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.


CONCLUSIONS
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.
Liu
L
L
Department of Ophthalmology, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
Hartwig
D
D
Harloff
S
S
Herminghaus
P
P
Wedel
T
T
Geerling
G
G
D000328:Adult; D000368:Aged; D002454:Cell Differentiation / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002498:Centrifugation / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002985:Clinical Protocols; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects / Q000648:ultrastructure; D005260:Female; D006133:Growth Substances / Q000097:blood; D006801:Humans; D008297:Male; D008875:Middle Aged; D009883:Ophthalmic Solutions / Q000008:administration & dosage* / Q000737:chemistry; D044967:Serum; D013918:Thrombin Time; D014801:Vitamin A / Q000097:blood; D014810:Vitamin E / Q000097:blood
D006133:Growth Substances; D009883:Ophthalmic Solutions; D014801:Vitamin A; D014810:Vitamin E