A C Riestra1, N Vazquez2, M Chacon2, S Berisa2, R M Sanchez-Avila2, G Orive3, E Anitua4, A Meana5, J Merayo-Lloves2. 1. Instituto Oftalmológico Fernández-Vega, Fundación de Investigación Oftalmológica, Universidad de Oviedo, Oviedo, Spain. Electronic address: ana.riestra@fio.as. 2. Instituto Oftalmológico Fernández-Vega, Fundación de Investigación Oftalmológica, Universidad de Oviedo, Oviedo, Spain. 3. Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country, Vitoria, Spain; Networking Biomedical Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, SLFPB-EHU, 01006 Vitoria-Gasteiz, Spain; BTI Biotechnology Institute, Vitoria, Spain. 4. BTI Biotechnology Institute, Vitoria, Spain. 5. Instituto Oftalmológico Fernández-Vega, Fundación de Investigación Oftalmológica, Universidad de Oviedo, Oviedo, Spain; U714 Centre for Biomedical Network Research on Rare Diseases (CIBERER), Oviedo, Spain.
Abstract
PURPOSE: Develop an autologous culture method for ex vivo expansion of human limbal epithelial progenitor cells (LEPCs) using Plasma Rich in Growth Factors (PRGF) as a growth supplement and as a scaffold for the culture of LEPCs. METHODS: LEPCs were cultivated in different media supplemented with 10% fetal bovine serum (FBS) or 10% PRGF. The outgrowths, total number of cells, colony forming efficiency (CFE), morphology and immunocytochemistry against p63- α and cytokeratins 3 and 12 (CK3-CK12) were analyzed. PRGF was also used to elaborate a fibrin membrane. The effects of the scaffold on the preservation of stemness and the phenotypic characterization of LEPCs were investigated through analysis of CK3-CK12, ABCG-2 and p63. RESULTS: LEPCs cultivated with PRGF showed a significantly higher growth area than FBS cultures. Moreover, the number of cells were also higher in PRGF than FBS, while displaying a better morphology overall. CFE was found to be also higher in PRGF groups compared to FBS, and the p63-α expression also differed between groups. LEPCs cultivated on PRGF membranes appeared as a confluent monolayer of cells and still retained p63 and ABCG-2 expression, being negative for CK3-CK12. CONCLUSIONS: PRGF can be used in corneal tissue engineering, supplementing the culture media, even in a basal media without any other additives, as well as providing a scaffold for the culture.
PURPOSE: Develop an autologous culture method for ex vivo expansion of human limbal epithelial progenitor cells (LEPCs) using Plasma Rich in Growth Factors (PRGF) as a growth supplement and as a scaffold for the culture of LEPCs. METHODS: LEPCs were cultivated in different media supplemented with 10% fetal bovine serum (FBS) or 10% PRGF. The outgrowths, total number of cells, colony forming efficiency (CFE), morphology and immunocytochemistry against p63- α and cytokeratins 3 and 12 (CK3-CK12) were analyzed. PRGF was also used to elaborate a fibrin membrane. The effects of the scaffold on the preservation of stemness and the phenotypic characterization of LEPCs were investigated through analysis of CK3-CK12, ABCG-2 and p63. RESULTS: LEPCs cultivated with PRGF showed a significantly higher growth area than FBS cultures. Moreover, the number of cells were also higher in PRGF than FBS, while displaying a better morphology overall. CFE was found to be also higher in PRGF groups compared to FBS, and the p63-α expression also differed between groups. LEPCs cultivated on PRGF membranes appeared as a confluent monolayer of cells and still retained p63 and ABCG-2 expression, being negative for CK3-CK12. CONCLUSIONS: PRGF can be used in corneal tissue engineering, supplementing the culture media, even in a basal media without any other additives, as well as providing a scaffold for the culture.
Authors: Eduardo Anitua; Mar Zalduendo; Maria Troya; Mohammad H Alkhraisat; Leticia Alejandra Blanco-Antona Journal: Int J Mol Sci Date: 2022-06-11 Impact factor: 6.208
Authors: Antonio J Villatoro; Viviana Fernández; Silvia Claros; Cristina Alcoholado; Manuel Cifuentes; Jesús Merayo-Lloves; José A Andrades; José Becerra Journal: Int J Mol Sci Date: 2017-10-28 Impact factor: 5.923
Authors: Ronald M Sanchez-Avila; Jesús Merayo-Lloves; Ana C Riestra; Silvia Berisa; Carlos Lisa; José Alfonso Sánchez; Francisco Muruzabal; Gorka Orive; Eduardo Anitua Journal: Medicine (Baltimore) Date: 2018-04 Impact factor: 1.889
Authors: Ronald M Sánchez-Ávila; Carlos A Robayo-Esper; Eva Villota-Deleu; Álvaro Fernández-Vega Sanz; Álvaro Fernández-Vega González; Borja de la Sen-Corcuera; Eduardo Anitua; Jesús Merayo-Lloves Journal: Clin Pract Date: 2022-01-10
Authors: Ronald M Sánchez-Ávila; Álvaro Fernández-Vega González; Álvaro Fernández-Vega Sanz; Jesús Merayo-Lloves Journal: Int Med Case Rep J Date: 2019-07-12