Bovine collagen type 1

The collagen films that are ultimately used as substrates for further processing were prepared prior to their fabrication and integration with the NE electronics. The process began with Type I bovine collagen in a 3 mg mL⁻¹ solution (Advanced BioMatrix, Carlsbad, CA, USA). The collagen was gently mixed with 10X phosphate-buffered saline (PBS) and 0.1 M NaOH at a ratio of 13:2:1 by volume until small fragments of collagen started to form. The mixed collagen solution was then cast into an acrylic mold, followed by polymerization at 37 °C and 96% humidity for four days, yielding uniform collagen gels (thickness of 40 mm). These collagen gels were then dried on glass slides in air at 37 °C for 24 h followed by multiple rinses with DI water and air drying, forming dried collagen films (thickness of 40 μm). The collagen films were subsequently removed from the acrylic mold and glass slides for subsequent use as substrates. The entire process was carried out under sterile conditions. Basement Membrane Matrix Matrigel (BD Bioscience, Frankline Lakes, NJ, USA) was diluted with PBS solution to a concentration of 2 mg mL⁻¹ and was used for subsequent fabrication of ECM-NEs.

Type I bovine collagen (6 mg/mL; Nutragen, Advanced Biomatrix) was mixed on ice with 10X DMEM (13.8 mg/mL) with or without phenol red, 10X reconstitution buffer (2.25mg/mL [22 mg/mL sodium bicarbonate and 20 mM HEPES in 0.062 N NaOH]), FBS (8.5% v/v) and L-Glutamine (2 mM). After mixing the solution carefully to avoid bubbles, the pH was adjusted to 7.4 with sterile sodium hydroxide. Cells at different concentrations and ratios were added to the solution and gently mixed. Cell concentration was 5 × 10⁵ cells/scaffold for each cell line (1:1 ratio); 5 × 10⁵ and 2.5 × 10⁵ cells/scaffold (2:1 ratio) and 5 × 10⁵ cells/scaffold for one cell line. Four different trials were tested for this group of scaffolds:
Once the solution was homogenous, it was distributed into tissue culture inserts (0.4 μm pore size, 24 mm diameter, VWR) in 6-well plates, using a volume of 2.5 mL/insert. Plates were transferred to a cell culture incubator (37°C, 5% CO₂) for 2h to allow polymerization of collagen. Once the constructs were solidified, 3 mL of warmed cell culture medium was added per well (1.5 mL on top of the gel/inside the insert and 1.5 mL outside/under it (i.e., bottom of the well). Plates were maintained in the cell culture incubator and the medium was changed every two days ^{35 (link)–37 (link)}.

To prepare collagen matrix, Type-I bovine collagen (Advanced Biomatrix, 5 mg ml⁻¹) was diluted to 1.5 mg ml⁻¹ in PBS, 1× RPMI and 10x MEM solution to obtain the final concentration. The acidic solution was neutralized with 1 M NaHC0₃ by titration based on the color of the indicator in RPMI. The pre-polymerized collagen was kept in ice until further use. Cold collagen gel solution was pipetted over the test devices and allowed to incubate at 37 °C for 45 min to gel. In some experiments, the collagen was doped with 100 ng ml⁻¹ of transforming growth factor (TGF)-β or 100 ng ml⁻¹ of Interleukin-8 (IL-8) to stimulate tumor invasion as required.