RESUMEN
A Anexina A1 (AnxA1) é uma proteína de 37 kDa que controla o desenvolvimento da reação inflamatória inata, e favorece a eferocitose e o reparo tecidual. Em doenças inflamatórias intestinais (DIIs), tanto a AnxA1 endógena, como a sintética e o peptídeo sintético mimético ao N-terminal da proteína (Ac2-26) inibem o desenvolvimento de doença e induzem a cicatrização. O presente projeto teve o objetivo de obter novas formulações para carrear a AnxA1 recombinante (rAnxA1) ou o Ac2-26 e testar suas eficácias no modelo de colite experimental induzida pelo dextram sulfato de sódio (DSS, 0-6 dias) em camundongos C57Bl/6 machos. A rAnxA1 foi funcionalizada em nanocápsulas de núcleo lipídico de parede múltipla (MLNC) pela ligação Zn2+, com alta eficência de incorporação (92%) e adminsitrada pelas vias oral, intravenosa ou intraperitoneal durante a fase latente da doença (6º-9º dia). Somente o tratamento intraperitoneal com MLNC-AnxA1 (12,5 µg/mL) reduziu significativamente os sinais clínicos da doença, restaurou a integridade da estrutura colônica e a proliferação celular, bem como aumentou expressão de junções celulares da barreira intestinal. Ainda, MLNC-AnxA1 induziu a polarização de macrófagos para o fenótipo M2 in vivo no tecido inflamado e in vitro após estímulo com lipopolissacarídeos (LPS) bacteriano. Na tentativa de obter uma formulação terapêutica com atividade por vial oral, o peptídeo Ac2-26 foi incorporado em sílica mesoporosa ordenada SBA-15 e revestidos com Eudragit® L30-D55. A incorporação do peptídeo foi efetiva (88%) e a administração oral de Eudragit-SBA15-Ac2-26 (6º-9º dia; 200 µg/camundongo; 8 mg/kg) reduziu significativamente os sintomas clínicos e inflamação. De fato, ensaios de PET-SCAN mostraram que o SBA-15 permaneceu no intestino por até 16 horas após a administração e promoveu a liberação do peptídeo no intestino inflamado. Em cultura celular de epitélio (Caco-2), Eudragit-SBA15-Ac2-26 favoreceu a internalização de Ac2-26. Em conjunto, as duas estratégias expermentais de entrega do rAnxA1 ou Ac2-26 foram eficientes e os resultados obtidos sugerem que mais estudos devem ser realizados para a confirmação das estratégias de tratamento. Com o intuito de buscar ferramentas para ampliar estes estudos, durante estágio BEPE foram realizados estudos em cultura de células epiteliais baseado em células-tronco adultas diferenciadas in vitro. Os resultados mostraram que rAnxA1 ou Ac2-26 protegeram a integridade epitelial após desafio com LPS, pela regulação positiva da expressão das junções oclusivas e aderentes e redução da expressão de claudina-2, responsável pelo aumento da permeabilidade intercelular; pela modulação negativa decitocinas pró-inflamatórias CXCL-1 e MCP-1, e positiva de citocina antiinflamatória IL-10. Desta forma, padronizamos um novo modelo de cultura celular ainda não testada para a AnxA1 ou Ac2-26, que poderá ser empregada para desvendar os mecanismos da MLNC-AnxA1 e do Eudragit-SBA15-Ac2-26
Annexin Al (AnxA1) is a 37 kDa protein that controls the development of the innate inflammatory reaction, and favors efferocytosis and tissue repair. In inflammatory bowel diseases (IBDs), both endogenous and synthetic AnxA1 and the synthetic peptide mimetic to the N-terminal of the protein (Ac2-26) inhibit the development of disease and induce healing. The present project aimed to obtain new formulations to carry recombinant AnxA1 (rAnxA1) or Ac2-26 and test their efficacy in the experimental colitis model induced by dextram sodium sulfate (DSS, 0-6 days) in C57Bl/6 mice. rAnxA1 was functionalized into multiwall lipid core nanocapsules (MLNC) by Zn2+ binding, with high incorporation efficiency (92%) and administered orally, intravenously or intraperitoneally during the latent phase of the disease (6º-9º day). Only intraperitoneal treatment with MLNC-AnxA1 (12.5 µg/mL) significantly reduced the clinical signs of the disease, restored the integrity of the colonic structure and cell proliferation, as well as increased the expression of intestinal barrier cell junctions. Furthermore, MLNC-AnxA1 induced macrophage polarization to the M2 phenotype in vivo in inflamed tissue and in vitro after stimulation with bacterial lipopolysaccharides (LPS). In an attempt to obtain a therapeutic formulation with oral activity, the Ac2-26 peptide was incorporated into ordered mesoporous silica SBA-15 and coated with Eudragit® L30-D55. Peptide incorporation was effective (88%) and oral administration of Eudragit-SBA15-Ac2-26 (6º-9º day; 200 µg/mice; 8 mg/kg) significantly reduced clinical symptoms and inflammation. PET-SCAN assays showed SBA-15 remained in the intestine for up to 16 hours after administration and promoted the release of the peptide in the inflamed intestine. In epithelial cell culture (Caco-2), SBA15-Ac2-26 favored the internalization of Ac2-26. Taken together, the two experimental delivery strategies for rAnxA1 or Ac2-26 were efficient and the results obtained suggest that more studies should be carried out to confirm the treatment strategies. In order to seek tools to expand these studies, during the BEPE internship, studies were carried out in epithelial cell cultures based on adult stem cells differentiated in vitro. The results showed rAnxA1 or Ac2-26 protected epithelial integrity after challenge with LPS, by upregulating the expression of tight and adherens junctions and reducing the expression of claudin-2, responsible for increasing intercellular permeability; by negative modulation of pro-inflammatory cytokines CXCL-1 and MCP-1, and positive modulation of anti-inflammatory cytokine IL-10. In this way, we standardized a new cell culture model that has not yet been tested for AnxA1 or Ac2-26, which could be used to unravel the mechanisms of MLNC-AnxA1 and Eudragit-SBA15-Ac2-26
Asunto(s)
Animales , Masculino , Ratones , Anexina A1/análisis , Colitis/patología , Inflamación/clasificación , Intestinos/anomalías , Técnicas In Vitro/instrumentación , Enfermedades Inflamatorias del Intestino/diagnóstico , Técnicas de Cultivo de Célula/instrumentación , Mascotas/anomalíasRESUMEN
Breast cancer (BC) is the most commonly occurring cancer and primary cause of cancerrelated mortality in women worldwide. Investigations into BC have been conducted in in vitro and in vivo models. Of these models, the cultivation of tumor cell lines in twodimensional models is the most widely employed in vitro model to study tumor physiology. However, this approach does not accurately model all aspects observed in tumors. To address these limitations, threedimensional (3D) in vitro models have been developed. In these, it is possible to reproduce the interaction between tumor cells and the extracellular matrix, as well as the interrelationship between tumor cells and stromal cells, in order to replicate the interactions observed within the 3D environment of in vivo tumors. The present review summarizes the most common 3D in vitro models used to study BC, including spheroid models, organonachip models, hydrogel models and bioprinted models, with a discussion of their particular advantages and limitations.
Asunto(s)
Bioimpresión/métodos , Neoplasias de la Mama/patología , Dispositivos Laboratorio en un Chip , Esferoides Celulares/patología , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Femenino , Humanos , Hidrogeles , Microambiente TumoralRESUMEN
Leishmaniasis, a neglected tropical disease (NTD), is a set of diseases caused by obligatory parasitic protozoa of the genus Leishmania. And it has cutaneous and visceral eishmaniasis as its main forms. Treatment includes pentavalent antimonials. These drugs have several disadvantages, such as the need for parenteral administration, use of high dosages, long duration of treatment, severe toxicity, resistance and variable efficacy. The candidate for hydroxymethylnitrofural drug (NFOH), a prodrug derived from nitrofural, showed high activity in cell cultures infected with Trypanosoma cruzi and less toxicity when compared to nitrofural. Due to its low solubility in water and reduced bioavailability, NFOH has failed the in vivo efficacy tests. Nanostructured drug delivery systems have the potential to overcome these challenges due to their evident advantages: greater therapeutic efficacy, less toxicity, modified drug release and increased gastrointestinal absorption of drugs with low water solubility. The objective of this project will be the preparation and evaluation of the physicochemical characteristics of a nanostructured lipid carrier containing hydroxymethylnitrofural (NLC-NFOH). The NFOH showed the highest solubility in Miglyol® 840 among the tested liquid lipids. For solid lipids, Gelucire® 50/13 and Precirol® ATO5 proved to be more suitable for the solubilization of NFOH. The optimized NLC-NFOH consisted of these three lipids. These lipids were selected using a quick Technobis Crystal 16TM methodology, microscopy and DSC. Different lipid selection tools provided scientific knowledge relevant to the development of NLC. The NLC-NFOH had an average z of 198.6 ± 5.4 nm, a PDI of 0.11 ± 0.01 and a zeta potential of -13.7 ± 0.7 mV. This study allowed a design space development approach of the first NLC-NFOH with the potential to treat leishmaniasis orally. The development of a sensitive bioanalytical method using HPLC and evaluation of some analytical figures of merit for the validation allowed the quantification of NFOH and NF. The bioanalytical method for analysis of NFOH and NF use Zorbax SB-C18, 5µm, (4.6x250mm) HPLC column. The mobile phase was consisted of acetonitrile:water (20:80 v/v) with flow rate of 1.2 ml/min, at UV detection of 370 nm. The linearity of NFOH and NF was found in the range 0.0253.0 µg/ml with a correlation coefficient of r > 0.98. The precision was 2.44 to 13.77% for NFOH and 2.61 to 18.42%; the accuracy was 2.66 to 14.28% for NFOH and 2.09 to 19.06% for NF. The method showed to be suitable for effectively evaluation of NFOH is serum. NLC-NFOH (2.8 mg/kg) was administered to animals by gavage, and the blocking flow of the chylomicrons model was performed with an intraperitoneal injection of cycloheximide. The presence of NFOH in serum was evaluated with and without cycloheximide. The cytotoxicity assay of NLC-NFOH and blank-NLC showed more than 90% viable cells at the maximum concentration used (2560 µM). NFOH and NF were detected at 1h after the gavage of DMSO-NFOH or NLC-NFOH, without the pretreatment with cycloheximide. The concentration found for DMSO-NFOH and NLC-NFOH were 0.0316 and 0.0291 µg/mL, respectively. The NLC presented the NFOH absorption by the lymphatic system, demonstrated by blocking chylomicrons flow
A leishmaniose, uma doença tropical negligenciada (DTN), é um conjunto de doenças causadas por protozoários parasitas obrigatórios do gênero Leishmania. E tem como formas principais a leishmaniose cutânea e visceral. O tratamento inclui antimoniais pentavalentes. Esses fármacos apresentam várias desvantagens, como necessidade de administração parenteral, uso de altas dosagens, longa duração do tratamento, toxicidade grave, resistência e eficácia variável. O candidato ao fármaco hidroximetilnitrofural (NFOH), um pró-fármaco derivado do nitrofural, apresentou alta atividade em culturas de células infectadas pelo Trypanosoma cruzi e menor toxicidade quando comparado ao nitrofural. Devido à sua baixa solubilidade em água e biodisponibilidade reduzida, o NFOH falhou nos testes de eficácia in vivo. Os sistemas nanoestruturados de liberação de fármacos têm potencial para superar esses desafios devido às suas vantagens evidentes: maior eficácia terapêutica, menor toxicidade, liberação modificada do fármaco e aumento da absorção gastrointestinal de fármacos com baixa solubilidade em água. O objetivo deste projeto será a preparação e avaliação das características físico-químicas de um carreador lipídico nanoestruturado contendo hidroximetilnitrofural (NLC-NFOH). O NFOH apresentou a maior solubilidade no Miglyol® 840 entre os lipídios líquidos testados. Para lipídios sólidos, Gelucire® 50/13 e Precirol® ATO5 se mostraram mais adequados para a solubilização de NFOH. O NLC-NFOH otimizado consistiu desses três lipídios. Esses lipídios foram selecionados usando Technobis Crystal 16TM, microscopia e DSC. Diferentes ferramentas de seleção de lipídios forneceram conhecimento científico relevante para o desenvolvimento de NLC. O NLC-NFOH teve z-average de 198,6 ± 5,4 nm, PDI de 0,11 ± 0,01 e potencial zeta de -13,7 ± 0,7 mV. Este estudo permitiu o desenvolvimento por abordagem de Design Space do primeiro NLC-NFOH com potencial para tratar a leishmaniose por via oral. O desenvolvimento de um VIII método bioanalítico sensível utilizando HPLC e a avaliação de algumas figuras analíticas de mérito para a validação permitiram a quantificação de NFOH e NF em soro. O método bioanalítico para análise de NFOH e NF usou coluna de HPLC Zorbax SB-C18, 5 µm, (4,6 x 250 mm). A fase móvel foi constituída por acetonitrila: água (20:80 v / v) com vazão de 1,2 ml / min, com detecção no UV de 370 nm. A linearidade de NFOH e NF foi encontrada na faixa de 0,0253,0 µg / ml com um coeficiente de correlação de r> 0,98. A precisão foi de 2,44 a 13,77% para NFOH e 2,61 a 18,42%; a precisão foi de 2,66 a 14,28% para NFOH e 2,09 a 19,06% para NF. O método mostrou-se adequado para avaliação efetiva do NFOH no soro. NLC-NFOH (2,8 mg / kg) foi administrado aos animais por gavagem, e o modelo de bloqueio do fluxo de quilomícrons foi realizado com injeção intraperitoneal de cicloheximida. A presença de NFOH no soro foi avaliada com e sem cicloheximida. O ensaio de citotoxicidade de NLC-NFOH e brancoNLC mostrou mais de 90% de células viáveis na concentração máxima utilizada (2560 µM). NFOH e NF foram detectados 1h após a gavagem de DMSO-NFOH ou NLC-NFOH, sem o pré-tratamento com cicloheximida. As concentrações encontradas para DMSO-NFOH e NLC-NFOH foram 0,0316 e 0,0291 µg / mL, respectivamente. O NLC apresentou a absorção do NFOH pelo sistema linfático, demonstrada pelo bloqueio do fluxo dos quilomícrons
Asunto(s)
Leishmaniasis/patología , Química Física/clasificación , Administración Oral , Medicina Tropical/clasificación , Técnicas In Vitro/instrumentación , Preparaciones Farmacéuticas/análisis , Cromatografía Líquida de Alta Presión/métodos , Técnicas de Cultivo de Célula/instrumentación , Metodología como un Tema , Liberación de Fármacos/efectos de los fármacos , Absorción Gastrointestinal/efectos de los fármacos , Sistema LinfáticoRESUMEN
Neste trabalho foram sintetizados e caracterizados três complexos de cobre com ligantes imínicos, com o objetivo de avaliar sua atividade tripanocida. Esses complexos foram caracterizados por diversas técnicas espectroscópicas, como UV-Vis, Infravermelho e EPR, além de análise elementar e espectrometria de massa. Juntamente com outros complexos similares previamente sintetizados pelo nosso grupo, tiveram suas atividades avaliadas frente à forma tripomastigota do parasita T. cruzi, responsável pela fase aguda da doença de Chagas, por ensaios de viabilidade celular, com determinação do valor de seus IC50, concentração em que observamos a morte de 50% da cultura celular, pela metodologia denominada MTT. Todos os complexos mostraram-se eficientes frente a tripomastigotas, apresentando valores de IC50 abaixo de 10 µM, com quatro deles obtendo índice de seletividade maior que 10, fator importante para definir agentes promissores antichagásicos. Complexos selecionados também tiveram sua atividade verificada frente à forma amastigota do parasita, responsável pela fase crônica da doença, utilizando método de imageamento por microscópio de fluorescência e contagem celular. Estudos de inibição da cruzaína, uma cisteíno-protease importante para o metabolismo do parasita foram conduzidos em colaboração com o laboratório do Prof. Wagner Alves de Souza Júdice, da Universidade de Mogi das Cruzes. Quatro dos compostos testados apresentaram atividade inibitória frente a cruzaína, sendo dois de cobre, um de zinco e um ligante livre. Os estudos também permitiram diferenciar os mecanismos de inibição dos compostos, com os complexos de cobre apresentando um mecanismo de inibição clássico e o composto de zinco e o ligante livre apresentando o mecanismo de inibição competitiva parabólica com cooperatividade
In this work, three copper complexes with iminic ligands were synthesized and characterized, with the objective of evaluating their trypanocidal activity. These complexes were characterized by several spectroscopic techniques, such as UV-Vis, Infrared and EPR, in addition to elementary analysis and mass spectrometry. Together with other similar complexes previously synthesized by our group, their activities were evaluated against the trypomastigote form of the parasite T. cruzi, responsible for the acute phase of Chagas disease, by cell viability tests, with determination of the value of their IC50, concentration in that we observed the death of 50% of the cell culture, by the methodology called MTT, all presenting IC50 values below 10 µM, with four of them obtaining a selectivity index greater than 10, important factor for defining promising antichagasic agents. Selected complexes also had their activity verified against the amastigote form of the parasite, responsible for the chronic phase of the disease, using a fluorescence microscope and cell counting imaging method. Inhibition studies of cruzain, a cysteine protease important for the metabolism of the parasite, were conducted in collaboration with the laboratory of Professor Wagner Alves de Souza Júdice at the University of Mogi das Cruzes. Four of the tested compounds showed inhibitory activity against cruzain, two of copper, one of zinc and a free ligand. The studies also allowed to differentiate the mechanisms of inhibition of the compounds, with the copper complexes presenting a classic inhibition mechanism and the zinc compound and the free ligand presenting the competitive parabolic inhibition mechanism with cooperativity
Asunto(s)
Enfermedad de Chagas/patología , Cobre/química , Iminas/agonistas , Antiparasitarios , Espectrometría de Masas/métodos , Tripanocidas/administración & dosificación , Técnicas de Cultivo de Célula/instrumentación , Proteasas de Cisteína/química , LigandosRESUMEN
Biomaterials may be useful in filling lost bone portions in order to restore balance and improve bone regeneration. The objective of this study was to produce polycaprolactone (PCL) membranes combined with two types of bioglass (Sol-Gel and melt-quenched) and determine their physical and biological properties. Membranes were produced through electrospinning. This study presented three experimental groups: pure PCL membranes, PCL-Melt-Bioglass and PCL-Sol-gel-Bioglass. Membranes were characterized using Scanning Electron Microscopy, Fourier Transform Infrared Spectrophotometry (FTIR), Energy-Dispersive Spectroscopy and Zeta Potential. The following in vitro tests were performed: MTT assay, alkaline phosphatase activity, total protein content and mineralization nodules. Twenty-four male rats were used to observe biological performance through radiographic, fracture energy, histological and histomorphometric analyses. The physical and chemical analysis results showed success in manufacturing bioactive membranes which significantly enhanced cell viability and osteoblast differentiation. The new formed bone from the in vivo experiment was similar to that observed in the control group. In conclusion, the electrospinning enabled preparing PCL membranes with bioglass incorporated into the structure and onto the surface of PCL fibers. The microstructure of the PCL membranes was influenced by the bioglass production method. Both bioglasses seem to be promising biomaterials to improve bone tissue regeneration when incorporated into PCL.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Cerámica/química , Poliésteres/química , Animales , Materiales Biocompatibles , Desarrollo Óseo , Diferenciación Celular , Electroquímica , Humanos , Células Madre Mesenquimatosas/fisiología , Osteogénesis , Ratas , Ingeniería de Tejidos/métodosRESUMEN
The present study was aimed to develop a membrane sparger (MS) integrated into a tubular photobioreactor to promote the increase of the carbon dioxide (CO2 ) fixation by Spirulina sp. LEB 18 cultures. The use of MS for the CO2 supply in Spirulina cultures resulted not only in the increase of DIC concentrations but also in the highest accumulated DIC concentration in the liquid medium (127.4 mg L-1 d-1 ). The highest values of biomass concentration (1.98 g L-1 ), biomass productivity (131.8 mg L-1 d-1 ), carbon in biomass (47.9% w w-1 ), CO2 fixation rate (231.6 mg L-1 d-1 ), and CO2 use efficiency (80.5% w w-1 ) by Spirulina were verified with MS, compared to the culture with conventional sparger for CO2 supply. Spirulina biomass in both culture conditions had high protein contents varying from 64.9 to 69% (w w-1 ). MS can be considered an innovative system for the supply of carbon for the microalgae cultivation and biomass production. Moreover, the use of membrane system might contribute to increased process efficiency with a reduced cost of biomass production.
Asunto(s)
Ciclo del Carbono/fisiología , Dióxido de Carbono/metabolismo , Técnicas de Cultivo de Célula/instrumentación , Microalgas/efectos de los fármacos , Biomasa , Dióxido de Carbono/farmacología , Membranas/química , Microalgas/crecimiento & desarrollo , Fotobiorreactores/microbiologíaRESUMEN
Acute pancreatitis is one of the first pathological processes where autophagy has been described in a human tissue. Autophagy, autodigestion, and cell death are early cellular events in acute pancreatitis. Recent advances in understanding autophagy highlight its importance in pathological conditions. However, methods for monitoring autophagic activity during complex diseases, involving highly differentiated secretory cells, are complicated, and the results are sometimes misinterpreted. Here, we describe methods used to identify autophagic structures and to measure autophagic flux in cultured cell models and animal models of pancreatitis. We also briefly describe the pancreas specific autophagy mouse model that was useful to understand the actual role of autophagy in pancreatitis and to identify a novel selective autophagy pathway named zymophagy. Lastly, we describe the immunomagnetic isolation of autophagosomes and the detection of autophagy in pancreatic tissue samples derived from humans.
Asunto(s)
Autofagosomas/patología , Autofagia , Precursores Enzimáticos/metabolismo , Páncreas/patología , Pancreatitis/patología , Células Acinares , Animales , Autofagosomas/ultraestructura , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular , Ceruletida/toxicidad , Modelos Animales de Enfermedad , Humanos , Lisosomas/metabolismo , Masculino , Ratones , Microscopía Electrónica/instrumentación , Microscopía Electrónica/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Páncreas/citología , Pancreatectomía , Pancreatitis/inducido químicamente , Pancreatitis/cirugía , Ratas , Vesículas Secretoras/patologíaRESUMEN
The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the parameters for one and two-color experiments and the required controls for these experiments. Finally, we review the statistical analysis of the FCS data and summarize the use of numerical simulations as a complementary approach that helps us to understand the complex matrix of molecular interactions network within the nucleus.
Asunto(s)
Núcleo Celular/metabolismo , Microscopía Intravital/métodos , Citometría de Barrido por Láser/métodos , Espectrometría de Fluorescencia/métodos , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Línea Celular , Técnica del Anticuerpo Fluorescente/instrumentación , Técnica del Anticuerpo Fluorescente/métodos , Microscopía Intravital/instrumentación , Citometría de Barrido por Láser/instrumentación , Rayos Láser , Mesocricetus , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodosRESUMEN
The cell cycle has fundamental effects on cell cultures and their products. Tools to synchronize cultured cells allow the study of cellular physiology and metabolism at particular cell cycle phases. However, cells are most often arrested by methods that alter their homeostasis and are then cultivated in poorly controlled environments. Cell behavior could then be affected by the synchronization method and culture conditions used, and not just by the particular cell cycle phase under study. Moreover, only a few viable cells are recovered. Here, we designed an integrated system where a large number of cells from a controlled bioreactor culture is separated by centrifugal elutriation at high viabilities. In contrast to current elutriation methods, cells are injected directly from a bioreactor into an injection loop, allowing the introduction of a large number of cells into the separation chamber without stressful centrifugation. A low pulsation peristaltic pump increases the stability of the elutriation chamber. Using this approach, a large number of healthy cells at each cell cycle phase were obtained, allowing their direct inoculation into fully instrumented bioreactors. Hybridoma cells synchronized and cultured in this system behaved as expected for a synchronous culture.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Animales , Técnicas de Cultivo de Célula/instrumentación , Línea Celular Tumoral , Diseño de Equipo , RatonesRESUMEN
Gamma radiation has been shown particularly useful for the functionalization of surfaces with stimuli-responsive polymers. This method involves the formation of active sites (free radicals) onto the polymeric backbone as a result of the high-energy radiation exposition over the polymeric material. Thus, a microenvironment suitable for the reaction among monomer and/or polymer and the active sites is formed and then leading to propagation to form side-chain grafts. The modification of polymers using high-energy irradiation can be performed by the following methods: direct or simultaneous, pre-irradiation oxidative, and pre-irradiation. The most frequently used ones correspond to the pre-irradiation oxidative method as well as the direct one. Radiation-grafting has many advantages over other conventional methods because it does not require the use of catalyst nor additives to initiate the reaction and usually no changes on the mechanical properties with respect to the pristine polymeric matrix are observed. This chapter is focused on the synthesis of smart polymers and coatings obtained by the use of gamma radiation. In addition, the diverse applications of these materials in the biomedical area are also reported, with focus in drug delivery, sutures, and biosensors.
Asunto(s)
Polímeros/química , Técnicas Biosensibles/métodos , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Portadores de Fármacos/química , Polímeros/síntesis química , Polímeros/efectos de la radiación , Radiación Ionizante , Propiedades de Superficie , Suturas , TemperaturaRESUMEN
This study aimed at evaluating the influence of magnetic field on the growth and biomass composition of Spirulina sp., cultivated in vertical tubular photobioreactors. Magnetic fields of 5, 30 and 60mT generated by electric current and ferrite magnets were applied at different lengths of time. The magnetic field of 30 and 60mT for 1hd(-1) stimulated the growth, thus leading to higher biomass concentration by comparison with the control culture. Increase in productivity, protein and carbohydrate contents were 105.1% (60mT for 1hd(-1)), 16.6% (60mT for 24hd(-1)) and 133.2% (30mT for 24hd(-1)), respectively. These values were higher than the ones of the control. Results showed that magnetic field may influence the growth of Spirulina sp., since it triggers a stimulating effect and can leads to twofold biomass concentration in equal cultivation time periods.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Campos Magnéticos , Microalgas/crecimiento & desarrollo , Spirulina/crecimiento & desarrollo , Biomasa , Metabolismo de los Hidratos de Carbono , Técnicas de Cultivo de Célula/instrumentación , Concentración de Iones de Hidrógeno , Microalgas/metabolismo , Fotobiorreactores , Spirulina/metabolismoRESUMEN
Digitalis purpurea L. is one of the main economically viable sources of cardenolides (cardiac glycosides) for the pharmaceutical industry. Nevertheless, production of cardenolides in plants grown by traditional agriculture is not always an efficient process and can be affected by biotic and abiotic factors. This chapter provides two biotechnology strategies for biomass and cardenolide production in D. purpurea. Firstly, we report biomass production using a temporary immersion system (TIS), combined with cardenolide extraction and quantification. Secondly, an efficient protocol for genetic transformation via Agrobacterium tumefaciens is provided. These strategies can be used independently or combined in order to increase the content of cardiac glycosides in D. purpurea and to unravel biosynthetic pathways associated to cardiac glycoside production.
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Biotecnología/métodos , Cardenólidos/metabolismo , Digitalis/metabolismo , Agrobacterium tumefaciens/genética , Biomasa , Vías Biosintéticas , Biotecnología/instrumentación , Cardenólidos/análisis , Cardenólidos/aislamiento & purificación , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Digitalis/química , Digitalis/genética , Digitalis/microbiología , Diseño de Equipo , Transformación GenéticaRESUMEN
Conventional approaches to bone regeneration rarely use multiwall carbon nanotubes (MWCNTs) but instead use polymeric matrices filled with hydroxyapatite, calcium phosphates and bioactive glasses. In this study, we prepared composites of MWCNTs/polycaprolactone (PCL) for bone regeneration as follows: (a) MWCNTs randomly dispersed on PCL, (b) MWCNTs aligned with an electrical field to determine if the orientation favors the growing of human dental pulp stem cells (HDPSCs), and (c) MWCNTs modified with ß-glycerol phosphate (BGP) to analyze its osteogenic potential. Raman spectroscopy confirmed the presence of MWCNTs and BGP on PCL, whereas the increase in crystallinity by the addition of MWCNTs to PCL was confirmed by X-ray diffraction and differential scanning calorimetry. A higher elastic modulus (608 ± 4.3 MPa), maximum stress (42 ± 6.1 MPa) and electrical conductivity (1.67 × 10(-7) S/m) were observed in non-aligned MWCNTs compared with the pristine PCL. Cell viability at 14 days was similar in all samples according to the live/dead assay, but the 21 day cell proliferation, measured by MTT was higher in MWCNTs aligned with BGP. Von Kossa and Alizarin red showed larger amounts of mineral deposits on MWCNTs aligned with BGP, indicating that at 21 days, this scaffold promotes osteogenic differentiation of HDPSCs.
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Regeneración Ósea , Pulpa Dental/citología , Nanotubos de Carbono/química , Poliésteres/química , Células Madre/citología , Andamios del Tejido/química , Adolescente , Adulto , Huesos/citología , Huesos/fisiología , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Pulpa Dental/fisiología , Humanos , Ensayo de Materiales , Osteogénesis/fisiología , Células Madre/fisiología , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodos , Adulto JovenRESUMEN
Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.
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Técnicas de Cultivo de Célula/métodos , Ganglios Espinales/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Microelectrodos , Microscopía Fluorescente/métodos , Neuronas/fisiología , Animales , Técnicas de Cultivo de Célula/instrumentación , Tamaño de la Célula , Células Cultivadas , Ganglios Espinales/citología , Masculino , Neuronas/citología , Reconocimiento de Normas Patrones Automatizadas/métodos , Ratas WistarRESUMEN
In this work, changes in Vero cell cultivation methods have been employed in order to improve cell growth conditions to obtain higher viable cell densities and to increase viral titers. The propagation of the 17DD yellow fever virus (YFV) in Vero cells grown on Cytodex I microcarriers was evaluated in 3-L bioreactor vessels. Prior to the current changes, Vero cells were repeatedly displaying insufficient microcarrier colonization. A modified cultivation process with four changes has resulted in higher cell densities and higher virus titers than previously observed for 17DD YFV.
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Reactores Biológicos , Recuento de Células , Técnicas de Cultivo de Célula , Cultivo de Virus/métodos , Virus de la Fiebre Amarilla/crecimiento & desarrollo , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Supervivencia Celular , Chlorocebus aethiops , Medio de Cultivo Libre de Suero , Células Vero , Carga ViralRESUMEN
UNLABELLED: Induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps) could provide a powerful tool for studying the mechanisms underlying human liver development and disease, testing the efficacy and safety of pharmaceuticals across different patients (i.e., personalized medicine), and enabling cell-based therapies in the clinic. However, current in vitro protocols that rely upon growth factors and extracellular matrices (ECMs) alone yield iHeps with low levels of liver functions relative to adult primary human hepatocytes (PHHs). Moreover, these low hepatic functions in iHeps are difficult to maintain for prolonged times (weeks to months) in culture. Here, we engineered a micropatterned coculture (iMPCC) platform in a multiwell format that, in contrast to conventional confluent cultures, significantly enhanced the functional maturation and longevity of iHeps in culture for at least 4 weeks in vitro when benchmarked against multiple donors of PHHs. In particular, iHeps were micropatterned onto collagen-coated domains of empirically optimized dimensions, surrounded by 3T3-J2 murine embryonic fibroblasts, and then sandwiched with a thin layer of ECM gel (Matrigel). We assessed iHep maturity by global gene expression profiles, hepatic polarity, secretion of albumin and urea, basal cytochrome P450 (CYP450) activities, phase II conjugation, drug-mediated CYP450 induction, and drug-induced hepatotoxicity. CONCLUSION: Controlling both homotypic interactions between iHeps and heterotypic interactions with stromal fibroblasts significantly matures iHep functions and maintains them for several weeks in culture. In the future, iMPCCs could prove useful for drug screening, studying molecular mechanisms underlying iHep differentiation, modeling liver diseases, and integration into human-on-a-chip systems being designed to assess multiorgan responses to compounds.
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Comunicación Celular , Hepatocitos/fisiología , Células Madre Pluripotentes Inducidas , Técnicas de Cultivo de Célula/instrumentación , Células Cultivadas , Humanos , Factores de TiempoRESUMEN
The particle and fluid dynamics in a rotating cylindrical filtration (RCF) system used for animal cell retention in perfusion processes was studied. A validated CFD model was used and the results gave numerical evidence of phenomena that had been earlier claimed, but not proven for this kind of application under turbulent and high mesh permeability conditions, such as bidirectional radial exchange flow (EF) through the filter mesh and particle (cells) lateral migration. Taylor vortices were shown to cause EF 10-100 times higher than perfusion flow, indicating that EF is the main drag source, at least in early stages of RCF operation. Particle lateral migration caused a cell concentration reduction (CCR) near the filter surface of approximately 10%, contributing significantly to cell separation in RCF systems and giving evidence that the mesh sieving effect is not the sole phenomenon underlying cell retention in RCF systems. Filter rotation rate was shown to significantly affect both EF and CCR. A higher separation efficiency (measured experimentally at 2,000-L bioreactor scale) and an enhanced CCR (predicted by the numerical simulations) were found for the same rotation rate range, indicating that there is an optimal operational space with practical consequences on RCF performance. Experimental data of a large-scale perfusion run employing the simulated RCF showed high cell viabilities for over 100 days, which is probably related to the fact that the computed shear stress level in the system was shown to be relatively low (below 20 Pa under all tested conditions).
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Técnicas de Cultivo de Célula/instrumentación , Filtración/instrumentación , Hidrodinámica , Modelos Biológicos , Perfusión/instrumentación , Animales , Recuento de Células , Línea Celular Tumoral , Proliferación Celular , Simulación por Computador , Diseño de Equipo , RatonesRESUMEN
Currently, there are several techniques for modified cell culture surfaces under research to improve cell growth and adhesion. Recently, different methods have been used for surface coating, using biomolecules that enhance cell attachment and growth of nerve cells from spinal cord, such as the use of Poly-DL-Ornithine/Laminin. Plasma-polymerized pyrrole (PPy)-treated surfaces have showed improvement on surfaces biocompatibility with the cells in culture since they do not interfere with any of the biological cell functions. In the present work, we present a novel mouse nerve cell culture technique, using PPy-treated cell culture surfaces. A comparative study of cell survival using Poly-DL-Ornithine/Laminin-treated surfaces was performed. Our results of cell survival when compared with data already reported by other investigators, show that cells cultured on the PPy-modified surface increased survival up to 21 days when compared with Poly-DL-Ornithine/Laminin-coated culture, where 8 days cell survival was obtained. There were electrical and morphological differences in the nerve cells grown in the different surfaces. By comparing the peak ion currents of Poly-DL-Ornithine/Laminin-seeded cells for 8 days with cells grown for 21 days on PPy, an increase of 516% in the Na(+) current and 127% in K(+) currents in cells seeded on PPy were observed. Immunofluorescence techniques showed the presence of cell synapses and culture viability after 21 days. Our results then showed that PPy-modified surfaces are an alternative culture method that increases nerve cells survival from lumbar spinal cord cell culture by preserving its electrical and morphological features.
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Técnicas de Cultivo de Célula/instrumentación , Neuronas/fisiología , Pirroles/química , Médula Espinal/citología , Potenciales de Acción , Animales , Supervivencia Celular , Células Cultivadas , Medios de Cultivo , Impedancia Eléctrica , Iones/metabolismo , Laminina , Vértebras Lumbares , Potenciales de la Membrana , Ratones , Neuronas/citología , Polimerizacion , Potasio/metabolismo , Sodio/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Sinapsis/fisiología , Factores de TiempoRESUMEN
Casing materials and practices used in the cultivation of Agaricus bisporus were evaluated in the cultivation of Agaricus subrufescens, using the best techniques for optimization of production, including the possibility of re-casing of the compost for the production of a second crop of mushroom. Casing based on peat moss, loam soil or coir was compared to casing material mixed with or without spawn-run compost. Based on the results, we conclude that the casing layer used in the cultivation of A. subrufescens should not necessarily be the same as that used in the cultivation of A. bisporus. For the tested strain cultivated with loam soil as casing layer, the ruffling technique is highly superior to CACing and should be pursued in further research. The re-casing of compost in new cycles showed good results suggesting that the currently used compost could be improved.
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Agaricus/crecimiento & desarrollo , Técnicas de Cultivo de Célula/métodos , Agaricus/fisiología , Técnicas de Cultivo de Célula/instrumentación , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Suelo/químicaRESUMEN
This work describes the preparation and characterization of porous 3D-scaffolds based on chitosan (CHI), chitosan/silk fibroin (CHI/SF) and chitosan/silk fibroin/hydroxyapatite (CHI/SF/HA) by freeze drying. The biomaterials were characterized by X-ray diffraction, attenuated total reflection Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, scanning electron microscopy and energy dispersive spectroscopy. In addition, studies of porosity, pore size, contact angle and biological response of SaOs-2osteoblastic cells were performed. The CHI scaffolds have a porosity of 94.2±0.9%, which is statistically higher than the one presented by CHI/SF/HA scaffolds, 89.7±2.6%. Although all scaffolds were able to promote adhesion, growth and maintenance of osteogenic differentiation of SaOs-2 cells, the new 3D-scaffold based on CHI/SF/HA showed a significantly higher cell growth at 7 days and 21 days and the level of alkaline phosphatase at 14 and 21 days was statistically superior compared to other tested materials.