Topical treatment of ocular surface defects: comparison of the epitheliotrophic capacity of fresh frozen plasma and serum on corneal epithelial cells in an in vitro cell culture model.

Accelerated healing of ocular surface disorders was reported using serum for topical application. It is supposed that growth factors, fibronectin and vitamins in serum support the proliferation of corneal epithelial cells. The use of fresh frozen plasma (FFP) instead of serum is theoretically attractive, as it is more easily available from blood banks. In this study, serum and FFP were investigated for composition of epitheliotrophic factors and effect on corneal epithelial cells. Whole blood was taken from five donors. Serum and FFP were prepared, and the concentrations of epithelial growth factor (EGF), Platelet-derived growth factor (PDGF), transforming growth factor-beta1, fibronectin and vitamin A were determined. Immortalized human corneal epithelial cells were used to investigate growth, migration and differentiation in response to both blood products. Significant differences were found regarding the mediator composition of serum and FFP. Serum rather than FFP was significantly superior in stimulating cell growth, migration and differentiation. The epitheliotrophic capacity of blood products depends upon the composition of growth factors and vitamins. Blood clotting strongly influences the growth factor pattern. The superior epitheliotrophic capacity of serum might be due to the higher concentration of proliferation mediators such as EGF and PDGF and its higher content of vitamin A.

An optimised protocol for the production of autologous serum eyedrops.

BACKGROUND: Serum eyedrops have been successfully used in the treatment of severe dry eye, persistent epithelial defects and other severe ocular surface disorders. A number of clinical studies showed a variable efficacy of this approach, but the parameters for the production of this blood product varied significantly. In order to establish an optimised protocol for the production of serum eyedrops, we examined the effect of various clotting times, centrifugation forces, types of diluent and dilutions on the concentration of growth factors, fibronectin, and vitamins in serum and tested the epitheliotrophic capacity of these serum modifications in a cell culture model of human SV-40-immortalised corneal epithelial cells (HCE-T). METHODS: Serum samples were prepared with a clotting time of 20, 60 or 120 min, a centrifugation force of 500 xg or 3,000 xg, and diluted with BSS or isotonic saline. The concentrations of EGF, TGF-beta1, PDGF-AB, FGF, HGF, fibronectin, vitamin A and vitamin E in these samples were evaluated with ELISA and HPLC. HCE-T cells were incubated for 24, 48, 72, 96 and 144 h with 100, 50, 25, 12.5, 6.25 and 3.125% serum in diluent, and cell proliferation, migration and differentiation were evaluated by means of a luminescence-based ATP assay, a colony-dispersion assay and scanning electron microscopy. RESULTS: Using a longer clotting time resulted in an increased concentration of all the epitheliotrophic factors examined in serum; the difference was statistically significant for EGF, TGF-beta1 and HGF. Increasing the g force of centrifugation from 500 xg to 3,000 xg resulted in significantly less TGF-beta1, but more EGF and vitamin A. Cell proliferation was better supported by serum prepared with 3,000 xg and diluted with BSS. Serum prepared with a longer clotting time yielded better cell migration and differentiation. CONCLUSION: Clotting time, centrifugation and diluents have a significant impact on the composition and epitheliotrophic effects of serum. A long clotting time (>or=120 min), a sharp centrifugation (3,000 xg for 15 min) and dilution with BSS improve the ability of serum eyedrops to support proliferation, migration and differentiation of corneal epithelial cells.

[Epitheliotrophic capacity of serum and plasma eyedrops. Influence of centrifugation]. / Epitheliotrophe Kapazität von Serum- und Plasmaaugentropfen. Einfluss der Zentrifugation.

BACKGROUND: Serum eyedrops are a new modality for the treatment of ocular surface disorders. We examined the influence of the preparation of blood products in a cell culture model and compared it with plasma. MATERIAL AND METHODS: Serum and plasma were obtained from full blood of ten healthy volunteers and centrifuged at 500 and 3000 G. EGF, PDGF, TGF-beta1, fibronectin, and vitamin A were quantified by means of ELISA and HPLC. Cultures of human corneal epithelial cells were incubated with the four blood products in dose-response experiments and the intracellular ATP quantified. RESULTS: EGF, PDGF, and vitamin A were present in serum in significantly higher concentrations than in plasma. The concentration of fibronectin was not influenced by the preparation. Support of proliferation was best by 25% platelet-poor serum. Serum supported the differentiation and migration of epithelial cells better than plasma. CONCLUSION: The biochemical character of serum eyedrops is determined by the parameters chosen to produce the blood product. Plasma does not seem to offer an epitheliotrophic capacity equivalent to serum eyedrops. Their production should be optimized before any meaningful randomized controlled clinical trial can be attempted.