The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.

Déclaration d&#39;éthique: l&#39;éthique de recherche conseils d&#39;administration de l&#39;Université du Manitoba et Kenyatta National Hospital / Université de Nairobi ont approuvé cette étude et du consentement éclairé écrit a été obtenu de tous les participants à l&#39;étude. 1. Préparation des milieux et CMC Collection Tubes <ol><li> Préparer une solution saline tamponnée au phosphate (PBS) (NaCl 137,93 mM, 2,67 mM de KCl, 8,1 mM Na 2 HPO 4, 1,47 mM de KH 2 PO 4). Autoclave pour la stérilité. Cela peut être conservé à 4 ° C pendant plusieurs mois. </li><li> Pré-aliquote de 5 ml de PBS dans 50 ml tube Falcon (un par échantillon à prélever) et conserver à 4 ° C jusqu&#39;au moment de la collecte de l&#39;échantillon. </li><li> Étiqueter 2-4 cryotubes ou 1,5 ml microtubes par échantillon pour recueillir lavage pour le stockage. </li><li> Préparer un milieu de culture cellulaire: RPMI 1640 + 1% de pénicilline / streptomycine / amphotéricine B (concentration finale de 100 unités / ml, 100 pg / ml et 250 ng / ml, respectively, sans FCS / FBS ajouté. </li></ol> 2. Prélèvement des échantillons cytobrosse Collection d&#39;échantillons de cytobrosse est une procédure non-invasive qui doit être effectuée par un MD ou un gynécologue qualifié. <ol><li> Recueillir les cellules mononucléaires du col utérin (CMC) en utilisant à la fois un cytobrosse et un grattoir en bois ou en plastique. Recueillir CMC du participant en cours d&#39;examen au spéculum en insérant le grattoir et tournant autour de l&#39;orifice cervical (Figure 1). Insérez le cytobrosse dans les os endocervical pivoter sur 360 ° et placez juste deux la cytobrosse racloir dans le tube falcon 50ml contenant 5 mL de PBS. </li><li> Conserver les échantillons sur glace / à 4 º C jusqu&#39;à leur traitement. Pour maintenir la viabilité des cellules, des échantillons de processus à l&#39;intérieur de deux heures de la collecte. </li><li> Si possible, prélever des échantillons de sang appariés en vert meilleurs vacutainers d&#39;héparine pour périphérique de cellules mononucléaires du sang périphérique (PBMC) d&#39;isolement pour servir un comparateur / contrôle pour l&#39;analyse CMC. </li&gt; </ol> 3. Isolation des CMC Effectuer le traitement des échantillons dans un laboratoire de niveau de biosécurité 2, dans une enceinte de sécurité biologique stérile avec des gants doubles. <ol><li> Lors de la collecte, les échantillons de CMC peuvent être contaminés avec du sang. Exclure des échantillons de contamination du sang visible de l&#39;étude afin d&#39;éviter l&#39;inclusion de confusion de PBMC dans l&#39;échantillon final. En raison du faible nombre de lymphocytes isolés à partir d&#39;échantillons de CMC, la contamination du sang mineur peut facilement submerger la composante lymphocytaire CMC. </li><li> Tubes vortex de faucon contenant à la fois le cytobrosse et grattoir pour 45 sec de dissocier les cellules des brosses. Les échantillons peuvent devenir mousseux cours de ce processus; ce qui est normal. </li><li> Utilisez le cytobrosse pour gratter les restes de hors la raclette, et jeter le racleur dans une solution d&#39;eau de Javel. Pour recueillir les cellules restant attachés à la brosse / grattoir, utilisez un gant frais de serrer la raclette entre le pouce et l&#39;index.Faites glisser la brosse, permettant aux cellules et PBS à collecter dans le même tube de 50 ml. Jeter le cytobrosse dans une solution d&#39;eau de Javel, et jeter le gant. IMPORTANT: Utilisez un gant neuf pour chaque échantillon. </li><li> Monter un 100 um cellule de nylon filtre à 50 ml tube frais de faucon. En utilisant une pipette de transfert, de recueillir la suspension du PBS-cellule à partir du tube d&#39;échantillon et filtrer à travers le filtre dans le tube frais. </li><li> Ajouter 5 ml de RPMI 1640 avec le tube de l&#39;échantillon initial. En utilisant la pipette de transfert, laver les parois du tube d&#39;échantillon avec le RPMI, et transférer à travers le filtre vers le nouveau tube de collecte. Laver le fond du filtre en nylon avec le RPMI ainsi. </li><li> Centrifuger les échantillons pendant 10 min à 514 x g. Il est essentiel de laisser le frein de la centrifugeuse au cours de cette étape de réduction, afin d&#39;empêcher le délogement de la pastille CMC. </li><li> Retirez délicatement le surnageant du tube, en prenant soin de ne pas perturber le culot. taille de Pellet sera très variable; en sortemoi les échantillons, la pastille est assez grande en raison de la présence de grands nombres de cellules epitheliales, tandis que dans d&#39;autres cas, le culot est à peine visible et très transparente. </li><li> Reprendre le culot par agitation douce du tube Falcon. Ajouter 5 ml de PBS pour laver l&#39;échantillon. </li><li> Centrifuger à nouveau pendant 10 min à 514 xg sans frein de la centrifugeuse. </li><li> Prenez soin de le surnageant et remettre en suspension le culot comme ci-dessus. Les cellules sont prêtes pour la cryoconservation soit (en utilisant un protocole de cryoconservation PBMC), la stimulation ou la cytométrie en flux phénotypage. </li></ol> 4. CMC coloration de surface et cytométrie en flux <ol><li> Reprendre le culot de l&#39;étape 3.10 dans 100 pi de solution de blocage et transférer soit dans une plaque de 96 puits ou FACS tube. La solution de blocage (pour bloquer les récepteurs Fcy) recette est la suivante: IgG de souris de 1,8 pl (concentration finale de 0,2 pg / pl), 5 pl de FBS et 93,2 ul de lavage FACS (PBS + 2% de FBS).La coloration peut être effectué dans une plaque de 96 puits si la taille des granulés sont assez petites, mais peut avoir besoin d&#39;être effectuée dans des tubes FACS si pastilles sont systématiquement grande. Il est important pour titrer les anticorps en conséquence. </li><li> Bloquer les échantillons pendant 10 min sur la glace / à 4 ° C. </li><li> Laver les cellules avec 100 pi de FACS de lavage (PBS + 2% de FBS) dans des plaques à 96 puits ou 500 pl dans des tubes FACS. Centrifugeuse à 600 g pendant 10 min à 4 ° C avec le frein à faible (ou hors tension, si un réglage du frein bas n&#39;est pas disponible). </li><li> Eliminer le surnageant et remettre en suspension le culot de cellules par agitation. Ajouter viabilité Stain (Live Dead colorant de viabilité réparable). Préparer viabilité colorant selon les instructions du fabricant. Il peut avoir besoin d&#39;être titrée pour une utilisation dans chaque configuration laboratoire / de cytomètre. Incuber pendant 30 min à l&#39;obscurité à 4 ° C. </li><li> Laver les cellules 100 μl/500 pi de PBS (pour tubes plaque / FACS 96 puits). Centrifuger à 600 g pendant 10 min à faible / pas de frein. Retirer supernatant et remettre les cellules. </li><li> Incuber les cellules avec le cocktail d&#39;anticorps de marqueur de surface, dans un volume total de 100 pl. IMPORTANT: Optimiser et titrer chaque cytométrie en flux panneau avant la coloration de l&#39;échantillon. Incuber les cellules pendant 30 min à l&#39;obscurité à 4 ° C. </li><li> Répétez l&#39;étape 4.5. si la coloration dans une plaque à 96 puits, les cellules de transfert à un tube FACS et diluer à un volume final de 750 ul de FACS pour lessive contenant 1% de paraformaldehyde (PFA) (ou Cytofix, dilué à 1 4). Passez à acquisition de données sur un cytomètre de flux. </li></ol> 5. Prélèvement et la préparation de col de l&#39;utérus vaginale Lavage (CVL) <ol><li> Avant cytobrosse échantillonnage, utiliser une seringue pour laver l&#39;endocol avec 2 ml de PBS stérile et aspirer le liquide de lavage du cul de sac postérieur. </li><li> Recueillir l&#39;échantillon de lavage aspiré dans un tube Falcon de 15 ml et garder sur la glace / à 4 º C jusqu&#39;à leur traitement. </li><li> Extrait Centrifugeuse à 400 g pendant 7 min pour éliminer les débris cellulaires. </li><li> Collect le surnageant, aliquote de l&#39;échantillon dans 1 ml ou 500 aliquotes et conserver à -70 ° C. Aliquotes peuvent être décongelés pour ELISA ou analyse de réseau de billes de protéines CVL, mais éviter de multiples cycles de gel / dégel. </li><li> Si l&#39;on désire remettre en suspension le culot de débris cellulaires dans la suite de l&#39;ARN pour mesurer l&#39;expression de l&#39;ARN en suivant le protocole du fabricant. </li></ol> 6. D&#39;acquisition de données <ol><li> Préparer une série de tubes de compensation qui correspond à la face d&#39;anticorps. Exécutez les tubes de compensation pour ajuster chevauchement spectral. Analyser les échantillons sur un cytomètre de flux multicolore qui est configuré pour les anticorps conjugués à un fluorochrome utilisés dans le panneau. </li><li> Acquérir un échantillon PBMC premier à ajuster Forward Scatter (FSC) et Side Scatter (SSC) tension afin de détecter la population de lymphocytes. Essayez de les garder à 100 sur chaque axe sur une échelle linéaire. Exécution d&#39;un contrôle PBMC, il sera beaucoup plus facile de trouver de la population de lymphocytes CMC. </li><li> Le seuil de FSC pour les échantillons de CMC peut être augmentée par rapport à des échantillons de PBMC en raison de salir l&#39;axe SSC à de faibles valeurs de FSC. </li><li> Vortex chaque tube avant d&#39;acquérir. Acquérir la totalité du tube afin de maximiser le nombre d&#39;événements de grille de lymphocytes recueillis. Surveillez attentivement la machine pour éviter le colmatage. </li><li> Exporter les données dans un programme d&#39;analyse de cytométrie de flux tel que FlowJo. </li></ol> 7. Stratégie gate <ol><li> Sélectionnez Transférer diffusion latérale haute (FSC-H) / Forward zone de diffusion latérale (FSC-A) pour déterminer la population de maillot. </li><li> Sélectionnez Temps par rapport marqueur fluorescent (comme par exemple FITC) pour contrôler la qualité de l&#39;acquisition. Changement dans le taux d&#39;écoulement peut introduire des artefacts dans les données. Exclure toute zone qui affiche un écart dans le débit. </li><li> Identifier la population de lymphocytes sur SSC-A/FSC-A terrain. Utilisez un contrôle CMSP pour faciliter l&#39;identification. Notez que la population de lymphocytes CMC pourrait ne pas être aussi clair que le contrôle des PBMC. &lt;/ Li&gt; <li> Porte sur SSC-A/viability colorant d&#39;exclure les cellules mortes de cellules vivantes. La mort des cellules non spécifiquement incorporer les anticorps, qui présentera des artefacts. </li><li> Porte sur la SSC-A/CD3 d&#39;identifier la population de cellules T. A partir de cette grille, identifier les populations CD4 + et CD8 +. </li></ol>

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The authors have nothing to disclose.

Compte tenu des grandes lacunes dans les connaissances à l&#39;égard de l&#39;immunité à l&#39;appareil génital féminin (FGT), analyse phénotypique de CMC peut fournir un large éventail de points de vue dans les populations de lymphocytes multiples au col. Couplée à l&#39;analyse protéomique et des mesures de la charge virale dans le lavage de l&#39;utérus, l&#39;immunité aux infections sexuellement transmissibles (IST s) et d&#39;autres agents pathogènes peut être disséqué dans diverses populations. </...

La majorité des nouvelles infections à VIH dans le monde se pose par transmission hétérosexuelle, les femmes représentent 47% des nouvelles infections en 2011 (ONUSIDA 1). Comprendre les voies génitales féminines (FGT), l&#39;un des principaux portails d&#39;entrée pour le VIH et d&#39;autres agents pathogènes transmissibles sexuellement, est d&#39;une grande importance sur la voie de trouver des stratégies efficaces pour prévenir l&#39;infection. Les réponses immunitaires à la muqueuse génitale sont bien unique et diffèrent de celles mesurées dans le sang périphérique 2. Cependant, les connaissances actuelles de la dynamique immunitaires à la FGT est limitée au mieux. À ce jour, les études de l&#39;environnement immunitaire muqueux ont surtout porté sur les tissus lymphoïdes associés à l&#39;intestin (GALT), où il est devenu clair que les événements précoces dans les muqueuses après l&#39;infection ont un fort impact sur ​​la suite progression de la maladie 3,4. Prélèvement d&#39;échantillons de la muqueuse génitale représente un grand défi et est au moins partiellement responsable de la lack de compréhension de l&#39;immunologie de la FGT. Résoudre le casse-tête de la dynamique immunitaire entre l&#39;hôte et l&#39;agent pathogène dans le contexte de l&#39;environnement distinct qui est la FGT nécessite des méthodes efficaces pour la collecte et l&#39;analyse des échantillons de ce lieu. La FGT est divisé en deux sections: l&#39;appareil reproducteur supérieur qui comprend les trompes de Fallope, de l&#39;endomètre et de l&#39;endocol, et la zone inférieure qui contient l&#39;exocol et du vagin (examiné par Kaushic et al 5). Il n&#39;est pas encore clair que la contribution relative de ces différents sites est de l&#39;infection à VIH, mais on pense que les deux sites pourraient contribuer à l&#39;entrée du VIH 6. Les cellules T représentent 40 à 50% des leucocytes dans l&#39;appareil reproducteur supérieure et inférieure, tandis que les macrophages constituent environ 10% (passage en revue dans Rodriguez-Garcia et al 2). Les cellules T peuvent être détectés dans le vagin, le col utérin et de l&#39;endomètre. Les macrophages sont plus fortement localized dans l&#39;endomètre et du tissu conjonctif du myomètre que le col de l&#39;utérus, mais ils peuvent être détectés dans les deux tissus. Enfin, les cellules dendritiques plasmacytoïdes (pDC) et les cellules de Langerhans peuvent également être détectés dans les tissus de traitement des fumées. Le phénotype et les proportions des populations immunitaires et leur susceptibilité à l&#39;infection à VIH peuvent varier surtout selon des cycles hormonaux, l&#39;utilisation de contraceptifs hormonaux, la vaginose bactérienne ou des activités sexuelles 5,7-9. Diverses méthodes ont été développées pour étudier les populations immunitaires et de l&#39;environnement de la FGT. Biopsie cervicale, cytobrosses col de l&#39;utérus et des lavages cervico-vaginales (CVL) 10-12 sont le plus couramment utilisé dans la littérature. Collection CVL par PBS lavage est la méthode la plus simple et permet l&#39;étude des protéines immunomodulateurs, mais les résultats du rendement des cellules extrêmement faible, et n&#39;est donc pas adapté à l&#39;étude des populations de cellules immunitaires de la FGT 13. Échantillons CVL sont, d&#39;autre part, vEry utile pour évaluer l&#39;environnement à l&#39;abri de la FGT par mesure de l&#39;expression de différentes cytokines, les chimiokines ou les facteurs anti-microbiens à l&#39;aide de méthodes telles que l&#39;ELISA, tableau cytokine de talon 14 ou la spectrométrie de masse 15,16. Caractérisation des fréquences des cellules immunitaires, les phénotypes et des fonctions peut être obtenue en collectant les cellules mononucléaires du col (CMC) par cytobrosse col de l&#39;utérus ou de l&#39;échantillonnage de biopsie cervicale. L&#39;échantillonnage de biopsie cervicale est une méthode invasive qui augmente l&#39;inconfort et le risque de saignement et prend de 2 à 11 jours pour guérir suivant le mode opératoire en fonction de l&#39;état immunitaire de la femme 12. D&#39;autre part, cytobrosses cervicales, en dépit de la baisse de rendement des cellules recueillies, est un moyen moins invasif et plus commode de recueillir les cellules immunitaires de l&#39;FGT. Les deux méthodes peuvent atteindre le même rendement de leucocytes CD45 +, mais deux cytobrosses cervicales séquentielles sont nécessaires pour obtenir la même quantité de cellules contenues in une biopsie 13. Néanmoins, cytobrosse échantillonnage fournit encore un nombre acceptable de cellules (environ 5000 CD45 + cellules / de cytobrosse) pour plus d&#39;ex vivo phénotypage en cytométrie de flux 14. De plus, la caractérisation fonctionnelle peut être réalisée sur ces échantillons, comme la stimulation et le flux intracellulaire par cytométrie ou qPCR ont été effectuées en utilisant les CMC de cytobrosse dérivé d&#39;identifier les réponses immunitaires spécifiques au VIH 17 ou la polarisation de la cellule Th 18. L&#39;expansion de la population de cellules T peut également faciliter les études fonctionnelles avec CMC 19. Il est important de noter que les biopsies et cytobrosses échantillonnent des parties distinctes de la FGT. Les biopsies sont obtenues à partir de la partie supérieure de l&#39;épithélium et le stroma de l&#39;exocol 12,13, tandis que cytobrosses cervicales échantillonnent l&#39;orifice utérin, la collecte des cellules dérivées de l&#39;épithélium de l&#39;endocol et vraisemblablement la zone de transformation. Échantillons cytobrosse donc goûter une nouvellerégion composée d&#39;une seule couche de l&#39;épithélium cylindrique, tandis que les biopsies, comprennent une région bordée par un épithélium pavimenteux stratifié 5. Par conséquent, la nature des populations leucocytaires recueillies par biopsie cervicale et cytobrosse diffère. Les biopsies de recueillir une plus grande proportion de cellules T CD3 +, alors que cytobrosses se traduisent par une collection de plus grande proportion de monocytes CD14 + / 13 macrophages. L&#39;étude de l&#39;immunologie de la FGT a eu un intérêt pour de nombreuses années 20-22 et nous avons accumulé beaucoup d&#39;expertise à l&#39;étude des CMC de cytobrosse dérivés. Nos études portent principalement sur l&#39;étude des infectés par le VIH, non infecté et exposés au VIH séronégatifs (Hesn) femmes travailleuses du sexe de Nairobi, au Kenya. Le VIH se réplique préférentiellement dans les cellules T activées 23 et inférieure du nombre de cellules activées qui peuvent être ciblées par le VIH dans le FGT pourraient contribuer à la protection contre la contamination par le VIH. En accord avec cette hypothèse, plusieurs studies ont décrit l&#39;activation immunitaire plus faible parmi les travailleurs du sexe de Hesn qui sont très exposés au VIH restent encore non infectées 24,25, et ce phénotype de repos est également observée dans le FGT 14. Ici, nous décrivons la méthodologie pour le traitement et l&#39;évaluation des cellules T activation dans des échantillons provenant de CMC cytobrosses col de l&#39;utérus par ex vivo cytométrie de flux.

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			<td height="21" style="height:21px;width:295px;">100uM Cell Strainer for 50 ml Falcon tube</td>
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			<td style="width:133px;">352360</td>
			<td>CMC processing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">RPMI 1640</td>
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			<td>CMC processing</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fetal Bovine serum&#160;</td>
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			<td>CMC processing</td>
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			<td>CMC processing</td>
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			<td height="21" style="height:21px;width:295px;">50ml Falcon tube</td>
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			<td height="21" style="height:21px;width:295px;">15 ml Falcon tube&#160;</td>
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			<td>CVL processsing</td>
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			<td height="21" style="height:21px;width:295px;">1.5ml tube ependroff</td>
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			<td height="21" style="height:21px;">LIVE/DEAD Fixable Cell Stain Kit</td>
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collection, isolation, and flow cytometric analysis of human endocervical samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

cytométrie de flux multiparamétrique est un outil puissant pour étudier les phénotypes et les fonctions de sous-ensembles de cellules dans les tissus non définies auparavant. L&#39;analyse des échantillons de CMC peut fournir des informations à la fois sur les lymphocytes et monocytes populations avec les stratégies de déclenchement appropriées. Une stratégie de déclenchement CMC représentant, par rapport à un profil PBMC identifié, est présenté à la figure 2.</stro...

Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Collection, isolement, et cytométrie en flux analyse des échantillons Endocervical humain

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

collection-isolation-flow-cytometric-analysis-human-endocervical

Research

JoVE Journal

Medicine

19.0K vues.  University of Manitoba. The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays. 

Vidéo: Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

L&#39;isolement des îlots humains à partir des patients partiellement pancréatectomisés

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Recherche

Médecine

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

Protocole de collecte des pancréas humain

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

L&#39;isolement, la culture, et de l&#39;imagerie de l&#39;homme causés par des amas de cellules pancréatiques

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

L&#39;isolement et la caractérisation fonctionnelle de Human ventriculaire cardiomyocytes à partir des échantillons chirurgicaux frais

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolement de cellules souches du cancer à partir d&#39;échantillons cancer de la prostate humaine

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Fractionnement rapide et isolement de composants de sang total dans les échantillons obtenus à partir d&#39;un milieu communautaire

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

L&#39;isolement et le profilage des exosomes de Bile humain contenant microARN-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Caractérisation cytométrique simultanée des types de cellules multiples récupérées du cerveau de la souris / moelle épinière grâce à différentes méthodes d'homogénéisation

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

Test d’hémocompatibilité des implants de contact sanguin dans un modèle de boucle de flux imitant le flux sanguin humain

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Comité de LC-MS/MS validé pour quantifier 11 médicaments antituberculeux pharmacorésistants dans de petits échantillons de cheveux

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...