Cell-imprint Surface Modification By Contact Photolithography Based

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Biological and Medical Applications of Materials and Interfaces

Cell-imprint Surface Modification By Contact Photolithography Based Approaches: Direct Cell Photolithography and Optical Soft Lithography Using PDMS Cell-imprints Hanie Kavand, Harald van Lintel, Soroush Bakhshi Sichani, Shahin Bonakdar, Hamed Kavand, Javad Koohsorkhi, and Philippe Renaud ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00523 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019

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ACS Applied Materials & Interfaces

Cell-imprint Surface Modification By Contact Photolithography Based Approaches: Direct Cell Photolithography and Optical Soft Lithography Using PDMS Cell-imprints

Hanie Kavand†, Harald van Lintel†, Soroush Bakhshi Sichani‡, Shahin Bonakdar§, Hamed Kavand‡, Javad Koohsorkhi‡*, and Philippe Renaud†*

†École

Polytechnique Fédérale de Lausanne, STI IMT LMIS4, Station 17, CH-1015 Lausanne, Switzerland

‡Advanced

Micro and Nano Devices Lab, Faculty of New Sciences and Technologies, University of Tehran, 14395-1561 Tehran, Iran

§National

*Corresponding

Cell Bank of Iran, Pasteur Institute of Iran, 13169-43551, Tehran, Iran

authors: [email protected]; [email protected]

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Keywords: surface modification, cell-imprint, direct cell photolithography, cell-mask, optical soft lithography

ABSTRACT:

New cell-imprint surface modification techniques based on direct cell photolithography and optical soft lithography using PDMS cell-imprints are presented for enhanced cell-based studies. The core concept of engineering materials for cell-based studies is the material’s ability to redesign the physicochemical characteristics of the cellular niche. There is a growing interest in direct molding from cells (cell-imprinting). These negative copies of cell surface topographies have been shown to affect cell shape and direct mesenchymal stem cells’ differentiation. Analyzing the results is however challenging as cells seeded on these substrates do not always end up in a cell-pattern, which leads to decreased effectiveness and biased quantification. To gain control over cell seeding into the patterns and avoid unwanted cell population outside of the patterns, the cell-imprinted surface needs to be modified. From this perspective, the standard optical contact lithography process was modified and cells were introduced to the cleanroom. Direct cell photolithography was used for a single step PDMS cell-imprint (chondrocytes as the molding template) surface modification down to single cell (approximately 5 m in diameter) resolution. As cells come in a variety of shapes, sizes, and optical profiles, a complementary optical soft lithography based photomask fabrication technique is also reported. The simplicity of the fabrication process makes this cell-imprint surface modification technique compatible with any adherent cell type and leads to efficient cell-based studies.

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1. INTRODUCTION Cell functions are tightly regulated by tissue-specific biochemical and biophysical signals as cells reside in spatially defined microenvironments.1 Gaining a clear understanding of how tissues regenerate, how a dysregulation leads to tissue malfunction, and how efficient drug treatments could restore pathologically altered tissues, is in need of a simulated condition that provides the physicochemical elements present in the cellular microenvironment. These include growth factors, extracellular matrix (ECM) proteins, mechanical loadings, and topological cues.2–5 Based on these elements, devise approaches have been developed to partially regulate the biological crosstalk between cells and their surroundings. The ability to transfer biological and molecular patterns to polymer surfaces has received much attention in the past two decades and has led to widespread applications.6–9 Application of poly(dimethylsiloxane) (PDMS) (and other materials)10–12 cell-imprinted substrates has been demonstrated to be an efficient method to influence cellular behavior, mediate cell capture, and guide differentiation of stem cells.9,11,13–15 PDMS is regarded as an interesting substrate for cell-based studies due to its biocompatibility, optical transparency, gas permeability, and easy yet cost effective manufacturing.16 Micro-/nano-structured topographies engender morphological changes (cell shapedependent modulation of focal adhesion, cytoskeletal tension, and nucleus shape) followed by cell function regulations.17–19 However, the cell-imprint recognition and signaling mechanism is still poorly understood with no general consensus. Several studies suggest that the cellimprint selectivity is principally controlled by chemical recognition or better said an efficient cell recognition is dependent on molecule-guided topographical interactions,12,20 whereas another study14 highlights the prominent role of physical recognition in stem cell differentiation. Importantly, it should be mentioned that most cell-imprint based studies use 3



well spread cells as the template to create surface topographies. These cells create less deep structures with larger active areas on the mold and thus the micro-/nano structures have a relatively higher chance of getting engaged in the cell-surface interactions. However, nonflat/spread cells, such as primary chondrocytes, create hollow microstructures on the mold and the probability of cells getting involved in the nano-structured topographies will not be easy or prevalent. In order to determine whether the cell-imprint recognition mechanism is chemically or physically driven, or if there is interplay between the two factors, new approaches should be considered. Previous studies14,15,21,22 have only focused on the applicability aspect of the cell-imprinted substrates; despite successful achievements (method reproduced by the authors in Figure 1), two critical problems still exist. Firstly, it is the lack of homogeneity over the imprinted surface. This problem originates from the conventional non-uniform cell seeding that later on, acts as the master for imprinting. This results in the co-existence of patterned and flat surfaces on the cell-imprints, thus not all cells will be seeded inside the cell-imprinted cavities and many cells will stay on the flat area and won't sense the same environment (Figure 1e,f). Without further gene analysis and by just comparing the morphological differences, it could be predicted that these cells will have different fates. Moreover, these cells may in fact influence the cells in the cavities through paracrine signaling and make the results difficult to interpret. The other drawback is neglecting the cells’ dynamics. Association of any cellular response to the cell-imprinted substrates, without an exclusive adherence and stabilization on the topographies all through the experiment, remains debatable and results should be treated with considerable caution. Hence, a better understanding of cell-imprinted coding and signaling can be achieved by developing a process that allows the control of cell placements inside the cell-imprinted cavities. 4


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Figure 1. Schematic and real images from cell-imprinted substrates. (a) Random cell (primary chondrocytes) seeding on a substrate. (b) Cell-imprints are fabricated by PDMS molding. (ci-iii) Shows optical, AFM, and SEM images of chondrocytes. (d) AFM image of a PDMS chondrocyte imprint. The scanned surface shows the topography of cells being transferred to the PDMS. Filopodia or the tiny cytoplasmic protrusions seen in (c-iii) can also be seen in the PDMS imprint. (e) Cells can adhere anywhere on the randomly patterned chondrocyte cellimprints, sense different microenvironments, and thus make analysis challenging. The schematic measurements were acquired from an AFM cross-sectional analysis. (f) Confocal images of stromal cells cultured on a PDMS chondrocyte-imprint. In order to increase the

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efficiency and cell guiding into the imprinted cavities, these surfaces should be modified. Arrow points to a cell, residing in a cell-imprinted cavity (Scale bar = 50 m).

Chemical modification of cell-imprinted surfaces (flat areas) with repelling molecules such as PEG23 can minimize the unwanted cell population on the imprinted surface and make data analysis authentic (Figure 2a). Furthermore, modifying the cell-imprinted cavities with for instance cell adhesive layers or signaling protein can make studies more efficient. Although cell-imprinted surfaces (the bulk surface) had been chemically modified for specific studies,12,20,24 to our knowledge no study has been conducted to surface modify cell-imprints down to single cell resolution (adherent and eukaryotic cells).

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Figure 2. Schematics illustrating the concept of the study. (a-i) Ascribing cell behavioral change to the cell-imprint cavities can be realistic if all cells experience the imprinted microenvironment. (a-ii) This can be achieved by surface modifying the flat (shown here) or the imprinted surface. (a-iii) A cell-repelling layer can inhibit the attachment of cells on unwanted (flat) areas. We introduce (b-i) direct cell photolithography and (b-ii) an optical soft lithography based technique for fabricating cell-imprint photolithographic masks. (Colorcode: fixed cells= purple, photoresist= orange) (c) The fabricated mask can be used for photolithography on the cell-imprinted substrate and further surface modification processes. 7



In order to have precise control and modification possibilities over cell-imprints, mapping the cell-seeding pattern is a prerequisite. The straightforward solution to do this is to pattern cell seeding according to a predefined design.25–28 Despite being successful, these approaches have their limitations such as being time-consuming, difficult in accessing exclusive materials, and in need of several costly microfabrication processes. To circumvent cell patterning, we propose innovative approaches (two complementary methods) to replicate the pre-seeded cell contour on photolithography masks (Figure 2b). These masks can then be used to pattern the layers that will define the flat areas and the cell cavities on the PDMS cellimprints through another photolithography process. For example a cell repellant surface (on the flat areas) can be achieved by an etched out gold layer in the cell-imprinted cavities and deposition of a thiol-PEG on the remaining gold (flat surfaces) or the cell cavities can be masked by photoresist prior to gold layer (or any other molecule) deposition and modification. These are just a few insights from the various modification possibilities provided by the proposed techniques that highlight their potential for cell-imprint surface modification applications. Chondrocyte is an exemplary cell type for the validation of the proposed methods as it covers almost all available cellular phenotypes (Figure 3d-1-3) as it tends to eventually change from a physiologically rounded shape (rabbit chondrocyte = approximately 5 m in diameter) to a fibroblast-like phenotype (rabbit chondrocyte = approximately 60 m in diameter) on 2D substrates.

2. EXPERIMENTAL SECTION Cell-mask preparation: Alignment marks were laser written (VPG 200 Heidelberg Instruments) on a Cr blank photomasks (Optical density = 2.8, Reflectivity = 12%, Nanofilm, Inc.). The patterned glasses were functionalized with an aminosilane monolayer to help cell 8


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adhesion. Briefly, glasses were treated with oxygen plasma for 30 s and kept under vacuum in a desiccator with a few drops of (3-Aminopropyl)triethoxysilane (Merck) for 30 min. Substrates were then autoclaved and used as a cell-seeding platform. Cell culture: Articular cartilage was attained from rabbits obtained from a slaughterhouse and chondrocyte isolation was carried out as described by Brittberg et.al.29 Briefly, the tissue was minced after excision, washed 3 times in washing media consisting of DMEM/Ham’s F12 (Gibco) + Penicililin-Streptomycin (3%, Sigma), digested in trypsin-EDTA solution (0.25%, Sigma) for 1 h, and finally suspended in collagenase type I (0.5 mg/ml, Gibco) solution overnight at 37C. Cells were washed and plated on the prepared glass substrates with DMEM/Ham’s F12 containing 10% FBS (Gibco). As chondrocytes lose their native morphology over time, freshly isolated cells were fixed 24 h after seeding and subsequent attachment using glutaraldehyde solution (4%, Sigma). Dedifferentiated chondrocytes (passage 3), MDA-MB-231, and MCF-7 breast cancer cell line (ATCC®) were also tested. Cell-imprint fabrication: Fixed cells were directly utilized as the master template for PDMS cast molding. PDMS base and curing agent (Sylgard 184, Dow Corning) were mixed in the ratio of 10:1, degased, and poured onto the master. The photolithographic aspect of this process requires a perfectly flat surface. A circular plate (102 mm in diameter) was cut out of PMMA (3 mm thick), creating a semi-confined boundary for molding. A secondary PMMA plate was placed on top of the circular plate upon which the construct was fixed to the cellmask using paper clips, and held vertical. The PDMS mixture was poured into the cavity and baked at 60C for 6 h. The resulting mold was then bonded to a float glass (102 mm in diameter) to create a stable platform for further process. A PDMS mask was also fabricated from a silicon (Si) wafer whose surface was patterned with micropillars. Patterns (diameter = 20 μm) were transferred to the photoresist (Heidelberg 9



MLA150) and the Si wafer was dry etched (Alcatel AMS 200 SE) to fabricate 5 μm tall pillars. The resist was stripped and to ease PDMS molding, wafers were activated by O2 plasma for 30 s and silanized using trimethylchlorosilane (TMCS) (Sigma). Direct cell photolithography: Cells on glass wafers are directly used to expose a photoresist in a hard contact mode. As this article is focused on the fundamentals, direct cell photolithography was used to modify the cell-imprinted surfaces with a gold layer to make analysis straightforward. To clarify that the surface modification is solely dependent on the photolithography process and the Au accumulation/concentration area in the cellular pits is not due to the physical shape of the surface, the Cr layer (adhesive layer between the PDMS and the gold) was also analysed. The mold and mask (M&M) technique: Cellular templates were created on aminosilanized glass wafers that were patterned to fabricate alignment marks. This is especially important for the alignment step between the cell-mask and the cell-imprint. The cell-imprints are produced by PDMS molding of chondrocytes. After Cr/Au deposition on the PDMS cell-imprints, a positive photoresist is used for the photolithography process with the cell-mask. The substrate is then put in the gold etch solution for a few seconds. The remained photoresist protects the cellular patterns and maintains the modified surfaces of these patterns unaffected. Imaging: Samples were inspected using optical microscope (Nikon PPTIPHOT 200). Scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDC) analyses were carried out using (Zeiss MERLIN). To minimize charging of the photoresist, accelerating voltages of around 1 keV were used. Atomic force microscopic (AFM) topographical images were acquired in a standard contact mode (Bruker). For confocal cell imaging, cells were fixed with paraformaldehyde (Sigma-Aldrich) for 20 min at ambient temperature and then permiabilized using Triton X100 (0.1%, Sigma) for 30 min. The actin 10


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cytoskeleton was dyed using Alexa Fluor 488 Phalloidin (Life technologies) for 45 min at 4C. The nucleus was dyed using DAPI (Life technologies). Cells were imaged using Zeiss LSM700 invert confocal microscope. Surface modification: Thin layer deposition was carried out by evaporation (AllianceConcept EVA 760). Photoresist layers were coated (sputtering, Alliance-Concept DP 650) with gold (20 nm) before SEM imaging. A carbon layer (20 nm) was sputtered on the surface of modified PDMS molds before SEM/EDC imaging of the surface modified PDMS samples. Photolithography: Photoresists AZ 1512 HS (thickness = 1.5 μm) and AZ 9221 (thickness = 2 μm) (Clariant GmbH) were used. Photoresist layers were exposed to 350-390 nm (i-line) wavelength filters by an exposure tool (Süss MA6Gen3). The light intensity on the MA6Gen3 mask-aligner was set to 20 mW/cm2 in CP and constant dose regulation modes with i-line filter installed. The exposure dose for direct cell photolithography and optical soft lithography using AZ 1512 HS and AZ 9221 photoresists coated on Cr deposited (thickness = 100 nm) float glass was 34 mJ/cm2 and 140 mJ/cm2, respectively. For exposure using binary masks, the exposure dose was set to 49 mJ/cm2 for AZ 1512 HS and 175 mJ/cm2 for AZ 9221 photoresist. 3. RESULTS AND DISCUSSION 3.1. Direct cell photolithography In direct cell photolithography, we propose fixed cells (Figure 1c) (same cells that were used for making the cell-imprint substrate (Figure 1d)) as a contour and a substitute for the Cr-glass mask in the photolithography exposure on a photoresist using collimated UV light of a mask aligner. To the best of our knowledge no report exists on cells being the subject of optical lithography. In direct cell photolithography, fixed cells on glass wafers (cell-mask) are used as a photomask for exposure on a positive photoresist (in a hard contact mode). The 11



patterns formed after photoresist development imitates the spatial distribution and replicates the areas occupied by cells on the glass wafer. Figures 3 represents the results obtained from this concept.

Figure 3. Direct-cell photolithography. In this approach fixed cells (purple) on glass slides are used for exposure on the photoresist (orange colored layer). This process creates gray scale images from cells (a). (b-i) MCF-7 cell line and its (b-ii) corresponding transferred pattern on the photoresist. (b-iii) Besides the 2D pattern transfer, direct-cell photolithography shows details from cells. (c-i) MDA-MB-231 cell line and (c-ii) its pattern on the photoresist. For further evaluations, chondrocytes were tested. (d) Isolated chondrocytes were observed using phase-contrast microscope after 6-day culture in a tissue culture polystyrene plate. Freshly 12


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isolated chondrocytes have a round morphology (1), but this morphology is gradually flattened (2), and finally (3) attains the dedifferentiated fibroblast-like phenotype (Scale bar = 50 μm). (e) Confocal image of dedifferentiated chondrocytes showing the nuclei (blue) and the actin cytoskeleton (green) (Scale bar = 50 μm). (f-i) Optical image of dedifferentiated chondrocytes and (f-ii-iii) their pattern transferred on the photoresist (arrows point to the nucleus in a real cell and its pattern on a photoresist). Cells partially absorb UV light and thus the photoresist is partially exposed. The spatial variation in the developed patterns created by direct cell photolithography is mainly caused by the different optical properties of the cells and their components.30,31 The parameters involved in direct cell photolithography mainly include refractive indices and absorption. Despite the loss of topographical information due to the lens and the projection effect, information regarding cell compartments, shapes, and localizations (contour) can be obtained. As seen in figure 3, changes in physiological parameters, like the shape or protein content, has a direct effect on the amount of absorption and diffraction of light. To achieve a good contrast and delineate cell shape, the exposure time should be adjusted. In case of inadequate exposure, the patterns are modified (for details see supporting information, Figure S1and S2). We used the M&M technique, as we name it, to directly carry out photolithography using a cell-mask on its relating cell-imprint (Figure 4). The results show that by choosing the appropriate exposure time and photoresist development, the cell contour can be replicated on a Cr-blank (to create Cr-glass mask) or directly on a PDMS cell imprint with fidelity.

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Figure 4. The M&M technique: 3D illustration and optical images. (a) PDMS cell-imprints. (b) Cr/Au layer is deposited on the surface. (c) The cell-master (previously used for cellimprint fabrication) now acts as the mask (cell-mask) for the photolithography process on the photoresist-coated cell-imprint. (d) After development and Au etching, the Au modified cellimprinted structures are obtained. (e) SEM/EDS result shows surface elemental analysis for Au, the underlying Cr, and the Si, which is the base element in PDMS. (f) To show the ability of cell-mask in patterning the photoresist on the PDMS cell-imprints, the chondrocyte cellmask was intentionally misaligned during photolithography (Scale bar = 100 m).

Despite being a potent method, the direct cell photolithography approach turned out to be challenging (optimizing the working distance between the cells and the photoresist, alignments between the cell-mask and the cell-imprint due to the micro-structured cell-mask, 14


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and also PDMS shrinkage after curing32) thus a supplementary photomask fabrication technique was further assessed.

3.2. Optical soft lithography using PDMS cell-imprints Optical soft lithography methods use soft elastomeric molds as a photomask. PDMS elastomers can be used as photomask as they are transparent to UV light from the near UV to NIR region of spectra.33 The first 2D optical soft lithography patterns were created using a binary elastomeric phase mask.34 Figure S3 represents the patterns created on the photoresist using a PDMS patterned by micropits. In this case, a sharp minimum transmitted intensity appears at the step edges of the relief structures and produces lines on photoresist. The sidewalls of the resist is patterned as the consequence of interference of standing waves, which is a sinusoidal (the sum of the downward-propagating image and the upwardpropagating reflected image) variation of dose through the thickness of the resist.35 The process of binary (Cr) mask (cell contour) fabrication with a soft lithography approach starts with a soft mold, i.e. PDMS cell-imprint patterns, which is placed against a photoresist coated Cr blank in a soft contact pressure mode (Figure 5). The created 3D photoresist patterns are mostly defined by the near field effects of refraction of the PDMS 3D cell shapes. According to the Snell’s law, when a ray is incident normally, the angle of incidence would be equal to 0 and the ray passes without refraction. However, very few points exist on the PDMS cell-imprint that lie perpendicular to the incident ray. Thus, refraction occurs during exposure. Regardless of how similar these complex patterns with sub-micron features are to the cells’ topography, these structures do not share the very same topographies to the cells, as this is not a true gray scale photolithography. In order to acquire true gray scale cell patterns, ink lithography could be used.34 15



Figure 5. Optical soft lithography with cell-imprints. (a) AFM image showing the topography of the PDMS cell-imprint. Photolithography is done using PDMS cell-imprints in a soft contact mode and gray scale patterns are created on the photoresist. (b) Optical images showing the developed photoresist patterns. Different photoresist colors indicate different heights (Scale bar = 25 m). (c-i,ii) shows the SEM images of the developed patterns. It is noted that cells come in different sizes (depending on the cell cycle phase), acquire different shapes after attachment, and also some cells are damaged during the fixation process, thus variant cell-imprint structures are created in the PDMS and also on the photoresist. In any case, the cell contour is transferred to the photoresist.

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To show conclusively that these fabricated masks could indeed be considered as typical photomasks, misaligned photolithography was carried out (Figure 6). SEM/EDC analysis shows effective single cell cavity modification (Figure 6d).

Figure 6. (a) Cell-imprint modification using the Cr mask with cell contour patterns fabricated from optical soft contact lithography using cell-imprints. (b) Optical image of a fabricated mask prior to photoresist stripping. The 3D structured photoresist prevents the underlying Cr layer to be etched in the Cr etchant solution. Regardless of the 3D photoresist structures created on the photoresist, the cell contour is transferred and the 2D Cr mask is obtained with fidelity. (c) Misaligned photolithography and gold layer patterning on the cellimprints. (d) SEM/EDC results showing the single cell-imprinted cavity modified with gold.

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By tuning the photolithography parameters, cell imprinted areas can be replicated on a Crglass mask for further cell-imprint modifications. This method is superior to the direct cell photography method as there are no lens effects (we work now in the near field), and the problem of inhomogeneity of the cell-mask surface is solved. Furthermore, as the Cr-mask is fabricated from a PDMS mold, which has already undergone shrinkage, the mask and the cellimprint patterns will have dimensionally identical patterns. Although the sub-micron gray scale features observed on the photoresist are not actual 3D replicates of cells due to the near field effects in creating interference patterns, the 2D patterns they create on a Cr-glass mask (after subsequent Cr-etch process) are acceptable as a replicate of the cell contour.

4. CONCLUSION We have presented a new concept for guiding cells into the cavities of cell-imprinted substrates by introducing direct cell photolithography and soft contact photolithography using cell-imprinted substrates. These techniques, like other optical lithographic techniques, use light as a pattern transfer element. The other element that the process requires is either the cell or an elastomeric mold patterned directly from the cell. Direct cell photolithography can be an interesting method to optically transfer, visualize and compare cells in regards to their intercellular components. Using this technique for cell-imprint surface modification is straightforward, but the surface inhomogeneity (mainly caused by cell aggregation during cell culture) as well as the cell’s morphology can make the photolithography process challenging. The optical soft lithography using PDMS cell-imprints is superior to the direct cell photography method as this process is not affected by cell inhomogeneity (in regards to height). Moreover, as the Cr-mask is fabricated from a PDMS mold, the mask and the cell-

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imprint patterns will have dimensionally identical patterns, as there would be no PDMS shrinkage involved in the process.

This study is the first to report the application of cells and their imprinted patterns for photolithography, as well as applying these techniques for surface modification. As we continue to divulge the knowledge about cell-based photolithography, which could be implemented in a variety of other applications, these methods can be used to validate the effectiveness of surface modification on improved cellular response in cell-imprinted substrates.

ASSOCIATED CONTENT Supporting Information: Supporting Information is available free of charge via Internet at http://pubs.acs.org. Optical images of direct cell photolithography using cells fixed on a non-photolithography grade glass, Direct-cell photolithography of primary chondrocytes, Optical soft lithography using micropatterned PDMS molds, SEM images showing patterned photoresist using PDMS cell-imprint optical soft lithography Author information Corresponding authors *Email: [email protected]; [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. 19



ACKNOWLEDGMENT The authors thank the EPFL Center of MicroNanoTechnology staff for their valuable assistance and support. We also thank Golfam Sadeghian (Advanced Micro and Nano Devices Lab, University of Tehran) for challenging discussions and Benoît Desbiolles (EPFL LMIS4) for help with SEM imaging.

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Cell-imprint Surface Modification By Contact Photolithography Based

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