PRGF-Endoret® in corneal tissue engineering

Abstract

Purpose: The objective of the present study is to analyze the potential use of PRGF-Endoret® in every step of corneal tissue engineering, supplementing the culture media and as a scaffold for limbal stem cells.

Methods: PRGF-Endoret® derivatives: 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 healthy donors 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 CaCl₂ and incubated at 37 0C for one hour until membrane coagulation. The released supernatants were collected, filtered, aliquoted and stored at - 800C until use in the following culture media: Basal medium (BM): DMEM/F12 supplemented with 1% antibiotics and 10% PRGF-Endoret® (BM_PRGF) or FBS (BM_FBS). Classic medium (CM): DMEM/F12 supplemented with 5mg/ml insulin, 8.33ng/ml cholera toxin, 24mg/ml adenine, 1.3ng/ml triiodothyronine, 0.4mg/ml hydrocortisone, 1% antibiotics and 10% PRGF-Endoret® (CM_PRGF) or FBS (CM_FBS). Proliferation assays: Limbal stem cells were obtained from explants of 2-3mm in diameter of the limbal region and cultured on the media described above. At 7 days, cells were fixed in ice-cold methanol and their proliferation was assessed by quantification of the growth area. Culture on PRGF-Endoret® membrane: Human limbal stem cells were cultured on PRGF-Endoret® membranes. Once confluent, membranes were fixed and analyzed by phase contrast microscopy, scanning electron microscopy and immunocytochemistry for p63 and cytokeratin in order to check their immunological markers.

Results: Growth area in cultures in CM_PRGF was significantly better (p<0.05) than cultures in CM_FBS, and, even though all cultures in BM were significantly lower (p<0.05) than cultures in CM, BM_PRGF shows a significantly better (p<0.01) growth area than cultures in BM_FBS, being as effective as CM_FBS. In the other hand, limbal stem cells seems to attach and proliferate on a PRGF-Endoret® membrane maintaining their cellular markers with PRGF-Endoret®, instead of FBS, as supplement in the culture media.

Conclusions: PRGF-Endoret® could be used in corneal tissue engineering, both, supplementing the culture media, even in a basal media without any other additives, as well as scaffold for the culture of the limbal stem cells.