FIG. 1.

New in vitro flow and diffusion assays. (A) Modified flow apparatus. Fibrin samples were made with 8–20 mg/mL fibrinogen concentration solutions mixed with a constant thrombin concentration solution at 2 IU/mL. (i) The apparatus was made using a 60-mL syringe as the reservoir for the permeation liquid, being serum-free DMEM. (ii) An insert containing the fibrin construct of varying fibrinogen concentration was attached via silicon glue to make a leak-proof seal. This insert was made from using modified Spin-X centrifuge tube filter inserts, where the membrane was removed from the inner base of the original insert. (iii) Fibrin constructs were prepared on mesh at the bottom of the inserts allowing eluate to come through with minimal obstruction. The eluate consisted of serum-free DMEM±FITC-dextran or Rhodamine-Dextran microparticles. The eluates were collected at various hydrostatic pressures. (B) Modified diffusion apparatus. (i) Uptake-diffusion system: Diffusion assessment is performed over various fibrin scaffolds. At 24 h, 100-μL samples were taken from the top of the fibrin scaffold (nondiffused dextran) and from the bottom of each well (diffused dextran). Samples were transferred to a black plate reader for minimal light exposure. (ii) Release-diffusion system: Fibrin gels were formed in inserts fitted with 8.0-μm membrane. Serum-free DMEM was used as the media. Media level in the well rests at the same level to the bottom of the fibrin sample. The volume of the fibrin, media above the fibrin, and media in the well are 1, 0.5, and 1.5 mL, respectively. DMEM, Dulbecco's modified Eagle's medium; FITC, fluorescein isothiocyanate.