaction between cells and the surrounding extracellular matrix (ECM) plays an important role in the regulation of many physiological and pathological processes such as tissue morphogenesis, healing, and tumor growth. Since the present invention can optically manipulate surface features, it enables real-time control of the extracellular niche, (multi)cellular spatial arrangement, orientation, and movement. This process is reversible, remotely controllable, and non-invasive, which is considered important for defining temporal programs such as cell differentiation, stem cell phenotype acquisition, tissue regeneration, induction of directional cell migration, and decoupling of topographical and chemical cues. In the body, cells are exposed to various types of biophysical cues that can be converted into biochemical activities in a process called mechanotransduction. These cues are important co-regulators such as cell alignment and migration. For example, muscle, nerve cells, and endothelial cells show highly aligned tissues in our body, and cell migration is greatly affected by the topographical features of the cell environment.

[0067] To demonstrate the feasibility of photoinduced patterning and its effect on cells, epithelial cells were cultured on a flat surface (Figure 5, left) and on the surface of a system containing a photoinduced azobenzene-containing material (Figure 5, right). As can be seen from the figure, the surface topography has a great influence on cell alignment and migration.

[0068] Results and Discussion Characterization of SRG writing and erasure in a DR1g-PDMS bilayer structure To assemble the platform, a cover glass was first coated with a photo-patternable amorphous thin layer of ethyl-N-(2-hydroxyethyl)-4-(4-nitrophenylazo)-aniline, namely a dispersion red 1-containing molecular glass (DR1g; thickness 480 ± 20 nm) further coated with PDMS. The resulting DR1g-PDMS bilayer structure was used as a photoreactive cell culture platform where DR1g functions as the photoreactive part.

[0069] To study the effect of PDMS (base: hardener ratio 10:1) on SRG formation, three different PDMS prepolymer dilutions (0.02, 1, and 50 wt-%) in n-hexane were tested with the same spin-coating parameters. These samples are herein denoted as DR1g-PDMSx. Here, x represents the PDMS concentration in hexane. The thickness of the PDMS layer was measured by ellipsometry and profilometry. Polarization analysis was used for the accurate measurement of the 0.02 and 1 wt% layers, and a surface profiler was used for the thickest PDMS layer (50 wt%). The thickness was 4.5 μm (DR1g-PDMS 50 ), 65 nm (DR1g-PDMS1), 20 nm (DR1g-PDMS 0.02) It was. The DR1g-PDMS bilayer was optically patterned using optical interference lithography in a Lloyd's mirror configuration, inducing mass transfer in DR1g and surface deformation of the PDMS coating to form SRG. The periodicity of the interference pattern was determined by the wavelength and the angle between the mirror and the laser beam. By changing the angle, SRGs with different periodicities can be realized (in the range of approximately 300 nm to 10 μm). Here, since this period had been previously used when controlling the arrangement of epithelial cells of the same material, the period was set to 1 μm. In-situ monitoring of SRG formation in different DR1g-PDMS bilayers was carried out by measuring the diffraction efficiency (DE). The thickness of the DR1g film (480 ± 20 nm) was selected to be thick enough so that SRG formation does not depend on small variations in the layer thickness. Therefore, the difference in DE was solely due to the difference in the PDMS layer. To confirm the formation of SR, the samples were imaged with an atomic force microscope (AFM).

[0070] The DE curves during SRG writing into the DR1g-PDMS bilayer are shown in Fig. 6a. From these images, it can be observed that the DE systematically decreases as the thickness of the PDMS layer increases. By AFM imaging, it was confirmed that the SRGs of DR1g-PDMS 0.02 and DR1g-PDMS1 were formed with a predicted period of 1 μm (Figs. 6b, c). The surface modulation depth was more than 400 nm in DR1g-PDMS 0.02 but in DR1g-PDMS1, the modulation depth significantly decreased and reached 160 nm. DR1g-PDMS 50In the case of AFM, since the sine wave pattern could not be observed, SRG was not formed on the outer surface of the PDMS coating. 8% DE is considered to be due to the formation of a diffraction grating at the DR1g / PDMS interface. When a film containing azobenzene is between the glass substrate and the protective coating, stronger constraints are imposed for efficient movement. Since the formation of SRG requires mass transfer of the material, the presence of a thick PDMS layer increases the obstacle to the complex stress field that DR1g undergoes during SRG formation. Therefore, a PDMS coating with a thickness of less than 100 nm on the DR1g thin film does not inhibit the formation of SRG, but changes the mechanics of its formation.

[0071] After the formation of SRG, the topography is stable for at least one year at a temperature lower than the glass transition temperature (71 °C) of DR1g and can be erased by heat or a uniform light beam with a wavelength matching the absorption band of DR1g. Since direct heating cannot be performed locally and is not compatible with cell culture conditions, it is preferable to erase SRG with visible light (such as a 530 nm LED). To study the dynamics of SRG erasure, DE was monitored during the erasure of samples showing the same initial DE value (about 7%, Figure 7a). The dynamics of erasure are shown in Figure 7b. Since all samples reached a similar DE value (about 0.5%) at the end of the process, the thickness of the PDMS layer does not seem to affect the effectiveness of the erasure process from the perspective of the DE value. However, obvious differences in erasure dynamics are observed when the thickness of PDMS is different. Interestingly, in DR1g-PDMS1, D decreases systematically faster than in other samples, suggesting that a PDMS layer with an appropriate thickness can even accelerate the erasure of the topography. DR1g-PDMS 50 In the case of DR1g-PDMS, the erasure dynamics were different from those of other samples, and DE decreased relatively slowly in a two-step process.

[0072] When the sample was imaged by AFM, it was confirmed that the topography had decreased to 85% of the initial value of DR1g-PDMS1, as shown in Fig. 7c. The surface profile in Fig. 7d further emphasizes the difference between the erased topography and the non-erased topography. The decrease in modulation depth was 70% in DR1g-PDMS 0.02 . Even when the DE value reached a similar value, AFM showed that in DR1g-PDMS1, the erased topography reached a lower modulation depth. This was the motivation for selecting DR1g-PDMS1 for cell culture studies. When the grating is deep, ablation by a uniform laser beam becomes less effective compared to a lower SRG. Therefore, as judged from the higher remaining gratings observed in this sample, complete ablation of the topography may be more difficult to achieve on the DR1g-PDMS 0.02 surface. In DR1g-PDMS1, the depth of the grating is advantageous for the high-speed ablation process, resulting in a low residual topography and a limited irradiation time required during cell growth experiments. The grating was also deep enough to induce a cell response at the point of alignment. A thicker PDMS layer may function as a better protective layer to separate the DR1g layer from the cells.

[0073] The DR1g layer was coated with parylene C (i.e., poly(chloro-para-xylylene)) by chemical vapor deposition (CVD). The CVD of parylene C was mediated by four secondary chambers inserted into the main deposition chamber for each deposition run. Such secondary chambers had small orifices at the top, whose function was to control the deposition rate of the reactive monomer and fine-tune the thickness of the deposited layer. Generally, the thickness of the deposited parylene film is directly proportional to the mass of the dimer loaded into the machine. However, in the case of ultra-thin films (<100 nm), the reduction in dimer mass leads to a deposition process with low controllability and reliability (due to very short and unstable pressures). By utilizing the outflow of parylene molecules through holes with sizes smaller than the mean free path of the parylene monomer (Knudsen number greater than 1), the thickness of the deposited layer can be reliably obtained by introducing only the exact portion of the reactive monomer into the secondary chamber.

[0074] The prepared substrates were characterized by stylus profilometry and showed thicknesses in the range of 13 nm to 415 nm. Then, the formation of SRGs was tested for the samples coated with PDMS as described above. The samples showed the formation of SRGs for all samples with a thickness less than 90 nm. The barrier properties of the parylene C layer were also tested; the permeability test was performed by dropping 1 μl of various organic solvents (acetone, ethanol, isopropanol) and water onto the surface of the samples. Within seconds to 3 minutes, the solvent droplets either dissolve part of the DR1g layer or remain on the surface of the sample until they finally evaporate. Samples over 55 nm were, as expected, resistant to water penetration and also provided optimal protection against organic solvents (especially ethanol and isopropanol). The most powerful solvent tested was acetone, which was used to define the upper limit of the parylene layer thickness, on which SRGs could be formed, while showing consistent and reliable protection against acetone penetration (Figure 8).

[0075] The polyacrylamide hydrogel layer was also used for coating the DR1g substrate (Figs. 9a, b). The polyacrylamide was filled with fluorescent microparticles that emit red light (607 nm). The thickness of the hydrogel was estimated to be 100 μm. Next, in the user-defined region of interest (ROI), the DR1g was photo-stimulated with a 488 nm laser using a laser scanning confocal microscope (LSM 780, Zeiss). In these ROIs, as shown by the fluorescent microparticles captured by time-lapse microscopy and analyzed by digital image correlation (DIC), the flow of the photosensitive material caused corresponding deformations within the hydrogel. In the hydrogel tested (2.8 kPa), the strain propagated to a depth of at least 50 μm inside the hydrogel. This experiment demonstrated, in principle, that the photo-induced surface deformation of DR1g can be used to locally and mechanically stimulate soft materials such as hydrogels. Such hydrogels can be filled with cells or cells can be cultured on the hydrogel surface.

[0076] Cell alignment by SRG guide Cells sense the physical properties and mechanical forces of the environment on their surface, and these forces are transmitted deep into the cell and even to the nucleus. The main site of healing is the contact between the cell and the ECM, mainly the focal adhesion, which is a multi-protein complex on the cell membrane. The formation of focal adhesions at the cell-ECM interface regulates cell adhesion, alignment, and migration. E-cadherin, a calcium-dependent transmembrane protein, is one of the molecules found at the cell-cell contact site. E-cadherin is particularly present in adherens junctions and plays an important role at the cell-cell interface during the formation of tight and polarized epithelia.

[0077] To investigate whether the fine topography on the DR1g-PDMS1 bilayer can induce collective cell alignment, Madin-Darby canine kidney type II (MDCKII) epithelial cells were seeded on the SRG and their alignment with the underlying microtopography was studied. This cell line provides an excellent model for studying collective cell behavior. Although the mechanical transduction of single cells on microtopography has been mainly studied, such behavior has not yet been fully characterized for cell aggregates where coordinated movement occurs without complete disruption of cell-cell contacts. Figure 10a shows a schematic diagram of the sample pretreatment. Briefly, DR1g and PDMS were subsequently spin-coated to form a bilayer structure, and the SRG was etched as described above. After patterning, the surface was made hydrophilic by oxygen plasma treatment to improve protein attachment to the surface. Next, the surface was coated with collagen I to improve cell adhesion to the surface, MDCKII cells were seeded on the sample, and cultured for up to 72 hours. The movement of cells along the microtopography was tracked by time-lapse microscopy. The cells had already aligned along the fine topography within the first 24 hours after seeding on DR1-PDMS1. After 24 hours, the cells formed small colonies and extended along the pattern direction on both bare DR1g and DR1g-PDMS1, indicating that the PDMS layer did not inhibit the sensing of the topography under the cells (Figure 10b). The cells formed a confluent cell monolayer at the 72-hour time point.

[0078] From the perspective of cell-material and cell-cell interactions, the cellular response to microtopography was further investigated by immunolabeling MDCKII cells at various time points. To distinguish single cells, the nuclei were stained with DAPI. Cell-cell interactions were studied by detecting the intracellular localization of E-cadherin. Twenty-four hours after cell seeding, the cell nuclei were circular, but the cells had an elongated morphology along the surface microtopography, as observed from the localization of E-cadherin (Figure 10c). Furthermore, E-cadherin was accumulated in the cytoplasm, and the cells had not yet formed mature cell-cell junctions. At the 72-hour time point, the cell morphology was not as elongated as that at the 24-hour time point. After 72 hours, the cells formed a uniform cell layer, and E-cadherin was localized at the cell-cell interface, showing strong cell-cell interactions on the bilayer surface. Loss of E-cadherin, instead, indicated epithelial-mesenchymal transition (EMT), in which epithelial cells lost their phenotypic characteristics and changed into more motile and invasive non-polarized mesenchymal cells. Since E-cadherin was localized at the cell-cell interface, the cells on the bilayer surface formed a tight epithelial layer after 72 hours.

[0079] Focal adhesion kinase (FAK) is one of the molecules initially present in the formation of focal adhesions, and its phosphorylation indicates the formation of mature focal adhesions. Therefore, the morphological parameters of focal adhesions were studied by immunolabeling phosphorylated FAK (pFAK). Focal adhesions were observed at the basal cell pole 24 hours after seeding, and their distribution was further analyzed using fast Fourier transform (FFT). FFT converts spatial image information into frequency space, emphasizing periodic features and generating specific frequency patterns. Analysis showed that a primary frequency peak could be detected 24 hours after cell seeding (Figure 9c, FFT of the pFAK image), indicating a periodic distribution of the image feature (pFAK). After 72 hours, pFAK was still observed at the cell pole, but after forming a uniform cell layer, cell movement was more restricted. In the FFT of the focal adhesion channel, the primary frequency peak was still visible, indicating that focal adhesions were periodically distributed and that the cells were still recognizing information from topographical cues.

[0080] Erasure of SRG topography by live cells. Instead of using an LED, the fluorescence lamp of a confocal microscope (filtered in the blue region of the visible spectrum) was used to erase the fine topography, enabling the observation of live cells immediately after measurement. Since most microscopes can be equipped with environmental control functions suitable for live cell culture, this setup was considered practical for the biological environment. To set the erasure parameters, first, erasure was performed in a dry liquid environment at room temperature without using cells. As can be seen from the bright-field image and the digital holographic microscopy (DHM) image, illumination with a fluorescence lamp yielded a clearly distinguishable circular region in both dry and aqueous environments, and quantitative results regarding the surface profile were obtained (Figs. 11a, b). By monitoring the surface using DHM, rapid and quantitative characterization of the surface topography over a wider area compared to AFM became possible. The DHM images showed that the modulation depth decreased reproducibly within 5 minutes after irradiation. Under dry conditions, the modulation depth decreased by 75% from the initial value (Fig. 11c), which is indicated by the decrease in surface roughness from 56 nm to 14 nm. In the liquid environment, the modulation depth of the erased region decreased by 50% (Fig. 11d), and the (partially) erased surface became significantly rougher, showing rounded surface features.

[0081] MDCKII cells were seeded onto the SRG topography and cultured for 24 hours before ablation to allow cell orientation along the microtopography. The medium was placed on top at 37 °C in a humid atmosphere, and the sample was irradiated with the fluorescence light of a confocal microscope for 5 minutes and fixed and immunolabeled 2 hours after ablation. Partial photoablation was confirmed by DHM after cell removal by trypsin treatment. In the presence of the PDMS layer, ablation was more uniform compared to bare DR1g, and the number of the above-mentioned round surface features was significantly reduced. The potential of phototoxicity to cells was also investigated. In this experiment, DR1g was spin-coated on the bottom surface of a coverslip, and cells were seeded onto the sample where the glass substrate was at the cell-material interface. In such control samples, the same light intensity as in the ablation process was guaranteed to reach the plane of the cells, but no topographical changes occurred at the cell adhesion sites. The control samples were irradiated with the fluorescence light for 5 minutes, and a viability / cytotoxicity assay was performed 3 hours after ablation. No significant acute phototoxic effect on cell viability was observed since no dead cells were seen in the ablated areas as well as in the non-ablated areas. When investigating the effect of phototoxicity on cell morphology, PDMS was spin-coated on the cell-material interface on the opposite side of the control samples to ensure the same adhesion properties. No significant difference in cell morphology was observed within 2 hours after irradiation.

[0082] The cell groups had less spreading in their post-ablation morphology and were smaller in size. This may indicate that substrate adhesion was partially lost after the topographical change. Furthermore, after ablation, pFAK was observed to be concentrated more at the center of the cells than at the cell edges. When ablation was performed under a monolayer of epithelial cells with uniform microtopography, no significant morphological changes were observed. This observation suggests that when strong cell-cell junctions are formed, at least within 2 hours after ablation, monolayer epithelial cells are not immediately repositioned in response to the loss of the induced surface topography. Quantification of focal adhesion orientation was performed as described above. The orientation data showed that after ablation in small cell groups, the focal adhesions were more randomly oriented. However, no difference was observed in the case of a confluent cell layer. This indicates that smaller cell groups can sense the photo-induced topographical change and change the orientation of their focal adhesions accordingly. Ablation did not seem to affect focal elongation and area. Even if the ablation of the topography was partial using the lamp of a confocal microscope in the presence of liquid, the microtopography and surface roughness may change. The topographical change affected the morphology and focal adhesion orientation of small cell groups. However, no collective morphological response or focal adhesion orientation was observed during at least a 2-hour period. The cells remained attached to the ablated surface and survived after irradiation.

[0083] Conclusion The platform introduced here is composed of a film containing photo-responsive azobenzene and a thin PDMS or parylene C coating, and can independently control photo-responsiveness and the stability of materials in a cell culture environment. These layers together form a bilayer structure, enabling the modification of surface topography by the photo-induced movement of the azobenzene-containing film. The SRG topography was efficiently written and erased in the presence of the PDMS and parylene C layers. When MDCKII epithelial cells were seeded onto the photo-patterning system, the SRG topography was able to guide local adhesion orientation along the surface topography even after the formation of a uniform epithelial layer. The surface topography can be modified using the fluorescence lamp of a confocal microscope in the presence of living cells, enabling non-invasive control of the surface topography. Despite the partial erasure of the SRG topography, the topography could be modified without causing cell detachment or cell death. Therefore, photo-mediated erasure is a strategy for dynamically controlling the topography of materials for real-time cell experiments that can be performed with a conventional microscope setup. This platform can be further patterned with proteins, enabling the individual control of topographical and biochemical cues and further functionalization for various applications.

Example

[0084] Experimental section Sample preparation The polymer is PDMS. A bilayer of azobenzene-containing Disperse Red 1 molecular glass (DR1g, Solaris Chem Inc.) and polydimethylsiloxane (PDMS, SYLGARD 184, Dow) was prepared on a square cover glass by spin coating (Laurell Technologies Corporation). First, the cover glass was sonicated twice in acetone for 10 minutes. A chloroform solution of DR1g with a concentration of 9% (w / v) was prepared. The solution (35 μl) was applied to the cover glass (22x22mm 2) It was deposited on top at 1500 rpm for 30 seconds. PDMS was prepared by mixing a prepolymer silicone elastomer base and a curing agent at a ratio of 10:1. The uncured PDMS was diluted with n - hexane to create solutions of 50, 1, and 0.02 wt%. The solutions were applied onto thin DR1g films at 6000 rpm for 150 seconds and cured at 55 °C for 1.5 hours. First, samples for thickness measurement were prepared by spin - coating a PDMS solution onto a silicon substrate as described above. The thickness of the resulting PDMS film was measured by reflectance ellipsometry (J.A, Woollam VASE). The 50 wt% PDMS solution formed a film that was too thick for ellipsometry measurement, so its thickness was measured with a stylus surface profilometer (Veeco Dektak 150). In both techniques, the resolution limit is in the sub - nanometer range.

[0085] Sample Preparation The polymer is parylene C. A bilayer of Disperse Red 1 molecule glass (DR1g, Solaris Chem Inc.) containing azobenzene and parylene C (Galentis Ltd.) was prepared on a square cover glass by spin - coating Disperse Red 1 as shown above, followed by chemical vapor deposition of parylene C (Para Tech Coating Inc.) using a leaching - based method described elsewhere. The orifices connecting the interior of each secondary deposition chamber to the larger main machine chamber were square holes with a lateral size in the range of 200 μm to 8000 μm. The final thickness of the film was estimated using a stylus roughness meter (Bruker Dektak XT). For each deposition run, 2 g of dichloro - p - cyclophane dimer was loaded into a deposition system that functions as four cylindrical secondary chambers with an internal surface area of 19210 mm 2 of the deposition system that functions as four cylindrical secondary chambers.

[0086] Sample Preparation The polymer is a polyacrylamide hydrogel. A circular glass coverslip (13 mm) was washed in a 2% Hellmanex solution in an ultrasonic bath for 30 minutes, washed with abundant deionized water, and carefully dried. Passivation of the glass coverslip was achieved by grafting PLL-PEG. One drop (10 - 30 μl) of a PBS solution of 0.1 mg / ml PLL-g-PEG was dropped onto the coverslip and reacted for 30 minutes. The substrate was then washed with a large amount of deionized water. The reagent solution was prepared as follows: acrylamide (10 wt%), bis-acrylamide (0.03 wt%), fluorescent microparticles (0.04 wt%), N,N,N‘,N’-tetramethylethylenediamine (TEMED, 0.02 vol%), and ammonium persulfate (0.1 wt%) were dissolved in PBS. Next, the gelling solution was pipetted onto the coverslip coated with DR1-g and covered with the passivated coverslip for 15 minutes. The expected elastic modulus of the hydrogel is 2.8 kPa and the thickness is 100 μm.

[0087] Writing and erasing of surface relief gratings. The bilayer structure was optically patterned using interference lithography in a Lloyd mirror configuration. Writing of the surface relief grating (SRG) was performed using a 488 nm continuous wave laser (Coherent Genesis CX488-2000) with circular polarization, intensity 500 mW cm -2 , area 0.50 cm 2 . The microtopography period Λ was set to 1 or 1.5 μm and determined by Λ = λ / 2sinθ, where λ is the wavelength of the laser and θ is the angle between the mirror and the laser beam. Erasing of the SRG was performed with a 530 nm LED and the beam was directly focused on the SRG topography at an intensity of 100 mW cm -2 . Writing and erasing of the SRG were monitored with a low-power (1 mW) 633 nm He-Ne laser and the diffraction efficiency of the first-order diffracted beam was measured.

[0088] Cell culture. In this study, Madin-Darby canine kidney type II (MDCKII) cells were used. Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 in a medium consisting of MEM GlutaMax (Gibco) supplemented with fetal bovine serum (10%) and penicillin / streptomycin (1%). Before seeding the cells, the samples were sterilized under UV light for 40 minutes. The samples were coated with a 50 μg ml-1 monomeric rat tail type I collagen solution (Thermo Fischer Scientific) in 0.02 N acetic acid for 40 minutes.

[0089] Immunolabeling. Cells were fixed with 4% paraformaldehyde for 10 minutes, washed with PBS, permeabilized with permeabilization buffer (0.5% BSA, 0.5% Triton®-X100 in PBS) for 10 minutes, and blocked for 1 hour using 3% bovine serum albumin in PBS. Samples were labeled with rabbit anti-pFAK (1:200, Abcam, #ab81298) and rat anti-uvomorulin / E-cadherin (1:100, Sigma-Aldrich). The secondary antibodies used were anti-rat Alexa568 (1:200, Thermo Fisher Scientific #A110077) and anti-rabbit Alexa 647 (1:200, Thermo Fisher Scientific #A21244). The actin cytoskeleton was labeled using 488-phalloidin (1:50, Sigma-Aldrich #49 409). Samples were mounted with ProLong Diamond Antifade Mountant containing 4‘,6-diamidino-2-phenylindole (DAPI) (Thermo-Fisher Scientific, #P36935) to stain the cell nuclei.

[0090] Optical imaging. Samples were imaged using an optical (Zeiss) and a confocal microscope (Nikon A1R laser scanning confocal microscope, Nikon Instruments Europe BV). In the confocal microscope, the laser lines used were 405, 488, 561, and 633 nm. For each image, the laser intensity was adjusted to avoid photobleaching and the detector sensitivity was adjusted so that the brightness of the image was optimized. Images of 1024 x 1024 pixels were obtained using a 60x / 1.4 Plan-Apochromat oil immersion objective lens and a 20x / 0.8 Plan-Apochromat air immersion objective lens. The data were in the form of 3D Z-stacks, containing 30 - 40 slices at intervals of 150 - 250 nm each. Time-lapse microscopy was performed using an EVOS FL auto (Thermo Fisher Scientific).

[0091] Topography elimination by confocal microscopy. SRG topography was eliminated using an LSM780 laser scanning confocal microscope (Zeiss). A Plan-Apochromat 20 / 1.4 water immersion objective lens was used during elimination. During irradiation, the sample was in either a dry environment, a liquid culture environment, or a cell culture environment. The sample was irradiated with a fluorescent lamp filtered in the blue region (470 ± 40 nm) with an intensity of 1.5 Wcm -2 for 5 minutes. Bright-field images of the topography were taken before and after elimination. In the case of MDCKII cells, the sample was irradiated with a fluorescent lamp and then the cells were either detached from the sample with trypsin for surface property evaluation or fixed 2 hours later for immunolabeling.

[0092] Viability assay. MDCKII cells were seeded on the photopatterned bilayer and cultured on the sample for 24 hours. The topography was eliminated as described above. Three hours after elimination, the cells were washed with PBS and a viability / cytotoxicity kit for mammalian cells * was used *Using (Thermo Fischer Science), 600 μl of live / dead reagent solution containing 0.50 μl / ml of calcein AM and 2 μl / ml of ethidium monomer dimer-1 in PBS was added to each sample for staining. The samples were incubated at 37 °C for 30 minutes in a humidified atmosphere containing 5% CO2. After incubation, the reagent solution was aspirated and 600 μl of PBS was added to prevent drying of the cells. The samples were imaged with a confocal microscope (Nikon A1R laser scanning confocal microscope) using laser lines at 488 nm and 561 nm. A 1024x1024 pixel image was obtained using a 20x / 0.8 Plan-Apochromat air immersion objective lens.

[0093] Image and statistical analysis. The distribution of focal adhesions was analyzed by performing a fast Fourier transform on the focal adhesion channel using the FFT plugin in ImageJ. Before generating the FFT image, a circular region of 900 pixels was cropped, and the FFT image was generated from this region. ImageJ was used to measure the elongation, area, and orientation of focal adhesions. Further analysis of the elongation and orientation of focal adhesions was performed using the MomentMacroJ v1.4B script (https: / / www.hopkinsmedicine.org / fae / mmacro.html). The graphs in Figures 3d-f represent the average of 100 focal adhesions quantified from 10 individual images. In Figures 5c, d and 7c, the graphs represent the average of 30 focal adhesions quantified from two separate images. Prior to analysis, focal adhesion images were processed to remove pixel noise. The principal moments of inertia (maximum and minimum values) were measured, and cell elongation was defined as the ratio of these values (maximum / minimum value). The higher the value, the more elongated the focal adhesion. The direction of the focal adhesion was defined as the angle between the direction of the surface pattern and the major axis. Statistical analysis was performed using Origin version 2019b (OriginLab Corporation) and MATLAB®. For experiments with less than 100,000 quantified focal adhesions, the statistical power of the test was estimated. If the actual power value exceeded 75%, the statistical difference was presumed to be significant. Since the data was found to be non-normally distributed, non-parametric Kruskal-Wallis tests with Bonferroni correction and Dunn-Sidak post hoc tests were used to evaluate statistical significance.

[0094] The specific examples provided in the above description should not be construed as limiting the scope of the appended claims and / or their applicability.

Claims

**Claim 1**: A patterned system (100, 200) for cell culture, comprising: - A support structure (101, 201), - An azobenzene-containing intermediate layer (102, 202), and - A top layer (103, 203) comprising a protective polymer selected from elastomers, hydrogels, and parylene, wherein one or more regions of the azobenzene-containing intermediate layer are obtained by exposing them to light generated by a laser, and the wavelength of the light is 430 nm to 530 nm, thereby creating topographical features in the system. A patterned system. **Claim 2**: The patterned system according to claim 1, wherein the intensity of the light is 1 W cm-2 to 5 W cm-2. **Claim 3**: The patterned system according to claim 1, wherein the thickness of the azobenzene-containing layer is 50 nm to 5 µm, and the thickness of the top layer is 20 nm to 100 µm. **Claim 4**: The patterned system according to claim 3, wherein the thickness of the top layer is 20 nm to 200 nm. **Claim 5**: The patterned system according to claim 1, wherein the thickness of the azobenzene-containing layer is 50 nm to 5 µm, and the thickness of the top layer is 50 nm to 100 µm. **Claim 6**: The patterned system according to claim 1, wherein the support structure is selected from cell culture disks such as Petri dishes, microscope cover slips, and well plates. **Claim 7**: The patterned system according to claim 1, wherein the elastomer is a siloxane. **Claim 8**: The patterned system according to claim 7, wherein the siloxane is PDMS. **Claim 9**: The patterned system according to claim 1, wherein the azobenzene is N-ethyl-N-(2-hydroxyethyl)-4-(4-nitrophenylazo)aniline. **Claim 10**: A method for reversibly engraving topography on an azobenzene-containing material of a system comprising a support structure (101, 201), an azobenzene-containing intermediate layer (102, 202), and a top layer (103, 203) comprising a protective polymer selected from elastomers, hydrogels, and parylene, the method comprising the following steps: Scanning a laser beam on the uppermost layer, where the wavelength of the light is from 400 nm to 600 nm and the intensity of the light is 1 W / cm －２ ~5 W / cm －２ ; or Projecting an interference pattern of laser light onto a material, where the wavelengths of the two interfering laser lights are from 400 nm to 600 nm, and the intensities of the two interfering laser lights are 100 mW / cm －２ ~600 mW / cm －２ ; A method comprising. **Claim 11**: The method according to claim 10, wherein the method comprises the step of scanning laser light over the top layer, and the wavelength of the light is 430 nm to 530 nm. **Claim 12**: The method according to claim 10, wherein the method includes a step of projecting an interference pattern of laser light onto a material, and the wavelengths of the two interfering laser lights are from 430 nm to 530 nm. **Claim 13** A method for erasing topographical features of a patterned system according to any one of claims 1 to 9, the method including exposing the topographical features to light from 400 nm to 600 nm generated by a laser, or generated by a fluorescent lamp or an LED. **Claim 14**: The method according to claim 13, wherein the method includes a step of exposing the topographical features to light from 460 nm to 530 nm generated by a laser. **Claim 15** The intensity of the light generated by a laser, a fluorescent lamp, or an LED is 1 W / cm －２ to 5 W / cm －２ The method according to claim 13, wherein the intensity is such. **Claim 16** Use of the patterned system according to any one of claims 1 to 9 as a cell culture platform. **Claim 17** A cell culture method of culturing cells on the patterned system according to any one of claims 1 to 9. **Claim 18** The following steps: a) A step of providing a patterned system, b) A step of coating the uppermost layer of the patterned system with a cell adhesion protein, and c) A step of seeding cells onto the cell adhesion protein, the method according to claim 17. **Claim 19** The method according to claim 17, wherein the cells are selected from epithelial cells, fibroblasts, endothelial cells, neurons, mesenchymal stem cells, astrocytes, cardiomyocytes, and cancer cells. **Claim 20** The method according to claim 18, wherein the cell adhesion protein is selected from collagen, fibronectin, and laminin. **Claim 21** The method according to claim 18, including oxygen plasma treatment before step b).

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