JOAO VICTOR ALMEIDA DE ASSIS
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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.Resumo IPEN-doc 30278 High-throughput production of tumor spheroids (melanoma and colon carcinoma) using simple plate treatment and automated fluorescence microscopy analysis2023 - 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.Resumo IPEN-doc 30277 Comparison of image deconvolution methods to improve image quality of 3D cell cultures acquired using HCS/HTS equipment2023 - 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.