Organic-Polymeric Radial Flow Biorreactor for Liver Models / Biorreactor de flujo radial orgánico-polimérico para modelos de hígado

Abstract An artificial liver support system is based on the functional hepatocytes being cultured inside a bioreactor; this technique has being used as an effective therapy for treating chronic liver diseases in recent times. This work evaluates different parameters such as cell viability and metabolic function of the hepatocytes when cultured on a hybrid scaffold. The scaffold was built using a polypyrrole plasma coated polymer layer seeded with endothelial matrix for efficient three-dimensional hepatocyte growth within a radial flow bioreactor. The flow rate inside the bioreactor was 7 ml / min. The parts for the bioreactor where either built using food-grade steel and/or glass or the scaffolds comprise a Poly (L-lactic acid)-coated polypyrrole iodine layer or not for HepG2 culture. The results show that the Poly (L-lactic acid)-coated scaffolds increased cell proliferation by 30%, protein production by 16% and albumin secretion by 40% compared with the non-coated scaffold. All experiments were performed thrice and data was analysed by ANOVA and Tukey statistic models with a p<0.05. The obtained results demonstrated that radial flow bioreactors in conjunction with hybrid scaffolds improve hepatocytes' physiological and functional properties and could be used as an alternative therapy for patients with liver diseases.

Resumen Un sistema de soporte hepático artificial se basa en utilizar hepatocitos funcionales cultivados en un biorreactor; esta técnica ha demostrado que se puede utilizar como una terapia eficaz para el tratamiento de enfermedades crónicas del hígado en los últimos tiempos. Este trabajo evalúa diferentes parámetros tales como la viabilidad celular y la función metabólica de los hepatocitos cuando se cultivan en un andamio híbrido. El andamio fue construido usando una capa de polímero recubierto de polipirrol plasma, se sembró con un cultivo tridimensional de células endoteliales y de hepatocitos dentro de un biorreactor de flujo radial. La velocidad de flujo en el interior del biorreactor fue de 7 ml / min. Las piezas para el biorreactor fueron construidas con acero de calidad alimentaria y / o vidrio. Los andamios control fueron de ácido L-poliláctico y a estos se les agrego un revestimiento de polipirrol-yodo para el cultivo de HepG2. Los resultados muestran que el ácido L-poliláctico recubierto, aumento la proliferación celular en un 30%, la producción de proteínas en un 16% y la secreción de albúmina por 40% en comparación con el andamio no recubierto. Todos los experimentos se llevaron a cabo tres veces y los datos se analizaron mediante modelos estadísticos ANOVA y Tukey con una p <0.05. Los resultados obtenidos demostraron que los biorreactores de flujo radial conjuntamente con andamios híbridos mejoran las propiedades fisiológicas y funcionales hepatocitos y podrían utilizarse como una terapia alternativa para los pacientes con enfermedades hepáticas crónicas.

Nerve cells culture from lumbar spinal cord on surfaces modified by plasma pyrrole polymerization.

Currently, there are several techniques for modified cell culture surfaces under research to improve cell growth and adhesion. Recently, different methods have been used for surface coating, using biomolecules that enhance cell attachment and growth of nerve cells from spinal cord, such as the use of Poly-DL-Ornithine/Laminin. Plasma-polymerized pyrrole (PPy)-treated surfaces have showed improvement on surfaces biocompatibility with the cells in culture since they do not interfere with any of the biological cell functions. In the present work, we present a novel mouse nerve cell culture technique, using PPy-treated cell culture surfaces. A comparative study of cell survival using Poly-DL-Ornithine/Laminin-treated surfaces was performed. Our results of cell survival when compared with data already reported by other investigators, show that cells cultured on the PPy-modified surface increased survival up to 21 days when compared with Poly-DL-Ornithine/Laminin-coated culture, where 8 days cell survival was obtained. There were electrical and morphological differences in the nerve cells grown in the different surfaces. By comparing the peak ion currents of Poly-DL-Ornithine/Laminin-seeded cells for 8 days with cells grown for 21 days on PPy, an increase of 516% in the Na(+) current and 127% in K(+) currents in cells seeded on PPy were observed. Immunofluorescence techniques showed the presence of cell synapses and culture viability after 21 days. Our results then showed that PPy-modified surfaces are an alternative culture method that increases nerve cells survival from lumbar spinal cord cell culture by preserving its electrical and morphological features.

Development of a neuromuscular junction model on surfaces modified by plasma polymerization / Desarrollo de un modelo de unión neuromuscular sobre superficies modificadas por polimerización por plasma

The aim of this work is to implement a biological model of neuromuscular junctions to study the mechanisms involved in intra and inter cellular processes using cell co-cultures. To optimize growth and development of the neuromuscular junction, cells were seeded on plasma polymerized pyrrole which has proven suitable for other types of cell cultures. The cell lines used were motor neuron NG108-15 and skeletal muscle C2C12. Cells were evaluated according to their morphology and electrophysiological characteristics. To observe the expression of specific proteins of the nerve synapse, immunocytochemical techniques were applied using dying antibodies. Proteins localized in nerve terminals were dyed and imaged by fluorescence microscopy. Images of cell co-cultures showed the formation of neuromuscular junctions. The preparation of neuromuscular junctions described in this work will allow the study of the mechanisms involved in their functions.

El objetivo de este trabajo es implementar un modelo biológico de unión neuromuscular para el estudio de los mecanismos involucrados en los procesos intra e intercelulares empleando co-cultivos celulares. Con el fin de optimizar el crecimiento y desarrollo de las uniones neuromusculares, las células se cultivaron sobre superficies de polipirrol obtenidas mediante polimerización por plasma que han mostrado ser adecuadas en otros tipos de cultivos celulares. Las líneas celulares que se emplearon fueron los modelos de motoneurona NG108-15 y muscular C2C12. Las células se evaluaron de acuerdo a su morfología y características electrofisiológicas. Para observar la expresión de proteínas clave de la sinapsis, se aplicaron técnicas inmunocitoquímicas utilizando anticuerpos específicos para la marcación de proteínas localizadas en las terminales nerviosas adquiriendo imágenes con microscopía de fluorescencia. Las imágenes de los co-cultivos celulares mostraron la formación de uniones neuromusculares. El método de preparación de uniones neuromusculares que se describe en este trabajo permitirá estudiar los mecanismos involucrados en sus funciones.