Development of a 3D in Vitro Disease Model for Multiple Myeloma

[ES] La ingeniería tisular ha evolucionado hacia el modelado de la fisiología humana in vitro. El microambiente de la médula ósea (BM) es también hogar de procesos malignos. El mieloma múltiple (MM) es una neoplasia hematológica caracterizada por proliferación y acumulación en la BM de células plasmáticas monoclonales. Los tratamientos han mejorado, sin embargo, sigue siendo incurable. Moléculas de la matriz extracelular como fibronectina (FN) o ácido hialurónico (HA) tienen un papel reconocido en la resistencia a fármacos (DR). La inadecuación de los modelos preclínicos bidimensionales es una de las bases del problema de DR. Se han intentado diferentes enfoques in vitro, sin embargo, se basan en hidrogeles y andamios celulares diseñados para células adherentes, mientras que las células de MM presentan crecimiento en suspensión. El objetivo principal de esta Tesis es desarrollar, optimizar y validar una plataforma de cultivo 3D, denominada microgel, basada en microesferas en un medio líquido y que coexisten con células de MM creciendo dinámicamente en suspensión. Se desarrollaron y caracterizaron diferentes microesferas con diferentes funcionalizaciones. Optimizamos un protocolo de polimerización en suspensión para la obtención de microesferas a base de acrilatos con dos composiciones diferentes (presencia (10%) o ausencia (0%) de ácido acrílico (AA)) i dos distribuciones de tamaño diferentes ( 70 ¿m). La FN se adsorbió en la superficie de la microesfera, mientras que el HA, colágeno I y diferentes secuencias peptídicas se injertaron covalentemente. Se modificaron las microesferas comerciales Cytodex 1 para adaptar sus características a la plataforma. Se utilizaron técnicas capa por capa (LbL) para introducir HA y sulfato de condroitina (CS) en su superficie. Por tanto, se ha generado un amplio repertorio de microesferas para desarrollar microgeles. Se optimizaron y validaron las condiciones de cultivo para la plataforma de microgel. Las condiciones óptimas se establecieron como 150 rpm de velocidad de agitación utilizando un agitador orbital y microesferas de < 60 ¿m. Los microgeles con diferentes composiciones y funcionalizaciones permitieron una buena proliferación de las líneas RPMI8226, U226 y MM1.S. Todos los sistemas respetaron el patrón de crecimiento en suspensión, factor que ha demostrado ser clave para su buen desempeño en cultivo 3D. En estudios iniciales de DR, la línea celular RPMI8226 cultivada en microgeles que contenían AA mostró una resistencia significativamente mayor a la dexametasona que sus cultivos en suspensión. Y las líneas RPMI8226, U226 y MM1.S cultivadas en microgeles que contenían AA mostraron una resistencia significativamente mayor a bortezomib que sus cultivos en suspensión. Por lo tanto, la presencia de AA en la matriz polimérica mostró un efecto positivo en la generación de DR in vitro y requerirá más estudios. Se ha validado la reducción de escala del sistema para trabajar con volúmenes más pequeños de microesferas y números reducidos de células, lo que es de gran relevancia para su traslación clínica. Finalmente, se han realizado cultivos preliminares con la línea celular RPMI8226 en los microgeles basados en Cytodex 1. Las microesferas de Cytodex 1 sin modificación tuvieron un efecto negativo sobre la viabilidad de las células de MM. La modificación mediante LbL con los pares quitosano/CS y quitosano/HA aumentó la viabilidad y proliferación. Sin embargo, estos sistemas no respetaron el carácter no adherente de las células MM. Hemos desarrollado y validado un novedoso sistema de cultivo basado en un medio 3D semisólido definido por microesferas y células de MM especialmente diseñado para células en suspensión. Este sistema constituye una herramienta versátil que debe explorarse más a fondo para el cultivo 3D de neoplasias hematológicas y para estudios de resistencia a fármacos in vitro.[CAT] L'enginyeria tissular ha evolucionat cap al modelat de la fisiologia humana in vitro. El complex microambient de la medul·la òssia (BM) és també llar d'alguns processos malignes. El mieloma múltiple (MM) és una neoplàsia hematològica caracteritzada per una proliferació i acumulació a la BM de cèl·lules plasmàtiques monoclonals. Els tractaments han millorat, no obstant, el MM segueix sent incurable. Molècules de la matriu extracel·lular com fibronectina (FN) o àcid hialurònic (HA) tenen un paper reconegut en la generació de resistència a fàrmacs (DR) en MM. La inadequació dels models preclínics bidimensionals és una de les bases del problema de DR. Per això, s'han intentat diferents aproximacions in vitro, tanmateix es basen en hidrogels i andamis cel·lulars dissenyats per a cèl·lules adherents, mentre que les cèl·lules de MM presenten creixement en suspensió. L'objectiu principal d'aquesta Tesi és desenvolupar, optimitzar i validar una plataforma de cultiu 3D, denominada microgel, basada en microesferes en un medi líquid i que coexisteixen amb cèl·lules de MM que creixen dinàmicament en suspensió. S'han produït i caracteritzat diferents microesferes amb diferents funcionalitzacions. S'ha optimitzat un protocol de polimerització en suspensió per a l'obtenció de microesferes d'acrilats amb dues composicions diferents (presència (10%) o absència (0%) d'àcid acrílic (AA)) i amb dos distribucions de diàmetres diferents ( 70 ¿m). La FN es va adsorbir, mentre que el HA, el col·lagen I i diferents seqüències peptídiques es van unir covalentment. S'han modificat microesferes comercials Cytodex 1 per tal d'adaptar les seves característiques a la plataforma del microgel. Mitjançant tècniques capa a capa (LbL) s'han introduït HA i sulfat de condroïtina (CS) a la seua superfície. Per tant, s'ha generat un ampli repertori de microesferes per desenvolupar microgels. Es van optimitzar i validar les condicions de cultiu per a la plataforma de microgel. Les condicions òptimes de cultiu es varen establir com a 150 rpm de velocitat d'agitació utilitzant un agitador orbital i microesferes de < 60 ¿m. Els microgels amb diferents composicions i funcionalitzacions van permetre una bona proliferació de les línies RPMI8226, U226 i MM1.S. Tots els sistemes van respectar el patró de creixement en suspensió, factor que ha demostrat ser clau per al seu bon rendiment en cultius 3D. En estudis inicials de DR línia cel·lular RPMI8226 cultivada en microgels que contenien AA va mostrar una resistència significativament major a la dexametasona que els seus cultius en suspensió convencionals. Línies RPMI8226, U226 y MM1.S cultivades en microgels que contenien AA mostraren una resistència significativament major a bortezomib que els seus cultius en suspensió convencionals. Per tant, la presencia d'AA a la matriu polimèrica de les microesferes va mostrar un efecte positiu en termes de generació de DR in vitro, cosa que requerirà estudis futurs. S'ha validat la reducció de l'escala del sistema per treballar amb volums més petits de microesferes i menys cèl·lules, el que és de gran rellevància per a la seva translació clínica. Finalment, s'han realitzat cultius preliminars amb la línia cel·lular RPMI8226 en els microgels basats en les Cytodex 1. Les microesferes de Cytodex 1 sense modificar van mostrar efecte negatiu sobre la viabilitat de les cèl·lules de MM. La modificació mitjançant LbL amb els parells quitosà/CS i quitosà/HA va augmentar la viabilitat i proliferació de cèl·lules MM. No obstant, aquests sistemes no respectaren el caràcter no adherent de les cèl·lules de MM. S'ha desenvolupat i validat un nou sistema de cultiu cel·lular basat en un medi 3D semisòlid definit per microesferes i cèl·lules de MM, especialment dissenyat per a cèl·lules no adherents. Aquest sistema constitueix una eina versàtil que ha de ser explorada per al cultiu 3D de neoplàsies hematològiques i per a estudis de resistència a fàrmacs in vitro.[EN] Tissue engineering has evolved towards modeling of human physiology in vitro. The bone marrow (BM) microenvironment is likewise the home of some malignant processes. Multiple myeloma (MM) is a hematological neoplasia characterized by proliferation and BM accumulation of monoclonal plasma cells. Treatments have improved; however, MM remains incurable. Extracellular matrix molecules such as fibronectin (FN) or hyaluronic acid (HA) have a recognized role in drug resistance (DR). The inadequacy of two-dimensional preclinical models is one cause of the DR problem, different in vitro approaches have been developed, however, all these studies are based on hydrogels and scaffolds designed for adherent cells while MM cells are suspension growing cells. The main objective of this Thesis is to develop, optimize and validate a 3D culture platform, termed as microgel, based on microspheres suspended in a liquid media and coexisting with MM cells growing dynamically in suspension. Different microspheres with different functionalities were developed and characterized. We optimized a suspension polymerization protocol for the obtention of acrylates-based microspheres with two different compositions: with presence (10%) or absence (0%) of acrylic acid (AA). We obtained two different size distributions ( 70 ¿m). FN was adsorbed on microsphere surface, while HA, collagen I and different peptide sequences were covalently grafted. Commercial Cytodex 1 microspheres were modified to adapt their characteristics to the microgel platform. Layer-by-layer (LbL) technics were used to introduce HA and chondroitin sulfate (CS) on Cytodex 1 surface. Therefore, a wide repertoire of microspheres has been generated to develop microgels. The culture conditions for the microgel platform were optimized and validated. Agitation is needed to keep microspheres and cells in suspension. Optimal culture conditions were 150 rpm of stirring speed using orbital shaker and < 60 ¿m diameter microspheres. Microgels with different compositions (0% AA, 10% AA) and functionalizations (none, HA, FN, collagen 1 and peptide sequences) allowed good proliferation of RPMI8226, U226 and MM1.S cells under 3D conditions. All the 3D systems respected the suspension growth pattern which appears as key factor for their good performance in 3D culture. In the initial DR studies, we found that MM cell line RPMI8226 cultured in microgels containing AA showed significantly higher resistance to dexamethasone than their conventional suspension cultures. And that MM cell lines RPMI8226, U226 and MM1.S cultured in microgels containing AA showed significantly higher resistance to bortezomib than their conventional suspension cultures. Thus, AA in the polymeric microsphere matrix showed a positive effect on the generation of DR in vitro and will require further studies. The scale-down of the system to work with smaller volumes of microspheres and reduced cell numbers has been validated, this is of great relevance for their clinical application. Finally, preliminary cultures with the cell line RPMI8226 have been performed with the Cytodex 1-based microgels. Cytodex 1 microspheres without modification had a negative effect on MM cells viability. LbL modification with the pairs chitosan/CS and chitosan/HA increased MM cells viability and proliferation. However, these systems did not respect the non-adherent character of MM cells. We have developed and validated a novel cell culture system based on a semi-solid 3D media defined by microspheres and MM cells which is specially designed for cells in suspension. It represents a versatile tool that should be further explored for the 3D culture of hematological malignancies and drug resistance studies in vitro.Me gustaría agradecer al Servicio de Microscopía de la UPV y a sus técnicos por su valiosa ayuda con las técnicas de microscopía electrónica, a la Agencia Estatal de Investigación (proyecto PID2019-106099RB-C41 / AEI / 10.13039/501100011033) y al Ministerio de Ciencia, Innovación y Universidades (ayuda predoctoral FPU17/05810) que han financiado esta Tesis.Clara Trujillo, S. (2022). Development of a 3D in Vitro Disease Model for Multiple Myeloma [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/186054TESI

In Vitro Modeling of Non-Solid Tumors: How Far Can Tissue Engineering Go?

[EN] In hematological malignancies, leukemias or myelomas, malignant cells present bone marrow (BM) homing, in which the niche contributes to tumor development and drug resistance. BM architecture, cellular and molecular composition and interactions define differential microenvironments that govern cell fate under physiological and pathological conditions and serve as a reference for the native biological landscape to be replicated in engineered platforms attempting to reproduce blood cancer behavior. This review summarizes the different models used to efficiently reproduce certain aspects of BM in vitro; however, they still lack the complexity of this tissue, which is relevant for fundamental aspects such as drug resistance development in multiple myeloma. Extracellular matrix composition, material topography, vascularization, cellular composition or stemness vs. differentiation balance are discussed as variables that could be rationally defined in tissue engineering approaches for achieving more relevant in vitro models. Fully humanized platforms closely resembling natural interactions still remain challenging and the question of to what extent accurate tissue complexity reproduction is essential to reliably predict drug responses is controversial. However, the contributions of these approaches to the fundamental knowledge of non-solid tumor biology, its regulation by niches, and the advance of personalized medicine are unquestionable.PROMETEO/2016/063 project is acknowledged. The CIBER-BBN initiative is funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. 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CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy. Blood, 113(18), 4341-4351. doi:10.1182/blood-2008-10-186668Jacamo, R., Chen, Y., Wang, Z., Ma, W., Zhang, M., Spaeth, E. L., … Andreeff, M. (2014). Reciprocal leukemia-stroma VCAM-1/VLA-4-dependent activation of NF-κB mediates chemoresistance. Blood, 123(17), 2691-2702. doi:10.1182/blood-2013-06-511527Hatano, K., Kikuchi, J., Takatoku, M., Shimizu, R., Wada, T., Ueda, M., … Ozawa, K. (2008). Bortezomib overcomes cell adhesion-mediated drug resistance through downregulation of VLA-4 expression in multiple myeloma. Oncogene, 28(2), 231-242. doi:10.1038/onc.2008.385Bourgine, P. E., Martin, I., & Schroeder, T. (2018). Engineering Human Bone Marrow Proxies. Cell Stem Cell, 22(3), 298-301. doi:10.1016/j.stem.2018.01.002Chramiec, A., & Vunjak-Novakovic, G. (2019). Tissue engineered models of healthy and malignant human bone marrow. Advanced Drug Delivery Reviews, 140, 78-92. doi:10.1016/j.addr.2019.04.003Tavakol, D. N., Tratwal, J., Bonini, F., Genta, M., Campos, V., Burch, P., … Braschler, T. (2020). Injectable, scalable 3D tissue-engineered model of marrow hematopoiesis. Biomaterials, 232, 119665. doi:10.1016/j.biomaterials.2019.119665Isern, J., Martín-Antonio, B., Ghazanfari, R., Martín, A. M., López, J. A., del Toro, R., … Méndez-Ferrer, S. (2013). Self-Renewing Human Bone Marrow Mesenspheres Promote Hematopoietic Stem Cell Expansion. Cell Reports, 3(5), 1714-1724. doi:10.1016/j.celrep.2013.03.041Jing, D., Fonseca, A. V., Alakel, N., Fierro, F. A., Muller, K., Bornhauser, M., … Ordemann, R. (2010). Hematopoietic stem cells in co-culture with mesenchymal stromal cells - modeling the niche compartments in vitro. Haematologica, 95(4), 542-550. doi:10.3324/haematol.2009.010736Butler, J. M., Gars, E. J., James, D. J., Nolan, D. J., Scandura, J. M., & Rafii, S. (2012). Development of a vascular niche platform for expansion of repopulating human cord blood stem and progenitor cells. Blood, 120(6), 1344-1347. doi:10.1182/blood-2011-12-398115Leisten, I., Kramann, R., Ventura Ferreira, M. S., Bovi, M., Neuss, S., Ziegler, P., … Schneider, R. K. (2012). 3D co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche. Biomaterials, 33(6), 1736-1747. doi:10.1016/j.biomaterials.2011.11.034Raic, A., Rödling, L., Kalbacher, H., & Lee-Thedieck, C. (2014). Biomimetic macroporous PEG hydrogels as 3D scaffolds for the multiplication of human hematopoietic stem and progenitor cells. Biomaterials, 35(3), 929-940. doi:10.1016/j.biomaterials.2013.10.038Severn, C. E., Macedo, H., Eagle, M. J., Rooney, P., Mantalaris, A., & Toye, A. M. (2016). Polyurethane scaffolds seeded with CD34+ cells maintain early stem cells whilst also facilitating prolonged egress of haematopoietic progenitors. Scientific Reports, 6(1). doi:10.1038/srep32149Mahadik, B. P., Bharadwaj, N. A. K., Ewoldt, R. H., & Harley, B. A. C. (2017). Regulating dynamic signaling between hematopoietic stem cells and niche cells via a hydrogel matrix. Biomaterials, 125, 54-64. doi:10.1016/j.biomaterials.2017.02.013Wilkinson, A. C., Ishida, R., Kikuchi, M., Sudo, K., Morita, M., Crisostomo, R. V., … Yamazaki, S. (2019). Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature, 571(7763), 117-121. doi:10.1038/s41586-019-1244-xSieber, S., Wirth, L., Cavak, N., Koenigsmark, M., Marx, U., Lauster, R., & Rosowski, M. (2017). Bone marrow‐on‐a‐chip: Long‐term culture of human haematopoietic stem cells in a three‐dimensional microfluidic environment. Journal of Tissue Engineering and Regenerative Medicine, 12(2), 479-489. doi:10.1002/term.2507Bourgine, P. E., Klein, T., Paczulla, A. M., Shimizu, T., Kunz, L., Kokkaliaris, K. D., … Martin, I. (2018). In vitro biomimetic engineering of a human hematopoietic niche with functional properties. Proceedings of the National Academy of Sciences, 115(25), E5688-E5695. doi:10.1073/pnas.1805440115De la Puente, P., Muz, B., Gilson, R. C., Azab, F., Luderer, M., King, J., … Azab, A. K. (2015). 3D tissue-engineered bone marrow as a novel model to study pathophysiology and drug resistance in multiple myeloma. Biomaterials, 73, 70-84. doi:10.1016/j.biomaterials.2015.09.017Torisawa, Y., Spina, C. S., Mammoto, T., Mammoto, A., Weaver, J. C., Tat, T., … Ingber, D. E. (2014). Bone marrow–on–a–chip replicates hematopoietic niche physiology in vitro. Nature Methods, 11(6), 663-669. doi:10.1038/nmeth.2938Reinisch, A., Hernandez, D. C., Schallmoser, K., & Majeti, R. (2017). Generation and use of a humanized bone-marrow-ossicle niche for hematopoietic xenotransplantation into mice. Nature Protocols, 12(10), 2169-2188. doi:10.1038/nprot.2017.088Theocharides, A. P. A., Rongvaux, A., Fritsch, K., Flavell, R. A., & Manz, M. G. (2015). Humanized hemato-lymphoid system mice. Haematologica, 101(1), 5-19. doi:10.3324/haematol.2014.115212Abarrategi, A., Mian, S. A., Passaro, D., Rouault-Pierre, K., Grey, W., & Bonnet, D. (2018). Modeling the human bone marrow niche in mice: From host bone marrow engraftment to bioengineering approaches. Journal of Experimental Medicine, 215(3), 729-743. doi:10.1084/jem.20172139Rose-Zerilli, M. J. J., Gibson, J., Wang, J., Tapper, W., Davis, Z., Parker, H., … Strefford, J. C. (2016). Longitudinal copy number, whole exome and targeted deep sequencing of «good risk» IGHV-mutated CLL patients with progressive disease. Leukemia, 30(6), 1301-1310. doi:10.1038/leu.2016.10Reinisch, A., Thomas, D., Corces, M. R., Zhang, X., Gratzinger, D., Hong, W.-J., … Majeti, R. (2016). A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells. Nature Medicine, 22(7), 812-821. doi:10.1038/nm.4103Vaiselbuh, S. R., Edelman, M., Lipton, J. M., & Liu, J. M. (2010). Ectopic Human Mesenchymal Stem Cell-Coated Scaffolds in NOD/SCID Mice: An In Vivo Model of the Leukemia Niche. Tissue Engineering Part C: Methods, 16(6), 1523-1531. doi:10.1089/ten.tec.2010.0179Groen, R. W. J., Noort, W. A., Raymakers, R. A., Prins, H.-J., Aalders, L., Hofhuis, F. M., … Martens, A. C. M. (2012). Reconstructing the human hematopoietic niche in immunodeficient mice: opportunities for studying primary multiple myeloma. Blood, 120(3), e9-e16. doi:10.1182/blood-2012-03-414920Chen, Y., Jacamo, R., Shi, Y., Wang, R., Battula, V. L., Konoplev, S., … Andreeff, M. (2012). Human extramedullary bone marrow in mice: a novel in vivo model of genetically controlled hematopoietic microenvironment. Blood, 119(21), 4971-4980. doi:10.1182/blood-2011-11-389957Holzapfel, B. M., Hutmacher, D. W., Nowlan, B., Barbier, V., Thibaudeau, L., Theodoropoulos, C., … Levesque, J.-P. (2015). Tissue enginee

Novel microgel culture system as semi-solid three-dimensional in vitro model for the study of multiple myeloma proliferation and drug resistance

[EN] Multiple myeloma (MM) is a hematological malignancy in which the patient's drug resistance is one of the main clinical problems. As 2D cultures do not recapitulate the cellular microenvironment, which has a key role in drug resistance, there is an urgent need for better biomimetic models. Here, a novel 3D platform is used to model MM. The semi-solid culture consists of a dynamic suspension of microspheres and MM cells, termed as microgel. Microspheres are synthesized with acrylic polymers of different sizes, compositions, and functionalities (fibronectin or hyaluronic acid). Optimal conditions for the platform in terms of agitation speed and microsphere size have been determined. With these parameters the system allows good proliferation of the MM cell lines RPMI8226, U226, and MM1.S. Interestingly, when used for drug resistance studies, culture of the three MM cell lines in microgels showed close agreement in revealing the role of acrylic acid in resistance to anti-MM drugs such as dexamethasone and bortezomib. This work presents a unique platform for the in vitro modeling of non-solid tumors since it allows keeping non-adherent cells in suspension conditions but in a 3D context that can be easily tuned with different functionalizations.This work was funded by the Spanish State Research Agency (AEI) through the PID2019-106099RB-C41/AEI/10.13039/501100011033 Project. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER Actions were financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was also supported by the Spanish Ministry of Science, Innovation and Universities through Grant NO FPU17/05810 awarded to Sandra Clara-Trujillo. The Microscopy Service of the UPV (UniversitatPolitecnica de València) is gratefully acknowledged for helping with FESEM characterization.Clara-Trujillo, S.; Tolosa, L.; Cordón, L.; Sempere, A.; Gallego Ferrer, G.; Gómez Ribelles, JL. (2022). Novel microgel culture system as semi-solid three-dimensional in vitro model for the study of multiple myeloma proliferation and drug resistance. Biomaterials Advances. 135:1-13. https://doi.org/10.1016/j.bioadv.2022.21274911313

Biomimetic microspheres for 3D mesenchymal stem cell culture and characterization

[EN] Stem cells reside in niches, specialized microenvironments that sustain and regulate their fate. Extracellular matrix (ECM), paracrine factors or other cells are key niche regulating elements. As the conventional 2D cell culture lacks these elements, it can alter the properties of naive stem cells. In this work we designed a novel biomimetic microenvironment for cell culture, consisting of magnetic microspheres, prepared with acrylates and acrylic acid copolymers and functionalized with fibronectin or hyaluronic acid as ECM coatings. To characterize cell proliferation and adhesion, porcine mesenchymal stem cells (MSCs) were grown with the different microspheres. The results showed that the 3D environments presented similar proliferation to the 2D culture and that fibronectin allows cell adhesion, while hyaluronic acid hinders it. In the 3D environments, cells reorganize the microspheres to grow in aggregates, highlighting the advantages of microspheres as 3D environments and allowing the cells to adapt the environment to their requirements.PROMETEO/2016/063 project is acknowledged. This work was partially financed with FEDER funds (CIBERONC (CB16/12/00284)). The CIBER-BBN initiative is funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was also supported by the Spanish Ministry of Science, Innovation and Universities trough Sandra Clara-Trujillo FPU17/05810 grant.Clara-Trujillo, S.; Marin-Paya, JC.; Cordón, L.; Sempere, A.; Gallego Ferrer, G.; Gómez Ribelles, JL. (2019). Biomimetic microspheres for 3D mesenchymal stem cell culture and characterization. Colloids and Surfaces B Biointerfaces. 177:68-76. https://doi.org/10.1016/j.colsurfb.2019.01.050S687617

El pensamiento crítico como un paso adelante en la Formación Profesional. Estudiantes de Formación Profesional inmersos en centros de alta tecnología

[EN] This communication summarizes the results of the Erasmus+ project "CRİTİCAL THİNKİNG AS A STEP FORWARD İN VET EDUCATİON: VET students immersed in high technology teams" in which european technologybased companies, higher research centers and vocational training centers have participated. For two years, strategies have been designed to develop training in critical thinking tools in vocational students during internships in high-tech centers. We have chosen a reduced set of critical thinking skills that we think can act as a catalyst for student training throughout their professional careers, and in creative work that contributes effectively to the company's objectives. The debate has crystallized in the writing of two tools: a template of help to the tutor in the company for the preparation of the work plan of the student and another template for the student's laboratory notebook. In one case and in another the writing of these notebooks requires the student and the tutor to reflect on the critical thinking skills that are intended to develop in the student.[ES] Esta comunicación resume parte de los resultados del proyecto Erasmus+ “CRİTİCAL THİNKİNG AS A STEP FORWARD İN VET EDUCATİON: VET students immersed in high technology teams” en el que han participado empresas de base tecnológica, centros superiores de investigación y centros de formación profesional europeos. Durante dos años se han diseñado estrategias para desarrollar la formación en herramientas de pensamiento crítico en los estudiantes de formación profesional durante prácticas realizadas en centros de alta tecnología. Se ha elegido un conjunto reducido de capacidades de pensamiento crítico que pensamos que pueden actuar de desencadenante de la formación del estudiante a lo largo de toda su carrera profesional, y en un trabajo creativo que contribuya de forma efectiva a los objetivos de la empresa. El debate ha cristalizado en la redacción de dos herramientas: una plantilla de ayuda al tutor en la empresa para la preparación del plan de trabajo del estudiante y otra plantilla para el cuaderno de laboratorio del estudiante. En un caso y en otro la redacción de estos cuadernos obliga al estudiante y al tutor a una reflexión relacionada con las capacidades de pensamiento crítico que se pretenden desarrollar en el estudiante.Este trabajo ha sido financiado por el programa Erasmus + de la dentro del proyecto 2017-1-ES01-KA202-038469, Servicio Español para la Internacionalización de la Educación (SEPIE) referencia nacional ZZ02Solano-Martínez, C.; Clara Trujillo, S.; Guillot Ferriols, MT.; Gómez Estrada, L.; Gómez Ribelles, JL. (2019). El pensamiento crítico como un paso adelante en la Formación Profesional. Estudiantes de Formación Profesional inmersos en centros de alta tecnología. En IN-RED 2019. V Congreso de Innovación Educativa y Docencia en Red. Editorial Universitat Politècnica de València. 1342-1353. https://doi.org/10.4995/INRED2019.2019.10340OCS1342135

Reviviendo los pasos de los abuelos del Municipio de Salgar, Antioquia

Following in the footsteps of grandparents is a sociocultural research project that applies a strategy to recover and promote oral tradition along with grandparents from Salgar in Antioquia, Colombia. To this end, it was proposed that orality transcends culture and traditions. As regards methodology, the ethnographic model was used. It allowed us to collect unprecedented and meaningful information for the municipality’s intangible cultural heritage. This process was developed in three phases: the ancestors that had oral fluency linked to ancestral experiences with a cultural and educational content were identified in order to promote such experiences among teenagers and children. The main conclusion was the importance of oral tradition as a two-way street because it brings dignity to grandparents, since they feel heard and embraced, and at the same time, it works as a strategy to foster educational processes.Reviviendo los pasos de los abuelos es una investigación sociocultural cualitativa que aplica un ejercicio de recuperación y difusión de la tradición oral con abuelos del Municipio de Salgar en Antioquia, Colombia. Para ello se planteó como hipótesis que la oralidad trasciende la cultura y costumbres de los pueblos. Desde lo metodológico se siguió el método etnográfico que permitió la recopilación de información inédita y significativa para el patrimonio cultural e inmaterial del Municipio. El proceso se llevó a cabo en tres fases: un diagnóstico para identificar cuales ancestros poseían fluidez oral, en relación a las vivencias ancestrales con un propósito cultural y educativo, finalizando con la difusión entre jóvenes y niños. La principal conclusión fue la importancia de la tradición oral en doble vía en tanto contribuye en la dignificación de los abuelos, al sentirse escuchados y acogidos, a la vez que funciona como estrategia de fortalecimiento de los procesos educativos

Autogestión comunitaria local, base de adaptación al cambio climático en la Orinoquia colombiana

El enfoque participativo local-regional complementa y da contenidos especiales al abordaje académico y técnico de la planeación estratégica regional, llena el enorme vacío de inclusión en la planificación y en la definición de políticas territoriales e incorpora las posiciones complejas y versátiles de actores locales y de la periferia regional. Esta participación es un factor diferencial, analítico y propositivo de las territorialidades, indispensable para la objetividad e integralidad de acciones y estrategias convergentes hacia un concepto común de desarrollo sostenible regional. Éste es un ejercicio clave de desarrollo concertado, basado en procesos participativos a nivel loca. Se siguieron metodologías de tipo cualitativo, tales como caracterización de actores, elaboración de línea de tiempo (de 1950 a 2014), 7 entrevistas semiestructuradas, 75 encuestas, establecimiento de 15 indicadores, análisis multicriterio (modelo de telaraña de sostenibilidad) y construcción de escenarios. Participaron 75 personas como representantes de la sociedad civil de las veredas La Gileña, Nueva Zelandia y el centro poblado de Aguaclara del municipio de Sabanalarga (Casanare). Se logró el conocimiento del uso de su territorio en su multidimensionalidad, la identificación de vulnerabilidades y la concertación de acciones estratégicas en torno a una prospectiva de sostenibilidad en el contexto local: en la dimensión social en los componentes de bienestar, gobernabilidad, asociatividad y saneamiento básico y en la dimensión productiva y en la dimensión ecológica. Este mecanismo de construcción de escenarios surgido de la participación local constituye un ejercicio de autogestión comunitaria que facilita la toma de decisiones, orientadas a la adaptación al cambio climático en un ámbito multisectorial e integral.Trabajo publicado en Acta Bioquímica Clínica Latinoamericana; no. 52, supl. 2, parte I, diciembre de 2018.Universidad Nacional de La Plat

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Tropical Data: Approach and Methodology as Applied to Trachoma Prevalence Surveys

PURPOSE: Population-based prevalence surveys are essential for decision-making on interventions to achieve trachoma elimination as a public health problem. This paper outlines the methodologies of Tropical Data, which supports work to undertake those surveys. METHODS: Tropical Data is a consortium of partners that supports health ministries worldwide to conduct globally standardised prevalence surveys that conform to World Health Organization recommendations. Founding principles are health ministry ownership, partnership and collaboration, and quality assurance and quality control at every step of the survey process. Support covers survey planning, survey design, training, electronic data collection and fieldwork, and data management, analysis and dissemination. Methods are adapted to meet local context and needs. Customisations, operational research and integration of other diseases into routine trachoma surveys have also been supported. RESULTS: Between 29th February 2016 and 24th April 2023, 3373 trachoma surveys across 50 countries have been supported, resulting in 10,818,502 people being examined for trachoma. CONCLUSION: This health ministry-led, standardised approach, with support from the start to the end of the survey process, has helped all trachoma elimination stakeholders to know where interventions are needed, where interventions can be stopped, and when elimination as a public health problem has been achieved. Flexibility to meet specific country contexts, adaptation to changes in global guidance and adjustments in response to user feedback have facilitated innovation in evidence-based methodologies, and supported health ministries to strive for global disease control targets