4.3. Fabrication of 3D Complex Tumour Models; “Tumouroids”

All ACMs were fabricated using monomeric Type I rat-tail collagen (First Link, Birmingham, UK) and the RAFT^TM protocol pages 8–9 (Lonza, Slough, UK) as previously described [28]. To summarise: 10X MEM (Sigma-Aldrich, Dorset, UK Sigma-Aldrich, Dorset, UK) was mixed with collagen and neutralising agent (N.A.) (17% 10 M NaOH (Sigma-Aldrich, Dorset, UK) in 1 M HEPES buffer Gibco^TM through Thermo Fisher Scientific, Loughborough, UK)) and mixed with cell suspension resulting in 80% collagen, 10% 10× MEM, 6% N.A., and 4% cells. To seed the ACMs, 1 × 10⁴ cells for initial drug screening or 5 × 10⁴ cells for later tumouroids were added to 240 µL of the collagen mix per ACM and set in a 96-well plate (Corning^® Costar^® through Sigma-Aldrich, Dorset, UK). The gel mix was polymerised at 37 °C for 15 min, followed by plastic-compression using the 96-well RAFT^TM absorbers (Lonza, Slough, UK). In order to produce simple or complex “tumouroids” [18], the ACMs were nested into a stroma. For the simple stroma, collagen solution as described above was prepared, and ACMs were directly embedded into a 24-well plate (Corning^® Costar^® through Sigma-Aldrich, Dorset, UK) containing 1.3 mL of the non-cross-linked collagen mix. The tumouroids were again polymerised at 37 °C for 15 min and plastic-compressed using the 24-well RAFT^TM absorbers (Lonza, Slough, UK). Tumouroids were continuously cultured at 5% CO₂ atmospheric pressure and 37 °C with 50% media changes every 48 h.