ABSTRACT
BACKGROUND: Autologous serum has been advocated for the treatment of persistent corneal epithelial defects and other ocular surface disorders which may be a local manifestation of a systemic disease. Many of these underlying disorders are cytokine-mediated and require immunosuppressive therapy. The systemic disease and medication could potentially influence the epitheliotrophic capacity of serum eye drops. We compared the effect of serum from healthy and immunosuppressed donors in a human corneal epithelial cell culture model. METHODS: Serum was prepared under standardised conditions from full blood samples of 10 healthy donors and 10 patients suffering from rheumatoid arthritis. All patients were treated with prednisolone and methotrexate, one also with azathioprine. In these serum samples EGF, FGF, HGF, PDGF-AB, TGF-beta1, fibronectin, vitamin A and E as well as IL-6 were quantified by means of routine ELISA or HPLC technology. SV-40 immortalised human corneal keratinocytes were cultured in 96-well plates at 37 degrees C, 5 % CO (2) with a fully defined culture medium. At 30 % confluency the culture medium was substituted by one of the test preparations. Proliferation of cell cultures was quantified by means of a luminescence-based ATP assay in dose-response experiments. A colony dispersion assay was used to examine the effect on cell migration and differentiation was assessed by means of scanning electron microscopy. RESULTS: Serum from healthy donors differed from serum of immunosuppressed individuals suffering from rheumatoid arthritis only in that it contained significantly higher amounts of fibronectin and TGF-beta1. Support of proliferation, migration and differentiation of corneal epithelial cells was dose-dependent, but no significant difference was observed between the serum of the two different groups of donors. At a dilution of 25 % serum of healthy donors showed a significantly higher stimulation of migration than serum of immunosuppressed patients. CONCLUSION: The effect of serum on migration but not proliferation is affected by systemic diseases requiring immunosuppression. If an epithelial defect of a patient with rheumatoid arthritis does not respond to treatment with diluted autologous serum, undiluted serum should be tried since the positive effect of serum on cell migration is positively correlated with dose.
Subject(s)
Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/immunology , Epithelium, Corneal/immunology , Immunosuppressive Agents/therapeutic use , Serum/immunology , Adult , Aged , Cell Differentiation/drug effects , Cell Division/drug effects , Cell Movement/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Female , Fibronectins/metabolism , Humans , In Vitro Techniques , Lymphotoxin-alpha/metabolism , Male , Microscopy, Electron, Scanning , Middle AgedABSTRACT
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.
