Este trabalho foi financiado pelo National Institutes of Health concessão R01DK080971 e Juvenile Diabetes Research Foundation concessão 37-2009-44. Agradecemos também o apoio recebido do Pediatric Diabetes Research Center UCSD (PDRC).

1. A transfecção de HEK293 Camada <ol><li> Prepara HEK293 meio celular por adição a 500 ml de frascos de meio DMEM (com 4,5 g / ml de glucose e de vermelho de fenol e sem glutamina): 50 ml de FBS, 5 mL de 100X solução de estreptomicina penicilina /, 5 ml de solução de L-glutamina e 500 100x anfotericina ul B. </li><li> Placa para células HEK293 em uma placa de 24 poços utilizando 0,5 ml de meio por poço HEK293. Assegure-se que as células são distribuídos uniformemente em toda a parte inferior da placa. </li><li> Quando as células HEK293 chegar a 100% de confluência, transfecção com 0,8 ug do plasmídeo que codifica a proteína de interesse (inserido num vector de expressão de mamífero) e 2 ul de Lipofectamina 2000, de acordo com o protocolo de lipofectamina. Optou-se por utilizar o pcDNA 3,3 backbone projetado para expressão em células de mamíferos. </li><li> Opcionalmente, após 6 horas de incubação, a troca da mídia transfecção descrito no protocolo Lipofectamine com HEK293 meios normais. </li><li> Para avaliar a expressão da prote transfectadasnum subconjunto dos poços de cultura de tecidos transfectados podem ser usadas para a detecção de imunofluorescência da proteína expressa ou para análise por Western blot da expressão da proteína, utilizando técnicas convencionais. </li></ol> 2. A fixação opcional de células HEK293 que expressam a proteína Transfectadas As células HEK293 transfectadas podem ser fixados suavemente, a fim de facilitar a co-cultura em meios que podem ser prejudiciais para a vida (por exemplo, células HEK293 passo 3.9 abaixo), ou para permitir que a preparação eficiente de antecedência de placas para várias experiências de uma só vez (ver também a discussão). <ol><li> Realizar estudos piloto para determinar a evolução no tempo da expressão da proteína nas células HEK293. </li><li> Após transfecção de células da camada HEK293, aspirar os meios de comunicação das células no ponto de tempo pós-transfecção associado com maior nível de expressão. Se isto ocorre dentro das primeiras 24 horas, não se aspirar até 24 horas após a transfecção. </li><li> Lave HEK293 células suavemente com D-PBS, em seguida adicionar 500 mL de 4% de paraformaldeído (PFA) e incubar a temperatura ambiente durante 30 min. (Para fazer a solução de PFA 4%, comece com uma solução de 16% em 1X PBS e diluir 1:04 com 1X PBS). </li><li> Utilizando PBS estéril, lavar suavemente as células três vezes em PBS e incubar O / N a 4 ° C. </li><li> Lavar as células 3 vezes com PBS estéril, em seguida, adicionar PBS e deixar a 4 ° C até ser necessário (tempo máximo de armazenamento poderá variar dependendo da proteína expressa. Nós armazenar células HEK293 transfectadas para expressar neuroligina isoformas de até 2 semanas, sem alterações significativas nos efeitos observados sobre a secreção de insulina). </li></ol> 3. Co-cultura de INS-1 beta células com células HEK293 <ol><li> Prepara INS-1 media adicionando a 500 ml de meio RPMI 1640 (com glicose e de vermelho de fenol e sem glutamina): 50 ml de FBS, 5 mL de 100X de penicilina / estreptomicina, 5 ml de solução de L-glutamina 100X, 5 mL de piruvato de sódio, 500 mL de anfotericina B, 500 ul de beta-mercaptoetanol (isto é quase idêntico ao do medihum normalmente usado para a cultura ilhéu principal, exceto para a adição de beta-mercaptoetanol). </li><li> Utilizando-celular de remoção (ou outro removedor de células não enzimática), INS-1 de colheita de células de aproximadamente 70-80% de confluência fora da parte inferior de um frasco de cultura T75. Cada frasco de células irá fornecer aproximadamente suficiente para 48 poços. </li><li> Depois girando as células na centrífuga durante 3 min a 1200 rpm, ressuspender em uma mistura 50/50 de INS-1 e meios de HEK. Para um frasco confluente de 70-80% de células INS-1, ressuspender em cerca de 25 ml de meios mistos. </li><li> Se estiver usando células HEK293 que foram fixados em paraformaldeído, em vez de células vivas HEK293, ressuspender as células INS-1 em 100% INS-1 media, em vez de mídia mista. </li><li> Utilizando uma pipeta de 10 ml, desalojar as células da pelota para fazer uma suspensão de células homogéneas. </li><li> Aspirar para remover os meios de comunicação sobre as células HEK293. </li><li> Usando uma pipeta P1000, adicionar cuidadosamente 500 ul de suspensão de célula INS-1 sobre a camada de células HEK ao longo dos lados of poço. Após cada 6 poços, usar uma pipeta de 10 ml para ressuspender as células INS-1 de novo para assegurar uma suspensão homogénea de células. O esquema geral da co-cultura set-up é mostrado na Figura 1. </li><li> Incubar as células durante 24-48 horas, dependendo do protocolo experimental. </li><li> Se for necessário mais de 48 período de co-cultura hr, use passos opcionais na seção 2 acima para corrigir as células HEK293. </li><li> Se estiver usando o roedor dissociada primária ou ilhotas humanas [eg ver protocolos anteriores em Jove 11-15], certifique-se de que uma mídia adequados, como RPMI com 5 ml pen / strep, 5 ml L-Glut, 0,5 ml anfotericina-B é usado em vez de media uma INS-. Embora se possa utilizar as células das ilhotas dissociadas numa placa de 24 poços, na prática, devido ao grande número de ilhotas potencialmente necessários, é recomendado que seja tida em consideração a redução de escala para uma placa com cavidades 48 (necessitando de menos células de ilhéus por poço). Em uma placa de 48, adicionar cerca de 100 ilhotas no valor de dissociaçãoted células por placa para começar. Dependendo da eficácia de dissociação e do método utilizado, menor número de ilhotas pode ser necessária. </li></ol> 4. Glicose estimulada secreção de insulina <ol><li> Pré-incubação de células em 250 uL de 2,5 mM de glucose em KRB (tampão de bicarbonato de Krebs-Ringer) durante um mínimo de 1 hora. Para pré-incubar, remova o meio de co-cultura e substituir imediatamente com KRB. Esta troca deve ser feito um poço de cada vez para minimizar a exposição de células INS-1 para oxigênio ambiental. Aspirar com uma mão enquanto a pipetagem baixa (2,5 mM) de KRB de glicose com a outra. </li><li> Taxas de pré-incubação de KRB a 250 ul de ambos 2,5 mM ou 20 mM de glucose em um tampão KRB assim de cada vez. </li><li> Se for apropriado para o ensaio específico, secretagogos adicionais, tais como 100 ^ M de IBMX pode ser adicionado para produzir uma resposta mais forte de secreção de insulina estimulada. Células beta das ilhotas principais dissociadas em particular, são pouco responsiva ao aumento da concentração de glicose alone, portanto, aqui especialmente a adição de IBMX ou outros secretagogos deve ser considerada 16,17. </li><li> Após 1 hora de incubação, transferir mídia para microcentrifugue tubos e girar 1.500 xg por 5 min. </li><li> Remover 200 ul do sobrenadante e utilizar este para a análise da insulina por RIA ou ELISA. </li><li> Para se preparar o ligado celular, adicionar 100 ul de tampão de lise celular RIPA (NaCl 150 mM, 1,0% IGEPALCA-630 ou Nonidet P-40, 0,5% desoxicolato de sódio, 0,1% de SDS, Tris a 50 mM, pH 8,0) com inibidores de protease para o placa imediatamente após a remoção de meios de comunicação e incubar a 4 ° C durante 20 min. </li><li> Girar lisado durante 15 minutos a 10000 xg e retirar 50 ul de sobrenadante para a análise da insulina por RIA ou ELISA. </li></ol>

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Não temos nada a divulgar.

O método de co-cultura aqui descrito fornece um meio eficaz para determinar a importância fisiológica das proteínas beta da superfície da célula, transmembranares específicos, e, especificamente, dos seus domínios extracelulares. Por cultura de células beta (ou células de insulinoma, tais como as células INS-1 utilizados aqui), em contacto com células HEK293 que indica uma proteína de interesse sobre a superfície celular, as experiências podem ser concebidas para determinar os efeitos da extracelulares in...

Descrevemos aqui um método para facilitar as investigações de como os domínios extracelulares de proteínas transmembrana específicos afetam a secreção de insulina. O método sondas os efeitos das interacções da proteína de interesse com proteínas (ou possivelmente de outras moléculas), na superfície das células beta do pâncreas. O método permite investigações de como as proteínas da superfície celular, expressas por células beta ou por outras células vizinhas (por exemplo, células endoteliais, neurónios, células alfa pancreáticas) afectam a função das células beta através de interacções transcelulares (isto é, através de interacções com os parceiros de interacção na superfície adjacente células beta). A membrana plasmática celular contém um conjunto complexo de proteínas estruturais e funcionais, que servem como ponte para o ambiente extracelular. Pela formação de ligações transcelular ou por abertura de eventos de sinalização de plástico, as interações entre proteínas de superfície celular pode ajudar a coordenar afunção das células vizinhas. Células beta do pâncreas são agrupados dentro das ilhotas pancreáticas e agir de forma coordenada para manter a homeostase da glicose 1. Como revelado, por exemplo, a importância da extracelular EPHA-ephrinA e neuroligin-2 interações na regulação da glicose estimulada a secreção de insulina, está se tornando cada vez mais claro que o aumento do conhecimento das interações entre proteínas extracelulares que ocorrem nas superfícies das adjacente células beta será de grande importância para a obtenção de uma compreensão completa da secreção de insulina, a maturação funcional das células beta e na manutenção da homeostase da glicose 1-3. O objectivo do método descrito aqui é o de permitir investigações dos efeitos sobre a função de interacções envolvendo transcelular transmembranar específica ou de outro modo, as proteínas de superfície celular associados à célula beta. Por células beta co-cultura com células HEK293 transfectadas com diferentes construções de expressão, os efeitos sobre a bfunção das proteínas da superfície das células diferentes ou variantes mutadas célula eta do mesmo pode ser eficientemente sondadas. Isto é conseguido sem a necessidade de transfectar as próprias células beta. Elucidação dos papéis particulares interações transcelulares por estudos knockdown, nocaute ou superexpressão em células beta cultura ou in vivo necessita de perturbação direta de mRNA das células beta e expressão da proteína, potencialmente afetando a saúde das células beta e / ou função de maneiras que poderiam confundir análise de os efeitos das interacções extracelulares específicos. Estas abordagens também alterar os níveis dos domínios intracelulares das proteínas alvo e, além disso, não permitem que os efeitos devido às interacções entre as proteínas sobre ou dentro da mesma célula para se distinguir os efeitos de interacção intracelular. Aqui, um método para determinar o efeito das interacções específicas transcelular na capacidade de segregar insulina e capacidade de resposta das células beta é described. Este método aplica-se a linhas de células beta secretoras de insulina, tais como as células INS-1, 4 e roedor primárias dissociadas ou células beta humanas. Baseia-se em modelos de co-cultura desenvolvidos por neurobiologists, que verificaram que a exposição de culturas de neurónios a proteínas neuronais específicos expressos em HEK293 (COS), as camadas de células conseguiu identificar as proteínas que conduzem a formação de sinapses 5,6. Dadas as semelhanças entre a maquinaria de secreção das sinapses neuronais e das células beta, que argumentou que a função das células beta e maturação funcional pode ser conduzido por interações transcelulares semelhantes 7-9. A fim de investigar estas interacções, foi desenvolvido o sistema aqui descrito, em que as células beta são co-cultivadas numa camada de células HEK293 expressando uma proteína de interesse 10. Este sistema permite que o citoplasma das células beta para permanecer intacta enquanto extracelulares interacções proteína-proteína são manipulados.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>pcDNA 3.3 vector/backbone</td>
			<td>Invitrogen</td>
			<td>K830001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>18324012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Mediatech</td>
			<td>45001-312</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen/strep solution</td>
			<td>Mediatech</td>
			<td>45001-652-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Mediatech</td>
			<td>45001-808-1/30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Mediatech</td>
			<td>45001-404</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Mediatech</td>
			<td>45001-434</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Mediatech</td>
			<td>45001-710-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985023</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell stripper</td>
			<td>Mediatech</td>
			<td>45000-668</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T75 Flask</td>
			<td>BD</td>
			<td>1368065</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>50980487</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Mediatech</td>
			<td>45001-130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Mediatech</td>
			<td>MT35010CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma</td>
			<td>I5879-100MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td>Sigma</td>
			<td>R0278</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

coculture analysis of extracellular protein interactions affecting insulin secretion by pancreatic beta cells

Interações entre proteínas de superfície celular ajudar a coordenar a função das células vizinhas. Células beta do pâncreas são agrupados dentro de ilhotas pancreáticas e agir de forma coordenada para manter a homeostase da glicose. Está se tornando cada vez mais claro que as interações entre proteínas transmembrana nas superfícies de células beta adjacentes são importantes determinantes da função das células beta. Elucidação dos papéis particulares interações transcelulares por estudos knockdown, nocaute ou superexpressão em células beta cultura ou in vivo necessita de perturbação direta de mRNA e expressão da proteína, potencialmente afetando a saúde das células beta e / ou função de maneiras que poderiam confundir análises dos efeitos de interacções específicas. Estas abordagens também alterar os níveis dos domínios intracelulares das proteínas alvo e podem evitar os efeitos devido às interacções entre as proteínas no interior da mesma membrana da célula para ser disdiferenciadas das efeitos das interacções transcelulares. Aqui, um método para a determinação do efeito de interacções específicas transcelular na capacidade de segregar insulina e capacidade de resposta das células beta é apresentado. Este método aplica-se a linhas de células-beta, tais como células INS-1, e para as células beta primárias dissociadas. Baseia-se em modelos de co-cultura desenvolvidas por neurobiólogos, que descobriu que a exposição de neurônios cultivados às proteínas neuronais específicos expressos em HEK293 (ou COS) camadas de células identificadas proteínas importantes para a condução de formação de sinapses. Dadas as semelhanças entre a maquinaria de secreção das sinapses neuronais e das células beta, que argumentou que a maturação funcional das células beta pode ser conduzido por interações transcelulares semelhantes. Foi desenvolvido um sistema em que as células beta são cultivadas sobre uma camada de células HEK293 expressando uma proteína de interesse. Neste modelo, o citoplasma da célula beta é tocada enquanto extracelular da proteína-proteína interactions são manipulados. Embora o foco aqui primariamente em estudos de glicose estimulada a secreção de insulina, outros processos podem ser analisados, por exemplo, as alterações na expressão do gene, como determinado por imunotransf erência ou qPCR.

Utilizando o método descrito aqui, testámos o efeito de diferentes variantes do neuroliguinas proteína na secreção de insulina. Isto complementa a obra publicada a investigar o efeito da neuroliguinas-2 em função das células beta 10. Figura 2, por exemplo, descreve os resultados obtidos a partir coculturing INS-1 células beta com células HEK293 transfectadas para expressar uma isoforma neuroliguinas referido aqui como NL- X. Esta experiência foi concebida para testar a hipótese de...

Watch this Scientific Journal Video about Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells at JoVE.com

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interações de proteínas intracelulares são determinantes importantes da função das células beta no pâncreas. Detalhado aqui é um método adaptado a partir de um modelo de co-cultura de synaptogenesis-para investigar como as proteínas transmembrana específicos influenciar a secreção de insulina. As células HEK293 transfectadas expressam as proteínas de interesse, células beta não precisa ser transfectadas directamente ou de outro modo perturbado.

Análise de co-cultura da Relação de proteínas extracelulares que afetam secreção de insulina por células beta pancreáticas se

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

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Research

JoVE Journal

Medicine

12.8K visualizações.  University of California, San Diego. Interações de proteínas intracelulares são determinantes importantes da função das células beta no pâncreas. Detalhado aqui é um método adaptado a partir de um modelo de co-cultura de synaptogenesis-para investigar como as proteínas transmembrana específicos influenciar a secreção de insulina. As células HEK293 transfectadas expressam as proteínas de interesse, células beta não precisa ser transfectadas directamente ou de outro modo perturbado.

Vídeo: Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

Melhorar IV A administração de insulina em um hospital comunitário

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

Medicina

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Sistema de co-cultura com uma fatia organotípicas Cérebro e esferóide 3D de carcinomas de células

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Dupuytren Humano do<em&gt; Ex Vivo</em&gt; Cultura para o Estudo da Miofibroblastos e Matriz Extracelular Interações

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

Neurodegeneração em um modelo animal de Chronic beta-amilóide Oligï¿½ero Infusion é contrariada pelo anticorpo Tratamento infundido com bombas osmóticas

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

Aplicação de anastomose to-end em robótica pancreatectomia Central

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Homogeneous Time-resolved F&amp;#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

Förster homogênea de tempo-resolvido ressonância energia baseada em transferência ensaio para detecção de secreção de insulina

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Isolamento dos hepatócitos primários de rato para análise de síntese de proteína nascente pelo método de rotulagem não-radioativo L-azidohomoalanine

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Investigação de bio-energética de Candida albicans usando real-time extracelular fluxo análise

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...