Navegando por Palavras-chave "Cell culture techniques"
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- ItemSomente MetadadadosARPE-19 Cell Uptake of Small and Ultrasmall Superparamagnetic Iron Oxide(Informa Healthcare, 2014-04-01) Grottone, Gustavo Teixeira [UNIFESP]; Loureiro, Renata Ruoco [UNIFESP]; Covre, Joyce [UNIFESP]; Rodrigues, Eduardo Buchele [UNIFESP]; Gomes, José Álvaro Pereira [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Purpose: To investigate the cytotoxicity, cellular intake and magnetic field interaction of three superparamagnetic iron oxide (SPIO) and one ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles on ARPE-19 cells.Methods: Two FDA-approved SPIO nanoparticles (Endorem and Lumirem), one commercial SPIO(FluidMag-L) and one FDA-approved USPIO (Feraheme) were tested. Nanoparticle suspensions were diluted and prepared in high- (1000 Fe-ug/ml) and low- (100 Fe-ug/ml) dose suspensions. ARPE-19 cells were incubated in four 24-well plates and the medium changed every other day until cells attained 80% confluence. Nanoparticle cytotoxicity was evaluated using the XTT cytotoxicity assay. Cellular attraction was tested after digestion of the cells in collagenase A (1 mg/ml) overnight. A 3500 Gauss neodymium magnet was used to attract cells to the well walls. ARPE-19 cell ultrastructure was evaluated by transmission electron microscopy (TEM) to determine the specific locations of nanoparticles within the cell membranes.Results: Cytotoxicity assessment by the XTT assay revealed that ARPE-19 cells that were exposed to either concentration of Endorem, FLuidMag-L, Feraheme non-conjugated with protamine and heparin or Lumirem demonstrated no statistically significant toxicity. Cells exposed to Feraheme when conjugated with protamine and heparin presented severe toxicity in both concentrations. When a magnetic field was applied, all nanoparticle-containing samples, except Feraheme non-conjugated form, were promptly attracted. TEM and prussian blue staining examination revealed that Feraheme alone was not initially capable of cellular uptake. This issue was solved by conjugating Feraheme with protamine and heparin (although cytotoxicity was found on those samples). Endorem, FLuidMag-L, Feraheme conjugated form were found within the cytoplasm of ARPE-19 cells.Conclusions: Ferahame when conjugated with protamine and heparin was cytotoxic at the higher and lower doses, as revealed by the XTT assay. Endorem and FluidMag-L were not toxic at the studied concentrations. Feraheme non-conjugated solutions and Lumirem solutions provided were harmless but were not internalized by ARPE-19 cells. All the studied nanoparticles were attracted to the magnetic field except Feraheme in the non-conjugated form.
- ItemAcesso aberto (Open Access)Comparação entre membrana amniótica com e sem epitélio como substrato para cultura de células epiteliais do limbo ex vivo(Conselho Brasileiro de Oftalmologia, 2011-04-01) Covre, Joyce Luciana [UNIFESP]; Loureiro, Renata Ruoco [UNIFESP]; Cristovam, Priscila Cardoso [UNIFESP]; Ricardo, José Reinaldo da Silva [UNIFESP]; Freymüller-Haapalainen, Edna [UNIFESP]; Gomes, José Álvaro Pereira [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)PURPOSE: To evaluate the efficacy and ultrastructural aspects of human limbal epithelial cells cultured on amniotic membrane (AM) with and without epithelium. METHODS: Limbal epithelial cell cultures were established from cadaveric cor neo-scleral rim explants derived from 6 different donors. The explants from each donor were placed under 3 different groups: on human preserved AM with epithelium (Group 1), AM deepithelialized with trypsin (Group 2) and control (Group 3). The epithelial cell migration was evaluated under phase contrast microscopy. After 15 days, the amniotic membrane with cells cultures were removed and submitted to scanning and transmission electron microscopy to check for epithelial migration and adhesion. RESULTS: All epithelial cell cultures from the controls grew over the botton of the culture plate wells until reaching confluence. Epithelial cultures grew over all but one denuded amniotic membrane. In the group amniotic membrane with epithelium, epithelial cell growing was observed only in 1 well. CONCLUSIONS: Using this model, denuded amniotic membrane appeared to be the best substrate for epithelial cell migration and adhesion comparing to amniotic membrane with epithelium. Removal of amniotic membrane epithelial seems to be an important step for establishing limbal epithelial cell culture on amniotic membrane.
- ItemAcesso aberto (Open Access)Hemoderivados como suplemento no meio de cultivo para células-tronco dentárias humanas(Universidade Federal de São Paulo (UNIFESP), 2011-07-27) Pisciolaro, Ricardo Luiz [UNIFESP]; Duailibi, Silvio Eduardo [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Introduction: One among many aims of medicine is to overcome injuries inflicted to the organism by diseases, aging and trauma, re-establishing the usual functions. About tissues losses, several authors claim that the ideal replacement is the healthy tissue itself, originated from the same source or developed by Tissue Engineering (TE). However, much research is needed before in vivo application. Objective: To evaluate three different kinds of sera supplies used in stem cell culture media, as to cellular proliferation and cellular injuries on dental stem cell. Methods: Five experiments were made utilizing incompletely developed third molar teeth. After enzymatic digestion, the adult stem cells were cultivated in four different kinds of culture media. Medium I, serum free (SF); medium II, supplied with FBS (heterologous serum- HeS); medium III, supplied with homologous human serum (homologous serum- HoS) and medium IV, supplied with autologous human serum (autologous serum . AuHS).These cultures were analyzed comparatively as to cellular proliferation; they were submitted Von Kossa (VK) and Alizarin Red (AR) markers tests for four weeks (checked weekly), and each two weeks checked for Colonies Forming Unities (CFUs). On the 28th day, all four cultures were submitted to comet assay, and were inspected for possible cellular DNA injuries. The results underwent a non-parametric statistical Friedman fs variance test, with significance (p) . 5%. Results: Culture medium IV reached a cellular proliferation rate higher than medium I, showing a significant result (p*=0,0074). Culture medium II presented a superior proliferation result than medium I, and similar to medium III, although neither of them presented significant result when compared to medium IV. The comet assay fs results showed minor cellular DNA injury in the medium IV cultures, when compared to medium II and III cultures. The CFUs were numerous in the media IV and III cultures, respectively, and there was higher mineralization rate in the medium IV than in the media II and III. Conclusion: The culture medium supplied with AuHS significantly improved cellular proliferation. Human sera proved to be a viable supply to human dental stem cell culture.
- ItemAcesso aberto (Open Access)Importância do co-cultivo com fibroblastos de camundongo 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo humano(Conselho Brasileiro de Oftalmologia, 2008-10-01) Cristovam, Priscila Cardoso [UNIFESP]; Glória, Maria Aparecida da [UNIFESP]; Melo, Gustavo Barreto de [UNIFESP]; Gomes, José Álvaro Pereira [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)PURPOSE: To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR). METHODS: Corneo-scleral rims from different donors (n=6) had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the segments was placed with the epithelial side up on the bottom of a 6-well culture plate (Group A). The other two fragments were trypsinized and the obtained cell suspension was cultured with (Group B) or without (Group C) irradiaded 3T3 cells. The cells were cultured in supplemental hormonal epithelial medium (SHEM), the epithelial migration and clone formation in groups A, B and C were evaluated with phase contrast microscopy and rodamine B staining. RESULTS: Epithelial cell growth was observed in 4/6 rims (Group A). All epithelial cell suspensions that were cultured with 3T3 cells (Group B) formed clones. No adhesion or true clone formation (holo- or meroclones) was observed in the cell suspensions that were cultivated without 3T3 (Group C) (p=0.009). CONCLUSIONS: Epithelial cell suspension obtained from corneo-scleral rims in this model needs to be cultivated with 3T3 cells in order to form clones and establish limbal epithelial cell colonies with the potential to be used for ocular surface reconstruction.
- ItemSomente MetadadadosKeloid explant culture: a model for keloid fibroblasts isolation and cultivation based on the biological differences of its specific regions(Wiley-Blackwell, 2010-10-01) Tucci-Viegas, Vanina Monique [UNIFESP]; Hochman, Bernardo [UNIFESP]; Franca, Jeronimo P. [UNIFESP]; Ferreira, Lydia M. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)In vitro studies with keloid fibroblasts frequently present contradictory results. This may occur because keloids present distinct genotypic and phenotypic characteristics in its different regions, such as the peripheral region in relation to the central region. We suggest an explant model for keloid fibroblasts harvesting, standardising the initial processing of keloid samples to obtain fragments from different regions, considering its biological differences, for primary cell culture. the different keloid regions were delimited and fragments were obtained using a 3-mm diameter punch. To remove fragments from the periphery, the punch was placed in one longitudinal line extremity, respecting the lesion borders. for the central region, it was placed in the intersection of lines at the level of the largest longitudinal and transversal axes, the other fragments being removed centrifugally in relation to the first one. Primary fibroblast culture was carried out by explant. Flow cytometry analysis showed cell cycle differences between the groups, confirming its different origins and biological characteristics. in conclusion, our proposed model proved itself efficient for keloid fibroblast isolation from specific regions and cultivation. Its simplicity and ease of execution may turn it into an important tool for studying the characteristics of the different keloid-derived fibroblasts in culture.
- ItemAcesso aberto (Open Access)Modelo de compressão contínua na cultura de fibroblastos derivados do ligamento periodontal humano(Universidade Federal de São Paulo (UNIFESP), 2015) Mattar, Marco Antonio [UNIFESP]; Ferreira, Lydia Masako [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Introdução: Modelos de estudos in vitro são considerados como padrão ouro para análises de atividades celulares que visam mimetizações da dinâmica das células in vivo, inclusive quando as amostras celulares são submetidas à compressão estática. Células quando cultivadas sob um substrato representam a estrutura natural e função dos tecidos in vivo no que diz respeito à fisiologia, forma da célula e seu ambiente. Objetivo: Avaliar a viabilidade e morte celular no modelo de compressão contínua na cultura de fibroblastos humanos derivados do ligamento periodontal. Métodos: Foram selecionados 10 pacientes, submetidos à extrações dos 4 terceiros molares inclusos por indicação ortodôntica. A amostra consistiu de 4 mm2 de tecido periodontal do terço médio das raízes. A mesma foi cultivada até a 6ª passagem e depois as células foram divididas em dois grupos: Grupo Controle (GC), com cultivo em monocamada e substrato sem aplicação de força durante 6h e Grupo Experimental (GE3 e GE4), com cultivo em monocamada e substrato com aplicação de carga de 3 e 4 g/cm2 durante 6h. Resultados: Tanto o GC quanto o GE3 e GE4, monocamada e substrato, não apresentaram diferença estatística nos valores de viabilidade celular e apoptose. Com o aumento da carga o GE4 indicou maior necrose em relação ao GC e o GE3. Conclusão: Não houve diferença na utilização de substrato de colágeno na cultura de fibroblastos periodontais em nenhum dos parâmetros avaliados, mas houve maior necrose com o aumento da carga na avaliação intragrupos. Descritores: Ligamento periodontal, Sobrevivência celular, Apoptose, Técnicas de cultura de células.