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

3 Results
3.1 Experimental results from lithographical techniques
In order to regulate cell shape and subsequently the cytoskeleton, adhesive islands consisting of ECM-proteins and of different geometric shapes were generated. For this approach, geometric shapes of different sizes were designed and written on a photomask during a photolithography process and subsequently transferred onto a silicon master (Fig. 6 A). The silicon master was used for casting PDMS stamps, which in turn were used for substrate patterning either by µCP (Fig. 6 B) or by stencil patterning (Fig. 6 B) through the fabrication of stencils.

fig 2 Pictures from the silicon master, µCP and stencil patterning

Fig. 6 Pictures from the silicon master, µCP and stencil patterning. A) Patterns produced by photolithography are transferred to the silicon master. Each square represents areas with different micropatterns in different sizes. E.g., all bowtie-shaped micropatterns with an average cell spreading area of 900 µm² can be found in the square marked by *. B) A PDMS stamp grasped with tweezers is placed on a glass coverslip in µCP. C) A stencil grasped with tweezers is placed onto an elastomer substrate.

The microstructures are shown in Figure 7. By way of illustration, the micropatterns are shown as they are observed on stencils. Two types of micropatterns were designed: bowtie (Fig. 7 A) and single (Fig. 7 B) patterns. Each pattern was designed in four different sizes allowing an average cell spreading area of 1600 µm², 1100 µm², 900 µm² and 700 µm². The distance between the patterns amounted to approx. 150 µm in order to prevent cells from bridging the gap between the patterns.

Fig. 7 Pictures of microstructured stencils.

Fig. 7 Pictures of microstructured stencils. A) A stencil with five different bowtie-shaped micropatterns in three different sizes. B) A stencil with seven different single-shaped micropatterns in three different sizes. Patterns marked by * are examples of patterns with incomplete through holes.

Most patterns were well molded and the holes spanned the entire stencil. However, some patterns (Fig. 7 *), especially those composed of thin lines, showed an insufficient or no through holes, which cause malformed or no micropatterns during stencil patterning.

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