Fibrin in vitro angiogenesis assay kit

The first layer of fibrin gel was prepared by mixing 30 μL of 7 wt% fibrinogen solution with 20 μL of thrombin solution provided by Fibrin In Vitro Angiogenesis Assay kit (Sigma Aldrich) into each of 96-well plate wells and incubating at 37 °C for 20 min. Relevant cells were passaged using trypsin/EDTA solution and resuspended into fresh MV2 media containing 100 ng/mL of VEGF-C at concentration of 50,000 cells/mL. 100 μL of the cell suspension was added on top of the first layer of the gel and allowed to settle for 24 h at 37 °C. Then the media was aspirated and another layer of 30 μL of fibrinogen and 20 μL of thrombin solution was added on top of the seeded cells and incubated at 37 °C for 5 min. Then 100 μL of MV2 media containing 100 ng/mL of VEGF-C was added to top of the gel. Cells were allowed to form networks for 48 h, then imaged. Fibrin gels were imaged on the confocal microscope (A1R Nikon) using Texas Red and FITC channels. We captured 31 z-stack images across 200 μm along the z-axis centered around the stack with the brightest fluorescence signals as determined visually.

To mimic blood and lymphatic sprouting into 3D matrices, we performed angiogenesis assay using IdenTX chip (AIM Biotech, Singapore) following manufacture protocol and previous studies.^{24 (link),29 (link)} Briefly, the middle channel was filled with fibrin gel by mixing 6 μL of 7 wt% fibrinogen solution with 4 μL of thrombin solution provided by Fibrin In vitro Angiogenesis Assay kit (Sigma Aldrich). To induce angiogenesis, 2 μM of S1P (Sphingosine-1 phosphate) and 100 ng/mL of VEGF-C were encapsulated into the fibrin gel.^{22 (link)} After gelation, an equal density of BEC and/or LEC (5 × 10⁶ cells/mL) were seeded on each chamber of the IdenTx Chips separated by the fibrin gel in the middle. After 24 h, BEC and/or LEC were found invading the fibrin gel. The two channels system enabled easy access to the fibrin gel region for angiogenesis study with BEC and/or LEC.