Development of a cell-based medicinal product: Regulatory structures in the European Union

IntroductionNew therapies with genes, tissues and cells have taken the emerging field for the treatment of many diseases. Advances on stem cell therapy research have led to international regulatory agencies to harmonize and regulate the development of new medicines with stem cells.Sources of dataEuropean Medicines Agency on September 15, 2012.Areas of agreementCell therapy medicinal products should be subjected to the same regulatory principles than any other medicine.Areas of controversyTheir technical requirements for quality, safety and efficacy must be more specific and stringent than other biologic products and medicines.Growing pointsCell therapy medicinal products are at the cutting edge of innovation and offer a major hope for various diseases for which there are limited or no therapeutic options.Areas timely for developing researchThe development of cell therapy medicinal products constitutes an alternative therapeutic strategy to conventional clinical therapy, for which no effective cure was previously available. © 2012 Published by Oxford University Press. All rights reserved.This work was supported by Fundación Progreso y Salud, Consejería de Salud, Junta de Andalucía (Grant PI-0022/2008); Consejería de Innovación Ciencia y Empresa, Junta de Andalucía (Grant CTS-6505; INP-2011-1615-900000); FEDER co-funded grants from Instituto de Salud Carlos III (Red TerCel-Grant RD06/0010/0025; PI10/00964) and the Ministry of Health and Consumer Affairs (Advanced Therapies Program Grant TRA-120). CIBERDEM is an initiative of the Instituto de Salud Carlos III.Peer Reviewe

Study of the stability of packaging and storage conditions of human mesenchymal stem cell for intra-arterial clinical application in patient with critical limb ischemia

Critical limb ischemia (CLI) is associated with significant morbidity and mortality. In this study, we developed and characterized an intra-arterial cell suspension containing human mesenchymal stem cells (hMSCs) for the treatment of CLI. Equally, the stability of cells was studied in order to evaluate the optimal conditions of storage that guarantee the viability from cell processing to the administration phase. Effects of various factors, including excipients, storage temperature and time were evaluated to analyze the survival of hMSCs in the finished medicinal product. The viability of hMSCs in different packaging media was studied for 60 h at 4 °C. The best medium to maintain hMSCs viability was then selected to test storage conditions (4, 8, 25 and 37 °C; 60 h). The results showed that at 4 °C the viability was maintained above 80% for 48 h, at 8 °C decreased slightly, whereas at room temperature and 37 °C decreased drastically. Its biocompatibility was assessed by cell morphology and cell viability assays. During stability study, the stored cells did not show any change in their phenotypic or genotypic characteristics and physicochemical properties remained constant, the ability to differentiate into adipocytes and osteocytes and sterility requirements were also unaltered. Finally, our paper proposes a packing media composed of albumin 20%, glucose 5% and Ringer's lactate at a concentration of 1 × 106 cells/mL, which must be stored at 4 °C as the most suitable to maintain cell viability (>80%) and without altering their characteristics for more than 48 h

Study of the stability of packaging and storage conditions of human mesenchymal stem cell for intra-arterial clinical application in patient with critical limb ischemia

Critical limb ischemia (CLI) is associated with significant morbidity and mortality. In this study, we developed and characterized an intra-arterial cell suspension containing human mesenchymal stem cells (hMSCs) for the treatment of CLI. Equally, the stability of cells was studied in order to evaluate the optimal conditions of storage that guarantee the viability from cell processing to the administration phase. Effects of various factors, including excipients, storage temperature and time were evaluated to analyze the survival of hMSCs in the finished medicinal product. The viability of hMSCs in different packaging media was studied for 60 h at 4 °C. The best medium to maintain hMSCs viability was then selected to test storage conditions (4, 8, 25 and 37 °C; 60 h). The results showed that at 4 °C the viability was maintained above 80% for 48 h, at 8 °C decreased slightly, whereas at room temperature and 37 °C decreased drastically. Its biocompatibility was assessed by cell morphology and cell viability assays. During stability study, the stored cells did not show any change in their phenotypic or genotypic characteristics and physicochemical properties remained constant, the ability to differentiate into adipocytes and osteocytes and sterility requirements were also unaltered. Finally, our paper proposes a packing media composed of albumin 20%, glucose 5% and Ringer's lactate at a concentration of 1 × 106 cells/mL, which must be stored at 4 °C as the most suitable to maintain cell viability (>80%) and without altering their characteristics for more than 48 h. © 2013 Elsevier B.V. All rights reserved.This study was supported by Grants RD08/0010/2005 (Red TERCEL) and PI10/00964 from Institute of Heath Carlos III and TRA-120 (Ministry of Health) to BS.Peer Reviewe

Movile Device Traffic analysis with Supervised Machine Learning Techniques

Trabajo de Fin de Doble Grado en Administración y Dirección de Empresas - Ingeniería Informática, Facultad de Informática UCM, Departamento de Ingeniería del Software e Inteligencia Artificial, Curso 2021/2022Este proyecto, está destinado a desarrollar una herramienta que implemente el Machine Learning y el Deep learning para el análisis de tráfico. Es decir, aportar una aplicación de investigación al análisis forense con el fin de perseguir los ciberataques. Dado a las ventajas que aporta Python, se ha optado por ser el lenguaje de desarrollo de este proyecto haciendose uso de las diferentes libreria que contiene como Keras y Tensor Flow.This project, is intended to develop a tool which implements machine learning and deep learning to analyze the network´s traffic. This means, contribute with an application that investigates the cyber attacks. Due to the advantages of Python, its have been selected to be the language in which the project has been develop, using the different libraries it contains such as Keras and Tensor Flow.Depto. de Ingeniería de Software e Inteligencia Artificial (ISIA)Fac. de InformáticaTRUEunpu

Preservation of critical quality attributes of mesenchymal stromal cells in 3D bioprinted structures by using natural hydrogel scaffolds

Three dimensional (3D) bioprinting is an emerging technology that enables complex spatial modeling of cell-based tissue engineering products, whose therapeutic potential in regenerative medicine is enormous. However, its success largely depends on the definition of a bioprintable zone, which is specific for each combination of cell-loaded hydrogels (or bioinks) and scaffolds, matching the mechanical and biological characteristics of the target tissue to be repaired. Therefore proper adjustment of the bioink formulation requires a compromise between: (i) the maintenance of cellular critical quality attributes (CQA) within a defined range of specifications to cell component, and (ii) the mechanical characteristics of the printed tissue to biofabricate. Herein, we investigated the advantages of using natural hydrogel-based bioinks to preserve the most relevant CQA in bone tissue regeneration applications, particularly focusing on cell viability and osteogenic potential of multipotent mesenchymal stromal cells (MSCs) displaying tripotency in vitro, and a phenotypic profile of 99.9% CD105(+)/CD45,(-) 10.3% HLA-DR,(+) 100.0% CD90,(+) and 99.2% CD73(+)/CD31(-) expression. Remarkably, hyaluronic acid, fibrin, and gelatin allowed for optimal recovery of viable cells, while preserving MSC's proliferation capacity and osteogenic potency in vitro. This was achieved by providing a 3D structure with a compression module below 8.8 +/- 0.5 kPa, given that higher values resulted in cell loss by mechanical stress. Beyond the biocompatibility of naturally occurring polymers, our results highlight the enhanced protection on CQA exerted by bioinks of natural origin (preferably HA, gelatin, and fibrin) on MSC, bone marrow during the 3D bioprinting process, reducing shear stress and offering structural support for proliferation and osteogenic differentiation

Progress in 3D Bioprinting Technology for Osteochondral Regeneration

Osteochondral injuries can lead to osteoarthritis (OA). OA is characterized by the progressive degradation of the cartilage tissue together with bone tissue turnover. Consequently, joint pain, inflammation, and stiffness are common, with joint immobility and dysfunction being the most severe symptoms. The increase in the age of the population, along with the increase in risk factors such as obesity, has led OA to the forefront of disabling diseases. In addition, it not only has an increasing prevalence, but is also an economic burden for health systems. Current treatments are focused on relieving pain and inflammation, but they become ineffective as the disease progresses. Therefore, new therapeutic approaches, such as tissue engineering and 3D bioprinting, have emerged. In this review, the advantages of using 3D bioprinting techniques for osteochondral regeneration are described. Furthermore, the biomaterials, cell types, and active molecules that are commonly used for these purposes are indicated. Finally, the most recent promising results for the regeneration of cartilage, bone, and/or the osteochondral unit through 3D bioprinting technologies are considered, as this could be a feasible therapeutic approach to the treatment of OA.This research was funded by the BASQUE COUNTRY GOVERNMENT/EUSKO JAURLARITZA (Department of Education, University and Research, Consolidated Groups IT907- 16). Author S.R.-A. thank the BASQUE COUNTRY GOVERNMENT for the granted fellowship (PRE_2021_2_0153)

Evaluation of Glycerylphytate Crosslinked Semi- and Interpenetrated Polymer Membranes of Hyaluronic Acid and Chitosan for Tissue Engineering

In the present study, semi- and interpenetrated polymer network (IPN) systems based on hyaluronic acid (HA) and chitosan using ionic crosslinking of chitosan with a bioactive crosslinker, glycerylphytate (G1Phy), and UV irradiation of methacrylate were developed, characterized and evaluated as potential supports for tissue engineering. Semi- and IPN systems showed significant differences between them regarding composition, morphology, and mechanical properties after physicochemical characterization. Dual crosslinking process of IPN systems enhanced HA retention and mechanical properties, providing also flatter and denser surfaces in comparison to semi-IPN membranes. The biological performance was evaluated on primary human mesenchymal stem cells (hMSCs) and the systems revealed no cytotoxic effect. The excellent biocompatibility of the systems was demonstrated by large spreading areas of hMSCs on hydrogel membrane surfaces. Cell proliferation increased over time for all the systems, being significantly enhanced in the semi-IPN, which suggested that these polymeric membranes could be proposed as an effective promoter system of tissue repair. In this sense, the developed crosslinked biomimetic and biodegradable membranes can provide a stable and amenable environment for hMSCs support and growth with potential applications in the biomedical field.The authors thanks to “La Caixa” Foundation (ID 100010434), which supported Ana Mora-Boza (scholarship code LCF/BQ/ES16/11570018) and to the Spanish Ministry of Economy and Competitiveness for financial support (project RTC-2016-5451-1) and the Fundación Mutua Madrileña (project FMM-AP17196-2019). M. R. Aguilar and B. Vázquez-Lasa are members of the SusPlast platform (Interdisciplinary Platform for Sustainable Plastics towards a Circular Economy) from the Spanish National Research Council (CSIC)

Development, characterization and sterilisation of Nanocellulose-alginate-(hyaluronic acid)- bioinks and 3D bioprinted scaffolds for tissue engineering

3D-bioprinting is an emerging technology of high potential in tissue engineering (TE), since it shows effective control over scaffold fabrication and cell distribution. Biopolymers such as alginate (Alg), nanofibrillated cellulose (NC) and hyaluronic acid (HA) offer excellent characteristics for use as bioinks due to their excellent biocompatibility and rheological properties. Cell incorporation into the bioink requires sterilisation assurance, and autoclave, β-radiation and γ-radiation are widely used sterilisation techniques in biomedicine; however, their use in 3D-bioprinting for bioinks sterilisation is still in their early stages. In this study, different sterilisation procedures were applied on NC-Alg and NC-Alg-HA bioinks and their effect on several parameters was evaluated. Results demonstrated that NC-Alg and NC-Alg-HA bioinks suffered relevant rheological and physicochemical modifications after sterilisation; yet, it can be concluded that the short cycle autoclave is the best option to sterilise both NC-Alg based cell-free bioinks, and that the incorporation of HA to the NC-Alg bioink improves its characteristics. Additionally, 3D scaffolds were bioprinted and specifically characterized as well as the D1 mesenchymal stromal cells (D1-MSCs) embedded for cell viability analysis. Notably, the addition of HA demonstrates better scaffold properties, together with higher biocompatibility and cell viability in comparison with the NC-Alg scaffolds. Thus, the use of MSCs containing NC-Alg based scaffolds may become a feasible tissue engineering approach for regenerative medicine.Author thanks the Basque Government for granted fellowship to S. Ruiz-Alonso (PRE_2020_2_0143). This study was financially supported by the Basque Country Government (IT907-16), the Ministerio de Economía, Industria y Competitividad (FEDER funds, project RTC-2016- 5451-1), Fundación Mutua Madrileña (project FMM-AP17196-2019), the Instituto de Salud Carlos III, ERDF funds (DTS19/00145) and by the Consejería de Economía, Conocimiento, Empresas y Universidad, Junta de Andalucía (project no. PY18-2470 and SOMM17/6109/UGR, FEDER Funds). Authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/ EHU

Sphingolipid extracts enhance gene delivery of cationic lipid vesicles into retina and brain

[EN]The aim was to evaluate relevant biophysic processes related to the physicochemical features and gene transfection mechanism when sphingolipids are incorporated into a cationic niosome formulation for non-viral gene delivery to central nervous system. For that, two formulations named niosphingosomes and niosomes devoid of sphingolipid extracts, as control, were developed by the oil-in water emulsion technique. Both formulations and the corresponding complexes, obtained upon the addition of the reporter EGFP plasmid, were physicochemically and biologically characterized and evaluated. Compared to niosomes, niosphingosomes, and the corresponding complexes decreased particle size and increased superficial charge. Although there were not significant differences in the cellular uptake, cell viability and transfection efficiency increased when human retinal pigment epithelial (ARPE-19) cells were exposed to niosphingoplexes. Endocytosis via caveolae decreased in the case of niosphingoplexes, which showed higher co-localization with lysosomal compartment, and endosomal escape properties. Moreover, niosphingoplexes transfected not only primary central nervous system cells, but also different cells in mouse retina, depending on the administration route, and brain cortex. These preliminary results suggest that niosphingosomes represent a promising non-viral vector formulation purposed for the treatment of both retinal and brain diseases by gene therapy approach.This work was supported by the Basque Country Government (Department of Education, University and Research, Consolidated Groups IT907-16) . Additional funding was provided by the CIBER of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) , an initiative of the Carlos III Health Institute (ISCIII) . I.V.B. and M.S.R. thank the University of the Basque Country (UPV/EHU) for the granted postdoctoral fellowship (ESPDOC19/47) and the granted pre-doctoral fellowship (PIF17/79) , respectively. Authors wish to thank the intel-lectual and technical assistance from the ICTS "NANBIOSIS," more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU) . Technical and human sup-port provided by SGIKER (UPV/EHU) is also gratefully acknowledged

El huerto didáctico de la Facultad de Educación-Centro de Formación del profesorado como espacio para trabajar el cambio climático y la educación para la sostenibilidad en futuros maestros de Educación Infantil y Primaria

Se detallan las actividades realizadas en el huerto didáctico de la facultad de educación en relación al proyecto 212 de la convocatoria 2019 de PIMCD de la UCM