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

3.2 Comparing µCP and stencil patterning
In the scope of this work, unmodified glass slides were used for µCP. Stamp was incubated with a FN/fluorescently labelled secondary antibody ink solution for 1 h. After aspirating the solution and drying the ink, the microstructured stamp surface was placed in contact with the non-treated glass substrate for 1 min, 30 min and 1 h respectively. The resulting patterns are illustrated in Figure 8. Local inhomogeneity of protein density and malformed patterns was observed frequently. In some cases, a faint imprint indicated a small displacement of the stamp during the process. Other regions were covered completely with the ink solution. Overall, µCP with unmodified glass surfaces show an insufficient protein patterning.

Fig. 8 Images of micropatterns produced using µCP.

Fig. 8 Images of micropatterns produced using µCP. A) The overview shows malformed or missing micropatterns visualized via fluorescent antibodies. B) An enlarged view of a printed area drenched in ECM-solution due to too much ink on the PDMS stamp. Bar, 50 µm.

In order to achieve better results, a second printing method was tested. Stencil patterning is based on the usage of thin sheets comprising through holes of the desired pattern and size. The stencil is deposited on a silicone elastomer substrate and a coating solution is placed on top of the microstructured stencil. Areas outside the through holes are protected from coming into contact with the coating solution whereas regions with through holes are exposed to the solution and are locally modify.
Compared with the results of µCP this approach shows better performance in homogeneity of protein density and in fabricating accurate features (Fig. 9).

Fig. 9 Images of micropatterns produced using stencil patterning

Fig. 9 Images of micropatterns produced using stencil patterning. A) Overview of micropatterns with stencil patterning shows accurate features and a better homogeneous coating of extracellular matrix protein. Bar, 50 µm. B-H) Detailed view of different single micropatterns. Bar, 10 µm.

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