Generación de un dispositivo de migración celular con encapsulación de factores
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2016
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es
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Universidad Andrés Bello
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Licencia CC
Resumen
Las células madres se están comenzando a utilizar cada vez más en diferentes terapias celulares pero los resultados mostrados suelen variar entre grupos celulares provenientes de diferentes donantes o tejidos. Esta variación ha provocado que organismos reguladores soliciten una evaluación de las células antes de usarlas en alguna terapia para así poder estandarizar su uso como producto. De acuerdo a los mecanismos de acción de las células madre adultas (MSC), donde las respuestas migratorias juegan un rol importante, se ha propuesto en este proyecto que un indicador de la efectividad potencial de estas células en tratamientos podría definirse en parte por su capacidad de migración inducida por determinados factores. Es por esto que planteamos el uso de técnicas de fotolitografía, manejo de biomateriales, encapsulación y liberación de factores para crear un dispositivo que permita observar el desarrollo de la migración celular estimulada por un factor. Una particularidad de este dispositivo es que el factor inductor de migración se encuentra encapsulado en el dispositivo para ser presentado a la célula en forma controlada, gradual y creciente. Primero diseñamos un dispositivo que permite la migración celular, restringiendo a la célula a un espacio limitado el cual la fuerza a avanzar en una sola dirección y en favor de un gradiente de concentración del factor. Este diseño fue creado en AutoCAD® e impreso, generando así un silicon wafer el cual es el molde físico primario del dispositivo. Posteriores pasos de generación de un molde secundario e inyección final del hidrogel dan forma al dispositivo en su versión final. Se comprobó la funcionalidad del dispositivo midiendo el potencial migratorio de dos subpoblaciones celulares viendo diferencias entre ellas, por otro lado se vio que el dispositivo muestra relación entre presencia de marcadores en la superficie de una muestra y la migración de ésta. Finalmente, utilizando un biopreservante que mantiene la viabilidad celular, comprobamos que las células pueden ser viables pero no necesariamente poseen un buen potencial migratorio. Con esto esperamos haber creado una herramienta de fácil uso para poder evaluar las MSC de un donante y obtener parámetros de desempeño comparativo traducibles en mejor eficiencia potencial para una terapia. En el futuro esperamos probar este dispositivo con muestras inyectadas a pacientes tratados y ver si existe una correlación entre la migración mostrada por el dispositivo y el efecto terapéutico.
Stem cells are being used every day often on many cell therapies but shown results may change among cells that comes from a different donor or tissue. This variation has prompted the regulatory entities to request a previous evaluation of the cells before being used on any therapy to ensure its safety and function as a product. According to adult stem cell action mechanism, where the migration response has a critical function, it has been proposed in this project that one of the possible indicators of the therapeutic potency of the cells could be their migration capability induced by specific factors. Here we use different techniques such as photolithography, biomaterials, encapsulation and factor release to create a device that allows to observe the cell migration induced by an encapsulated factor in a stable gradient. First we designed a device that allows cell migration, restricting the cell environment to a confined space forcing it to move towards the factor. This design was created on AutoCAD® and printed to generate a silicon wafer which corresponds to the primary mold of the device. Further steps of secondary mold fabrication and hydrogel injection/polimerization are needed to take the device to its final form. The device functionality has been proven through experimentations by measuring the migration potential of two stem cells subpopulations where we observe different migration profiles, on the other hand we correlated the presence of a surface marker through flow cytometry with the migration of the sample. Finally, using a biopreservan that maintains cell viability, we show that cells can be viable but that doesn‟t mean they have a good migration potential. With this we hope to offer an easy-to-use tool to evaluate MSC from a donor and obtain a performance index to compare with other samples and improve the efficiency of a cell therapy. In the future we hope to test this device with samples that were into patients and see if exists a correlation between the sample migration and the therapeutic effect.
Stem cells are being used every day often on many cell therapies but shown results may change among cells that comes from a different donor or tissue. This variation has prompted the regulatory entities to request a previous evaluation of the cells before being used on any therapy to ensure its safety and function as a product. According to adult stem cell action mechanism, where the migration response has a critical function, it has been proposed in this project that one of the possible indicators of the therapeutic potency of the cells could be their migration capability induced by specific factors. Here we use different techniques such as photolithography, biomaterials, encapsulation and factor release to create a device that allows to observe the cell migration induced by an encapsulated factor in a stable gradient. First we designed a device that allows cell migration, restricting the cell environment to a confined space forcing it to move towards the factor. This design was created on AutoCAD® and printed to generate a silicon wafer which corresponds to the primary mold of the device. Further steps of secondary mold fabrication and hydrogel injection/polimerization are needed to take the device to its final form. The device functionality has been proven through experimentations by measuring the migration potential of two stem cells subpopulations where we observe different migration profiles, on the other hand we correlated the presence of a surface marker through flow cytometry with the migration of the sample. Finally, using a biopreservan that maintains cell viability, we show that cells can be viable but that doesn‟t mean they have a good migration potential. With this we hope to offer an easy-to-use tool to evaluate MSC from a donor and obtain a performance index to compare with other samples and improve the efficiency of a cell therapy. In the future we hope to test this device with samples that were into patients and see if exists a correlation between the sample migration and the therapeutic effect.
Notas
Tesis (Magíster en Biotecnología)
Palabras clave
Células Madre, Células, Análisis