Cell Handling

Human dermal fibroblasts (CF-31) were cultured in Dulbecco’s Modified Eagle’s Medium containing 10 % fetal bovine serum and 100 U/ml penicillin, streptomycin, glutamine (DMEM). Tissue cultures were incubated at 37 ºC in a humidified incubator (Napco Scientific Company, Tualatin, Oregon) with a 5 % CO₂ atmosphere. All cell work was performed under sterile conditions (SterilGard Hood, The Baker Company, Incorporated, Stanford, Maine). Tissue culture dishes were used from Becton Dickinson Labware (FalconÔ Franklin Lakes, NJ) and Corning Inc. (Corning, NY)

Cell Freezing

Cell medium was removed and cells rinsed with PBS. Cells detached from plate through addition of 0.05 % Trypsin in 0.53 mM EDTA. Cells were spun at 185 g in a Sorvall GLC-2B Centrifuge (Dupont Instruments) and the supernatant removed via aspiration. The pellet was resuspended in 1ml cell freezing media (90 % Fetal bovine serum (FBS) and 10 % dimethyl sulfoxide (DMSO)) and placed in Styrofoam box within -80 ºC freezer.

Cell Recovery

Cuvette containing CF-31 (passage 5) was removed from a -80 ºC freezer. The cap was partially unscrewed to prevent pressure buildup during thaw. Cells were thawed by gently agitating cuvette in a 37 ºC water bath for about one minute until completely thawed. Pipette 500 ml of cells into two tissue culture plates each containing 10 ml DMEM preheated to 37 ºC. Cells allowed to attach for 2-24 hours at 37 ºC in a humidified incubator. Tissue culture plates rinsed three times with DMEM to remove all DMSO and returned to incubator with 10 ml DMEM.

Cell Attachment to Cytodex-3 Beads

Pharmacia (Peapack, NJ) Cytodex-3 beads were swelled in 50 ml PBS per gram of dry Cytodex-3 beads for at least 1 hour to swell to d₅₀ of 175 mm. The beads were autoclaved for 20 minutes at 120 °C. CF-31 cells were detached with Trypsin-EDTA and rinsed twice in SF-DMEM. The cells were counted with Bright-Line hemacytometer (Hausser Scientific, Horsham, PA) and suspended with Cytodex-3 beads in 8 ml SF-DMEM in a 15ml polypropylene tube at a ratio of 40 cells/bead. The suspension was rotated at 6 rpm for three days at 37°C in a National Labnet Company Hybaid oven (Woodbridge, NJ). Operator opened cap every 24 hours in a sterile hood to allow for gas transfer.