LEONARDO WILANS PEREIRA DE SOUZA ROCHA
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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 28613 Effective methodology for maintaining Toxoplasma gondii in vitro using paramagnetic iron nanoparticles to support three-dimensional cell culture2021 - NASCIMENTO, ANA C.G.; GALISTEO JUNIOR, ANDRES J.; SILVA, GIOVANA D. da; ROCHA, LEONARDO W.P. de S.; VIEIRA, DANIEL P.INTRODUCTION Toxoplasma gondii is a protozoan parasite that infects approximately one billion people worldwide. Upon infection, the host may die due to latent infection or presence with chronic cysts in brain, retina or muscle tissue. Humans can become infected consuming water or foods contaminated with oocysts or eating undercooked meat. Its virulent form is difficult to replicate in vitro, requiring additional steps using experimental animals. The use of nanotechnology can contribute to this in vitro production, through the three-dimensional cultivation of mouse fibroblast cells (NIH / 3T3 ATCC ® CRL-1658™) and nanoparticles synthesized with radiation. OBJECTIVES The objective of this work was to demonstrate the three-dimensional culture of fibroblast cells aggregated to nanoparticles for inoculation the T. gondii. MATERIALS AND METHODS This methodology was created to facilitate parasite management and replication. For the production of nanoparticles, the work used concentrations of iron sulfate II heptahydrate (Fe2SO4.7H2O, CAS 7782-63-0) and glycine (NH2CH2COOH, CAS 56-40-6) diluted in ultrapure water free ofO2 at pH 12. This solutionwas irradiated by electron beam of the IPEN / CNEN-SP Radiation Technology Center in doses of at least 15 and at most 30kGy. Paramagnetic iron oxide nanoparticles (PION’s) were then adsorbed on cell membranes, and cells were kept together by a magnetic field. Structured spheroids (4 day of culture) were infected with 106 parasites (RH strain) and the infection was evaluated by transmission electron microscopy. DISCUSSION AND RESULTS Tachyzoiteswere found inside 3T3 cells, assuring that the spheroid can be a suitable culture substrate to T. gondii in vitro propagation. CONCLUSION A three-dimensionalmethodology for in vitro cultivation of the parasite is perhaps the key for applications in the study of toxoplasmosis, as it has a fast, cheap, efficient production (yield and reduction of contamination).Resumo IPEN-doc 28597 Effective methodology for maintaining Toxoplasma gondii in vitro using paramagnetic iron nanoparticles to support three-dimensional cell culture2021 - NASCIMENTO, ANA C.G.; GALISTEO JUNIOR, ANDRES J.; SILVA, GIOVANA D. da; ROCHA, LEONARDO W.P. de S.; VIEIRA, DANIEL P.Toxoplasma gondii is a protozoan parasite that infects approximately one billion people worldwide. Upon infection, the host may die due to latent infection or presence with chronic cysts in brain, retina or muscle tissue. Humans can become infected consuming water or foods contaminated with oocysts or eating undercooked meat. Its virulent form is difficult to replicate in vitro, requiring additional steps using experimental animals. The use of nanotechnology can contribute to this in vitro production, through the three-dimensional cultivation of mouse fibroblast cells (NIH/3T3 ATCC® CRL-1658™) and nanoparticles synthesized with radiation. The objective of this work was to demonstrate the three-dimensional culture of fibroblast cells aggregated to nanoparticles for inoculation the T. gondii. This methodology was created to facilitate parasite management and replication. For the production of nanoparticles, the work used concentrations of iron sulfate II heptahydrate (Fe2SO4.7H2O, CAS 7782-63-0) and glycine (NH2CH2COOH, CAS 56-40-6) diluted in ultrapure water free of O2 at pH 12. This solution was irradiated by electron beam of the IPEN / CNEN-SP Radiation Technology Center in doses of at least 15 and at most 30kGy. Paramagnetic iron oxide nanoparticles (PION’s) were then adsorbed on cell membranes, and cells were kept together by a magnetic field. Structured spheroids (4 day of culture) were infected with 106 parasites (RH strain) and the infection was evaluated by transmission electron microscopy. Tachyzoites were found inside 3T3 cells, assuring that the spheroid can be a suitable culture substrate to T. gondii in vitro propagation. A three-dimensional methodology for in vitro cultivation of the parasite is perhaps the key for applications in the study of toxoplasmosis, as it has a fast, cheap, efficient production (yield and reduction of contamination).Resumo IPEN-doc 28383 Produção de esferóides mistos de células epiteliais prostáticas humanas e de adenocarcinoma prostático para testes de antitumorais2021 - ROCHA, LEONARDO W.P. de S.; MARUMO, MARIA H.B.