Extracellular matrix remodeling during adipogenesis in 2D and 3D cultures on chip

In adipose tissue, the ECM undergoes constant remodeling to allow changes in cell shape\n(from fibroblastic to spherical) in a process called adipogenesis during the switch between\nthe proliferative and differentiated states. Although the transcriptional program of this\nprocess has been revealed i...

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Autor principal:	Ardila Riveros, Jessi Carolina
Otros Autores:	Guberman, Alejandra
Formato:	Tesis de maestría acceptedVersion
Lenguaje:	Inglés
Publicado:	Facultad de Farmacia y Bioquímica 2019
Materias:	Matriz extracelular Tejido adiposo Adipose extracellular matrix ECM 2D 3D Culture conditions Ciencias de la vida
Acceso en línea:	http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_5942 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_5942.dir/5942.PDF
Aporte de:	Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires

id	I28-R145-HWA_5942
record_format	dspace
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-145
collection	Repositorio Digital de la Universidad de Buenos Aires (UBA)
language	Inglés
orig_language_str_mv	eng
topic	Matriz extracelular Tejido adiposo Adipose extracellular matrix ECM 2D 3D Culture conditions Ciencias de la vida
spellingShingle	Matriz extracelular Tejido adiposo Adipose extracellular matrix ECM 2D 3D Culture conditions Ciencias de la vida Ardila Riveros, Jessi Carolina Extracellular matrix remodeling during adipogenesis in 2D and 3D cultures on chip
topic_facet	Matriz extracelular Tejido adiposo Adipose extracellular matrix ECM 2D 3D Culture conditions Ciencias de la vida
description	In adipose tissue, the ECM undergoes constant remodeling to allow changes in cell shape\n(from fibroblastic to spherical) in a process called adipogenesis during the switch between\nthe proliferative and differentiated states. Although the transcriptional program of this\nprocess has been revealed in great detail, the micro-environmental factors triggering and\ncontrolling the cell differentiation process are not. For instance, has been previously shown\nthat MMP-14 deficiency does not interfere with adipogenesis under conventional two\ndimensions (2D) culture in vitro. However, its loss inhibits completely the differentiation\nprocess when tested within a three-dimensional (3D) collagen gel. Therefore, this protein\nrepresents an ideal model for establishing a new cell culture technology. This however is\nnot a simple task due to the fact that our standard 2D cell cultures are not closely enough\nreflecting ECMs found under in vivo conditions. To overcome the disadvantages of current\ncell assay platforms for screening ECM remodeling processes in general the goal in this\nthesis was to investigate EMC remodeling in 3D tissue model system. To approach this\ncomplex task I first established the protein analytics in standard 2D cell culture technology\nand then transfered the knowledge to the 3D approach. I used a microfluidic large-scale\nintegration (mLSI) chip platform to integrate the steps of formation a 3D spheroid cell\nculture, long-term cultivation of stem cell spheroids, differentiation, and protein analytics.\nAdditionally to the known behavior of MMP14 I investigated in detail MMP-2, which is\nactivated by MMP-14 but its precise function during the ECM remodeling process in 2 and\n3D cell cultures is unknown. Upon measuring quantitatively the protein expression pattern\nof MMP-14 and MMP-2 together with collagens an fibronectin in 2D standard cell cultures,\nmy results confirmed that MMPs play a central role during early stages (from 0 to 6 days)\nof white adipocyte differentiation. PLA results showed that MMP-2 has no direct impact on\nthe expression levels of MMP-14 and FN. However, FN regulation is more dependent on\nthe expression of MMP-14 as observed with siRNA silencing strategy. In the second part\nof my thesis I aimed to confirm the results found in the 2D cell culture system within a new\n3D spheroid culture model for hASCs. Although, I was not able to transfer the experiments\nfrom the 2D approach in 3D with the developed workflows this approach is now in reach to\nunderstand the entire signaling network activity and spatial information of the involved\nproteins. In case of the MMPs this means we will be able to determine at which time point\nthe proteinases are activated and find their corresponding ECM target.
author2	Guberman, Alejandra
author_facet	Guberman, Alejandra Ardila Riveros, Jessi Carolina
format	Tesis de maestría Tesis de maestría acceptedVersion
author	Ardila Riveros, Jessi Carolina
author_sort	Ardila Riveros, Jessi Carolina
title	Extracellular matrix remodeling during adipogenesis in 2D and 3D cultures on chip
title_short	Extracellular matrix remodeling during adipogenesis in 2D and 3D cultures on chip
title_full	Extracellular matrix remodeling during adipogenesis in 2D and 3D cultures on chip
title_fullStr	Extracellular matrix remodeling during adipogenesis in 2D and 3D cultures on chip
title_full_unstemmed	Extracellular matrix remodeling during adipogenesis in 2D and 3D cultures on chip
title_sort	extracellular matrix remodeling during adipogenesis in 2d and 3d cultures on chip
publisher	Facultad de Farmacia y Bioquímica
publishDate	2019
url	http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_5942 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_5942.dir/5942.PDF
work_keys_str_mv	AT ardilariverosjessicarolina extracellularmatrixremodelingduringadipogenesisin2dand3dculturesonchip
_version_	1766017555511640064
spelling	I28-R145-HWA_59422022-04-18 In adipose tissue, the ECM undergoes constant remodeling to allow changes in cell shape\n(from fibroblastic to spherical) in a process called adipogenesis during the switch between\nthe proliferative and differentiated states. Although the transcriptional program of this\nprocess has been revealed in great detail, the micro-environmental factors triggering and\ncontrolling the cell differentiation process are not. For instance, has been previously shown\nthat MMP-14 deficiency does not interfere with adipogenesis under conventional two\ndimensions (2D) culture in vitro. However, its loss inhibits completely the differentiation\nprocess when tested within a three-dimensional (3D) collagen gel. Therefore, this protein\nrepresents an ideal model for establishing a new cell culture technology. This however is\nnot a simple task due to the fact that our standard 2D cell cultures are not closely enough\nreflecting ECMs found under in vivo conditions. To overcome the disadvantages of current\ncell assay platforms for screening ECM remodeling processes in general the goal in this\nthesis was to investigate EMC remodeling in 3D tissue model system. To approach this\ncomplex task I first established the protein analytics in standard 2D cell culture technology\nand then transfered the knowledge to the 3D approach. I used a microfluidic large-scale\nintegration (mLSI) chip platform to integrate the steps of formation a 3D spheroid cell\nculture, long-term cultivation of stem cell spheroids, differentiation, and protein analytics.\nAdditionally to the known behavior of MMP14 I investigated in detail MMP-2, which is\nactivated by MMP-14 but its precise function during the ECM remodeling process in 2 and\n3D cell cultures is unknown. Upon measuring quantitatively the protein expression pattern\nof MMP-14 and MMP-2 together with collagens an fibronectin in 2D standard cell cultures,\nmy results confirmed that MMPs play a central role during early stages (from 0 to 6 days)\nof white adipocyte differentiation. PLA results showed that MMP-2 has no direct impact on\nthe expression levels of MMP-14 and FN. However, FN regulation is more dependent on\nthe expression of MMP-14 as observed with siRNA silencing strategy. In the second part\nof my thesis I aimed to confirm the results found in the 2D cell culture system within a new\n3D spheroid culture model for hASCs. Although, I was not able to transfer the experiments\nfrom the 2D approach in 3D with the developed workflows this approach is now in reach to\nunderstand the entire signaling network activity and spatial information of the involved\nproteins. In case of the MMPs this means we will be able to determine at which time point\nthe proteinases are activated and find their corresponding ECM target. Fil: Ardila Riveros, Jessi Carolina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Guberman, Alejandra Facultad de Farmacia y Bioquímica Meier, Matthias Ardila Riveros, Jessi Carolina 2019-03-26 En el tejido adiposo, la MEC se somete a una remodelación constante para permitir cambios en la forma de la célula (de fibroblástica a esférica) en un proceso llamado adipogénesis durante el paso de estados proliferativos a diferenciados. Aunque el programa transcripcional de este proceso se ha revelado con gran detalle, los factores microambientales que desencadenan y controlan dicho proceso nos son has sido estudiados en detalle. Por ejemplo, se ha demostrado previamente que la deficiencia de MMP-14 no interfiere con la adipogénesis en cultivos convencionales de dos dimensiones (2D). Sin embargo, su pérdida inhibe por completo el proceso de diferenciación cuando se prueba dentro de un gel de colágeno tridimensional (3D). Por lo tanto, esta proteína es un modelo ideal para establecer una nueva tecnología de cultivo celular. Sin embargo, esto no es una tarea sencilla debido al hecho de que nuestros cultivos de células 2D estándar no reflejan suficientemente la estructura de la MEC encontrada en condiciones in vivo.\nPara superar las desventajas de las plataformas de ensayo de células actuales en el estudio de procesos de remodelación de MEC en general, el objetivo en esta tesis fue investigar la remodelación de MEC en el sistema de modelo de tejido 3D. Para abordar esta compleja tarea, primero establecí el análisis de proteínas en la tecnología estándar de cultivo de células 2D y luego transferí el conocimiento al enfoque 3D. Utilicé una plataforma de chip microfluídico de integración a gran escala (mLSI) para integrar los pasos de la formación de un cultivo de células esferoidales 3D, el cultivo a largo plazo de esferoides de células madre, la diferenciación y el análisis de proteínas. Además del comportamiento conocido de MMP14, investigué en detalle MMP-2, la cual es activada por MP-14 y cuya función durante el proceso de remodelación de MEC en cultivos de células 2 y 3D es desconocida. Al medir cuantitativamente el patrón de expresión proteica de MMP-14 y MMP-2 junto con colágenos y fibronectina en cultivos celulares estándar 2D, mis resultados confirmaron que las MMPs desempeñan un papel central durante las etapas tempranas (de 0 a 6 días) de la diferenciación de adipocitos blancos. Los resultados de PLA mostraron que MMP-2 no tiene un impacto directo en los niveles de expresión de MMP-14 y FN. Sin embargo, la regulación de FN es más dependiente de la expresión de MMP-14 como se observó con la estrategia de silenciamiento con siRNA.\nEn la segunda parte de mi tesis, mi objetivo fue confirmar los resultados encontrados en el sistema de cultivo de células 2D dentro de un nuevo modelo de cultivo de esferoides 3D para hASCs. Aunque no pude transferir los experimentos desde el enfoque 2D en 3D. Los flujos de trabajo desarrollados con esté enfoque nos permiten ahora encaminarnos en comprender toda la actividad de la red de señalización y la información espacial de las proteínas involucradas. En el caso de las MMP, esto significa que podremos determinar en qué momento las proteinasas se activan y encontrar su objetivo de ECM correspondiente. application/pdf Zotta, Elsa Miriuka, Santiago Wäsch, Ralph Matriz extracelular Tejido adiposo Adipose extracellular matrix ECM 2D 3D Culture conditions eng Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Extracellular matrix remodeling during adipogenesis in 2D and 3D cultures on chip info:eu-repo/semantics/masterThesis info:ar-repo/semantics/tesis de maestría info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_5942 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_5942.dir/5942.PDF

Extracellular matrix remodeling during adipogenesis in 2D and 3D cultures on chip

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