Rhodamine fibronectin

A flat PDMS stamp was cleaned and then incubated with 50 μg/ml rhodamine fibronectin (Cytoskeleton, Denver, CO, USA) in deionized water for 1 hour. The stamp was dried under sterile airflow and deposited gently on the micropost array, previously subjected to UV ozone treatment. A gentle pressure was applied to the stamp. The contact between the microposts and the stamp was ensured for at least 1 minute. Subsequently the substrates were sterlized in 70% ethanol and immersed in 0.4% Pluronics F127 (Sigma–Aldrich, St. Louis, MO) in PBS for 1 hour to prevent non-specific protein absorption to the non-functionalized surface of the PDMS microposts. Finally the substrates were rinsed with sterile MilliQ water and kept in PBS before use46 (link).

PDMS stamps were fabricated using standard photolithography techniques.23 (link) The stamps contain features of either 10 µm wide lines with 10 µm spacing in between or a fishnet pattern with 5 µm wide lines with 10 µm spacing at an angle of ±15°, 30° or 45° with respect to the 0° axis (Fig. 1). PDMS stamps were incubated with 50 mg/mL rhodamine fibronectin (Cytoskeleton, Denver, CO) in PBS for one hour, after which they were dried using compressed air. The thin film constructs were treated with UV-ozone (PDS UV-ozone cleaner; Novascan, Ames, IA) for 8 minutes just before transfer of the fibronectin onto the constructs. The stamps were positioned in such a way that the 0° axis of the stamp coincided with the length direction of the to be cut films. After 10 minutes of conformal contact, the constructs were rinsed three times with PBS and stored in PBS at 4 °C until use.1 (link),23 (link)
Figure 1

Schematic overview of the micro-contact printing layout of the four different fibronectin patterns. The fibronectin lines are depicted in grey, the spacing in black and the angle α is depicted in the top left corner. The short arrows represent 5 µm and the long arrows represent 10 µm.

In preparation for the experiments, the scaffolds were first affixed to the glass bottom of 6‐well dishes (MatTek Corp., Ashland, MA) using sterile, high‐vacuum grease (Dow Corning, Midland, MI). The scaffolds were then soaked in 70% ethanol for disinfection, followed by two phosphate‐buffered saline (PBS) washes (Thermo Fisher Scientific). Subsequently, the fibers were coated with either 4 µg ml⁻¹ fibronectin (Invitrogen, Carlsbad, CA) or 4 µg ml⁻¹ rhodamine fibronectin (Cytoskeleton Inc., Denver, CO) for 2 h prior to cell seeding to aid cell attachment to the fibers. For the quantification of how fibronectin concentration mediates cell migration, two additional fibronectin concentrations of 1 and 16 µg ml⁻¹ were used. Once the cell culture reached ≈80% confluency, 0.25% Trypsin (ATCC, Manassas, VA) was added, and the culture was incubated for ≈5 min. After the cells detached from the flask surface, 3 ml of fresh cell media was added to dilute the effect of the Trypsin. The entire solution was then placed in a centrifuge at 1000 RPM for 5 min. Following the centrifugation, the media was aspirated, and the cells were resuspended in fresh media. Finally, cells were seeded at a density of ≈3 000 000 cells ml⁻¹ on the scaffolds.