Evaluation of cell culture in microfluidic chips for application in monoclonal antibody production

Microfluidic chips are useful devices for cell culture that allow cell growth under highly controlled conditions, as is required for production of therapeutic recombinant proteins. To understand the optimal conditions for growth of cells amenable of recombinant protein expression in these devices,we culturedHEK-293T cells under different microfluidic experimental conditions. The cells were cultured in polymethyl methacrylate (PMMA) and polydi-methylsiloxane (PDMS)microdevices, in the absence or presence of the cell adhesion agent poly-D-lysine. Different microchannel geometries and thicknesses, as well as the influence of the flow rate have also been tested, showing their great influence in cell adhesion and growth. Results show that the presence of poly-D-lysine improves the adhesion and viability of the cells in continuous or discontinuous flow. Moreover, the optimal adhesion of cells was observed in the corners of themicrochannels, as well as in wide channels possibly due to the decrease in the flow rate in these areas. These studies provide insight into the optimal architecture of microchannels for long-term culture of adherent cells in order to use microfluidics devices as bioreactors for monoclonal antibodies production.Fil: Peñaherrera Pazmiño, Ana Belén. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Payés, Cristian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Sierra Rodero, Marina. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vega, M.. Universidad Tecnológica Nacional; ArgentinaFil: Rosero, G.. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; ArgentinaFil: Helguera, Gustavo Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pérez, M. S.. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; Argentin

Design and analysis of different models of microfluidic devices evaluated in enhanced oil recovery (EOR) assays

Microfluidic devices are a new platform for Enhanced Oil Recovery (EOR) assays. A successful oil recoveryin a reservoir can be different in another, hence the importance of a methodology for assessment prior toinjection. In the current study, micro and nanotechnology techniques were applied to develop EOR assays.The EOR chips simulated the phenomena occurred in micro-nano scale reservoirs. In general, the first step ofthe experiments corresponded to fill the microchannels with oil, then was injected water at constant flow rateuntil oil recovery ceases and finally was injected polymer or CDG. The recovery behaviors of the fluids werestudied by digital image analysis. Results allowed obtaining oil recovery for each evaluated fluid. Theoptimum configuration of the microchannels showed 80% of oil residual saturation after water injection.Keywords: EOR, poral volume, microgels and polymers, microfuidic devices.Fil: Rosero Yánez, Gustavo Ivan. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; ArgentinaFil: Peñaherrera Pazmiño, Ana Belén. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olmos Carreno, Carol Maritza. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; ArgentinaFil: Boschan, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería; ArgentinaFil: Granell, Pablo Nicolás. Instituto Nacional de Tecnología Industrial; ArgentinaFil: Golmar, Federico. Instituto Nacional de Tecnología Industrial; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lasorsa, Carlos Alberto. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería; Argentin

Epoxy resin mold and PDMS microfluidic devices through photopolymer flexographic printing plate

Photopolymer flexographic printing plate is a new photopolymeric material used for microdevices fabrication. This work demonstrates that a photopolymer flexographic master mold can be used for the fabrication of PDMS (polydimethylsiloxane) microdevices by a multi-step manufacturing process. The methodology entails three main fabrication steps: (1) a photopolymer flexographic printing plate mold (FMold) is generated by UV exposure through a transparent film, (2) an epoxy resin mold (ERmold) is fabricated by transferring the features of the photopolymer mold and (3) a PDMS microdevice is manufactured from the epoxy resin mold. The characterization of the manufactured PDMS microdevices was performed using scanning electron microscopy (SEM) and profilometry. Results showed high accuracy in the replication of the profiles. To show the feasibility of the fabrication process a microdevice for microdroplet generation was designed, manufactured and tested. Hence, the manufacturing process described in this work provides an easy, robust, and low-cost strategy that facilitates the scaling-up of microfluidic devices without requiring any sophisticated equipment.Fil: Olmos Carreno, Carol Maritza. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vaca Mora, Andrea Vanessa. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rosero, Gustavo. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Peñaherrera Pazmiño, Ana Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Pérez Sosa, Camilo José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: de Sá Carneiro, Igor. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Vizuete, Karla. Universidad de Las Fuerzas Armadas Espe; EcuadorFil: Arroyo, Carlos R.. Universidad de Las Fuerzas Armadas; EcuadorFil: Debut, Alexis. Universidad de Las Fuerzas Armadas; EcuadorFil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Instituto de Ingeniería Biomédica; ArgentinaFil: Cumbal, Luis. Universidad de Las Fuerzas Armadas; EcuadorFil: Lerner, Betiana. Universidad de Buenos Aires. Facultad de Ingeniería. Instituto de Ingeniería Biomédica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentin

Mechanical matching between a ligand and receptor

Interactions between ligands and receptors and subsequent ?locking? must involve some resistance to unbinding, manifesting itself as an interaction force. At body temperature, spontaneous unbinding will occur, however, external forces are required to accelerate this process. Bearing in mind the potential forces that the receptor?ligand complex is likely to be subjected to in a biological environment, it might be hypothesised that there is some mechanical matching between the receptor and ligand. To test this hypothesis, various receptor and ligand pairs were unfolded in their entirety in order to determine their total unfolding force. In this way, the total force to unfold the protein could be determined, allowing a comparison between ligand and receptor pairs. The interest of this work is to examine the interaction between five proteins and a mica surface by AFM without any modification to preserve the natural elastic properties of the protein molecules during the force measurements. The results showed a mechanical matching between GP120 (ligand) and CD4 (receptor) when analysing the total force required to unfold the same number of domains or events shown by the force distance curves of these proteins.Fil: Peñaherrera Pazmiño, Ana Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Newcastle; Reino Unid

Novel Reproducible Manufacturing and Reversible Sealing Method for Microfluidic Devices

Conventional manufacturing methods for polydimethylsiloxane (PDMS)-based microdevices require multiple steps and elements that increase cost and production time. Also, these PDMS microdevices are mostly limited to single use, and it is difficult to recover the contents inside the microchannels or perform advanced microscopy visualization due to their irreversible sealing method. Herein, we developed a novel manufacturing method based on polymethylmethacrylate (PMMA) plates adjusted using a mechanical pressure-based system. One conformation of the PMMA plate assembly system allows the reproducible manufacture of PDMS replicas, reducing the cost since a precise amount of PDMS is used, and the PDMS replicas show uniform dimensions. A second form of assembling the PMMA plates permits pressure-based sealing of the PDMS layer with a glass base. By reversibly sealing the microdevice without using plasma for bonding, we achieve chip on/off configurations, which allow the user to open and close the device and reuse it in an easy-to-use way. No deformation was observed on the structures of the PDMS microchannels when a range of 10 to 18 kPa pressure was applied using the technique. Furthermore, the functionality of the proposed system was successfully validated by the generation of microdroplets with reused microdevices via three repetitions

Analysis of tumoral spheres growing in a multichamber microfluidic device

Lab on a Chip (LOC) farming systems have emerged as a powerful tool for single cell studies combined with a non‐adherent cell culture substrate and single cell capture chips for the study of single cell derived tumor spheres. Cancer is characterized by its cellular heterogeneity where only a small population of cancer stem cells (CSCs) are responsible for tumor metastases and recurrences. Thus, the in vitro strategy to the formation of a single cell‐derived sphere is an attractive alternative to identify CSCs. In this study, we test the effectiveness of microdevices for analysis of heterogeneity within CSC populations and its interaction with different components of the extracellular matrix. CSC could be identify using specific markers related to its pluripotency and self‐renewal characteristics such as the transcription factor Oct‐4 or the surface protein CD44. The results confirm the usefulness of LOC as an effective method for quantification of CSC, through the formation of spheres under conditions of low adhesion or growing on components of the extracellular matrix. The device used is also a good alternative for evaluating the individual growth of each sphere and further identification of these CSC markers by immunofluorescence. In conclusion, LOC devices have not only the already known advantages, but they are also a promising tool since they use small amounts of reagents and are under specific culture parameters. LOC devices could be considered as a novel technology to be used as a complement or replacement of traditional studies on culture plates.Fil: Belgorosky, Denise. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández Cabada, Tamara. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Peñaherrera Pazmiño, Ana Belén. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Langle, Yanina Verónica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Booth, Ross. Millipore Sigma Corporation; Estados UnidosFil: Bhansali, Shekhar. University of Florida; Estados UnidosFil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. University of Florida; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Eijan, Ana Maria. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lerner, Betiana. Universidad Tecnologica Nacional. Facultad Regional Haedo. Centro de Ingenieria de Recubrimientos Especiales y Nanoestructuras.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Desarrollo de nuevas metodologías para estudios de Recuperación Asistida de Petróleo (EOR)

El reto de las nuevas metodologías de estudio EOR reside en la compleja interacción de los agentes inyectados con los fluidos que se encuentra en el yacimiento, bajo las condiciones físicas y químicas del mismo. Es así como las metodologías de estudio EOR deben proveer una evaluación completa de los fluidos inyectados.En el área de los dispositivos de microfluídica, los microsistemas analíticos se conciben para integrar varias operaciones de laboratorio: inyección de muestras y reactivos, mezclado, incubación, reacción, separación, detección, aislamiento, etc. Estos microsistemas se identifican como lab-on-a-chip (LOC).La tecnología de Lab-on-a-chip es ideal para estudiar la recuperación asistida de petróleo y su eficacia. Es posible la fabricación de microchips que simulen medios porosos con diseños personalizados, incluyendo poros, canales y la interconexión de diferentes zonas con distintas permeabilidades que se reproducen con precisión, además de proveer transparencia óptica para la visualización de fluidos.Fil: Olmos Carreno, Carol Maritza. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; ArgentinaFil: Rosero Yánez, Gustavo Ivan. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Peñaherrera Pazmiño, Ana Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; ArgentinaFil: Lasorsa, Carlos Alberto. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; ArgentinaFil: Boschan, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perez, Maximiliano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo. Grupo de Ingeniería de Recubrimientos Especiales y Nanotecnología; Argentin

Production of monoclonal antibodies in microfluidic devices

Herein, a microfluidic device with cistern design for cultivation of adherent eukaryotic cells for the production of recombinant proteins is presented. The geometric configuration of the microchannels in the device provided laminar flow with reduced velocity profiles in the cisterns, resulting in an adequate microenvironment for long-term adherent cell growth with passive pumping flow cycles of 24 hours. CHO-ahIFNα2b and HEK-ahIFNα2b adherent cell lines expressing a novel anti-hIFN-α2b recombinant monoclonal antibody (MAb) for the treatment of systemic lupus erythematosus were cultured on the surface of PDMS/glass microchannels coated with poly-d-lysine. A 24 day culture of CHO-ahIFNα2b cells resulted in MAb concentrations up to 166.4 μg mL -1 per day. The productivity of CHO-ahIFNα2b and HEK-ahIFNα2b cell lines was higher in the microdevice compared to that obtained using the adherent cell culture method (T-flask), with a 5.89- and 7.31-fold increase, respectively. Moreover, biological analysis of the MAbs produced in the microdevice showed no significant differences in the neutralizing antiproliferative activity of the hIFN-α2b or the cytokine cell signaling compared to the MAbs produced with cell adherent methods. These results suggest that this microfluidic device is suitable for long-term culture of mammalian cells and can improve the productivity of cells expressing recombinant MAbs with potential for therapeutic use without affecting the quality attributes of the product.Fil: Bourguignon, Natalia. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Attallah, Carolina Veronica. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Karp, Paola Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Booth, Ross. No especifíca;Fil: Peñaherrera Pazmiño, Ana Belén. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Payés, Cristian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Oggero Eberhardt, Marcos Rafael. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Helguera, Gustavo Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin

Influence of thermal treatment and aging time on the stability and swelling properties of nano/micron-sized polymeric spheres for improved oil recovery application

Understanding the knowledge of swelling and stability properties of polymeric spheres are essential for improved oil recovery; these properties will allow guaranteeing an efficient block of high permeability channels and large pore throats. These properties of nano/micron-sized polymeric spheres commercially available for improved oil recovery applications were investigated. The polymeric spheres emulsion was characterized by scanning electron microscopy (SEM). Data analysis reveals uniform and spherical structures with an average diameter of 47 ± 4.8 nm. After hydration, polymeric spheres can swell due to water absorption reaching an average particle diameter of 3.3 ± 0.2 μm. The effect of aging time, thermal treatment, the removal of the oil phase, and the dispersing stabilizer on the swelling and stability was studied. It has been found that polymeric spheres structure suffers deterioration with the progressive aging time, temperature increase, and with the removal of the oil phase and dispersing stabilizer. As a result, crosslinked polymer aggregates are formed which subsequently acquiring fibrillar morphologies. Further, this study provides an understanding of the stability and swelling behavior of polymeric microspheres using polydimethylsiloxane microdevices. The results evidenced that the behavior of the polymeric spheres can be beneficial to improve oil recovery.Fil: Rosero, Gustavo. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Olmos Carreno, Carol Maritza. Universidad Nacional de Colombia; Colombia. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Peñaherrera Pazmiño, Ana Belén. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vaca Mora, Andrea Vanessa. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vizuete, Karla. Universidad de Las Fuerzas Armadas; EcuadorFil: Debut, Alexis. Universidad de Las Fuerzas Armadas; EcuadorFil: Cumbal, Luis. Universidad de Las Fuerzas Armadas; EcuadorFil: Masiero, Diana. YPF - Tecnología; ArgentinaFil: Hernandez, Maria I.. YPF - Tecnología; ArgentinaFil: Lasorsa, Carlos. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Perez, Maximiliano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Instituto de Ingeniería Biomédica; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Universidad de Buenos Aires. Facultad de Ingeniería. Instituto de Ingeniería Biomédica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Accessible and Cost-Effective Method of PDMS Microdevices Fabrication Using a Reusable Photopolymer Mold

This work describes a novel and cost-effective method of polydimethylsiloxane (PDMS) microchips fabrication by using a printing plate photopolymer called Flexcel as a master mold (Fmold). This method has demonstrated the ability to generate multiple devices from a single master, reaching a minimum channel size of 25 μm, structures height ranging from 53 to 1500 μm and achieving dimensions of 1270 × 2062 mm2, which are larger than those obtained by the known techniques to date. Scanning electron microscopy, atomic force microscopy, and profilometry techniques have been employed to characterize the Fmold and PDMS replicas. The results showed high replication fidelity of Fmold to the PDMS replica. Furthermore, it was proved the reusability of the Fmold. In our study, up to 50 PDMS replicas have been fabricated without apparent degradation of the mold. The feasibility of the resulting PDMS replica was effectively demonstrated using a microfluidic device for enhanced oil recovery analysis.Fil: Bourguignon, Natalia. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olmos Carreno, Carol Maritza. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sierra Rodero, Marina. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Peñaherrera Pazmiño, Ana Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Rosero Yánez, Gustavo Ivan. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pineda Ramos, Pedro Antonio. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Vizuete, Karla. Universidad de Las Fuerzas Armadas; EcuadorFil: Arroyo, Alejandro Carlos. Universidad de Las Fuerzas Armadas; EcuadorFil: Cumbal Flores, Luis. Universidad de Las Fuerzas Armadas; EcuadorFil: Lasorsa, Carlos Alberto. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin