Collagen Gel Preparation and Cell Seeding

Atelopeptide fibrillar bovine dermal collagen (no. 5005-B; PureCol, Advanced BioMatrix) was prepared at 1.7 mg ml⁻¹ in DMEM: 100 μl per well in 96-well plates; 300 μl per well in 24-well plates, 700 μl per well in 12-well plates. Telopeptide-intact rat tail collagen (no. 5153-A; PureCol, Advanced BioMatrix) was prepared at 0.5 mg ml ⁻¹ following the manufacturer’s instructions (700 μl per well in 24-well plates). Bovine collagen gels at 1.7 mg ml⁻¹ and rat tail collagen gels at 0.5 mg ml⁻¹ have comparable stiffness, pore diameter and pore cross section as previously described^{22 (link)}. After collagen gel polymerization (4 h for bovine collagen, 2 h for rat tail collagen), cells were seeded on top of collagen in medium containing 10% FCS, allowed to adhere for 24 h and medium changed to 1% serum for 24 h. Except where indicated, imaging and immunoblotting were performed on cells cultured on a thick layer of collagen I.