Development Of An Automated System For The Preparation Of Fibronectin Eyedrops From Human Blood Plasma

Abstract

Purpose: : To develop an automated system for the preparation of autologous fibronectin eyedrops from blood plasma and to evaluate the purity and biological activity of the eyedrops for clinical application.

Methods: : The amount and purity of fibronectin in eyedrops were evaluated by enzyme–linked immunosorbent assay and immunoblot analysis. The biological activity of fibronectin in vitro was examined by measurement of the attachment of BHK cells or SV40–transformed human corneal epithelial (HCE) cells to a fibronectin matrix. Cytotoxicity was assessed by measurement of the release of lactate dehydrogenase into the culture medium of these cells. The effects of fibronectin eyedrops on corneal epithelial wound healing in vivo were examined by measurement of the size of the defective area in the mechanically scraped corneal epithelium of rabbits.

Results: : A computerized mechanical system based on affinity chromatography and gel filtration was developed for the purification of fibronectin. All the necessary columns, solutions, and containers were packaged as a set and sterilized. The mean recovery of fibronectin from plasma was 19.9%, yielding a concentration of 141.6 µg/ml. The number of BHK or HCE cells that attached to fibronectin–coated dishes increased in proportion to the concentration of fibronectin used for coating (0 to 20 µg/ml). Fibronectin did not induce the release of lactate dehydrogenase from the cells. The administration of fibronectin eyedrops significantly increased the rate of epithelial wound closure in vivo compared with that observed after administration of phosphate–buffered saline.

Conclusions: : We have developed an automated system for the preparation of fibronectin eyedrops from human plasma. The prepared eyedrops are biologically active, as revealed by their stimulatory effect on corneal epithelial wound closure in rabbits. This automated system is suitable for clinical application to the treatment of persistent corneal epithelial defects.

Keywords: extracellular matrix • wound healing