Ce travail a été financé par les Instituts de la Santé subvention R01DK080971 et Juvenile Diabetes Research Foundation subvention 37-2009-44 nationale. Nous apprécions également le soutien reçu de la Pediatric Research Center Diabetes UCSD (CEDE).

1. La transfection de cellules HEK293 couche <ol><li> Préparer milieu cellulaire HEK293 en ajoutant à bouteilles de 500 mL de DMEM (avec 4,5 g / ml de glucose et rouge de phénol et sans glutamine): 50 ml de FBS, 5 ml 100X pénicilline / solution de streptomycine, 5 ml de solution de L-glutamine 100x et 500 amphotéricine ul B. </li><li> Plate sur les cellules HEK293 dans une plaque de 24 puits en utilisant 0,5 ml de HEK293 médias par puits. S&#39;assurer que les cellules sont réparties uniformément sur le fond de la plaque. </li><li> Lorsque les cellules HEK293 atteindre 100% de confluence, transfect avec 0,8 ug de plasmide codant la protéine d&#39;intérêt (inséré dans un vecteur d&#39;expression mammalien) et 2 ul de Lipofectamine 2000, selon le protocole Lipofectamine. Nous avons choisi d&#39;utiliser le pcDNA 3.3 épine dorsale conçu pour l&#39;expression dans les cellules de mammifères. </li><li> Eventuellement, après 6 heures d&#39;incubation, échange les médias de transfection décrit dans le protocole Lipofectamine avec HEK293 médias normales. </li><li> Pour évaluer l&#39;expression de la prote transfectéesdans un sous-ensemble des puits de culture tissulaire transfectées peut être utilisé pour la détection par immunofluorescence de la protéine exprimée ou pour l&#39;analyse par Western blot de l&#39;expression de protéines en utilisant des techniques standard. </li></ol> 2. Fixation facultative de cellules HEK293 exprimant la protéine transfectées Cellules HEK293 transfectées peuvent être fixés doucement afin de faciliter co-culture dans les médias susceptibles de nuire à la vie des cellules HEK293 (par exemple étape 3.9 ci-dessous) ou pour permettre la préparation efficace à l&#39;avance des plaques de plusieurs expériences à la fois (voir également la discussion). <ol><li> Mener des études pilotes afin de déterminer l&#39;évolution temporelle de l&#39;expression de la protéine dans les cellules HEK293. </li><li> Après transfection de la couche de cellules HEK293, aspirer le support à partir des cellules au point de temps après la transfection associé à plus haut niveau d&#39;expression. Si cela se produit dans les 24 premières heures, ne pas aspirer jusqu&#39;à 24 heures après transfection. </li><li> Lavez HEK293 cellules doucement avec D-PBS puis ajouter 500 ul paraformaldéhyde 4% (PFA) et incuber à température ambiante pendant 30 min. (Pour faire une solution à 4% PFA, commencer avec une solution de 16% dans du PBS 1X et diluer à 1:4 avec du PBS 1X.) </li><li> Utiliser PBS stérile, laver délicatement les cellules 3 fois et les incuber dans du PBS O / N à 4 ° C. </li><li> Laver les cellules 3 fois avec du PBS stérile, puis ajouter PBS et laisser à 4 ° C jusqu&#39;à ce que nécessaire (durée de stockage maximale peut varier en fonction de la protéine exprimée. Nous stockons des cellules HEK293 transfectées pour exprimer isoformes neuroligin pendant 2 semaines sans changements importants dans les effets observés sur la sécrétion d&#39;insuline). </li></ol> 3. Co-culture de cellules INS-1 bêta avec cellules HEK293 <ol><li> Préparer INS-1 média en ajoutant à 500 ml de milieu RPMI 1640 (avec le glucose et le rouge de phénol et sans glutamine): 50 ml de FBS, 5 ml 100X pénicilline / streptomycine, 5 ml de solution 100X L-glutamine, 5 ml de pyruvate de sodium, 500 amphotéricine pl B, 500 pl bêta-mercaptoéthanol (ce qui est presque identique à la médecineum généralement utilisé pour la culture des îlots primaire à l&#39;exception de l&#39;ajout de bêta-mercaptoéthanol). </li><li> Utilisation de Cell-strip-teaseuse (ou un autre solvant de cellule non enzymatique), INS-1 récolte des cellules à environ 70-80% de confluence du fond d&#39;un flacon de culture T75. Chaque flacon fournira suffisamment de cellules pour environ 48 puits. </li><li> Après filer vers le bas des cellules dans la centrifugeuse pendant 3 min à 1200 rpm, remettre en suspension dans un mélange 50/50 de l&#39;INS-1 et médias HEK. Pour un flacon confluent de 70-80% des cellules INS-1, remettre en suspension dans environ 25 ml de médias mixtes. </li><li> Si vous utilisez des cellules HEK293 qui ont été fixés dans du paraformaldéhyde plutôt que des cellules vivantes HEK293, remettre en suspension les cellules INS-1 à 100% INS-1 médias plutôt que de techniques mixtes. </li><li> En utilisant une pipette de 10 ml, de déloger des cellules de la pastille pour faire une suspension de cellules homogène. </li><li> Aspirer à retirer le support sur les cellules HEK293. </li><li> Avec une pipette P1000, ajouter délicatement 500 pl de suspension cellulaire INS-1 sur la couche de cellules HEK le long des côtés of le puits. Après tous les 6 puits, utiliser une pipette de 10 ml pour remettre à nouveau les cellules INS-1 pour assurer une suspension de cellules homogène. L&#39;économie générale de la co-culture set-up est représenté dans la figure 1. </li><li> Incuber les cellules pendant 24-48 heures selon le protocole expérimental. </li><li> Si plus de 48 période de coculture h est nécessaire, utiliser des étapes facultatives de la section 2 ci-dessus pour fixer les cellules HEK293. </li><li> Si vous utilisez rongeur dissocié primaire ou îlots humains [voir par exemple les protocoles antérieurs dans JoVE 11-15], veiller à ce que des médias appropriés, tels que RPMI avec stylo 5 ml / streptomycine, 5 ml L-Glut, 0,5 ml d&#39;amphotéricine-B est utilisé à la place de INS-1 médias. Même si on peut utiliser des cellules d&#39;îlots dissociées dans une plaque à 24 puits, dans la pratique, en raison du grand nombre d&#39;îlots potentiellement nécessaires, il est recommandé que l&#39;on envisage de mise à l&#39;échelle vers le bas sur une plaque à 48 puits (nécessitant moins de cellules des îlots pancréatiques par puits). Sur une plaque à 48 puits, ajouter environ 100 îlots de dollars de dissociationted cellules par plaque pour commencer. Selon l&#39;efficacité de la dissociation et la méthode utilisée, moins d&#39;îlots peuvent être nécessaires. </li></ol> 4. Glucose sécrétion d&#39;insuline stimulée <ol><li> Préincuber cellules dans 250 ul de 2,5 mM de glucose dans KRB (tampon bicarbonate de Krebs-Ringer) pour un minimum de 1 heure. Pour préincuber, éliminer le milieu de co-culture et remplacer immédiatement avec KRB. Cet échange devrait se faire un bien à la fois pour minimiser l&#39;exposition des cellules INS-1 à l&#39;oxygène de l&#39;environnement. Aspirer avec une main pendant le pipetage le bas (2,5 mm) KRB de glucose avec l&#39;autre. </li><li> Bourse pré-incubation KRB à 250 pi de 2,5 mM ou 20 mM de glucose dans KRB tampon d&#39;un bien à un moment. </li><li> Le cas échéant, pour l&#39;expérience spécifique, sécrétagogues supplémentaires telles que 100 um IBMX peuvent être ajoutés pour produire une réponse de sécrétion d&#39;insuline stimulée plus robuste. Les cellules dissociées bêta des îlots pancréatiques primaires en particulier, sont mal répondre à l&#39;augmentation des concentrations de glucose alone, alors voici surtout plus de IBMX ou autres sécrétagogues doit être considérée 16,17. </li><li> Après 1 heure d&#39;incubation, le milieu de transfert à Microfuge tubes et filent 1500 g pendant 5 min. </li><li> Retirer 200 pi du surnageant et l&#39;utiliser pour l&#39;analyse par l&#39;insuline RIA ou ELISA. </li><li> Pour préparer lysat cellulaire, ajouter 100 ul de tampon de lyse cellulaire RIPA (NaCl 150 mM, 1,0% IGEPALCA-630 ou Nonidet P-40, 0,5% de désoxycholate de sodium, SDS 0,1%, Tris 50 mM, pH 8,0) avec les inhibiteurs de protéase pour le plaque immédiatement après avoir retiré les médias et incuber à 4 ° C pendant 20 min. </li><li> Isoler lysat pendant 15 min à 10000 xg et éliminer 50 pi de surnageant pour analyse par l&#39;insuline RIA ou ELISA. </li></ol>

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Nous n&#39;avons rien à révéler.

La méthode décrite ici co-culture est un moyen efficace pour déterminer l&#39;importance physiologique de protéines beta-cell-surface, transmembranaires spécifiques, et en particulier de leurs domaines extracellulaires. Par les cellules bêta de culture ou de cellules d&#39;insulinome (comme les cellules INS-1 utilisés ici) en contact avec les cellules HEK293 affichant une protéine d&#39;intérêt sur la surface de la cellule, les expériences peuvent être conçues pour déterminer les effets des interactions pr...

Nous décrivons ici une méthode pour faciliter les enquêtes sur la façon dont les domaines extracellulaires de protéines transmembranaires spécifiques affectent la sécrétion d&#39;insuline. La méthode des sondes les effets des interactions de la protéine d&#39;intérêt avec des protéines (ou éventuellement d&#39;autres molécules) sur la surface des cellules bêta pancréatiques. Le procédé permet des enquêtes de comment les protéines de surface cellulaire exprimée par des cellules bêta ou par d&#39;autres cellules voisines (par exemple des cellules endothéliales, des neurones, des cellules alpha du pancréas) affectent la fonction des cellules bêta par des interactions transcellulaires (soit par des interactions avec des partenaires d&#39;interaction sur la surface de côté cellules bêta). La membrane de plasma cellulaire contient un ensemble complexe de protéines structurales et fonctionnelles servant de ponts pour le milieu extracellulaire. Par la formation de connexions transcellulaires ou par l&#39;initiation des événements de signalisation en plastique, les interactions entre les protéines de surface cellulaire peuvent aider à coordonner l&#39;la fonction des cellules voisines. les cellules bêta du pancréas sont regroupés dans les îlots pancréatiques et d&#39;agir de manière coordonnée pour maintenir l&#39;homéostasie du glucose 1. Comme l&#39;a révélé, par exemple, par l&#39;importance des extracellulaire EPHA-ephrinA et neuroligin-2 interactions dans la régulation du glucose stimulée par la sécrétion d&#39;insuline, il devient de plus en plus clair que l&#39;augmentation de la connaissance des interactions extracellulaires qui se produisent entre les protéines sur les surfaces de côté cellules bêta seront d&#39;une grande importance pour acquérir une compréhension complète de la sécrétion d&#39;insuline, les bêta maturation fonctionnelle des cellules et le maintien de l&#39;homéostasie du glucose 1-3. Le but de la méthode décrite ici est de permettre à des enquêtes sur les effets sur la fonction des cellules bêta des interactions transcellulaires impliquant transmembranaire spécifique ou des protéines non-surface cellulaire associés. Par les cellules bêta co-culture avec des cellules HEK293 transfectées avec différentes constructions d&#39;expression, les effets sur la bla fonction des cellules ETA de différentes protéines de surface cellulaire ou des variantes mutantes de celui-ci peut être sondé efficacement. Ceci est accompli sans avoir à transfecter les cellules bêta eux-mêmes. L&#39;élucidation du rôle des interactions particulières transcellulaires par effet de choc, coup de grâce ou la surexpression des études dans les cellules bêta en culture ou in vivo nécessite perturbation directe de l&#39;ARNm des cellules bêta et l&#39;expression des protéines, ce qui pourrait affecter la santé des cellules bêta et / ou la fonction d&#39;une manière qui pourrait fausser l&#39;analyse des les effets des interactions spécifiques extracellulaires. Ces approches également modifier les niveaux des domaines intracellulaires des protéines ciblées et, de plus, ne laissez pas les effets dus aux interactions entre protéines sur ou dans la même cellule à se distinguer des effets des interactions transcellulaires. Ici, un procédé pour déterminer l&#39;effet des interactions spécifiques transcellulaires sur la capacité de sécréter de l&#39;insuline et de la réactivité des cellules bêta est Described. Cette méthode est applicable aux lignes des cellules bêta qui sécrètent l&#39;insuline, tels que les cellules INS-1, 4 et rongeurs primaire dissocié ou des cellules bêta humaines. Il est basé sur des modèles développés par coculture neurobiologistes, qui ont trouvé que l&#39;exposition des neurones en culture à des protéines neuronales spécifiques exprimés sur HEK293 (ou COS) des couches de cellules pourrait identifier les protéines qui conduisent à la formation des synapses 5,6. Étant donné les parallèles entre les mécanismes de sécrétion des synapses neuronales et des cellules bêta, nous avons pensé que la fonction des cellules bêta et la maturation fonctionnelle pourraient être conduits par des interactions similaires transcellulaires 7-9. Afin de sonder ces interactions, nous avons développé le système décrit ici, dans lequel les cellules bêta sont co-cultivées sur une couche de cellules HEK293 exprimant une protéine d&#39;intérêt 10. Ce système permet le cytoplasme des cellules bêta de rester intact pendant des interactions protéine-protéine extracellulaire sont manipulés.

<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

Les interactions entre les protéines de surface cellulaire aider à coordonner la fonction des cellules voisines. les cellules bêta du pancréas sont regroupés au sein des îlots pancréatiques et d&#39;agir de manière coordonnée pour maintenir l&#39;homéostasie du glucose. Il devient de plus en plus clair que les interactions entre protéines transmembranaires à la surface des cellules bêta adjacents sont des déterminants importants de la fonction des cellules bêta. L&#39;élucidation du rôle des interactions particulières transcellulaires par effet de choc, coup de grâce ou la surexpression des études dans les cellules bêta en culture ou in vivo nécessite perturbation directe de l&#39;ARNm et l&#39;expression des protéines, ce qui pourrait affecter la santé des cellules bêta et / ou la fonction d&#39;une manière qui pourrait fausser les analyses des effets des interactions spécifiques. Ces approches modifient également les niveaux des domaines intracellulaires des protéines ciblées et peuvent prévenir les effets dus aux interactions entre protéines dans la même membrane cellulaire pour être affichéedistingué des effets des interactions transcellulaires. Ici, un procédé pour déterminer l&#39;effet des interactions spécifiques transcellulaires sur la capacité de sécréter de l&#39;insuline et de la réactivité des cellules bêta est présenté. Cette méthode est applicable aux lignes des cellules bêta, comme les cellules INS-1, et pour les cellules bêta primaires dissociées. Il est basé sur des modèles développés par coculture neurobiologistes, qui ont trouvé que l&#39;exposition des neurones en culture de protéines neuronales spécifiques exprimés sur HEK293 (ou COS) des couches de cellules protéines identifiées sont déterminantes pour stimuler la formation des synapses. Étant donné les parallèles entre les mécanismes de sécrétion des synapses neuronales et des cellules bêta, nous avons pensé que la maturation fonctionnelle des cellules bêta peut être entraînée par des interactions transcellulaires similaires. Nous avons développé un système où les cellules bêta sont cultivées sur une couche de cellules HEK293 exprimant une protéine d&#39;intérêt. Dans ce modèle, le cytoplasme des cellules bêta est intact tandis extracellulaire INTERACTIONS de protéine-protéinens sont manipulés. Bien que nous nous concentrons ici principalement sur les études de glucose stimulée par la sécrétion d&#39;insuline, d&#39;autres procédés peuvent être analysés, par exemple, des changements dans l&#39;expression des gènes tels que déterminés par immuno ou qPCR.

En utilisant la méthode décrite ici, nous avons testé l&#39;effet de différentes variantes de la neuroligin de protéines sur la sécrétion d&#39;insuline. Cela complète notre travail publié étudier l&#39;effet de neuroligin-2 sur la fonction des cellules bêta 10. Figure 2, par exemple, décrit les résultats obtenus à partir INS-1 co-culture de cellules bêta avec des cellules HEK293 transfectées pour exprimer un neuroligin isoforme appelée ici NL- X. Cette expérience a été co...

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

Les interactions entre protéines transcellulaires sont des déterminants importants de la fonction des cellules bêta du pancréas. Détaillé ici est une méthode adaptée à partir d&#39;un modèle de co-culture de synaptogénèse-pour étudier comment les protéines transmembranaires spécifiques influencent la sécrétion d&#39;insuline. Cellules HEK293 transfectées expriment des protéines d&#39;intérêt, les cellules bêta n&#39;ont pas besoin d&#39;être transfectées ou non directement perturbé.

Analyse Coculture des interactions protéiques extracellulaires affecter la sécrétion d&#39;insuline par les cellules bêta du pancréas

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 vues.  University of California, San Diego. Les interactions entre protéines transcellulaires sont des déterminants importants de la fonction des cellules bêta du pancréas. Détaillé ici est une méthode adaptée à partir d'un modèle de co-culture de synaptogénèse-pour étudier comment les protéines transmembranaires spécifiques influencent la sécrétion d'insuline. Cellules HEK293 transfectées expriment des protéines d'intérêt, les cellules bêta n'ont pas besoin d'être transfe...

Vidéo: 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.

Amélioration de l&#39;administration d&#39;insuline IV dans un hôpital communautaire

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

Recherche

Médecine

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.

Différenciation isolement, caractérisation et comparative de l&#39;homme dentaires cellules souches de pulpe Dérivé de dents permanentes à l&#39;aide de deux méthodes différentes

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.

Système de co-culture avec un cerveau organotypique et 3D sphéroïde de cellules de carcinome

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.

Human Dupuytren de<em&gt; Ex Vivo</em&gt; Culture pour l&#39;étude de myofibroblastes et de matrice extracellulaire 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 ...

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.

Etudier le cancer du pancréas Stem Cell Caractéristiques pour le développement de nouvelles stratégies de traitement

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.

Neurodégénérescence dans un modèle animal de chronique bêta-amyloïde Oligomère Infusion est contrecarré par Antibody traitement Infused avec les pompes osmotiques

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.

Demande d’une anastomose bout à bout en robotique pancréatectomie centrale

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.

Homogène Time-resolved Förster Resonance Energy axée sur le transfert test pour la détection de l’insulinosécrétion

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.

Isolation des hépatocytes de souris primaire pour analyse-synthèse protéine naissante par méthode d’étiquetage non-radioactifs 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.

Enquête bio-énergétique en temps réel à l’aide de Candida albicans analyse extracellulaire de Flux

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