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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| Formato: | Tesis de maestría acceptedVersion |
| Lenguaje: | Inglés |
| Publicado: |
Facultad de Farmacia y Bioquímica
2019
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| 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: |
| Sumario: | 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. |
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