Frozen 2 % stock of low gelling temperature agarose (FMC BioProducts, Rockland, ME) in PBS was remelted at 95 ºC in Fisher Scientific Company dry bath incubator for 5 minutes. Agarose was then diluted to 0.2 % in serum-free DMEM (SF-DMEM) and preheated to 37 ºC in hot water bath.

CF-31 cells were cultured until 80-95 % confluent. Cell medium was removed and cells rinsed with PBS. Cells were detached from plate via addition of 0.0 5% Trypsin in 0.53 mM EDTA and then diluted with SF-DMEM and spun down at 185 g in a Sorvall GLC-2B Centrifuge (Dupont Instruments). Using wide- mouth pipette tips, 1.1X10⁶ cells were resuspended in 100 mL 0.2 % agarose in SF-DMEM to a final cell concentration of 1.1X10⁷ cells/ml and retained at 37 ºC in a Fisher Scientific Isotemp 202 hot water bath. 1 mL droplets were placed in the center of a well on a 96-well Immulon 4 HBX flat bottom plate (Dynex Technologies, Inc., Chantilly, VA). Diameter of the droplet was about 1.4 mm. The plate was then covered and refrigerated at 4 ºC for 10 minutes to allow the agarose droplet to set. No more than 20 wells were ever used on one plate to eliminate variability in droplet-to-droplet temperatures. Each experimental group contained five replicates.

The proteins for coating the plate surface around the droplet were diluted to appropriate concentration in PBS at 4 ºC. Using a gel-loading tip, 75 mL of coating solution (BSA, intact FN, FN fragments) was pipetted around the agarose gel. Intact FN was diluted from 1mg/ml stock of human plasma fibronectin (FN) (Chemicon International, Temecula, CA). The plate was incubated at 37 ºC for two hours to allow for the proteins to adsorb to the polystyrene surface.

The PBS solutions were removed from the wells via aspiration and wells rinsed with 100 mL PBS to remove unadsorbed protein. 100 mL of SF-DMEM containing 2 % BSA and 100 ng/ml recombinant platelet derived growth factor-BB (PDGF) (ZymoGenetics, Seattle, WA) was added carefully through a gel-loading tip. The BSA in the medium was present to adhere to any bare regions on the plate. Since BSA exhibits anti-adhesive properties with regard to fibroblasts, it was used to ensure that the cells do not migrate on any polystyrene that has not been covered by FN or its constructs. Wells coated only with BSA were used as negative controls. The plate was then incubated at 37 ºC for 24 hours.

The cells were fixed through addition of glutaraldehyde to a 1 % solution with the media and left for 10 minutes at room temperature. The liquid was aspirated out and the wells were rinsed with PBS. The cells were stained with filtered 0.1 % crystal violet in 200 mM boric acid (pH 8.0) at room temperature for 10 minutes. The wells were then rinsed twice with PBS to remove background dye from plate. 100 mL of PBS was then added to prevent drying.

Analysis of Agarose Droplet Migration Assay

PBS was removed from the wells immediately prior to observation to improve clarity and focus of images. Observations were made using a Nikon SMX800 zoom stereomicroscope (Japan) with a Diagnostic Instruments Spot RT camera Attachment (Sterling Heights, MI). Images were captured with Spot 3.0 software running on a Dell Dimension 8100 PC. Using the bundled Spot 3.0 software, migration area was determined by outlining the boundaries of the fibroblast migration and the agarose gel. The area of migration (in square pixels) was then determined by the formula Total Area - Area of Droplet. The average migration area of all replicate conditions from each experiment was normalized against the average migration area of the positive control (intact FN) of the same experiment. In this manner, data from experiments on different days were pooled together. The averages and standard deviations of each pooled condition were obtained and reported. In addition, ANOVA tests were run on the data to determine whether the variations between conditions were random.

Introduction

Product Design

Materials

Methods

Recombinant FN Fragment Purification

Cell Culture