JOAO VICTOR ALMEIDA DE ASSIS

JOAO VICTOR ALMEIDA DE ASSIS

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Sodium alginate and nanocellulose hydrogel as scaffold to in vitro 3D prostate cancer irradiated model
2023 - 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.
Production of a double-network hydrogel using sodium alginate and nano-structured cellulose to 3D cell cultures
2023 - 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.
High-throughput production of tumor spheroids (melanoma and colon carcinoma) using simple plate treatment and automated fluorescence microscopy analysis
2023 - PRUDENTE, SULEYNA R.; ASSIS, JOAO V.A. de; SANTOS, ESTHER C. dos; SILVA, GIOVANA D. da; RODRIGUES, ALEX A.; ROCHA, LEONARDO W.P. de S.; FALCAO, PATRCIA L.; VIEIRA, DANIEL P.
Introduction and objective: Cancer is currently one of the leading causes of death in the world. The objective of this work is the formation of viable spheroids from cells of melanoma (SKMEL-37) and colon adenocarcinoma (HT29-MTX) cell lines and their evaluation regarding cell viability to enable the use of threedimensional cell culture as an alternative to the use of experimental animal models. Methodology: Cells were maintained in RPMI 1640 medium and kept in an incubator at 37°C, 5% CO₂ with controlled humidity. Upon reaching 60-70% confluence, cells were washed with phosphate buffered saline (PBS) and detached using trypsin solution. Afterwards, they were seeded in 24-well plates pre-treated with PluronicⓇ F-127 (0.5g/mL in 2-propanol) and turned back in incubator for 72 hours. Then, the formed spheroids were stained with Hoechst 33342 and SYTOX® Green solution, incubated for 60 minutes and images were acquired automatically in a HTS equipment (INCell 2500 HS, Cytiva). Results and discussion: Properly cohesive spheroids were obtained for both lineages, 20-30 per well. After 72h, only a small fraction of cells (about 5%) were considered unviable by SYTOX® staining. Principal Component Analysis (PCA) using 13 variables, and further Principal Component Regression (PCR) showed that nuclei mean and maximum intensities (Hoechst), and nuclei volume are the most relevant variables, corelated to number of plated cells. Days in culture appeared to not correlate with other variables. Conclusions: It was concluded that the methodology for the production of spheroids for melanoma and colon adenocarcinoma cell lines presented is simple, fast and cheap, in which, in 72 hours, the spheroids form freely, without restriction of shape and size and presenting low cell death, being also compatible with the high throughput screening technique (HTS). Nuclei volume and intensity can be used in future analysis to assess cell global viability in spheroids.
Comparison of image deconvolution methods to improve image quality of 3D cell cultures acquired using HCS/HTS equipment
2023 - ASSIS, JOAO V.A. de; VIEIRA, DANIEL P.
Introduction and objective: Accurate imaging analysis of 3D in vitro cell cultures became critical to evaluation of morphological and some physiological aspects of cells in these aggregates. Fluorescence microscopy protocols allow analysis of a plethora of molecular markers, DNA content and integrity and assessment of morphological changes upon different conditions. Light diffraction by plastic (or glass) substrate, and through sample and mounting/culture media distorts events, and thus images must require deconvolution methods to proper visualization and analysis. The work used 2 different algorithms and 3 iteration numbers to deconvolve images of spheroids acquired in large scale using HTS/HCS equipment. Methodology: Murine fibroblasts (NIH/3T3) were seeded on non-adhesive 96-well plates in RPMI 1640 medium. After 72 hours in culture, formed spheroids received pan-nuclear (Hoescht 33342) and nucleus of dead cells (SYTOX® Green) staining and imaged using HCS/HTS equipment (INCell 2500 HS, Cytiva). PSF files were generated using fluorescent beads in same cultured medium. Using DeconvolutionLab2 [1], a Fiji [2] plugin, spheroid images were deconvolved using the Tikhonov-Miller or Richardson-Lucy algorithms, with 10, 20 or 30 iterations. Orthogonal views of deconvolved stacks were analysed to find best results. Results and discussion: Both algorithms rendered similar results, with good reproducibility and resolution of light distortions. No real advantages were perceived in 30-iterations experiments, although 20 iterations rendered best images, regarding shape of nuclei (must be as spherical as possible) and drastic reduction of light refraction between slices. Conclusions: The work found that both methods were very easy to apply to stacks, with similar results. 20 iterations must be the better option to deconvolve spheroid images, uniting efficiency and saving computational resources. Richardson-Lucy can be the method of choice, as produced slightly better results.