MMP2 and type-1 collagen matrix influence in vitro lymphangiogenesis in the lymphatic ring assay. (A) Mouse lymphatic duct explants embedded in type I collagen gel were cultured for 9 days in the absence (control) or presence of recombinant MMP2 (rmMMP2 50 ng/mL). For quantification, grid corresponding to successive increments at fixed intervals of TD boundary was used on binarized images (right panel). The number of microvessel–grid intersections (N_i) was quantified on binarized images as described in “Lymphatic ring assay.” (B) Lymphatic rings issued from Mmp2-deficient mice were cultured without (Mmp2 KO) or with rmMMP2 (Mmp2 KO + rmMMP2). Rings issued from WT mice were used as control (WT). (C) Lymphatic duct explants were embedded in pepsinized or native collagen gel (final concentration, 1.5 mg/mL). (D) Native collagen gels contained 1.5, 2, or 3 mg/mL, revealing optimal migration at 2 mg/mL and less migration at higher and lower concentrations. Graphs represent computerized quantifications of LECs distribution around the ring at day 9. The histogram corresponds to LECs density at a distance (d) = 0.25 mm from the ring border. N_i = Number of intersections between microvessels and quantification grid. Values were normalized by taking the maximum of the control curve as 1. For all assays, the images presented are a representative 1 of all rings used for quantification (n ≥ 5; *P < .05; **P < .001). Bars represent 500 μm. Unstained live tissues were used in their culture medium, and images were captured with a phase-contrast microscope (Axiovert 25; Carl Zeiss Microscopy) coupled to an Axiocam color digital camera (Carl Zeiss), at room temperature using acquisition software KS400 3.0 (Carl Zeiss Vision) and a 5× NA 0.12 A-Plan lens (Carl Zeiss).