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

3.3 Behavior of cells on micropatterns
Due to the superior performance in protein patterning, stencil patterning was chosen for all subsequent experiments. After protein patterning, a subsequent incubation with the antifouling agent Pluronic® F-127 was performed in order to passivate non-printed regions and to allow specific adhesion of cells to the micropatterns. AK13-1 and HaCaT B10 cells were seeded at a density of 104 cells per cm2 and were maintained at 37°C and 5% CO2. Because cell attachment and full cell spreading time vary between the different cell lines, an adequate incubation time needed to be found accordingly. Generally, cells were checked in 2-4 h steps up to 24 h for cell attachment and spreading.
Both cell types on printed silicone elastomer substrates differed in shape and spreading behavior from cells on other substrates. While most cells attached rapidly on silicone elastomer substrates coated wholly with FN, on FN coated glass coverslips and on non-coated glass coverslips and reached about 50 µm in diameter, cells on micropatterned silicone elastomer substrates needed >6 h for cell attachment.
In order to minimize cell attachment time the passivation step with the antifouling agent Pluronic® F-127 was excluded since only a negligible number of attached cells were observed on non-coated silicone elastomer substrates and because Pluronic® F-127 is suspected to attach to micropatterns and thus prevent proper cell attachment and spreading. Cell micropatterning without the usage of Pluronic® F-127 resulted in a faster cell attachment (The time needed for cells to spread on micropatterns was approx. 24 h and remained unchanged despite the usage of conditioned cell culture media. To find out whether cells favored printed glass substrates over printed silicon elastomer substrates cells were tested for spreading time on CYTOOchips™ and on printed elastomer silicon substrates. A comparison revealed no notable changes in cell spreading time.

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