Tumor growth analysis by magnetic resonance imaging of the C6 glioblastoma model with prospects for the assessment of magnetohyperthermia therapy.

OBJECTIVE: The objective was to establish a pattern of tumor growth of the C6 model of glioblastoma multiform in Wistar rats via magnetic resonance imaging (MRI) for the subsequent verification of tumor volume reduction due to magnetic hyperthermia therapy. METHODS: Young male Wistar rats weighing between 250 and 300 g were used for the C6 model. After the rats were anesthetized (55 mg/ kg ketamine and 11 mg/kg xylazine), C6 lineage tumorigenic cells suspended in culture medium (10(5) cells in 10 microl) were stereotaxically injected into the right frontal cortex (bregma coordinates: 2.0 mm anteroposterior, 3.0 mm laterolateral, and 2.5 mm depth) of the rats using a Hamilton syringe. For the control group, the rats were injected with culture medium without cells. MRI scans were performed at 14, 21, and 28 d after the injection using a 2.0 T MRI scanner (Bruker BioSpec, Germany). The animals were anesthetized with 55 mg/kg ketamine and 11 mg/kg xylazine before being examined. Coronal multilayers were acquired using a standard spin echo sequence with the following parameters: repetition/echo time = 4.000 ms/67.1 ms, field of view = 3.50, matrix = 192, slice thickness = 0.4 mm, and slice separation = 0 mm. RESULTS: The MRI analysis enabled a clear visualization of the tumor mass, and it was possible to establish the tumor volume parameters on the various days that were examined. The volume at 14 d after induction was 13.7 +/- 2.5 mm3. On days 21 and 28, the tumor volumes were 31.7 +/- 6.5 mm3 and 122.1 +/- 11.8 mm3, respectively. CONCLUSION: These results demonstrated that it is possible to evaluate the C6 model tumor volume in rats, which will allow for the future implementation and verification of magnetic hyperthermia therapy.

Isolation, cultivation and characterization of CD133+ stem cells from human glioblastoma.

OBJECTIVE: To establish the method of isolation and culture of human glioblastoma neurospheres, and the purification of their stem cells, followed by the process of obtaining tumor subspheres, immunophenotypically characterizing this clonogenic set. METHODS: Through the processing of glioblastoma samples (n=3), the following strategy of action was adopted: (i) establish primary culture of glioblastoma; (ii) isolation and culture of tumor neurospheres; (iii) purify cells that initiate tumors (CD133+) by magnetic separation system (MACS); (iv) obtain tumor subspheres; (v) study the expression of the markers nestin, CD133, and GFAP. RESULTS: The study successfully described the process of isolation and culture of glioblastoma subspheres, which consist of a number of clonogenic cells immunophenotypically characterized as neural, which are able to initiate tumor formation. CONCLUSION: These findings may contribute to a better understanding of the process of gliomagenesis.

Evaluation of umbilical cord mesenchymal stem cell labeling with superparamagnetic iron oxide nanoparticles coated with dextran and complexed with Poly-L-lysine.

OBJECTIVE: The objective of this study was to evaluate the effect of the labeling of umbilical cord vein derived mesenchymal stem cells with superparamagnetic iron oxide nanoparticles coated with dextran and complexed to a non-viral transfector agent transfector poly-L-lysine. METHODS: The labeling of mesenchymal stem cells was performed using the superparamagnetic iron oxide nanoparticles/dextran complexed and not complexed to poly-L-lysine. Superparamagnetic iron oxide nanoparticles/dextran was incubated with poly-L-lysine in an ultrasonic sonicator at 37°C for 10 minutes for complex formation superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine by electrostatic interaction. Then, the mesenchymal stem cells were incubated overnight with the complex superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine and superparamagnetic iron oxide nanoparticles/dextran. After the incubation period the mesenchymal stem cells were evaluated by internalization of the complex superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine and superparamagnetic iron oxide nanoparticles/dextran by Prussian Blue stain. Cellular viability of labeled mesenchymal stem cells was evaluated by cellular proliferation assay using 5,6-carboxy-fluorescein-succinimidyl ester method and apoptosis detection by Annexin V- Propidium Iodide assay. RESULTS: mesenchymal stem cells labeled with superparamagnetic iron oxide nanoparticles/dextran without poly-L-lysine not internalized efficiently the superparamagnetic iron oxide nanoparticles due to its low presence detected within cells. Mesenchymal stem cells labeled with the complex superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine efficiently internalized the superparamagnetic iron oxide nanoparticles due to greater presence in the cells interior. The viability and apoptosis assays demonstrated that the mesenchymal stem cells labeled and not labeled respectively with the superparamagnetic iron oxide nanoparticles/dextran/poly-L-lysine continue to proliferate over seven days and the percentage of cells in early or late apoptosis is low compared to the percentage of live cells over the three days. CONCLUSION: Our results showed that the use of poly-L-lysine complexed with superparamagnetic iron oxide nanoparticles/dextran provides better internalization of these superparamagnetic iron oxide nanoparticles in mesenchymal stem cells Thus, we demonstrated that this type of labeling is not cytotoxic to the mesenchymal stem cells, since the viability and apoptosis assays showed that the cells remain alive and proliferating. The efficiency of this type of labeling in mesenchymal stem cells can provide non-invasive methods for monitoring these cells in vivo.

Study of internalization and viability of multimodal nanoparticles for labeling of human umbilical cord mesenchymal stem cells.

OBJECTIVE: To analyze multimodal magnetic nanoparticles-Rhodamine B in culture media for cell labeling, and to establish a study of multimodal magnetic nanoparticles-Rhodamine B detection at labeled cells evaluating they viability at concentrations of 10µg Fe/mL and 100µg Fe/mL. METHODS: We performed the analysis of stability of multimodal magnetic nanoparticles-Rhodamine B in different culture media; the mesenchymal stem cells labeling with multimodal magnetic nanoparticles-Rhodamine B; the intracellular detection of multimodal magnetic nanoparticles-Rhodamine B in mesenchymal stem cells, and assessment of the viability of labeled cells by kinetic proliferation. RESULTS: The stability analysis showed that multimodal magnetic nanoparticles-Rhodamine B had good stability in cultured Dulbecco's Modified Eagle's-Low Glucose medium and RPMI 1640 medium. The mesenchymal stem cell with multimodal magnetic nanoparticles-Rhodamine B described location of intracellular nanoparticles, which were shown as blue granules co-localized in fluorescent clusters, thus characterizing magnetic and fluorescent properties of multimodal magnetic nanoparticles-Rhodamine B. CONCLUSION: The stability of multimodal magnetic nanoparticles-Rhodamine B found in cultured Dulbecco's Modified Eagle's-Low Glucose medium and RPMI 1640 medium assured intracellular mesenchymal stem cells labeling. This cell labeling did not affect viability of labeled mesenchymal stem cells since they continued to proliferate for five days.

Intracellular labeling and quantification process by magnetic resonance imaging using iron oxide magnetic nanoparticles in rat C6 glioma cell line.

OBJECTIVE: To assess intracellular labeling and quantification by magnetic resonance imaging using iron oxide magnetic nanoparticles coated with biocompatible materials in rat C6 glioma cells in vitro. These methods will provide direction for future trials of tumor induction in vivo as well as possible magnetic hyperthermia applications. METHODS: Aminosilane, dextran, polyvinyl alcohol, and starch-coated magnetic nanoparticles were used in the qualitative assessment of C6 cell labeling via light microscopy. The influence of the transfection agent poly-L-lysine on cellular uptake was examined. The quantification process was performed by relaxometry analysis in T1 and T2weighted phantom images. RESULTS: Light microscopy revealed that the aminosilane-coated magnetic nanoparticles alone or complexed with poly-L-lysine showed higher cellular uptake than did the uncoated magnetic particles. The relaxivities of the aminosilane-coated magnetic nanoparticles with a hydrodynamic diameter of 50nm to a 3-T field were r1=(6.1±0.3)×10(-5) ms-1mL/µg, r2=(5.3±0.1)× 10(-4) ms(-1)mL/µg, with a ratio of r2 / r1 ≅ 9. The iron uptake in the cells was calculated by analyzing the relaxation rates (R1 and R2) using a mathematical relationship. CONCLUSIONS: C6 glioma cells have a high uptake efficiency for aminosilane-coated magnetic nanoparticles complexed with the transfection agent poly-L-lysine. The large ratio r2 / r1 ≅ 9 indicates that these magnetic nanoparticles are ideal for quantification by magnetic resonance imaging with T2-weighted imaging techniques.

Evaluation of umbilical cord mesenchymal stem cells labeling with superparamagnetic iron oxide nanoparticles coated with dextran and complexed with Poly-L-Lysine / Avaliação da marcação de células-tronco mesenquimais de cordão umbilical com nanopartículas superparamagnéticas de óxido de ferro recobertas com Dextran e complexadas a Poli-L-Lisina

Objetivo: O objetivo deste estudo foi avaliar o efeito da marcação de células-tronco mesenquimais obtidas da parede da veia do cordão umbilical com nanopartículas de óxido de ferro superparamagnéticas recobertas com dextran e complexadas a um agente transfector não viral denominado de Poli-L-Lisina. Métodos: A marcação das células-tronco mesenquimais foi realizada utilizando as nanopartículas de óxido de ferro superparamagnéticas recobertas com dextran complexadas e não complexadas a Poli-L-Lisina. As nanopartículas de óxido de ferro superparamagnéticas recobertas com dextran foram incubadas com o Poli-L-Lisina em um sonicador ultrassonico a 37ºC por 10 minutos, para a formação do complexo através de interação eletrostática. Em seguida, as células-tronco mesenquimais foram incubadas overnight com as nanopartículas de óxido de ferro superparamagnéticas complexadas e não com Poli-L-Lisina. Após o período de incubação as células-tronco mesenquimais foram avaliadas quanto à internalização do complexo nanopartícula de óxido de ferro superparamagnéticas /dextran/Poli-L-Lisina e nanopartícula de óxido de ferro superparamagnéticas /dextran através de ensaio citoquímico com azul de prússia. A viabilidade celular das célulastronco mesenquimais marcadas foi avaliada através do ensaio de proliferação celular utilizando o método de 5,6-carboxy-fluoresceinsuccinimidyl-ester e de morte celular através do método de anexinaiodeto de propídeo, ambos utilizando o recurso de citometria de fluxo. Resultados: Observamos nos ensaios citoquímicos que as célulastronco mesenquimais que foram marcadas com as nanopartícula de óxido de ferro superparamagnéticas /dextran sem a Poli-L-Lisina, não internalizaram com eficiência as nanopartículas devido pouca detecção de sua presença no interior das células. As células-tronco mesenquimais marcadas com o complexo nanopartícula de óxido de ferro superparamagnéticas /dextran/Poli-L-Lisina internalizaram com eficiência as nanopartículas devido à maior presença destas no interior das células. Os ensaios de viabilidade e morte celular demonstraram respectivamente que as células-tronco mesenquimais marcadas com as nanopartícula de óxido de ferro superparamagnéticas /dextran/Poli-L-Lisina continuam proliferando ao longo de sete dias e a porcentagem de células em apoptose inicial e tardia é baixa em relação à porcentagem de células vivas ao longo de três dias. Conclusão: Evidenciamos através de nossos resultados a necessidade da utilização da Poli-L-Lisina complexada com a nanopartícula de óxido de ferro superparamagnéticas /dextran para melhor internalização nas célulastronco mesenquimais. Paralelamente, demonstramos que este tipo de marcação não é citotóxico para as células-tronco mesenquimais já que os testes de morte e viabilidade celular mostraram que as células continuam vivas e proliferando.

Study of internalization and viability of multimodal nanoparticles for labeling of human umbilical cord mesenchymal stem cells / Estudo de internalização e viabilidade de nanopartículas multimodal para marcação de células-tronco mesenquimais de cordão umbilical humano

Objective: To analyze multimodal magnetic nanoparticles-Rhodamine B in culture media for cell labeling, and to establish a study of multimodal magnetic nanoparticles-Rhodamine B detection at labeled cells evaluating they viability at concentrations of 10mug Fe/mL and 100mug Fe/mL. Methods: We performed the analysis of stability of multimodal magnetic nanoparticles-Rhodamine B in different culture media; the mesenchymal stem cells labeling with multimodal magnetic nanoparticles-Rhodamine B; the intracellular detection of multimodal magnetic nanoparticles-Rhodamine B in mesenchymal stem cells, and assessment of the viability of labeled cells by kinetic proliferation. Results: The stability analysis showed that multimodal magnetic nanoparticles-Rhodamine B had good stability in cultured Dulbecco's Modified Eagle's-Low Glucose medium and RPMI 1640 medium. The mesenchymal stem cell with multimodal magnetic nanoparticles-Rhodamine B described location of intracellular nanoparticles, which were shown as blue granules co-localized in fluorescent clusters, thus characterizing magnetic and fluorescent properties of multimodal magnetic nanoparticles-Rhodamine B. Conclusion: The stability of multimodal magnetic nanoparticles-Rhodamine B found in cultured Dulbecco's Modified Eagle's-Low Glucose medium and RPMI 1640 medium assured intracellular mesenchymal stem cells labeling. This cell labeling did not affect viability of labeled mesenchymal stem cells since they continued to proliferate for five days.

Objetivo: Analisar a estabilidade das nanopartículas magnéticas multimodais-Rhodamine B em meios de cultura para marcação celular e, consequentemente, estabelecer o estudo de detecção intracelular de nanopartículas magnéticas multimodais-Rhodamine B nas células marcadas, avaliando a viabilidade celular nas concentrações de 10mig Fe/mL e 100mig Fe/mL. Métodos: Foram realizados: análise da estabilidade das nanopartículas magnéticas multimodais-Rhodamine B em meios de cultura diferentes; marcação das células-tronco mesenquimais com nanopartículas magnéticas multimodais-Rhodamine B; detecção intracelular das nanopartículas magnéticas multimodais-Rhodamine B nas células-tronco mesenquimais e avaliação da viabilidade das células marcadas por meio da cinética de proliferação. Resultados: A análise de estabilidade determinou que as nanopartículas magnéticas multimodais-Rhodamine B presentes nos meios de cultura Dulbecco's Modified Eagle's-Low Glucose e RPMI Medium 1640 apresentaram boa estabilidade. A marcação das células-tronco mesenquimais com nanopartículas magnéticas multimodais-Rhodamine B descreveu localização intracelular das nanopartículas, as quais se mostraram como grânulos azulados colocalizados nos grumos fluorescentes, caracterizando, assim, as propriedades magnéticas e fluorescentes das nanopartículas magnéticas multimodais-Rhodamine B. Conclusão: A estabilidade das nanopartículas magnéticas multimodais-Rhodamine B, presentes nos meios de cultura Dulbecco's Modified Eagle's-Low Glucose e RPMI Medium 1640, garantiu a eficiente marcação intracelular das células-tronco mesenquimais. Esse tipo de marcação não afetou viabilidade das células-tronco mesenquimais marcadas, já que as mesmas continuaram proliferando ao longo de 5 dias.

Isolation, cultivation and characterization of CD133+ stem cells from human glioblastoma / Isolamento, cultivo e caracterização de células-tronco CD133+ de glioblastoma humano

Objetivo: Estabelecer o método de isolamento e cultivo das neuroesferas de glioblastoma humano, bem como purificação de suas células-tronco, seguido do processo de obtenção de subesferas tumorais, caracterizando imunofenotipicamente esse conjunto clonogênico. Métodos: Por meio do processamento de amostras de glioblastomas (n=3), cumpriu-se a seguinte estratégia de ação: (i) estabelecimento da cultura primária de glioblastoma; (ii) isolamento e cultura de neuroesferas tumorais; (iii) purificação das células que iniciam os tumores (CD133+) por sistema de separação magnética (MACS); (iv) obtenção subesferas tumorais; (v) estudo da expressão de marcadores GFAP, CD133 e nestina. Resultados: Este estudo descreveu com sucesso o processo de isolamento e cultivo de subesferas de glioblastoma, as quais são constituídas por um conjunto clonogênico de células caracterizadas imunofenotipicamente como neurais, capazes de iniciar a formação tumoral. Conclusão: Estes achados poderão contribuir para a compreensão do processo de gliomagênese.

Intracellular labeling and quantification process by magnetic resonance imaging using iron oxide magnetic nanoparticles in rat C6 glioma cell line / Marcação intracelular e processo de quantificação por imagem por ressonância magnética utilizando nanopartículas magnéticas de óxido de ferro em células da linhagem C6 de glioma de rato

Objective: To assess intracellular labeling and quantification by magnetic resonance imaging using iron oxide magnetic nanoparticles coated with biocompatible materials in rat C6 glioma cells in vitro. These methods will provide direction for future trials of tumor induction in vivo as well as possible magnetic hyperthermia applications. Methods: Aminosilane, dextran, polyvinyl alcohol, and starch-coated magnetic nanoparticles were used in the qualitative assessment of C6 cell labeling via light microscopy. The influence of the transfection agent poly-L-lysine on cellular uptake was examined. The quantification process was performed by relaxometry analysis in T1 and T2weighted phantom images. Results: Light microscopy revealed that the aminosilane-coated magnetic nanoparticles alone or complexed with poly-L-lysine showed higher cellular uptake than did the uncoated magnetic particles. The relaxivities of the aminosilane-coated magnetic nanoparticles with a hydrodynamic diameter of 50nm to a 3-T were r1=(6.1±0.3)×10-5 ms-1mL/mug, r2=(5.3±0.1)× 10-4 ms-1mL/mug, with a ratio of r2 / r1 approximately equal to 9. The iron uptake in the cells was calculated by analyzing the relaxation rates (R1 and R2) using a mathematical relationship. Conclusions: C6 glioma cells have a high uptake efficiency for aminosilane-coated magnetic nanoparticles complexed with the transfection agent poly-L-lysine. The large ratio r2 / r1 approximately equal to 9 indicates that these magnetic nanoparticles are ideal for quantification by magnetic resonance imaging with T2-weighted imaging techniques.

Objetivo: Avaliar a marcação intracelular e o processo de quantificação por imagem por ressonância magnética usando nanopartículas magnéticas à base de óxido de ferro recobertas com materiais biocompatíveis em células da linhagem de glioma de rato C6 em experimentos in vitro. Esses métodos visam orientar ensaios futuros de indução de tumor in vivo, bem como possíveis aplicações da técnica de magneto-hipertermia. Métodos: Na avaliação qualitativa da marcação de células C6, realizada mediante microscopia óptica comum, foram utilizadas nanopartículas magnéticas recobertas com aminosilana, dextrana, álcool polivinílico e amido. A influência do agente de transfecção poly-L-lisine na captação celular foi analisada. O processo de quantificação foi realizado mediante a análise de relaxometria em imagens ponderadas em T1 e T2 do phantom. Resultados: A avaliação por microscopia óptica comum mostrou que nanopartículas magnéticas recobertas com aminosilana complexadas e não complexadas com poly-L-lisine apresentam melhor captação pelas células. As relaxatividades de nanopartículas magnéticas recobertas com aminosilana com diâmetro hidrodinâmico de 50nm para um campo de 3T foram: r1=(6,1±0,3)×10-5ms-1mL/mig, r2=(5,3±0,1)×10-4ms-1mL/mig; com uma razão de r2 / r1 aproximadamente igual a 9. O ferro captado pelas células foi calculado pela análise das taxas de relaxação (R1 e R2) mediante relação matemática. Conclusões: Linhagem de células C6 marcadas com nanopartículas magnéticas revestidas com aminosilana e complexadas com o agente de transfecção poly-L-lisine tem uma alta eficiência de captação das nanopartículas magnéticas. A grande razão r2 / r1 aproximadamente igual a 9 determina que essas nanopartículas magnéticas sejam ideais para estudar o processo de quantificação por imagem por ressonância magnética com técnicas de imagem ponderadas em T2.