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1.
Molecules ; 27(7)2022 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-35408484

RESUMO

Cell culturing methods in its classical 2D approach have limitations associated with altered cell morphology, gene expression patterns, migration, cell cycle and proliferation. Moreover, high throughput drug screening is mainly performed on 2D cell cultures which are physiologically far from proper cell functions resulting in inadequate hit-compounds which subsequently fail. A shift to 3D culturing protocols could solve issues with altered cell biochemistry and signaling which would lead to a proper recapitulation of physiological conditions in test systems. Here, we examined porous ultra-high molecular weight polyethylene (UHMWPE) as an inexpensive and robust material with varying pore sizes for cell culturing. We tested and developed culturing protocols for immortalized human neuroblastoma and primary mice hippocampal cells which resulted in high rate of cell penetration within one week of cultivation. UHMWPE was additionally functionalized with gelatin, poly-L-lysine, BSA and chitosan, resulting in increased cell penetrations of the material. We have also successfully traced GFP-tagged cells which were grown on a UHMWPE sample after one week from implantation into mice brain. Our findings highlight the importance of UHMWPE use as a 3D matrix and show new possibilities arising from the use of cheap and chemically homogeneous material for studying various types of cell-surface interactions further improving cell adhesion, viability and biocompatibility.


Assuntos
Técnicas de Cultura de Células , Polietilenos , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Camundongos , Peso Molecular , Polietileno/química , Polietilenos/química , Porosidade
2.
Neurogenetics ; 19(3): 189-204, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29982879

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that leads to the eventual death of motor neurons. Described cases of familial ALS have emphasized the significance of protein misfolding and aggregation of two functionally related proteins, FUS (fused in sarcoma) and TDP-43, implicated in RNA metabolism. Herein, we performed a comprehensive analysis of the in vivo model of FUS-mediated proteinopathy (ΔFUS(1-359) mice). First, we used the Noldus CatWalk system and confocal microscopy to determine the time of onset of the first clinical symptoms and the appearance of FUS-positive inclusions in the cytoplasm of neuronal cells. Second, we applied RNA-seq to evaluate changes in the gene expression profile encompassing the pre-symptomatic and the symptomatic stages of disease progression in motor neurons and the surrounding microglia of the spinal cord. The resulting data show that FUS-mediated proteinopathy is virtually asymptomatic in terms of both the clinical symptoms and the molecular aspects of neurodegeneration until it reaches the terminal stage of disease progression (120 days from birth). After this time, the pathological process develops very rapidly, resulting in the formation of massive FUS-positive inclusions accompanied by a transcriptional "burst" in the spinal cord cells. Specifically, it manifests in activation of a pro-inflammatory phenotype of microglial cells and malfunction of acetylcholine synapse transmission in motor neurons. Overall, we assume that the highly reproducible course of the pathological process, as well as the described accompanying features, makes ΔFUS(1-359) mice a convenient model for testing potential therapeutics against proteinopathy-induced decay of motor neurons.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Modelos Animais de Doenças , Camundongos Transgênicos , Proteína FUS de Ligação a RNA/genética , Animais , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Masculino , Camundongos , Neurônios Motores/fisiologia , Deficiências na Proteostase/genética , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Transdução de Sinais/genética , Medula Espinal/metabolismo , Medula Espinal/patologia
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