A cell culture system

Abstract

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

Description

Technical Field

[0001] The present disclosure relates to a cell culture system, and in particular to a photoreconfigurable system comprising a bilayer structure including an azobenzene-containing layer and a protective coating. Background Art

[0002] In drug development, over 90% of new molecules developed fail in clinical-stage trials. This low success rate is primarily due to the cell models used in in vitro studies. These model systems fail to recreate natural conditions in the human body: in vivo, the cellular microenvironment regulates a wide range of cellular functions. Therefore, there is a clear need for better cell-based models—and to achieve this, it is crucial to mimic dynamic in vivo conditions, including the cellular microenvironment, as closely as possible in in vitro cell culture.

[0003] Currently, cells are mostly cultured in cell culture discs, flasks, and plates, without properly simulating the cells' dynamic in vivo microenvironment, the cellular niche. A major drawback of these conditions is the lack of surface features, i.e., topography, which does not accurately reflect the environment that cells encounter in the human body. In addition, adherent cells in our bodies constantly change and interact with their surroundings. This behavior creates a dynamic extracellular environment that is difficult to reproduce in vitro.

[0004] Different microengineering approaches have been used to construct micropatterned cell culture substrates, but they typically have square topography rather than smooth structures, which do not resemble the cellular environment. Furthermore, they still lack reconfigurability or dynamic surface changes, thus only providing a static environment for cells.

[0005] Therefore, there remains a need for reconfigurable cell culture surfaces. Summary of the Invention

[0006] The present invention is based on the observation that by generating light-induced surface features using a bilayer structure comprising an azobenzene-containing layer and a protective coating, the surface topography can be repeatedly changed, erased or generally reconfigured.

[0007] Therefore, an object of the present invention is to provide a patterned cell culture system.

[0008] It is also an object of the present invention to provide a method for reversibly writing a topography on the surface of an azobenzene-containing material of said system, said method comprising focusing a light beam onto said material or projecting a laser interference pattern onto said material.

[0009] The present invention also aims to provide a method for erasing topographical features of a patterning system, said method comprising subjecting said topographical features to light of 430 nm to 600 nm generated by a laser, preferably a continuous wave laser, or by a fluorescent lamp or by an LED.

[0010] The present invention also aims to provide use of the system or the patterned system as a cell culture platform.

[0011] It is also an object of the present invention to provide a cell culture method, wherein cells are cultured on a patterned system.

[0012] The invention, including exemplary non-limiting embodiments both as to constructions and methods of operation, together with further objects and advantages thereof, will best be understood from the following description of specific exemplary embodiments when read in connection with the accompanying drawings.

[0013] The verbs "to comprise" and "to include" are used in this document as open limitations that neither exclude nor require the existence of unrecited features. Furthermore, it is to be understood that the use of references without a specific number throughout this document does not exclude a plurality. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A system according to an exemplary, non-limiting embodiment of the present invention is shown.

[0015] Figure 2 The trans-cis isomerization reaction of azobenzene derivatives induced by light is shown.

[0016] Figure 3 Graphical representations of azobenzene-driven a) surface relief grating formation and b) erasure of an exemplary system of the present invention are shown.

[0017] Figure 4 Sinusoidal surface relief gratings written by interference lithography, atomic force microscopy 3D projection, average cross-sectional profile and topography written by laser scanning are shown for an exemplary system of the present invention.

[0018] Figure 5 Shown are the morphological differences between Madin Darby canine kidney epithelial cells on flat (left) and 1 μm periodic sinusoidal topography (right) of an exemplary system of the present invention comprising an azobenzene-based membrane.

[0019] Figure 6a The results show that the power consumption of 300 mW cm -2Diffraction efficiency (DE) curves at different PDMS thicknesses during surface relief grating (SRG) writing (488 nm, circular polarization, probe beam wavelength 633 nm) with a high intensity (each curve is the average of three measurements). The standard deviation of the DE value at the end of SRG writing is ±7% (DR1g), 0.5% (DR1g-PDMS 50 ), ±5% (DR1g-PDMS1) and ±6% (DR1g-PDMS 0.02 ).

[0020] Figure 6b Shows DR1g-1PDMS after SRG writing 0.02 AFM image of the surface morphology.

[0021] Figure 6c AFM images of the surface morphology on DR1g-PDMS1 after SRG writing are shown.

[0022] Figure 7 The diffraction efficiency curves of PDMS layers with different thicknesses are shown in the following processes: a) at 500 mW cm -2 b) SRG writing with a 530 nm LED; c) AFM images of the surface morphology of DR1g-PDMS1 after writing (top) and erasing (bottom); d) cross-sectional profile of the SRG modulation depth during photolithographic writing (solid line) and erasing (dashed line) of DR1g-PDMS1.

[0023] Figure 8 Shown are the acetone resistance of uncoated DR1g (left) and DR1g coated with a 90 nm layer of parylene C (right).

[0024] Figure 9 Shown are a) graphic representation of polyacrylamide hydrogel coating on surface-modulated DR1g; b) DIC analysis of fluorescent microparticles showing lateral strain induced in the hydrogel by DR1g photostimulation.

[0025] Figure 10Shown are a) schematic diagram of DR1g-PDMS1 sample preparation for cell culture experiments; b) optical microscope images of MDCK II cells on flat glass substrates and surface-patterned films of DR1g and DR1g-PDMS1 24 hours after cell seeding. The black arrow indicates the direction of SRG morphology. Scale bar: 50 μm; c) immunolabeled MDCK II cells on surface-patterned DR1g-PDMS1 bilayers at different time points (24 hours, 72 hours). The markers used are DAPI (chromatin), E-cadherin (cell-cell junctions), and pFAK (mature focal adhesions). The organization of focal adhesions was analyzed, where the fast Fourier transform (FFT) of the pFAK image shows the period of the image (indicated by the 1st-order peak). The black arrow indicates the direction of SRG morphology. Scale bar 20 μm.

[0026] Figure 11 DHM images of the SRG topography on DR1g-PDMS1 after erasure using confocal microscopy fluorescent light filtered in the blue region (470 ± 40 nm) in a liquid environment (a) in a dry environment and b) in a liquid environment. Irradiation time: 5 minutes. Scale bar: 10 μm. c) Surface profile of the SRG topography on DR1g-PDMS1 after erasure in a dry environment and d) in a liquid environment. DETAILED DESCRIPTION

[0027] According to one aspect, the present disclosure relates to a cell culture system. Figure 1 An exemplary system 100 is shown in FIG. The system includes a support structure 101 , an azobenzene-containing middle layer 102 , and a top layer 103 .

[0028] The support structure can be any support structure used for cell culture. Exemplary support structures are cell culture dishes, such as culture dishes, microscope coverslips, and well plates. The support structure is typically made of plastic or glass. Exemplary culture dish forms are poly(styrene) and glass-bottom culture dishes.

[0029] Azobenzene-containing materials are photoreconfigurable. As defined herein, a photoreconfigurable material is a material whose shape can be reconfigured upon exposure to light. The azobenzene molecules can be substituted or unsubstituted. Exemplary photoinduced transformations of azobenzene units are as follows: Figure 2 As shown, R and R' refer to different para-substituents. Different substituents can also be added to the meta and ortho positions. An exemplary azobenzene suitable for use in the present technology is ethyl-N-(2-hydroxyethyl)-4-(4-nitrophenylazo)aniline.

[0030] The top layer comprises a protective polymer, such as an elastomer or a hydrogel. An exemplary elastomer is a silicone-containing polymer, such as polydimethylsiloxane (PDMS).

[0031] According to another embodiment, the protective polymer comprises parylene, preferably parylene C, i.e. poly(chloro-p-xylene). There are a variety of substituted [2.2] para-cyclophanes in which functional groups can be introduced into the phenyl ring. These functional groups allow the deposition of functionalized parylene films, or they can undergo further functionalization, thereby allowing the immobilization of bioactive molecules.

[0032] PDMS and parylene C are preferred coatings due to their good mechanical and barrier properties, hydrophobicity, chemical resistance, and biocompatibility. Another advantage of parylene C is that it can be deposited to produce pinhole-free ultrathin films. Furthermore, despite being semicrystalline, parylene C is highly transparent within the targeted thickness range.

[0033] 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.

[0034] 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 exemplary thickness of the azobenzene-containing layer is 500 nm.

[0035] When the protective polymer is PDMS or parylene C, the thickness of the top layer is preferably below 90 nm.

[0036] When the protective polymer is a hydrogel, the thickness of the top layer is preferably less than 50 μm.

[0037] The present disclosure also relates to a method for manufacturing a cell culture system 100, the method comprising:

[0038] a) providing a support structure 101,

[0039] b) coating the support structure with an azobenzene-containing layer 102, and

[0040] c) Coating the azobenzene-containing layer with a top layer 103 comprising a protective polymer.

[0041] The support structure is preferably selected from a culture dish, a microscope cover slip, or a well plate. According to one embodiment, the protective polymer comprises a hydrogel or an elastomer. An exemplary elastomer is a silicone. A specific silicone is PDMS. According to another embodiment, the protective polymer comprises parylene. A specific parylene is parylene C.

[0042] According to one embodiment, the coating of step b) comprises spin coating.

[0043] According to another embodiment, the coating of step c) comprises spin coating. Spin coating is preferred when the protective polymer is a silicone such as PDMS.

[0044] According to another embodiment, the coating of step c) comprises chemical vapor deposition polymerization. This is the preferred method when the protective polymer comprises parylene, such as parylene C.

[0045] Micrometer-scale and submicrometer-scale topography can be reversibly inscribed on the surface of azobenzene-containing materials, for example, by optical interference lithography, digital micromirror devices, microlens arrays, or simply by scanning a laser beam (e.g., from a laser scanning microscope) across the film surface. In the presence of a focused beam, thin azobenzene-based coatings tend to accumulate within or escape from the beam's focal volume. Thus, the scanning motion of the beam allows the inscription of any shape, much like a drawing tool.

[0046] A schematic diagram of generating and erasing topographic features to and from an exemplary system of the present invention is shown in FIG. Figure 3 shown.

[0047] Thus, one aspect of the present disclosure also provides a method for reversibly inscribing a topography on a surface of an azobenzene-containing material of the system. According to one embodiment, the method comprises focusing a light beam onto the material. According to a specific embodiment, the method comprises scanning a laser beam across the azobenzene-containing layer.

[0048] According to another embodiment, the method of reversibly writing a topography on a surface of an azobenzene-containing material of the system utilizes interference lithography. According to this embodiment, the method comprises projecting an interference pattern of laser light onto the material.

[0049] Since the middle layer is coated with the top layer, topography is also formed on the top layer.

[0050] The reversible writing includes patterning and erasing. The wavelength of light used for patterning and erasing is generally 400nm to 600nm, preferably 430nm to 530nm. The appropriate intensity range depends on the technology used. For example, 100mW cm -2 Up to 600mW cm -2 sufficient for interference lithography, while about 1W cm -2 Up to 5W cm -2 Patterning / erasing for laser scanning confocal microscopy.

[0051] According to one embodiment, for patterning, the method comprises subjecting one or more regions of the azobenzene-containing layer to light generated by a laser, preferably a continuous wave laser, at a wavelength of 400 nm to 600 nm, preferably 430 nm to 530 nm, thereby generating the topographical features of the system. An exemplary intensity of the light generated by the laser is preferably 1 W cm -2 Up to 5W cm -2 .

[0052] According to an embodiment, for erasing, the topographical features of the patterning system are subjected to light generated by a laser, preferably a continuous wave laser, at 400 nm to 600 nm, preferably 430 nm to 530 nm. An exemplary intensity of the light generated by the laser is 1 W cm -2 Up to 5W cm -2 .

[0053] The exemplary system 300 of the present disclosure that can be obtained by the above disclosed method is as follows: Figure 4 The figure also shows the sinusoidal surface relief grating written by interference lithography, atomic force microscopy 3D projection, average cross-sectional profile and topography written by laser scanning of the system.

[0054] According to another aspect, the present disclosure relates to a method of patterning the system by subjecting one or more regions of the azobenzene-containing layer of the system to light generated by a laser, preferably a continuous wave laser, at a wavelength of 400 nm to 600 nm, preferably 430 nm to 530 nm, thereby generating topographical features in the system. The intensity of the light is typically 1 W cm -2 Up to 5W cm -2 An exemplary wavelength is 488 nm, which is a preferred wavelength when the azobenzene-containing material is ethyl-N-(2-hydroxyethyl)-4-(4-nitrophenylazo)-aniline. An exemplary intensity is 1 W cm -2 .

[0055] The present disclosure also relates to a method for erasing a topographical feature from the system by subjecting the topographical feature to light generated by a laser, preferably a continuous wave laser, in the range of 460 nm to 530 nm. Exemplary wavelengths are 470 nm, 488 nm, and 530 nm. According to another embodiment, the erasing is performed by using light generated by a fluorescent lamp filtered in the range of 430 nm to 530 nm. An exemplary intensity generated by the laser is 1 W cm -2 The intensity of light produced by a fluorescent lamp is typically 1W cm -2 Up to 5W cm -2 Erasure can be performed using the aforementioned laser and fluorescent lamp, and can also be performed using LED.

[0056] The present invention allows for the generation of free-form topographic patterns on cell culture substrates. Even when cells are already growing on the substrate, the topography can be erased with a uniform light source (e.g., fluorescent light, LED light, or laser) and rewritten to create new patterns on the culture dish, making it possible to create dynamic topography that better mimics the dynamic conditions in the human body.

[0057] Therefore, one aspect of the present disclosure is to provide a method for culturing cells on a system comprising topographical features on a top surface. The system comprising topographical features can be obtained as disclosed above.

[0058] According to an exemplary embodiment, the method comprises the following steps:

[0059] a) providing the patterning system,

[0060] b) coating the top layer of the patterned system with a cell adhesion protein, and

[0061] c) seeding cells on the cell adhesion protein.

[0062] According to a preferred embodiment, the method comprises subjecting the system to an 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] Exemplary cell adhesion proteins suitable for use in the method are selected from collagen, fibronectin and laminin. The choice of cell adhesion protein depends on the cell type to be seeded.

[0065] Based on the use of azobenzene-containing bilayers to create light-induced surface features, the present system allows for robust and reconfigurable control of surface topography. According to one embodiment, a cell culture dish is coated with an azobenzene-containing polymer film in the presence of a protective silicone layer, allowing for easy chemical modification of the surface, ensuring biocompatibility, and fully supporting existing protein deposition techniques. According to another embodiment, a cell culture dish is coated with an azobenzene-containing polymer film in the presence of a protective parylene C layer, allowing for easy chemical modification of the surface, ensuring biocompatibility, and fully supporting existing protein deposition techniques.

[0066] In vivo, the dynamic interaction of cells with the surrounding extracellular matrix (ECM) plays a key role in the regulation of many physiological and pathological processes such as tissue morphogenesis, healing and tumor growth. Due to the ability to manipulate surface features with light, the present invention allows real-time control of extracellular niches, (multiple) cell spatial arrangement, orientation and migration. The process is reversible, remotely controllable, and non-invasive, which is very important for the definition of time programs such as cell differentiation, stem cell phenotype acquisition, tissue regeneration and triggered cell directional migration and decoupling morphology and chemical signals. In vivo, cells are exposed to different types of biophysical signals that can be converted into biochemical activities in a process known as mechanical transduction. These signals are important co-regulators of, for example, cell alignment and migration. For example, muscle cells, neuronal cells and endothelial cells show highly aligned tissues in our body, and cell migration is greatly affected by the morphological features of the cell environment.

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

[0068] Results and discussion

[0069] Characterization of SRG writing and erasing on DR1g-PDMS bilayer structures

[0070] To assemble the platform, a glass coverslip was first coated with a photopatternable, amorphous layer of ethyl-N-(2-hydroxyethyl)-4-(4-nitrophenylazo)-aniline, a molecular glass containing Disperse Red 1 (DR1g; thickness 480 ± 20 nm), which was also coated with PDMS. The resulting DR1g-PDMS bilayer structure served as a photoresponsive cell culture platform, with DR1g serving as the photoresponsive component.

[0071] To investigate the effect of PDMS (matrix:curing agent ratio 10:1) on SRG formation, three different PDMS prepolymer dilutions (0.02 wt%, 1 wt%, and 50 wt%) were tested in n-hexane using identical spin coating parameters. These samples are designated here as DR1g-PDMSx, where x represents the concentration of PDMS in hexane. The thickness of the PDMS layer was measured by ellipsometry and profilometry. Ellipsometry was used for precise measurements of the 0.02 wt% and 1 wt% layers, while profilometry was used for the thickest PDMS layer (50 wt%). The thickness of the PDMS layer was 4.5 μm (DR1g-PDMS 50), 65nm(DR1g-PDMS1) and 20nm(DR1g-PDMS 0.02 ). The DR1g-PDMS bilayer was photopatterned using optical interference lithography in a Lloyd mirror configuration, which induced mass migration in the DR1g and surface deformation of the PDMS coating, thereby forming SRGs. The period of the interference pattern is determined by the wavelength and the angle between the mirror and the laser beam. By varying the angle, SRGs with different periods can be obtained (roughly in the range of 300nm to 10μm). Here, the period was set to 1μm because such a period has previously been used to control epithelial cell alignment of the same material. SRG formation within different DR1g-PDMS bilayers was monitored in situ by diffraction efficiency (DE) measurements. The thickness of the DR1g film (480±20nm) was chosen to be large enough to make SRG formation independent of small variations in layer thickness. Therefore, differences in DE are only due to differences in the PDMS layer. The samples were imaged with atomic force microscopy (AFM) to confirm SR formation.

[0072] The DE curve during SRG writing on the DR1g-PDMS double layer is shown in Figure 2. Figure 6a As shown in the images, it can be observed that DE decreases systematically with increasing PDMS layer thickness. AFM imaging confirmed that DR1g-PDMS 0.02 and DR1g-PDMS1 formed an SRG with an expected period of 1 μm ( Figure 6b , Figure 6c ). DR1g-PDMS 0.02 The surface modulation depth of DR1g-PDMS1 is more than 400nm, while the modulation depth of DR1g-PDMS1 is significantly reduced to 160nm. 50 In the case of PDMS coating, no SRGs formed on the outer surface, as no sinusoidal patterns could be observed using AFM. The 8% DE can be attributed to grating formation at the DR1g / PDMS interface. When the azobenzene-containing film is positioned between the glass substrate and the protective coating, it imposes stronger constraints on efficient movement. SRG formation requires mass transfer, so the presence of a thick PDMS layer increases the hindrance to the complex stress field experienced by DR1g during SRG formation. Therefore, a PDMS coating less than 100 nm thick on top of a thin DR1g film does not inhibit SRG formation, but rather alters its formation dynamics.

[0073] After SRG formation, the morphology is stable for at least one year at temperatures below the glass transition temperature of DR1g (71°C), but can be erased using heat or a uniform light beam with a wavelength matching the absorption band of DR1g. Since direct heating cannot be localized and is incompatible with cell culture conditions, SRG erasure with visible light (e.g., a 530 nm LED) is preferred. To study the dynamics of SRG erasure, the DE (DE) of samples exhibiting the same initial DE value (approximately 7%) was monitored during the erasure process. Figure 7 a). Erase dynamic Figure 7 At the end of the process, all samples reached similar DE values ​​(about 0.5%), so the PDMS layer thickness does not seem to affect the effectiveness of the erasure process in terms of DE value. However, clear differences in the erasure dynamics can be observed between different PDMS thicknesses. Interestingly, the DE of DR1g-PDMS1 decreases systematically faster than that of the other samples, indicating that a PDMS layer of appropriate thickness can even accelerate topographical erasure. 50 In the case of , the erasure dynamics is different from that of other samples, and the DE decreases relatively slowly and proceeds in a two-step process.

[0074] The samples were imaged using AFM, which confirmed that the morphology of DR1g-PDMS1 was reduced to 85% of the initial value, as shown in Figure 2. Figure 7 As shown in c. Figure 7 The surface profile in d further highlights the difference between the erased and unerased morphologies. DR1g-PDMS 0.02 The modulation depth of the sample decreased by 70%. Even with similar DE values, AFM showed that the erased topography reached a lower modulation depth in the case of DR1g-PDMS1. This was the motivation for choosing DR1g-PDMS1 for the cell culture studies. At deeper gratings, erasing with a uniform laser beam is less effective than at lower SRGs. Therefore, the higher residual grating observed on this sample confirms that for DR1g-PDMS 0.02 On the surface, complete erasure of topography can be more difficult to achieve. Under DR1g-PDMS1, the grating depth facilitates a fast erasure process, resulting in low residual topography, thus limiting the required irradiation time during cell growth experiments. The grating depth is also sufficient to induce cellular responses related to alignment. The thicker PDMS layer acts as a protective barrier, separating the DR1g layer from the cells.

[0075] The DR1g layer was also coated with parylene C (i.e., poly(chloro-p-xylene)) by chemical vapor deposition (CVD). For each deposition run, the CVD of parylene C was mediated by four secondary chambers inserted into the main deposition chamber. These secondary chambers have a small hole at the top, whose function is to control the deposition rate of the reactive monomer, thereby fine-tuning the thickness of the deposited layer. In general, the thickness of the deposited parylene film is proportional to the mass of dimer loaded into the machine. However, for ultra-low thickness films (<100nm), the reduction in dimer mass will lead to an uncontrollable and unreliable deposition process (due to very short and unstable pressure). By taking advantage of the percolation of parylene molecules through pores with a size 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 allowing only a precise fraction of the reactive monomer to enter the secondary chamber.

[0076] The prepared substrates were characterized by stylus profilometry and showed thicknesses ranging from 13nm to 415nm. The PDMS coated samples were then tested for SRG formation as described above. In all samples with a thickness less than 90nm, the samples showed SRG formation. The barrier properties of the parylene C layer were also tested; permeability tests were performed by depositing 1μl drops of different organic solvents (acetone, ethanol and isopropanol) and water on the sample surface. Over a period of seconds to three minutes, the solvent droplet either dissolved a portion of the DR1g layer or remained intact on top of the sample surface until it finally evaporated. As expected, samples above 55nm were resistant to water penetration and also provided the best resistance to organic solvents (especially ethanol and isopropanol). The most aggressive solvent tested was acetone, which was used to define the upper limit of the thickness of the parylene layer on which SRGs can form while showing consistent and reliable resistance to acetone penetration ( Figure 8 ).

[0077] A polyacrylamide hydrogel layer was also used to coat the DR1g substrate ( Figure 9 a, Figure 9 b). Polyacrylamide was loaded with red-emitting (607 nm) fluorescent microparticles. The thickness of the hydrogel was estimated to be 100 μm. DR1g was then photostimulated with a 488 nm laser in user-defined regions of interest (ROIs) under a laser scanning confocal microscope (LSM780, Zeiss). In those ROIs, the flow of photosensitive material induced corresponding deformations in the hydrogel, as shown by the fluorescent microparticles captured by time-lapse microscopy and analyzed by digital image correlation (DIC). For the tested hydrogel (2.8 kPa), the strain propagated inside the hydrogel, at least as far as 50 μm deep. This experiment shows that, in principle, the photoinduced surface deformation of DR1g can be used to locally and mechanically stimulate soft materials such as hydrogels. Such hydrogels can be loaded with cells, or cells can be cultured on the hydrogel surface.

[0078] SRG-guided cell alignment

[0079] Cells sense the physical properties of their environment and the mechanical forces on their surface, but these forces are also transmitted deeper into the cell, even to the nucleus. The primary site of perception is the cell-ECM contact, primarily at focal adhesions, multiprotein complexes at the cell membrane. The formation of focal adhesions at the cell-ECM interface regulates cell attachment, alignment, and migration. One of the molecules found at cell-cell contact sites is the calcium-dependent transmembrane protein E-cadherin. E-cadherin is particularly present at adherens junctions and plays a key role at the cell-cell interface in the formation of tight, polarized epithelia.

[0080] To investigate whether microtopography on the DR1g-PDMS1 bilayer can guide collective cell alignment, Madin-Darby canine kidney type II (MDCKII) epithelial cells were seeded on SRG and their alignment to the underlying microtopography was studied. This cell line provides a good model for studying collective cell behavior. While mechanotransduction by individual cells on microtopography has been extensively studied, the behavior of collective cells that undergo coordinated movement without disrupting their cell-cell contacts has not been fully characterized. Figure 10 a shows a schematic diagram of the sample preparation. Briefly, DR1g and PDMS were then spin-coated to form a bilayer structure, and SRG was written as described previously. After patterning, the surface was made hydrophilic with oxygen plasma treatment to improve protein attachment to the surface. The surface was then coated with type I collagen to improve cell adhesion to the surface, and MDCKII cells were seeded on the sample and cultured for up to 72 hours. Cell migration along the microtopography was tracked by time-lapse microscopy. During the first 24 hours after seeding on DR1-PDMS1, the cells were already aligned along the microtopography. After 24 hours, the cells formed small colonies on bare DR1g and DR1g-PDMS1 that were elongated in the direction of the pattern, indicating that the PDMS layer did not inhibit the cells from sensing the underlying topography ( Figure 10 b) At the 72 hour time point, cells formed a confluent monolayer.

[0081] The cellular response to microtopography was further investigated in terms of cell-material interaction and cell-cell interaction by immunolabeling MDCKII cells at different time points. Cell nuclei were stained with DAPI to distinguish individual cells. Cell-cell interactions were investigated by detecting the intracellular localization of E-cadherin. E-cadherin localization showed that 24 hours after cell seeding, the nuclei were round, but the cells showed an elongated morphology along the surface microtopography ( Figure 10C). In addition, E-cadherin accumulates in cytoplasm, so cells have not yet formed mature cell-cell connection.At 72 hours time point, cell morphology is less elongated than 24 hours time point.After 72 hours, cells form a uniform cell layer, and E-cadherin is positioned at cell-cell interface, and strong cell-cell interaction is shown on double-layer surface.On the contrary, E-cadherin loss shows epithelial-mesenchymal transition (EMT), wherein epithelial cells lose their phenotypic characteristics and are converted into more motile and possibly invasive non-polarized mesenchymal cells.Because E-cadherin is positioned at cell-cell interface, the cells on double-layer surface form a tight epithelial layer after 72 hours.

[0082] Focal adhesion kinase (FAK) is one of the earliest molecules present in the development 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). 24 hours after seeding, focal adhesions were observed at the edge of the basal cells, and their distribution was analyzed by using fast Fourier transform (FFT). FFT converts spatial image information into frequency space, in which periodic features are highlighted, resulting in a specific frequency pattern. The analysis showed that the first-order frequency peak ( Figure 10 c, FFT of the pFAK image, demonstrating the periodic distribution of the image feature (pFAK). After 72 hours, pFAK was still observed at the cell edges, but cell movement became more restricted after a uniform cell layer had formed. In the FFT of the localized focal adhesion channel, a first-order frequency peak was still visible, indicating that the localized focal adhesions are periodically distributed and that the cells are still sensing information from the topographic cues.

[0083] Erasing of SRG topography with living cells. Instead of using an LED, the microtopography was erased using fluorescent light from a confocal microscope (filtered in the blue region of the visible spectrum), allowing for observation of living cells immediately after the measurement. This setup is considered suitable for biological environments because most microscopes can be equipped with environmental controls suitable for living cell culture. Erasing was first performed in the absence of cells at room temperature in a dry environment and in a liquid environment to set the erasing parameters. As can be seen from the bright field images and digital holographic microscopy (DHM) images, illumination with fluorescent light clearly produces a recognizable circular area in both dry and aqueous environments, allowing for quantitative results regarding the surface profile ( Figure 11 a, Figure 11 b) Monitoring the surface using DHM allows for rapid quantitative characterization of the surface topography over a larger area compared to AFM. DHM images show a reproducible decrease in the modulation depth within 5 minutes of irradiation. Under dry conditions, the surface roughness decreases from 56 nm to 14 nm, indicating a 75% decrease in the modulation depth compared to the initial value ( Figure 11c). In liquid environment, the modulation depth of the erased area decreases by 50% ( Figure 11 d), Furthermore, the (partially) erased surface is significantly rougher and exhibits rounded surface features.

[0084] MDCKII cells were seeded on SRG topography and cultured for 24 hours before being erased to orient the cells along the microtopography. The samples were illuminated with fluorescent light on a confocal microscope for 5 minutes at 37°C in a humidified atmosphere with culture medium on top. Two hours after erasure, they were fixed and immunolabeled. After cells were removed by trypsinization, partial photoerasure was confirmed by DHM. In the presence of the PDMS layer, erasure was more uniform compared to bare DR1g, resulting in significantly fewer of the aforementioned circular surface features. Possible phototoxicity to the cells was also investigated. For this experiment, cells were seeded on a sample in which DR1g was spin-coated onto the underside of a glass coverslip, with the glass substrate at the cell-material interface. This control sample ensured that light intensities similar to those during erasure reached the cell plane, but did not produce topographical changes at the cell adhesion site. The control sample was illuminated with fluorescent light for 5 minutes, and a LIVE / DEAD cell viability / cytotoxicity assay was performed 3 hours after erasure. No significant acute phototoxic effects on cell viability were observed, as no dead cells were observed in the erased areas, similar to those in the unerased areas. When studying the effect of phototoxicity on cell morphology, PDMS was spin-coated on top of the control sample at the cell-material interface on the other side to ensure similar adhesion properties. No significant differences in cell morphology were observed within 2 hours of irradiation.

[0085] After erasing, the cell clusters had a less diffuse morphology and smaller size, which may indicate a partial loss of substrate attachment after topographic changes. In addition, pFAK was observed to be more concentrated in the center of the cells rather than at the edges after erasing. No significant morphological changes were observed when the microtopography was erased beneath a uniform epithelial cell layer. This observation suggests that when strong cell-cell connections are formed, epithelial cells in the monolayer do not immediately rearrange in response to the loss of the guiding surface topography, at least for 2 hours after erasing. Quantification of local focal adhesion orientation was performed similarly to the above. The orientation data showed that local focal adhesions were more randomly oriented after erasing with small cell clusters. However, no differences were observed in the case of confluent cell layers. This suggests that smaller cell clusters can sense light-induced topographic changes and reorient local focal adhesions accordingly. Erasing did not appear to affect the elongation and area of ​​local focal adhesions. In the presence of liquid, even partial erasing of the surface topography using the confocal microscope light altered the surface microtopography and surface roughness. In the case of small cell clusters, topographic changes affected morphology and local focal adhesion orientation. However, no collective morphological response or local focal adhesion orientation was observed, at least over a 2-hour time span. After irradiation, cells remained attached to the erased surface and were viable.

[0086] in conclusion

[0087] The platform presented here consists of a photoreactive azobenzene-containing membrane and a thin PDMS or parylene C coating, allowing independent control of the material's photoreactivity and stability in cell culture environments. Together, these layers form a bilayer structure, which allows for surface topography modification using light-induced motion of the azobenzene-containing membrane. In the presence of the PDMS and parylene C layers, SRG topography can be efficiently photowritten and erased. When MDCK II epithelial cells are seeded on the photopatterning system, even after a uniform epithelial layer has formed, the SRG topography can still guide localized focal adhesion orientation along the surface topography. In the presence of living cells, the surface topography can be altered using the fluorescent light of a confocal microscope, enabling non-invasive control of surface topography. Although SRG topography erasure is only partial, it can still alter the topography without causing cell detachment or cell death. Therefore, light-mediated erasure is a strategy for dynamically controlling material topography in real-time cell experiments, which can be performed using conventional microscopy settings. The platform can also be patterned with proteins, enabling independent control of topography and biochemical signaling, and further functionalized for diverse applications.

[0088] Experimental part

[0089] Sample preparation. The polymer was PDMS. A double layer of disperse red 1 molecular glass containing azobenzene (DR1g, Solaris Chem) and polydimethylsiloxane (PDMS, SYLGARD 184, Dow) was prepared on a square glass cover slip by spin coating (Laurell Technologies). The glass cover slip was first sonicated twice in acetone for 10 minutes. A chloroform solution of DR1g was prepared at a concentration of 9% (w / v). The solution (35 μl) was deposited on a glass cover slip (22×22 mm) at 1500 rpm. 2 ) on a silicon substrate for 30 seconds. PDMS is prepared by mixing a pre-polymerized silicone elastomer matrix with a curing agent in a ratio of 10: 1. Uncured PDMS is diluted in n-hexane to produce 50 wt%, 1 wt% and 0.02 wt% solutions. The solution is dispensed on a thin DR1g film at 6000 rpm for 150 seconds and cured at 55°C for 1.5 hours. A sample for thickness measurement is first prepared by spin coating the PDMS solution on the above-mentioned silicon substrate. The thickness of the manufactured PDMS film is measured using a reflective ellipsometer (JA, Woollam VASE). The film formed by the 50 wt% PDMS solution is too thick to be measured using an ellipsometer, so the thickness is measured using a stylus profiler (Veeco Dektak 150). For both techniques, the resolution limit is in the sub-nanometer range.

[0090] Sample preparation. The polymer was parylene C.

[0091] A bilayer of azobenzene-containing Disperse Red 1 molecular glass (DR1g, Solaris Chem) and parylene C (Galentis Ltd.) was prepared on square glass cover slips by spin coating Disperse Red 1 as described above, followed by chemical vapor deposition (Para Tech Coatings) of parylene C using the effusion-based method described elsewhere. The orifice connecting the interior of each secondary deposition chamber to the larger main chamber was a square hole with lateral dimensions ranging from 200 μm to 8000 μm. The final thickness of the film was estimated using a stylus profilometer (Bruker Dektak XT). For each deposition run, 2 g of dichloroparacyclophane dimer was loaded onto four plates with an inner surface area of ​​19210 mm. 2 In the deposition system of the cylindrical secondary chamber.

[0092] Sample preparation. The polymer is a polyacrylamide hydrogel: Circular glass coverslips (13 mm) were washed with a 2% Hellmanex solution in an ultrasonic bath for 30 minutes, rinsed with copious amounts of deionized water, and carefully dried. The coverslips were passivated with grafted PLL-PEG. A drop (10 to 30 μl) of 0.1 mg / ml PLL-g-PEG in PBS was deposited on the coverslip and allowed to react for 30 minutes. The substrate was then washed with copious amounts of deionized water. The reagent solution was prepared as follows: acrylamide (10 wt %), bisacrylamide (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. The gelling solution was then pipetted onto a DR1-g-coated glass coverslip and covered with a passivated coverslip for 15 minutes. The hydrogel has an expected elastic modulus of 2.8 kPa and a thickness of 100 μm.

[0093] Surface relief grating writing and erasing. The bilayer structure was photopatterned using interference lithography in a Lloyd mirror configuration. A 488 nm circularly polarized continuous wave laser (Coherent Genesis CX488-2000) and 500 mW cm -2 The strength at 0.50cm 2 The surface relief grating (SRG) is inscribed on an area of ​​​​1.5 μm. The microtopography period Λ is set to 1 or 1.5 μm, which is determined by Λ = λ / 2sinθ, where λ is the laser wavelength and θ is the angle between the mirror and the laser beam. SRG erasure is performed using a 530 nm LED, with the beam focused directly on the SRG topography and an intensity of 100 mW cm -2 The SRG writing and erasing were monitored using a low-power (1 mW) 633 nm He-Ne laser, and the diffraction efficiency of the first-order diffracted beam was measured.

[0094] Cell culture. Madin-Darby canine kidney type II (MDCK II) epithelial cells were used in this study. They were cultured at 37°C in a humidified atmosphere containing 5% CO2 in a medium consisting of MEMGlutaMax (Gibco) supplemented with fetal bovine serum (10%) and penicillin / streptomycin (1%). Before cell inoculation, the samples were sterilized under UV light for 40 minutes. 50 μg ml -1 A solution of monomeric rat tail type I collagen (Thermo Fischer Scientific) in 0.02 N acetic acid was applied to the samples for 40 minutes.

[0095] Immunolabeling. Cells were fixed with 4% paraformaldehyde for 10 minutes, washed with PBS, permeabilized with permeabilization buffer (0.5% BSA, 0.5% Triton-X 100 in PBS) for 10 minutes, and blocked with 3% bovine serum albumin in PBS for 1 hour. Samples were labeled with rabbit anti-pFAK (1:200, Abcam, No. ab81298) and rat anti-Uvomorulin / E-cadherin (1:100, Sigma-Aldrich). The secondary antibodies used were anti-rat Alexa 568 (1:200, Thermo Fisher Scientific, No. A110077) and anti-rabbit Alexa 647 (1:200, Thermo Fisher Scientific, No. A21244). 488-phalloidin (1:50, Sigma-Aldrich, No. 49409) was used to label the actin cytoskeleton. Samples were mounted with ProLong Diamond antifade mounting medium (Thermo-Fisher Scientific, No. P36935) containing 4',6-diamidino-2-phenylindole (DAPI) to stain cell nuclei.

[0096] Optical imaging. The samples were imaged using an optical microscope (Zeiss) and a confocal microscope (Nikon A1R laser scanning confocal microscope, Nikon Instruments Europe BV). For the confocal microscope, the laser lines used were 405 nm, 488 nm, 561 nm, and 633 nm. For each image, the laser intensity was adjusted to avoid photobleaching, and the detector sensitivity was adjusted to optimize image brightness. 1024 × 1024 pixel images were captured using a 60 × / 1.4 Plan-Apochromat oil immersion objective and a 20 × / 0.8 Plan-Apochromat air immersion objective. The data were presented in the form of 3D z stacks consisting of 30 to 40 slices, each separated by 150 to 250 nm. Time-lapse microscopy was performed using an EVOS FL auto (Thermo Fisher Scientific).

[0097] Morphological erasure using confocal microscopy. SRG morphologies were erased using an LSM780 laser scanning confocal microscope (Zeiss). A Plan-Apochromat 20 / 1.4 water immersion objective was used during the erasure process. During the irradiation process, the samples were placed in a dry environment, a liquid environment, or a cell culture environment. The samples were illuminated with a fluorescent lamp filtered in the blue region (470 ± 40 nm) at 1.5 W cm -2The samples were illuminated with a fluorescent light at an intensity of 100 nm for 5 minutes. Bright-field images of the topography were captured before and after erasure. In the case of MDCK II cells, the samples were illuminated with fluorescent light and then detached from the samples with trypsin for surface characterization or fixed after 2 hours for immunolabeling.

[0098] LIVE / DEAD cell viability assay. MDCK II cells were seeded on the photopatterned bilayer and cultured on top of the sample for 24 hours. The topography was erased as described above. Three hours after erasure, the cells were washed with PBS and stained using the LIVE / DEAD Cell Viability / Cytotoxicity Kit* for Mammalian Cells* (Thermo Fischer Scientific) by adding 600 μl of LIVE / DEAD reagent solution, i.e., 0.50 μl / ml Calcein AM and 2 μl / ml Ethidium Monodimer-1 in PBS, to each sample. The samples were incubated for 30 minutes at 37°C in a humidified atmosphere containing 5% CO2. After incubation, the reagent solution was aspirated and 600 μl of PBS was added to prevent the cells from drying out. The samples were imaged using a confocal microscope (Nikon A1R laser scanning confocal microscope) using 488 nm and 561 nm laser lines. 1024 × 1024 pixel images were captured using a 20× / 0.8 Plan-Apochromat air immersion objective.

[0099] Image and Statistical Analysis. The distribution of focal adhesions was analyzed by fast Fourier transform of the focal adhesion channel using the FFT plugin in ImageJ. Before generating FFT images, a 900-pixel circular region was cropped and an FFT image was generated from this region. The elongation, area, and orientation of focal adhesions were measured using ImageJ. The elongation and orientation of focal adhesions were further analyzed using the MomentMacroJ v1.4B script (https: / / www.hopkinsmedicine.org / fae / mmacro.html). Before analysis, focal adhesion images were processed to remove pixel noise. The principal moments of inertia (i.e., maximum and minimum values) were measured, and cell elongation was defined as the ratio of these values ​​(maximum / minimum). Higher values ​​indicate greater elongation of focal adhesions. The orientation of focal adhesions was defined as the angle between the surface pattern direction and the axis of maximum. Statistical analyses were performed using Origin version 2019b (OriginLab, Inc.) and MATLAB. We estimated the test power for experiments quantifying fewer than 100 focal adhesions. We estimated that the difference was statistically significant, with actual statistical power exceeding 75%. Our data were found to have a non-normal distribution, so the non-parametric Kruskal-Wallis test with Bonferroni and Dunn-Sidak post hoc tests was used to assess statistical significance.

Claims

1. A patterned cell culture system (100, 200), comprising a support structure (101, 201), an azobenzene-containing intermediate layer (102, 202), and a top layer (103, 203) comprising a protective polymer, the patterned cell culture system being obtained by subjecting one or more regions of the azobenzene-containing intermediate layer to light generated by a laser, wherein the wavelength of the light is between 430 nm and 530 nm, thereby generating topographical features in the system, and wherein (i) the protective polymer is PDMS or parylene C, and the thickness of the top layer is less than 90 nm; or (ii) The protective polymer is a hydrogel and the thickness of the top layer is less than 50 μm.

2. The patterned cell culture system of claim 1, wherein the laser is a continuous wave laser.

3. The patterned cell culture system according to claim 1, wherein the intensity of the light is 1 W cm -2 Up to 5W cm -2 .

4. The patterned cell culture system according to claim 1, wherein the thickness of the azobenzene-containing layer is 50 nm to 5 μm, and wherein (i) the protective polymer is PDMS or parylene C, and the thickness of the top layer is from 20 nm to less than 90 nm, or (ii) The protective polymer is a hydrogel, and the thickness of the top layer is from 20 nm to less than 50 μm.

5. The patterned cell culture system according to claim 1, wherein the thickness of the azobenzene-containing layer is 50 nm to 5 μm, and wherein (i) the protective polymer is PDMS or parylene C, and the thickness of the top layer is 50 nm to less than 90 nm, or (ii) The protective polymer is a hydrogel, and the thickness of the top layer is 50 nm to less than 50 μm.

6. The patterned cell culture system of claim 1, wherein the support structure is selected from the group consisting of a cell culture dish, a microscope cover slip, and a well plate. The patterned cell culture system according to claim 6 , wherein the cell culture dish is a culture dish. 8 . The patterned cell culture system according to claim 1 , wherein the azobenzene is N-ethyl-N-(2-hydroxyethyl)-4-(4-nitrophenylazo)aniline.

9. A method for reversibly writing a topography on an azobenzene-containing material in 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, the method comprising: A laser beam is scanned on the top layer, wherein the wavelength of the light is 400 nm to 600 nm and the intensity of the light is 1 W cm -2 Up to 5W cm -2 or projecting a laser interference pattern onto the material, wherein the wavelength of the two interfering laser beams is 400 nm to 600 nm and the intensity of the two interfering laser beams is 100 mW cm -2 Up to 600mW cm -2 , and among them (i) the protective polymer is PDMS or parylene C, and the thickness of the top layer is less than 90 nm; or (ii) The protective polymer is a hydrogel and the thickness of the top layer is less than 50 μm.

10. The method of claim 9, wherein the wavelength of the laser beam scanned on the top layer is 430 nm to 530 nm. The method according to claim 9 , wherein the wavelength of the two interfering laser beams is 430 nm to 530 nm.

12. A method for erasing a topographical feature of a patterned cell culture system according to any one of claims 1 to 8, the method comprising subjecting the topographical feature to light of 400 nm to 600 nm generated by a laser, a fluorescent lamp or an LED.

13. The method of claim 12, wherein the method comprises subjecting the topographical features to light of 460 nm to 530 nm.

14. The method of claim 12, wherein the laser is a continuous wave laser.

15. The method according to any one of claims 12 to 14, wherein the intensity of the light generated by the laser, fluorescent lamp or LED is 1 W cm -2 Up to 5W cm -2 .

16. Use of the patterned cell culture system according to any one of claims 1 to 8 as a cell culture platform.

17. A cell culture method, wherein the cells are cultured on the patterned cell culture system according to any one of claims 1 to 8.

18. The method according to claim 17, comprising the following steps: a) providing the patterned cell culture system, b) coating the top layer of the patterned cell culture system with a cell adhesion protein, and c) seeding cells onto the cell adhesion protein.

19. The method of claim 18, wherein the cells are selected from the group consisting of epithelial cells, fibroblasts, endothelial cells, neurons, mesenchymal stem cells, astrocytes, cardiomyocytes, and cancer cells.

20. The method of claim 18, wherein the cell adhesion protein is selected from the group consisting of collagen, fibronectin, and laminin.

21. The method according to any one of claims 17 to 20, comprising performing an oxygen plasma treatment before step b).

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