Vázquez, N. (Natalia)

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    Mini Cleanroom for the Manufacture of Advanced Therapy Medicinal Products (ATMP): Bioengineered Corneal Epithelium
    (MDPI, 2021) Persinal, M. (Mairobi); Berisa, S. (Silvia); Merayo-Lloves, J. (Jesús); Chacón, M. (Manuel); Meana, A. (Álvaro); Fernández-Vega-Cueto, L. (Luis); Alfonso, J.F. (José F.); Baamonde, B. (Begoña); Alonso, S. (Sergio); Vázquez, N. (Natalia)
    Among several requirements for the manufacture of Advanced Therapy Medicinal Products (ATMP) are: following the guidelines of a pharmaceutical quality system, complying with Good Manufacturing Practice (GMP) and access to a cleanroom fulfilling strict environmental conditions (Class A work area and Class B environment). This makes ATMP expensive. Moreover, the production of many of these therapeutic products may also be unprofitable, as in most cases their use is limited to a few patients and to a single batch per manufacturing unit. To reduce costs, ATMP may be produced in a scaled-down system isolated from the external environment (isolator), allowing for placement of this facility in a Class D environment, which is much more permissive and less costly. In this work, we confirm that it is possible to manufacture bioengineered corneal epithelium inside an isolator while fulfilling all the safety assurance standards at an affordable cost for patients. This small-scale ultra-clean working environment complies with GMP guidelines and could be a solution for the high costs associated with conventional cleanroom ATMP production.
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    Autologous method for ex vivo expansion of human limbal epithelial progenitor cells based on plasma rich in growth factors technology
    (Elsevier, 2017) Berisa, S. (Silvia); Merayo-Lloves, J. (Jesús); Chacón, M. (Manuel); Meana, A. (Álvaro); Anitua, E. (Eduardo); Riestra, A.C. (A.C.); Orive, G. (Gorka); Sánchez-Ávila, R.M. (Ronald M.); Vázquez, N. (Natalia)
    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.
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    QobuR – A new in vitro human corneal epithelial model for preclinical drug screening
    (Elsevier, 2019) Persinal, M. (Mairobi); Berisa, S. (Silvia); Merayo-Lloves, J. (Jesús); Chacón, M. (Manuel); Meana, A. (Álvaro); Fernández-Vega-Cueto, L. (Luis); Alfonso, J.F. (José F.); Sanchez, M. (Manuel); Baamonde, B. (Begoña); Vázquez, N. (Natalia)
    A new in vitro human corneal epithelial model (QobuR) obtained from normal limbal tissue has been developed to study ocular irritancy of different ophthalmic compounded drugs. Phenotypical characterization and transepithelial electrical resistance (TEER) of QobuR revealed essential similarities compared with a native human cornea, displaying functional markers and TEER values near 1500 Ωcm2 at day 7th of cellular differentiation. Using this model, ocular irritancy and barrier integrity alterations were evaluated using MTT reaction and variations in TEER. We found that some of the Non-Irritant products evaluated still damage the corneal epithelial integrity and current protocols for ocular irritancy should therefore include a barrier integrity evaluation. Moreover, in order to comprehensively evaluate corneal permeability of the active ingredients, we propose the use of QobuR as an all-in-one alternative method for evaluating ocular irritancy, barrier disruptions and permeability rates of topically applied ocular drugs to improve current in vitro drug testing procedures.
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    Fibrin-Plasma Rich in Growth Factors Membrane for the Treatment of a Rabbit Alkali-Burn Lesion
    (MDPI, 2021) Persinal, M. (Mairobi); Berisa, S. (Silvia); Merayo-Lloves, J. (Jesús); Chacón, M. (Manuel); Meana, A. (Álvaro); Anitua, E. (Eduardo); Fernández-Vega-Cueto, L. (Luis); Sánchez-Ávila, R.M. (Ronald M.); Vázquez, N. (Natalia); Brea-Pastor, A. (Agustín)
    Abstract: The purpose of this work is to describe the use of Fibrin-Plasma Rich in Growth Factors (PRGF) membranes for the treatment of a rabbit alkali-burn lesion. For this purpose, an alkali-burn lesion was induced in 15 rabbits. A week later, clinical events were evaluated and rabbits were divided into five treatment groups: rabbits treated with medical treatment, with a fibrin-PRGF membrane cultured with autologous or heterologous rabbit Limbal Epithelial Progenitor Cells (LEPCs), with a fibrin-PRGF membrane in a Simple Limbal Epithelial Transplantation and with a fibrin-PRGF membrane without cultured LEPCs. After 40 days of follow-up, corneas were subjected to histochemical examination and immunostaining against corneal or conjunctival markers. Seven days after alkali-burn lesion, it was observed that rabbits showed opaque cornea, new blood vessels across the limbus penetrating the cornea and epithelial defects. At the end of the follow-up period, an improvement of the clinical parameters analyzed was observed in transplanted rabbits. However, only rabbits transplanted with cultured LEPCs were positive for corneal markers. Otherwise, rabbits in the other three groups showed positive staining against conjunctival markers. In conclusion, fibrin-PRGF membrane improved the chemically induced lesions. Nonetheless, only fibrin-PRGF membranes cultured with rabbit LEPCs were able to restore the corneal surface.