JOAO VICTOR ALMEIDA DE ASSIS
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Resumo IPEN-doc 30297 Sodium alginate and nanocellulose hydrogel as scaffold to in vitro 3D prostate cancer irradiated model2023 - SILVA, G.D.; SAKATA, S.K.; ASSIS, J.V.A.; VIEIRA, D.P.Introdução: Recently, traditional cell culture systems structured in 2 dimensions using monolayers of cells in culture media are being replaced by 3D structures, in which cells can be organized in spheroids. To obtain these structures, hydrogels can be used as permeable to gas and nutrients scaffolds, also providing physical support to cells. This work aimed to produce a double network hydrogel containing sodium alginate (SA) and nanocellulose (NC), obtained by irradiation of microcrystalline cellulose, and its ability to maintain in culture of human prostate adenocarcinoma. Objetivos: To analyze whether SA+NC gels can keep viable 3D LNCap (prostatic carcinoma) in vitro, with and without exposure to radiation (0 and 2Gy gamma). Métodos: 0.75g of microcrystalline cellulose (Sigma-Aldrich, 435236) dispersed in water were irradiated (300 kGy) (25 mm column height) in an electron beam source (Dynamitron® Job 188 ,RDI- Radiation Dynamics Inc.). The precipitated powder was washed in water by centrifugation. Nanocellulose pellet was added to a sodium alginate (2.5%) dissolved in PBS. LNCaP cells were maintained in RPMI 1640 medium in monolayers in culture flasks and controlled atmosphere (37º, 5% CO2). 24-well plates were used, pre-treated with Pluronic® F-127 solution (0.5g/mL in 2-propanol). The hydrophobic portions of Pluronic molecules were directed towards the center of the well, thus preventing cell adhesion to the culture plastic. In each well 1x105 cells were added, forming clusters of cells after 3 days. Clusters were removed and added to the hydrogel seeded in 96-well plates. Crosslinking was achieved using 100 μL of 2mM CaCl2 solution on top of the gels. After gelation, the saline solution was removed and the wells received 100μL of culture medium and were submitted to gamma irradiation with doses of 0 and 2 Gy (GammaCell, Canada), and further kept in incubator for 24h. Medium was replaced by fresh medium with Hoescht 33342 (10mg/mL) and SYTOX? Green (5mM) and kept in an incubator for 30 minutes. Plates were imaged in an INCell Analyzer 2500HS and images were obtained to determine the dead cell count. Resultados: Visual evidence of spheroids enclosed in gels showed increased cell viability in SA+NC comparing to SA gels only. No visual differences were observed in irradiated (2Gy) spheroids. Conclusão: SA+NC gels can sustain cell viability and cause no changes in cell radioresistance, being a suitable model to in vitro studies.Resumo IPEN-doc 30292 Production of a double-network hydrogel using sodium alginate and nano-structured cellulose to 3D cell cultures2023 - SILVA, GIOVANA D. da; SAKATA, SOLANGE K.; ASSIS, JOAO V.A. de; SANTOS, ESTHER C. dos; PRUDENTE, SULEYNA R.; RODRIGUES, ALEX A.; FALCAO, PATRICIA L.; VIEIRA, DANIEL P.Introduction and objective 2D cell cultures have limitations regarding on tissue representativity. 3D cell cultures can use hydrogels of alginate with cellulose with adequate viscoelasticity properties for cell growth, being from plant sources, abundant and low cost. This work consisted of producing a biocompatible gel from plant sources for threedimensional cultures, promoting polymeric matrices for cells, helping in cell interactions and nutrient transport, providing mechanical support, self-assembly capacity, biodegradation, ability to reticulation, stability control and mechanical resistance. Methodology Transformation of microcrystalline cellulose into nanofibers was achieved freezing aqueous suspensions on presence of 4M NaOH to proper dissociation of fibers. To obtain suitable dispersion, sodium citrate was added to prevent aggregation. Suspensions were analyzed by Scanning Electron Microscope (SEM), Fourier Transform Infrared Spectroscopy (FTIR), Zeta Potential. For cell viability analysis, murine fibroblastic cell lines (NIH/3T3) were plated (2.5 x 105 cells per well) 24-well plate embedded in gel (100µL). Results and discussion The analysis of cellulose suspensions through SEM, showed a significant change in the size and shape of the structures after hydrolysis, indicating the obtention of structures on a nanometric scale. For the analysis of cellulose aggregation, the zeta potential values indicated that after the addition of sodium citrate, greater dispersion was obtained between the cellulose structures, enabling resistance to the structure in a uniform way. FTIR analysis showed changes in the covalent bonds of the products. Cell viability assay showed structures containing fibroblast cells, alginate and cellulose with 1 cycle of freezing with citrate showed an intact gel structure, with cell aggregates indicating possible cell growth, while the one with only alginate showed dead cells and showed that the hydrogel did not induce cellular toxicity. These results suggest that the hydrolysis of microcrystalline cellulose can lead to obtaining cellulose nanofibers with potential for applications in tissue engineering. Conclusions Hydrogels, they have potential for applications in tissue engineering, since they have mechanical resistance and cell viability. In addition, hydrogels from exclusively vegetable sources, since these are in large quantity, low cost and environmental impact, given that the alginate comes from brown algae found in several coastal regions and the cellulose can be extracted from renewable sources or various vegetable waste from agroindustry.