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Jesus Merayo-Lloves, Ana Cristina Riestra, Alvaro Meana, Natalia Vazquez, Manuel Chacon, Mairobi Persinal, Gorka Orive, Eduardo Anitua; PRGF-Endoret®: A new approach in the treatment of limbal stem cell deficiency.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4350.
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© ARVO (1962-2015); The Authors (2016-present)
The objective of the present study is to develop a complete autologous therapy for the treatment of limbal stem cell deficiency using PRGF® technology.
Blood collection: 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 9mL tubes containing sodium citrate as anticoagulant. Blood was centrifuged at 580g for 8 minutes until blood fractionation.F1 serum supplement: F1 fraction was used as supplement of the culture medium. For this purpose, F1 fraction was activated with CaCl2, incubated at 37C for one hour, and finally, the released supernatants were collected by aspiration, aliquoted and stored at – 80C until use.F2 membrane: F2 fraction was used for the development of scaffolds. Briefly, 5mL of F2 fraction was collected, activated with CaCl2 and incubated at 37C. Once a gel was formed, it was flattened under mechanical pressure obtaining a 100µm membrane.Cellular culture: Corneoscleral tissue was attained from a local eye bank after penetrant-keratoplasty surgery. Human limbal stem cells (hLSC) were obtained from explants of 2-3mm in diameter of the limbal region and cultured in DMEM/F12 supplemented with 100U/mL penicillin, 0.1mg/mL streptomycin and 10% F1 serum supplement. When the culture reached confluence, cells were seeded onto F2 fraction membrane, cultured and fixed with ice-cold methanol when confluent.Cellular analysis: Cellular growth was assessed by phase contrast microscopy and scanning electron microscopy (SEM). Immunocytochemistry for p63 and cytokeratin was also performed in order to check their immunological markers.
Using F2 serum fraction we were able to achieve a manageable membrane of 100µm. This membrane was able to support cellular growth of hLSC, which were cultured with F1 serum fraction as an only supplement of the culture media. In these conditions, hLSC displays their typical polyhedral morphology while expressing their characteristic cellular markers, p63 and cytokeratin.
PRGF-Endoret® technology provides an autologous environment for the isolation of hLSC as well as an autologous membrane for their growth. This strategy allows the development of a complete autologous cell therapy for the treatment of limbal stem cell deficiency.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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