A cell toxicity assay was devised to test for the effect of leachable chemicals and break-down products of the HA construct on CF-31 adhesion and proliferation. This was accomplished by placing the HA constructs into a traswell that was inserted into a 24-well plate that contained cultured fibroblasts. Free materials in the HA constructs could then diffuse across the 8mm pores filter at the bottom of the insert. If such materials were toxic they would adversely affect cell number, which was measured by a colorometric assay.

The inserts were first prepared by adding 200 mL of HA construct (HA, HAFN) to transwell inserts (Corning Costar Corporation, Cambridge, MA) using positive displacement pipette. Those inserts, as well as empty inserts, were then frozen for 30 minutes at -80 °C and lyophilized in a Freezemobile 6ES at -53.7 °C and under a 57 mT vacuum for 3 days.

CF-31 cells were cultured until 80-95% confluent. Cell medium was removed and cells rinsed with PBS. The cells were detached from the culture plate via addition of 0.05% Trypsin in 0.53 mM EDTA-4Na and then diluted with DMEM. The cell suspension was spun down at 185 g in Sorvall GLC-2B centrifuge, the supernatant was aspirated, and then the cells were resuspended in DMEM to a concentration of 2.4x10⁴ cell/ml. 300 mL of cell solution (7.2 x10³ cells)was pipettedinto the bottom of 24-well plates (one for each experimental day). Transwell inserts containing HA or HAFN were inserted into each well. In addition, empty transwell inserts and wells without inserts at all as were used as positive controls. Four replicate wells of each condition for each day were used. 1ml of DMEM was added into wells through the transwell inserts.

For three to four successive days, the experimental group for that day was fixed by removing the inserts and adding 1 ml of 2 % glutaraldehyde. The plate was incubated at room temperature for 10 minutes. The glutaraldehyde was removed and the wells rinsed 5 times by slowly submerging the plate into dH₂O. Some dH₂O was retained to avoid drying until staining.

After the last experimental day, dH₂O was removed and the plate allowed to dry in hood. 200 mL of fresh 0.5 % crystal violet in 200 mM Boric Acid (pH 8.0) was added and allowed to stain the cells for 15 minutes on a vibrator at 600 rpm. The stain was aspirated, the wells rinsed with dH₂O, and the plate allowed to dry in the hood. 300 mL of 10% acetic acid was added to each well to destain. The cells were destained for 15 minutes on a vibrator at 600 rpm. The optical density (OD₅₉₀) of the destain solution was read and recorded from each well. This reading allows for a quantitative comparison between the extent of adhesion and proliferation of the cells exposed to different HA constructs.

Introduction

Product Design

Materials

Methods

Recombinant FN Fragment Purification

Cell Culture