An optimised protocol for the production of autologous serum eyedrops

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.

Use of Autologous Serum in the Treatment of Ocular Surface Disorders

“…[36][37][38] Although there is much evidence in the literature regarding the success in the treatment of various types of ocular surface disorders with the use of autologous serum eyedrops, most of these studies [36][37][38] are retrospective in nature and run the gambit in terms of the protocol used for the production of the serum. In other words, in these studies, 36-38 the concentrations of serum varied (from 20% to 100%), and the actual technique of processing the serum (clotting time, centrifugation time, and speed) varied as well.…”

Suero autólogo en el tratamiento del síndrome de ojo seco: Aspectos tecnológicos

“…También la contaminación del producto puede tener lugar durante su almacenamiento. En este sentido hay datos contradictorios ya que mientras determinados autores describen contaminación después de 30 días de uso otros aseguran la ausencia de la misma durante un período de hasta 12 semanas para el suero autólogo al 20% (4,5). Por esta razón cuando se entregan los envases de colirio de SA, se recomienda que se mantengan todas las formulaciones en el congelador salvo la que está en uso.…”

Aplicaciones del suero autólogo en oftalmología

“…Este tiempo de coagulación varía también según los autores, así los hay que centrifugan la muestra directamente mientras que otros esperan hasta dos días (33). En estudios experimentales realizados en cultivos celulares, se ha visto que un mayor tiempo de coagulación está relacionada con un mayor efecto del suero autólogo sobre la migración y diferenciación de las células epiteliales, recomendando, estos autores, un tiempo de coagulación de al menos dos horas (39). Estos mismos autores encuentran una mayor concentración de todos los factores examinados, siendo significativos para EGF, TGF-β cuando la sangre se dejó en reposo más de dos horas.…”

“…El filtro Millipore Express™ presenta una membrana de poliéster sulfota que la hace especialmente útil para el filtrado de estos productos biológicos. La mayoría de los autores utilizan suero fisiológico para hacer la dilución del suero, sin embargo, autores como Liu et al encuentran en cultivos de células epiteliales como estas proliferan más cuando la dilución se hace con BSS (39).…”