Patterned and switchable surfaces for biomaterial applications

Andrew Hook – Patterned and switchable surfaces for biomaterial applications4.2.3. Characterisation of polymer crosslinkingArrays were imaged before and after washing using a GenePix 4000A microarrayscanner (Axon Instruments) using a 17 mW 532 nm laser for excitation and a 570 nmsuppression filter. The thickness and profile of the arrayed spots was determined byprofilometry using a Dektak 6M Stylus Profiler (Veeco). A diamond stylus of radius12.5 µm was moved over the surface at a resolution of 0.25 µm/sample and a stylusforce of 5 mg. Each spot was measure 3 times. The rims of spots were ignored forheight measurements.4.2.4. Cell cultureSK-N-SH neuroblastoma and human embryonic kidney (HEK-293) cell lineswere used for cell attachment experiments. Cells were cultured in Dulbecco’smodified eagle media (DMEM) containing 10% fetal bovine serum, 5 mM Glutamaxand penicillin and streptomycin and incubated at 37 °C, 5% CO 2 and 60-70%humidity. For cell attachment studies, SK-N-SH cells were seeded onto surfaces at aseeding density of 5x10 4 cells/cm 2 and allowed to attach to the surface after whichthey were incubated at 37 °C, 5% CO 2 and 60-70% humidity for 24 hr. Cells werethen stained by incubation in Hoechst 33342 dye (10 mg/ml) for a further 5 minbefore analysis by fluorescence microscopy. Cells were visualised with an IX81Olympus fluorescence microscope and analysed using analySIS LS Research v2.5software using a 360-370 nm excitation filter and a 420 nm suppression filter todetect Hoechst 33342 fluorescence. Formation of a TCM was achieved by seedingHEK-293 cells onto the pEGFP-N1 and pDsRed2 checkerboard pattern. Cells wereseeded onto surfaces at a seeding density of 5x10 4 cells/cm 2 and allowed to attach tothe surface after which they were incubated at 37 °C, 5% CO 2 and 60-70% humidity4-137