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Establishment of a cell micropatterning method for the quantitative assessment of the organization of the keratin filament network

2 Materials and Methods
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2.2 Methods
2.2.1 Lithographic techniques
2.2.1.1 Fabrication of photolithography mask
The following method was done in cooperation with the Institute of Complex Systems 7: Biomechanics at the Jülich Research Centre. Different micropatterns for single cell analysis and bowtie-shaped micropatterns for cell-cell contact analysis were designed using the CleWin software.
The micropatterns were written on a blank photomask coated with a chromium layer and the negative photoresist layer SAL 601 by using the electron-beam lithograph EBPG-5H. The post-exposure bake of photomask was performed for 2 minutes at 105°C. Uncross-linked photoresist was removed using a 1 to 2 dilution of AZ 400K in DI water. Photomask was hard-baked for 15 min at 100°C, 15 min at 130°C and 15 min at 150°C successively. To remove residual layers of photoresist areas photomask was descummed for 10 to 120 s. Wet etching followed for 1 min in a chrome etching solution with a subsequent wash step in DI water. Finally, photomask was dried under a stream of nitrogen gas and exposed to oxygen plasma.

2.2.1.2 Fabrication of microstructured silicon master
The following method was performed by the Institute of Complex Systems 7: Biomechanics at the Jülich Research Centre. A silicon wafer was preheated at 180°C for 30 min and overlayed with a 25 µm thick layer of SU-8-25 photoresist (Fig. 3). Subsequently, the photoresist layer was soft-baked for 2 min at 65°C followed by slow ramp heating to 90°C within 5 min and additional baking of the photoresist at 90°C for 2 min. Using ultraviolet-photolithography at 346 nm with a source power of 7 mW for 25 s, the structures of the photolithography mask were transferred into the photoresist layer. The wafer was post-baked for 1 min at 65°C followed by slow ramp heating to 90°C within 5 min and additional baking for 1 min at 90°C. Uncross-linked photoresist was removed using SU-8 developer for 6 min. Finally, the photoresist was hard-baked for 30 min at 180°C. The SU-8-25 wafer was then silanized by exposure to vapor of FDTS in vacuum for 30 min at room temperature in order to prevent it from adhering to the PDMS during stamp fabrication.

Fig. 3 Schematic procedure for fabricating a microstructured master. Photoresist is spin-coated on a silicon wafer (a and b) and is locally exposed with UV light through a photomask (c and d). Photoresist is developed (e), rinsed and silanized (f).

Fig. 3 Schematic procedure for fabricating a microstructured master. Photoresist is spin-coated on a silicon wafer (a and b) and is locally exposed with UV light through a photomask (c and d). Photoresist is developed (e), rinsed and silanized (f).

2.2.2 Micropatterned substrate fabrication
2.2.2.1 Micro-contact printing
The first step for the fabrication of microstructured substrates using the µCP method consists of the fabrication of a microstructured master (see 2.2.1.2). The silicon master was cleaned under a stream of nitrogen. Sylgard® 184 Silicone Elastomer Base and Sylgard® 184 Silicone Elastomer Curing agent were mixed in a 10:1 ratio by thoroughly stirring. After removing air bubbles by degassing the solution in a vacuum desiccator the elastomer was poured over the master and cured at 60°C for 2 h (Fig. 4). The elastomer was peeled off the silicon master and small stamps were excised from the regions of interest. A drop of 50 µg/ml FN in PBS or 45µg/ml Collagen I in acetic acid mixed with 0.3% of an antibody of choice in PBS and acetic acid respectively was placed onto the microstructured surface of the PDMS-stamp. The ink was incubated for 1 h at RT. Subsequently, the ink drop was aspirated and the inked surface dried under a stream of nitrogen gas for 30 s. The PDMS-stamp was inverted and placed onto a gamma irradiated glass coverslip of a glass bottom dish immediately for 3 s and 30 min respectively. A 2 g weight was placed onto the PDMS-stamp in order to promote good contact with substrate and to stabilize the construct. The construct was carefully disassembled. Non-printed areas were blocked with a 1% Pluronic® F-127 in ddH2O for 1 h at 4°C in order to prevent cell adhesion. Subsequently, the substrate was carefully rinsed tree times with PBS.

Fig. 4 Schematic procedure for fabrication PDMS stamps and µCP. Liquid prepolymer mixture is casted on silicon master and cured (a). PDMS layer is subsequently peeled off the silicone master and excised into small stamps (b and c). For µCP the microstructured surface of the stamp is coated with an extracellular matrix protein solution and dried (d and e). PDMS stamp is placed in contact with a glass coverslip and removed after promoting good contact by applying gentle pressure.

Fig. 4 Schematic procedure for fabrication PDMS stamps and µCP. Liquid prepolymer mixture is casted on silicon master and cured (a). PDMS layer is subsequently peeled off the silicone master and excised into small stamps (b and c). For µCP the microstructured surface of the stamp is coated with an extracellular matrix protein solution and dried (d and e). PDMS stamp is placed in contact with a glass coverslip and removed after promoting good contact by applying gentle pressure.

2.2.2.2 Stencil patterning
In order to fabricate microstructured substrates through the usage of stencils PDMS-stamps were fabricated as described above (see 2.2.2.1). In the first step, coverslips were spin coated with a thin layer of PDMS (base to curing agent 10:1) for 7 s at 2500 rpm. Coated coverslips were mounted on the outer side of culture dishes with holes of approx. 20 mm diameter in a leak proof manner, the coated side facing the interior part of culture dishes. PDMS was cross-linked for 16 h at 60°C. Alternatively, cross-linked PDMS-coated coverslips were places in microwell plates. In the second step, PDMS-stamps were placed onto a sellotape adhered to a glass slide. Uncured epoxy resin was placed at the stamp boundaries and dispensed by capillary suction between stamp and sellotape (Fig. 5). Curing was performed through exposure to UV on a transilluminator table for 40 min. Stamps were removed and the microstructured stencils were peeled off the sellotape. Next, the stencil was placed on a PDMS-coated coverslip. A drop of 50 µg/ml FN in PBS or 45 µg/ml Collagen I in acetic acid mixed with 0.3% of an antibody was placed in top for 60 min. Trapped air bubbles were removed by degassing the construct for 2min. Afterwards, the solution was discarded and the membrane peeled off carefully from the elastomer substrate. Non-printed areas were blocked with 1% Pluronic® F-127 in ddH2O for 10 min at 37°C in order to prevent cell adhesion. Finally, the substrate was carefully rinsed tree times with ddH2O.

Fig. 5 Schematic procedure of stencil patterning. The initial step is to place a PDMS stamp on a substrate and to disperse epoxy by capillary suction between the stamp and the substrate (a and b). The stamp is subsequently removed (c) and the microstructured stencil peeled off the substrate (d). Following this, the stencil is placed on an elastomer substrate and coated with extracellular matrix proteins (e-h). After incubation and discarding the extracellular matrix protein solution, the stencil is removed. Areas exposed by through holes to the ECM solution are locally modified (h).

Fig. 5 Schematic procedure of stencil patterning. The initial step is to place a PDMS stamp on a substrate and to disperse epoxy by capillary suction between the stamp and the substrate (a and b). The stamp is subsequently removed (c) and the microstructured stencil peeled off the substrate (d). Following this, the stencil is placed on an elastomer substrate and coated with extracellular matrix proteins (e-h). After incubation and discarding the extracellular matrix protein solution, the stencil is removed. Areas exposed by through holes to the ECM solution are locally modified (h).

2.2.2.3 CYTOOchips™
A Starter’s CYTOOchip bears four different fluorescently labeled FN micropatterns (disc, crossbow, H and Y) in three sizes (1600 µm², 1100 µm², 700 µm²). The CYTOOchips were placed in independent wells of a 6 well plate. It was made sure that the CYTOOchips were placed correctly so that the CYTOO logo could be read the right way in the lower right of the chips. Since CYTOOchips were already patterned and coated by the manufacturer cells were ready to be dispensed into each well containing a CYTOOchip.

2.2.3 Cell culture
2.2.3.1 Cultivation and passaging adherent cells
Human vulva carcinoma-derived A431 subclone AK13-1 cells, producing human keratin 13-EGFP, and immortalized human keratinocyte-derived subclone HaCaT B10 cells, producing human keratin 5-EYFP cells, were grown in DMEM supplemented with 10% FCS Gold at 37°C and 5% CO2. In addition, 500 µg/ml G418 was added to the culture medium of HaCaT B10 cells. For subculturing purposes, cells were passaged after reaching 70-90% confluence, taking approximately up to 2 to 3 d. Medium was aspirated and cells were washed shortly with DPBS. Cells were trypsinized by adding a trypsin/EDTA solution and incubating them for about 15 min at 37°C and 5% CO2. Fresh medium was added and trypsinized cells were pipetted up and down several times to ensure a uniform cell suspension. To ensure a uniform, single-cell suspensions cell strainers with a pore size of about 40 µm were used. Cells were pelleted by 1000 rpm for 3 min, the supernatant was aspirated and cells were resuspended in fresh media, and transferred into a fresh flask containing fresh medium, ensuring an appropriate dilution concentration.

2.2.3.3 Cryopreservation of mammalian cells
For freezing procedures, medium was aspirated and confluent grown cells washed shortly with DPBS and trypsinized with trypsin/EDTA at 37°C and 5% CO2 until cells detached. Fresh DMEM medium supplemented with 10% FCS Gold was added and cells were pipetted up and down several times to ensure a uniform cell suspension. Next, cells were pelleted by low speed centrifuging at 1000 rpm for 3 min. Medium was discarded and cells were resuspended in pre-cooled freezing medium and aliquoted into cyrotubes. The cyrotubes were incubated in a vessel containing isopropanol at -80°C overnight and transferred into liquid nitrogen for long-term storage.
For thawing cells, cyrotubes were swayed in a 37°C pre-warmed water bath for rapid thawing. Subsequently, cells were resuspended in pre-warmed fresh DMEM medium supplemented with 10% FCS Gold and seeded into a new flask.

2.2.3.4 Cell seeding on micropatterns
After the fabrication of micropatterned substrates cells were collected by trypsinization as described above. Cells were diluted to a concentration of 15000 cells/ml or less in fresh DMEM medium supplemented with 10% FCS Gold, dispensed onto micropatterned substrates and incubated at 37°C and 5% CO2. Depending on the cell line, after about 2-7 h non-adherent cells were removed by gently aspirating the medium without letting the coverslips dry out entirely. DPBS was added and aspirated off. It was checked under the microscope for floating cells. If a large number of floating cells were observed, washing procedure was repeated. DPBS was replaced with fresh medium. Thereafter, it was checked repeatedly for full cell spreading.

2.2.4 Immunofluorescence staining
After cell seeding and spreading on micropatterns, medium was aspirated and substrates washed briefly in pre-warmed PBS. Cells were fixed for 5 min at -20°C in methanol and subsequently for 20 s at -20°C in acetone. After fixation, substrates were air-dried until acetone evaporated and then rinsed with PBS for 10 min at RT. Immunostaining was performed at RT for 1 h in primary antibody. Next, cells were rinsed tree times with PBS and then incubated for 1 h at RT with the secondary antibody in the dark. After three times washing with with PBS for 10 min cells were rinsed in ddH2O and finally mounted with Mowiol supplemented with 1 µg/ml DAPI on microscope slides.

2.2.5 Image acquisition and analysis
Immunofluorescence staining was assessed by an Axio Imager M.2 microscope equipped with an ApoTome.2 unit using an Axiocam MRm camera and with EC Plan-Neofluar 10x/0.30 Ph 1, Plan-Apochromat 20x/0.8, EC Plan-Neofluar 40x/0.75 and Plan-Apochromat 63x/1.40 Oil DIC M27 objectives. In general, best automatic settings were chosen for exposure.
Fluorescence recording was also done by confocal laser scanning microscopy using a LSM 710 Duo. When more than one fluorophore was used in a single sample, the emission spectra were adjusted to prevent significant overlap. Except for the comparison of fluorescence intensity, best automatic settings were chosen for laser intensity and gain. The diameter of the pinhole amounted to 1 AU.
The evaluation of pictures was done using the Zeiss ZEN 2009 software and the AxioVision software. Image processing was performed using the ImageJ-based image processing program FIJI. For generating heat maps CYTOO tooL for Image Processing-Reference cell, an ImageJ macro provided by CYTOO Inc., was tested.

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