Abstract
Purpose:
We analyze the potential use of PRGF-Endoret® as a substitute of xenogeneic sera in corneal cell cultures.
Methods:
Plasma rich in growth factors (PRGF) eye drop was obtained using the Endoret (PRGF) kit in Ophthalmology (BTI Biotechnology Institute, S.L., Vitoria, Spain). Briefly, blood was collected from human blood of healthy donors, after informed consent, into 9 mL tubes containing sodium citrate as anticoagulant. Blood was centrifuged at 580g for 8 minutes, whole plasma column was drawn off avoiding the buffy coat, activated with calcium chloride, incubated at 37 degrees for one hour, and finally, the released supernatants were collected by aspiration, filtered and aliquoted and stored at - 80 degrees until use. Corneoscleral tissue was attained from a local eye bank after penetrant-keratoplasty surgery. Limbal stem cells were obtained from explants of 2-3mm in diameter of the limbal region and cultured in culture medium containing 10% PRGF-Endoret® or 10% of fetal bovine serum for 7 days. Afterwards, cells were fixed in ice-cold methanol and their proliferation was assessed by quantification of the growth area. Immunocytochemistry for p.63 and cytokeratin was also performed in order to check their immunological markers.
Results:
Both, PRGF-Endoret® and Fetal Bovine Serum, cell cultures showed a positive expression in p.63 and cytokeratin, however some differences were found between both cultures, while cultures supplemented with Fetal Bovine Serum showed a positive stain for p63 in 73% of the nuclei, PRGF-Endoret® cultures showed a 97% of nuclei stained for p63. Nevertheless, limbal stem cell cultures supplemented with PRGF-Endoret® showed a better growth area in contrast with the ones supplemented with fetal bovine serum. Moreover, there was cellular growth in 80% of the explants cultured in PRGF-Endoret® while only 65% of fetal bovine serum explants had grown.
Conclusions:
PRGF-Endoret® seems to improve the cellular growth and, not only maintain the p63 expression, but improve it compared to Fetal Bovine Serum. Therefore, PRGF-Endoret® could be used as a substitute of xenogeneic sera, not only as an allogeneic product but also as an autologous product for the culture and expansion of corneal limbal stem cells.
Keywords: 765 wound healing •
482 cornea: epithelium