This work was supported in part by grants from NHRI (CS-104-PP-06 to B.L.Y.).

Le consentement éclairé du patient, tel qu&#39;approuvé par le conseil d&#39;examen institutionnel doit être obtenue pour l&#39;utilisation de cellules humaines. 1. Densité isolement Gradient de cellules mononucléaires du sang périphérique humain (CMSP) <ol><li> Ajouter 25 ml du sang total hépariné dans un tube de 50 ml par 25 ml d&#39;une pipette. <ol><li> Diluer les cellules avec 25 ml de solution saline tamponnée au phosphate (PBS). </li></ol></li><li> Ajouter 15 ml d&#39;un gradient de densité Ficoll-Paque dans un nouveau tube de 50 ml, et tout en inclinant le tube, très lentement et avec précaution ajouter à 25 ml de la suspension cellulaire diluée sur le gradient de densité de sorte qu&#39;il n&#39;y a pas de mélange du sang entier avec le gradient de densité, à savoir., pas de perturbation de l&#39;interface gradient de densité du sang. </li><li> Centrifugeuse la suspension de l&#39;étape 1.2 à 400 g pendant 30 à 40 min dans un balancement rotor libre à température contrôlée, sans frein à 20 ° C. Remarque: Trois couches distinctes doivent être apparente après centrifugation: ee couche supérieure étant plasma; la couche inférieure étant clairement le gradient de densité Ficoll-Paque; et une mince couche cellulaire, du milieu étant les CMSP </li><li> Aspirer et jeter la couche supérieure par aspiration avec une pipette Pasteur, avec un soin de ne pas perturber la couche d&#39;interphase de cellules mononucléaires (ie, lymphocytes, monocytes et les plaquettes). </li><li> Recueillir soigneusement cette couche de cellules mononucléaires avec une pipette de 10 ml à 50 ml d&#39;un nouveau tube de centrifugation. </li><li> Remplir le tube avec 20 ml de PBS, bien mélanger, et centrifuger à 300 g pendant 10 min à 20 ° C. Retirer le surnageant complètement après centrifugation. </li><li> Remettre en suspension le culot cellulaire dans 20 ml de PBS et on centrifuge à 200 g pendant 10 à 15 min à 20 ° C. Retirer le surnageant complètement après centrifugation. Remarque: Cette étape élimine les plaquettes qui sont-indésirables au sein de la CMSP; cette étape peut être répétée pour assurer une élimination complète des plaquettes. </li><li> Si aucune sélection de populations spécifiques eston le désire, culot de cellules de remettre en suspension dans des leucocytes du milieu complet (10% de sérum bovin fœtal (FBS), 1% de L-glutamine et 1% de pénicilline / streptomycine dans un milieu RPMI-1640) pour la culture après avoir effectué une numération des cellules 16 de suspension 1 ml de moyen par 10 7 PBMC avant de passer à l&#39;étape suivante. </li></ol> 2. marquage magnétique des leucocytes populations (passez à l&#39;étape 4 si aucune sélection des populations spécifiques est requis) <ol><li> Centrifugeuse CMSP suspension à 300 g pendant 10 min et aspirer le surnageant complètement. </li><li> Resuspendre le culot cellulaire dans 80 pi de PBS par 10 7 cellules totales. </li><li> Ajouter 20 pi de billes magnétiques spécifiées (c.-à-billes magnétiques CD14 pour l&#39;isolement des monocytes, des billes magnétiques CD4 pour l&#39;isolement de lymphocytes T) par 10 7 cellules totales. </li><li> Bien mélanger et incuber pendant 15 minutes à 2 à 8 ° C au réfrigérateur. </li><li> Ajouter 1-2 ml de PBS par 10 7 cellules pour se laver, et centrifugerà 300 xg pendant 10 min. </li><li> Aspirer le surnageant complètement et remettre en suspension jusqu&#39;à 10 8 cellules dans 500 pi de PBS. </li></ol> 3. Sélection magnétique des sous-populations leucocytaires Remarque: Une variété de séparateurs et des colonnes de billes magnétiques sont disponibles pour l&#39;isolement de la population spécifique de leucocytes par le marqueur de surface cellulaire à partir d&#39;un certain nombre de fabricants. L&#39;isolement peut être par sélection positive à base d&#39;un ou de quelques marqueurs positifs ou négatifs de sélection après l&#39;épuisement des populations non désirées. Une approche générale pour effectuer la sélection positive en utilisant un marqueur (c.-à-CD4 des lymphocytes T auxiliaires, ou pour les monocytes CD14) est décrit ci-dessous. <ol><li> Préparer et séparateur magnétique PRIME, notamment la colonne de séparation selon les instructions du fabricant. </li><li> Appliquer tube contenant l&#39;échantillon de PBMC étiquetés. Fournir deux tubes de 15 ml de centrifugeuses pour la collecte et de l&#39;unlabel marqué positivementfractions cellulaires ed, en suivant les instructions du fabricant. </li><li> Compter le nombre de cellules 15 et remettre en suspension les leucocytes sélectionnés positive (soit lymphocytes T CD4 + ou cellules CD14 +) dans leucocytes milieu complet, en utilisant 1 ml de milieu par 10 7 cellules. </li></ol> 4. CFSE coloration des leucocytes pour l&#39;évaluation de la prolifération Remarque: CFSE a été largement utilisé dans les enquêtes immunologiques, à la fois in vivo et des études in vitro. Notre protocole a été optimisé pour l&#39;étude in vitro de MSC / CMSP (ou d&#39;autres leucocytes) interactions. Bien que les étapes générales impliquées dans la conduite de CFSE marquage in vitro sont similaires, il peut y avoir des différences dans la dose et le calendrier spécifique du protocole 14. Cela peut être dû à un certain nombre de facteurs, à savoir, le type de cellule spécifique utilisé, le nombre de cellules utilisées qui peuvent probablement-affecter l&#39;intensité du signal de fluorescence CFSE. <ol&gt; <li> Diluer la solution stock CFSE (10 mM) dans du PBS à la concentration de travail désirée de 10 pm (solution CFSE-travail). Préparer 10 7 cellules (c.-à-CMSP ou cellules T) pour l&#39;étiquetage de la solution CFSE-travail. </li><li> Centrifuger à 300 g pendant 10 min pour obtenir un culot cellulaire et aspirer le surnageant. </li><li> Remettre les cellules doucement dans 1 ml de solution préchauffé (37 ° C) CFSE-travail et incuber les cellules pendant 10 min à 37 ° C. </li><li> Pour laver l&#39;excès de CFSE, diluer la suspension cellulaire avec 10x (en volume) de pré-refroidie (4 ° C) du milieu RPMI contenant 10% de FBS. Sédimenter les cellules par centrifugation à 300 g pendant 5 min et jeter le surnageant. Laver deux fois plus le culot cellulaire de cette manière. </li><li> Re-sédimenter les cellules par centrifugation et de compter le nombre de cellules 15. Reprendre 10 7 cellules (soit PBMC ou cellules T) dans 1 ml de milieu complet frais de leucocytes préchauffé. </li></ol> 5. Co-culture de MSC avec des leucocytes et l&#39;activation des leucocytes <ol><li> Moyen de pré-chaud MSC complet (FBS à 10% (pré-testé pour la croissance des MSC optimal), 1% de L-glutamine et 1% de pénicilline / streptomycine dans un milieu DMEM-bas milieu de glucose) à 37 ° C pendant pas plus de 30 min. </li><li> MSC de semences à 50.000 cellules dans 1 ml de MSC du milieu complet dans des plaques de 24 puits (densité de MSC: 25.000 cellules / cm 2) pour la fixation O / N dans un incubateur à 37 °, ce qui permet aux cellules souches pour atteindre 80% de confluence. </li><li> Milieu de Aspirer, et sur la base des numéros de MSC graines, ajouter CMSP CFSE-marqués (préalablement marquée-s&#39;il vous plaît se référer à protocole Étape 4 ci-dessus) à la CSM cultivées (ensemencées la veille dans des plaques à 24 puits) dans 1 ml de leucocytes milieu complet à un 01:10 (rapport des cellules) de rapport de co-culture de cellules souches mésenchymateuses à PBMC. </li><li> Ajouter la phytohémagglutinine mitogène (PHA), un activateur de leucocytes non spécifique, à une concentration finale de 10 pg / ml dans un volume total de 1 ml de milieu complet leucocytes par puits. <ol><li> Alternatively, afin de stimuler l&#39;activation des lymphocytes T en particulier: utiliser des a-CD3 / 28 microbilles et ajouter à la co-culture de deux cellules pour obtenir un rapport de 1 à billes-T-lymphocyte: 1. </li></ol></li><li> Pour le contrôle négatif, plaque 500.000 PBMC / puits (ou une population spécifique effecteur des leucocytes; densité cellulaire: 250.000 leucocytes / cm 2) dans une plaque de 24 puits avec 1 ml de milieu complet de leucocytes seulement; pour le contrôle positif, en plus de placage le même nombre de cellules PBMC / puits, ajouter PHA à une concentration finale de 10 pg / ml. </li><li> Dans le troisième et cinquième jours de l&#39;expérience de co-culture, d&#39;évaluer la prolifération des leucocytes marqués par CFSE (placées dans des tubes à fond rond) par analyse de cytométrie en flux 17 avec 488 nm excitation et d&#39;émission appropriés pour les filtres de fluorescéine. Note: coloration des cytokines intracellulaires pour l&#39;analyse par cytométrie en flux peut également être effectuée à ces leucocytes CFSE marqué à ce point pour évaluer les changements de leucocytes cytokine profil d&#39;expression comme modulated par les CSP. Depuis CFSE est évaluée avec un filtre approprié à la fluorescéine de, les anticorps choisis pour évaluer diverses cytokines ont besoin d&#39;être conjugués à des fluorochromes autres que la fluorescéine ou spectre similaire (à savoir, la phycoérythrine, péridinine complexe protéique de la chlorophylle (PerCP)). </li></ol> 6. Variation: effecteur répression Assay Magnetic Bead-sélectionné, MSC-induite immunomodulateur leucocytes sur CFSE-étiquetés cellules T CD4 + effecteurs activés <ol><li> MSC de semences à 250.000 cellules dans 3 ml de MSC du milieu complet dans des plaques 6 puits (densité MSC: 25.000 cellules / cm 2) pour la fixation O / N dans un incubateur à 37 °, ce qui permet aux cellules souches pour atteindre 80% de confluence. Au moins 3 plaques de 6 puits sont nécessaires pour assurer suffisamment de PBMC MSC-cocultivés pour la sélection des sous-population. </li><li> Aspirer le milieu et ajouter CMSP séparés que par étape 1, mais dans des plaques 6 puits à 2,5 x 10 6 cellules / puits (densité cellulaire: 250.000 leukocytes / cm 2) avec 3 ml de milieu complet leucocytes. Co-culture pour les 48-72 heures dans un incubateur à 37 ° C. </li><li> Billes magnétiques sélectionner population spécifique de leucocytes immunomodulateurs MSC-induites (c.-à-cellules CD14 +), conformément aux articles 2-3. Notes: coloration des cytokines intracellulaires pour l&#39;analyse par cytométrie en flux peuvent être effectuées à ce stade d&#39;évaluer les changements de leucocytes cytokine profil d&#39;expression, à savoir, l&#39;expression de l&#39;interleukine-10 comme modulés par MSC. </li><li> Ajout CFSE marqué cellules T CD4 + allogéniques générés par les sections 2-4 dans des plaques à 24 puits dans 1 ml de complète (densité de cellules T: 250.000 cellules / cm 2) moyen leucocytes aux leucocytes MSC-induites perles sélectionnées selon divers rapports , à savoir, 1:10, 1: 5, 1: 2 et 1: 1 (cellule à cellule) ratios. </li><li> Pour stimuler les cellules T CD4 +, CD3 ajouter a- / 28 microbilles conjuguées à obtenir un rapport bille à cellule de 1: 1. </li><li> Pour un contrôle négatif, plaque 500.000 cellules T CD4 + / puits dans un (densité de cellules T: 250.000 cellules / cm 2) plaque de 24 puits avec 1 ml de milieu complet de leucocytes seulement; pour le contrôle positif, en plus de placage le même nombre de cellules T CD4 + / puits, ajouter des a-CD3 / 28 microbilles conjuguées à obtenir un rapport bille à cellule de 1: 1. </li><li> Sur le 3 ème jour de co-culture, d&#39;évaluer la prolifération des cellules T CD4 + CFSE-marqués (placées dans des tubes à fond rond) par l&#39;écoulement analyse cytométrique 16 avec 488 nm excitation et d&#39;émission des filtres appropriés pour la fluorescéine. </li></ol>

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We have nothing to disclose.

Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application. 
One of the most critical technical aspects for success in these assays is having adequate PBMC...

Les cellules souches mésenchymateuses humaines (MSC) sont somatiques progéniteurs qui peuvent se différencier en lignées mésodermiques paraxiaux de l&#39;os, le cartilage, le tissu adipeux et 4/1, ainsi que de quelques lignées extramesodermal 5. Première isolé de la moelle osseuse adulte, ces progéniteurs multilignée ont été trouvés dans de nombreux tissus 6-8 et, de façon inattendue, montré pour avoir des propriétés immunomodulatrices fortes qui apparaissent prête très bien à l&#39;application clinique 9-12. Mécanismes détaillés impliqués dans les effets immunomodulateurs sont activement à l&#39;étude pour une application efficace des entités pathologiques spécifiques. Une des façons les plus simples pour évaluer l&#39;immunomodulation est en évaluant pour la suppression de la prolifération des leucocytes effecteur 13. La plupart des leucocytes effectrices telles que les monocytes et les lymphocytes T prolifèrent prodigieusement lorsqu&#39;elles sont stimulées ou activées. Fonction immunomodulatrice peut être évaluée lorsque la suppression deprolifération est mis en évidence. Traditionnellement, les leucocytes effecteur prolifération a été évaluée par détection de la [3H] thymidine dans l&#39;ADN. Cependant, cette méthode présente des inconvénients importants en raison des préoccupations de rayonnement et de l&#39;utilisation post-élimination, ainsi que l&#39;équipement complexe nécessaire. Bien qu&#39;il existe des dosages non radioactifs pour évaluer la prolifération cellulaire, l&#39;ester carboxyfluorescéine succinimidyl (CFSE) dosage présente d&#39;autres avantages tels que permettant l&#39;identification de populations cellulaires spécifiques, ce qui est particulièrement utile dans des expériences de co-culture impliquant plusieurs types de cellules. CFSE est un colorant fluorescent cellulaire qui peut être évaluée par analyse de cytométrie en flux. Comme les cellules se divisent, l&#39;intensité de cette étiquette cellulaire est diminuée proportionnellement; cette détermination permet non seulement de la prolifération cellulaire en général, mais aussi permet d&#39;évaluer le nombre de divisions cellulaires jusqu&#39;à 8 divisions avant la fluorescence devient difficile de detect contre signal de fond. En outre, la stabilité du CFSE fluorescent permet un suivi in vivo de cellules marquées telles que les cellules peuvent être trouvées à de nombreux mois 14. Ce test peut également être modifiée pour évaluer des types spécifiques de leucocytes effectrices ou la fonction immunomodulatrice de populations spécifiques de MSC induite par les leucocytes, tels immunomodulateurs comme l&#39;interleukine-10 (IL-10) la production de monocytes CD14 + 15 en effectuant magnétique bourrelet sélection de marqueur de surface de populations de cellules d&#39;intérêt avant ou après la co-culture selon le cas. Notre protocole décrit l&#39;essai de base de l&#39;évaluation des effets immunomodulateurs de MSC sur les leucocytes effectrices (organigramme représenté sur la figure 1) et une variation sur ce test de base pour l&#39;évaluation du MSC-induite immunomodulation de leucocytes sur CD4 + lymphocytes T effecteurs allogéniques (organigramme représenté la figure 4).

<table border="0" cellpadding="0" cellspacing="0" width="1205">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Ficoll-Paque PLUS</td>
			<td style="width:259px;">GE Healthcare</td>
			<td style="width:121px;">71-7167-00 AG</td>
			<td style="width:549px;">Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Vibrant CFDA-SE Cell Tracer Kit (CFSE)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">V12883</td>
			<td>Cellular label for detection of cell division</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Phytoagglutinin (PHA)</td>
			<td style="width:259px;">Sigma</td>
			<td>L8902</td>
			<td>Activation of human PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Dynabeads Human T-Activator CD3/28</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">111.32D</td>
			<td style="width:549px;">Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">autoMACS&#8482; Separator</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">autoMACS&#8482; Separator</td>
			<td>Magnetic based cell separator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">autoMACS&#174; Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-021-101</td>
			<td>separation columns</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:276px;">CD14 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td style="width:549px;">For positive selection of CD14+ human monocytes and macrophages from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">CD4 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">130-045-101</td>
			<td style="width:549px;">For positive selection of CD4+ human T lymphocytes from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">RPMI 1640 Medium</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11875</td>
			<td>Human PBMC/leukocyte culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">DMEM, Low glucose, pyruvate</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11885</td>
			<td style="width:549px;">Human mesenchymal stem cell (MSC) culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">L-glutamine</td>
			<td style="width:259px;">Life Technologies</td>
			<td>25030-081</td>
			<td>Supplementation for MSC complete medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Penicillin/Streptomycin</td>
			<td style="width:259px;">Life Technologies</td>
			<td>15070-063</td>
			<td>Supplementation for PBMC/leukocyte and MSC complete medium</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:276px;">Fetal bovine serum (FBS)</td>
			<td style="width:259px;">1) Hyclone, for MSC culture&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations)</td>
			<td style="width:121px;">1) SH30070.03M 2) 10091-148</td>
			<td>Pre-test lots for support of MSC in vitro culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">24-well cell culture plate</td>
			<td style="width:259px;">Corning</td>
			<td>COR3524</td>
			<td>Co-culture plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">50 mL centrifuge tube</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430291</td>
			<td>Isolation PBMCs from whole blood by Ficoll-Paque PLUS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">15 mL centrifuge tube&#160;</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430766</td>
			<td>Collection of the labeled and unlabeled cell fractions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round-bottom tubes</td>
			<td style="width:259px;">BD Falcon&#160;</td>
			<td style="width:121px;">352008</td>
			<td>Collection of cells for flow cytometric analysis</td>
		</tr>
	</tbody>
</table>

assessment of the immunomodulatory properties of human mesenchymal stem cells (mscs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

La figure 1 représente le schéma d&#39;ensemble de l&#39;expérience, et la figure 2 illustre l&#39;aspect des différentes conditions de culture cellulaire en tant que visualisées par contraste de phase inversé microscopie. MSC sont des cellules adhérentes avec un fibroblastique, la morphologie en forme de fuseau, tandis que les PBMC et les leucocytes sont de petites cellules non adhérentes rondes. Ces deux types de cellules morphologiquement différents peuvent être clairement vus dans la co-cultur...

Watch this Scientific Journal Video about Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs at JoVE.com

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

Les propriétés immunomodulatrices des cellules souches mésenchymateuses humaines (MSC) apparaissent de plus en plus pertinentes pour l&#39;application clinique. L&#39;utilisation d&#39;un système de co-culture de cellules souches mésenchymateuses et leucocytes du sang périphérique pré-colorées avec le succinimidyl ester de carboxyfluorescéine fluorescent du colorant (CFSE), nous décrivons l&#39;évaluation de l&#39;immunomodulation MSC in vitro sur la prolifération des leucocytes et effectrice des sous-populations spécifiques.

Évaluation de la propriétés immunomodulatrices des mésenchymateuses humaines cellules souches (MSC)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

assessment-immunomodulatory-properties-human-mesenchymal-stem-cells

Research

JoVE Journal

Immunology and Infection

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

12.1K vues.  National Health Research Institutes (NHRI).  Les propriétés immunomodulatrices des cellules souches mésenchymateuses humaines (MSC) apparaissent de plus en plus pertinentes pour l'application clinique. L'utilisation d'un système de co-culture de cellules souches mésenchymateuses et leucocytes du sang périphérique pré-colorées avec le succinimidyl ester de carboxyfluorescéine fluorescent du colorant (CFSE), nous décrivons l'évaluation de l'immunomodulation MSC in vitro sur la...

Vidéo: Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansion, purification et d&#39;évaluation fonctionnelle des cellules NK humaines périphérique Sang

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Recherche

Immunologie et infection

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Évaluation quantitative des cellules immunitaires dans le tissu blessés médullaires par cytométrie en flux: une nouvelle utilisation pour un procédé de purification cellulaire

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

PRP comme une nouvelle approche pour prévenir l&#39;infection: Préparation et<em&gt; In vitro</em&gt; Propriétés antimicrobiennes de PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Développement de cellules souches dérivées de cellules T régulatrices spécifiques de l&#39;antigène contre Autoimmunité

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextrane augmente l’efficacité de la Transduction des gènes des cellules NK primaires murines et humaines

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Méthode Agitateur magnétique pour la détection des<em&gt; Trichinella</em&gt; Larves dans les échantillons de muscle

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Un test de cytotoxicité axée sur la cytométrie de flux pour l’évaluation de l’activité des cellules NK humaine

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Évaluation de la cytotoxique et effets immunomodulateurs de Substances dans des tranches de poumon humain de coupe de précision

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Évaluation de l’Interface synaptique des cellules primaires humaines T du sang périphérique et le tissu lymphoïde

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

Différenciation induite des cellules m Cell-like dans les monocouches entéroïdes ileal dérivées de cellules souches humaines

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...