Hyaluronan gel migration assay was performed in HA gels molded in Falcon 24-well tissue culture treated plates. Before the gels are molded, however, the plate must be prepared. The polystyrene bottom of the plate was first blocked by coating with 175 mL of heat denatured 2 % BSA (HD-BSA). Previous work in our lab has shown that fibroblasts fail to adhere to surfaces coated with HD-BSA. HD-BSA was obtained by incubating 2 % BSA solution in a hot water bath boiling at 100 °C for 4 minutes. The plate was refrigerated overnight at 4 °C to allow for the HD-BSA to adhere to the plate surface.

The various constructs of crosslinked HA (HA, HA+FN, HAFN) were then molded to the wells. This was important to provide the cells a medium of uniform thickness that was thin enough to penetrate with a microscope. Using a positive displacement pipette, 400 µL of hydrogel was pipetted into each well. Three wells were used for each replicate condition. The gels were molded into the bottom of each well by centrifugation in a Sorvall RT6000B Refrigerated Centrifuge at 411 g and 4 °C for 5 minutes. The gels were then frozen for 30 minutes at –80 °C and lyophilized in a Freezemobile 6ES (Virtis, Gardiner, NY) at -53.7 °C and under a 57 mT vacuum for 3 days. Lyophilization was necessary to remove the water dialysate so that an appropriate salt solution for cell migration (SF-DMEM) could be substituted.

400 mL of SF-DMEM containing 100 ng/ml PDGF was added to rehydrate each gel. The plates were refrigerated for 8 hours to allow for complete hydration and then spun in a Sorvall RT6000B Refrigerated Centrifuge at 2000 rpm and 4 °C for 5 minutes to remove additional bubbles that became trapped during rehydration.

30 mg/ml human fibrinogen stock (Calbiochem, San Diego, CA) was diluted to 300 mg/ml in SF-DMEM with 100 ng/ml PDGF. The fibrinogen concentration corresponds to the fibrin concentration in the in vivo clot. FN solution was mixed in to yield a final concentration of 30 mg/ml FN. 400 mL of the fibrinogen solution was then pipetted into wells in the Falcon 24-well tissue culture treated plates. 200 U/ml stock thrombin (New York Blood Center, New York, NY) was diluted to 100 U/ml in dH₂O. 4mL of 100 U/ml was then added into the 400mL of fibrinogen solution to begin the polymerization reaction that converts fibrinogen into fibrin. The plate was incubated for 90 minutes at 37 °C to complete the polymerization. Fibrinogen was tested for FN content using Western blotting, and a negligible inherent concentration confirmed (1.4 mg/ml FN).

The fibroblast-seeded cytodex-3 beads were rinsed three times in SF-DMEM by allowing the beads to settle, aspirating the liquid, and resuspending the beads in SF-DMEM. These washes removed unattached cells, as well as proteins from the culture media. Roughly 100 beads/well were distributed to the top of each of the gels and spread around the surface with gently agitation. The plate was then returned to the 37 °C incubator for a 48-hour migration window.

Analysis of Hyaluronan Gel Three-Dimensional Migration Assay

At 24 hours, the plate was removed from 37 °C incubator and analyzed using phase contrast microscopy on a Nikon (inverted) Diaphot microscope in a 37 °C atmosphere (Nikon incubator, Japan). Cell outmigration was quantified by counting the number of cells that migrated out of ten randomly selected beads per well.

At 48 hours, the plate was removed from 37 °C incubator and the cells fixed with glutaraldehyde. The cells were then stained by the addition of 50 mL 0.1 % crystal violet, which was allowed 20 minutes to fully diffuse through the gel. Cell outmigration was quantified by counting the number of cells that migrated out of ten randomly selected beads per well. The cells were counted using a Nikon SMX800 zoom stereomicroscope.

Introduction

Product Design

Materials

Methods

Recombinant FN Fragment Purification

Cell Culture