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USING HUMAN ADIPOSE STEM CELLS SPHEROIDS AS NOVEL APPROACH FOR ASSESSMENT OF SILVER CHLORIDE NANOPARTICLES CHRONIC EXPOSURE
Human mesenchymal stem cells (hMSC), such as adipose stem cells (ASCs), play a crucial role in tissue regeneration, mainly through direct differentiation or secreting anti-inflammatory/repair molecules at the injury site. Hence, they are widely used in tissue bioengineering. Due its antimicrobial properties, silver-based nanoparticles (AgNPs) have been extensively used as coating for biomedical implants, including scaffolds seeded with hMSC. Chronic ASCs exposure to nanoparticles -at sub-lethal levels- may occur due to NPS coating in skin implants or even from sunscreens and cosmetics that contains it. Compared with monolayer culture, 3D culture system is able to enhance stemness, anti-inflammatory and proangiogenic properties of ASCs. Indeed, the use of 3D culture in toxicology assays has shown an increase of accuracy of in vitro drug assessment, mainly due to cell-matrix architecture seen in 3D cultures, that promotes biological barriers, resulting in diffusion and transport conditions more similar with those found in vivo.
Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados
To standardize 3D spheroids culture system as new methodology for nanotoxicological in vitro assays. For this purpose, we evaluated the effect of sub-lethal (5, 10 and 25µg/mL) concentrations of silver chloride nanoparticles (AgCl-NPs), on ASCs spheroid cultures, throughout 21 days of treatment.
Human lipoaspirate samples were obtained from healthy donors. This study was approved by the Research Ethics Committee of the Clementino Fraga Filho University Hospital (UFRJ-Protocol 145-09). After the ASCs isolation, the monolayer was maintained in culture flasks (37ºC and 5%CO2) until to reach the confluence of 2x106 cells. Then, ASCs were harvested and seeded into a micro-molded non-adhesive hydrogel with 81 circular recesses (2% agarose -UltrapureAgarose, Invitrogen- in 0.9% NaCl), according to the manufacturer’s recommendations (Microtissue Inc). ASCs spheroids were treated with sub-lethal concentrations (5,10and 25µg/mL) of AgCl-NPs for 21days. Cell morphology was accessed by phase contrast microscopy. Oxidative stress(ROS) and cells viability were accessed by flow cytometry. The ultrastructural morphology was examined by scanning and transmission electron microscopy. The quantification of cytokines in ASCs supernatants was performed by Cytometric Bead Array (BD) and a multiplex immunoassay BioPlex MAGPIX (BioRad), respectively. Also, the qualitative analysis of viability was examined by fluorescence microscopy, using the kit LIVE/DEAD (Thermo Fisher).
ASCs spheroids morphology and diameter remained widely unaltered after light microscopy evaluation. No statistical significance was found for cell viability. ROS levels increased significantly for the group treated with 5 and 10µg/mL at day 7 (p=0.0395, p=0.0266, respectively). Ultrastructural analysis showed cell damage only on the surface of ASCs spheroids treated with 10µg/mL. Nevertheless, it was observed that -all ASCs spheroids treated with AgCl-NPs- had significant (p<0.05) alterations on their secretory profile, mainly IL-6, IL-8, and IL-1β, at specific concentrations and kinetics. Transforming growth factor - β1 and -β2 was also altered significantly (p<0.05) throughout the treatment period.
Considerações Finais/Final considerations/Consideraciones finales
Our results indicate that the sub-lethal concentrations of AgCl-NPs tested altered the secretory profile of ASC spheroids, which may lead to long-term disorder in tissue homeostasis (e.g. subcutaneous adipose tissue exposed by products containing these nanoparticles).
Palavras-chave/Key words/Palabras clave
Spheroids, Adipose Stem Cells, 3D culture, Interleukin secretion, Silver Chloride Nanoparticles, alternative method.
Mesenchymal stem cells/adultas
LETICIA EMILIANO CHARELLI, Nathalia Vieira Muller, Karina Ribeiro Silva, Celso Sant'Anna, Leandra Santos Baptista