No abstract

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×g or 3,000×g, and diluted with BSS or isotonic saline. The concentrations of EGF, TGF-β1, 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 luminescencebased 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 diffference was statistically significant for EGF, TGF-β1 and HGF. Increasing the g force of centrifugation from 500×g to 3,000×g resulted in significantly less TGF-β1, but more EGF and vitamin A. Cell proliferation was better supported by serum prepared with 3,000×g 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 (≥120 min), a sharp centrifugation (3,000×g for 15 min) and dilution with BSS improve the ability of serum eyedrops to support proliferation, migration and differentiation of corneal epithelial cells.

Platelet releasate but not FFP may offer additional potential for the treatment of severe ocular surface disease. Platelet releasate may be suitable as a novel treatment option for ocular surface disease with a superior effect on cell growth.

Possibly owing to its high content of growth factors, PLT releasate has a distinct superior effect on cell growth. Stimulation of migration and differentiation was slightly inferior but still acceptable. PLT releasate could therefore be a novel treatment option for ocular surface defects.

These data show clear evidence for de novo synthesis of cytokines in PC. The cytokine pattern supports the hypothesis that activated monocytes are responsible for this cytokine synthesis. PC with a WBC contamination of > or = 108 contain inflammatory mediators in clinically relevant concentrations.