Collagen Reconstitution and Cell Migration Assays

Bovine dermal collagen was obtained as acidified, pepsinized solution (stock concentration 3.1 mg/ml; PureCol, Advanced BioMatrix); non-pepsinized (telopeptide intact) rat tail collagen was purified by acid extraction, stored in acidified lyophilized form (Hotary et al., 2000 (link)), and re-solubilized before use in 0.2% acetic acid. Fibrillar collagen matrices were reconstituted in vitro by raising the pH to 7.4 using either final concentrations of 0.345 N NaOH and buffering by 25 µM Hepes (Sigma-Aldrich) for rat tail matrices, or 0.75% Na-bicarbonate solution (Gibco) for bovine dermal lattices, together with mimimum essential Eagle’s medium (Sigma-Aldrich), and polymerized at 37°C, unless indicated otherwise (Hotary et al., 2000 (link); Wolf et al., 2007 (link)). High-density matrices of bovine dermal collagen were reconstituted after concentration of the collagen solution (20 mg/ml; speed vac concentrator; Savant). Delayed polymerization speed of rat tail collagen was achieved by lowering the temperature during polymerization to 20, 14, or 9°C. Cell-free collagen lattices were used for bright-field microscopy (vertical invasion studies; Fig. S1 A), 3D confocal and electron microscopy, and atomic force microscopy. For cell migration assays, cells (20,000/100 µl) after detachment with EDTA (2 mM) or multicellular spheroids (2–7 spheroids/100 µl) were suspended in neutralized collagen solution before polymerization and incorporated into a self-constructed chamber (see Fig. S1 A). After addition of medium (containing 10% FCS) spontaneous migration was monitored, except for PMNs which were stimulated by 100 ng/ml IL-8. Inhibition experiments were performed using 20 µM GM6001, a five-component protease inhibitor cocktail, function-perturbing anti-β1 mAb 4B4, or Y-27632. Inhibitors were added to both the cell–collagen mixture and supernatant. For collagen contraction assays, cells (8 or 16 × 10⁴, for incubation with Y-27632 or 4B4, respectively) were incorporated into non-anchored bovine collagen lattices (300 µl) in 48-well plates.