Le travail a été soutenu en partie par l&#39;Institut national de la Santé (R01GM079688, R21RR024439 et R21GM075838), la National Science Foundation (EFAC 0.941.055), et la Fondation MSU.

1. L&#39;isolement des cellules souches mésenchymateuses Rat Les cellules souches mésenchymateuses ont été isolés de 6-8 semaines Sprague-Dawley rats femelles, comme décrit précédemment 1, 2. Isolée MSC peuvent adhérer à la surface en plastique et facilement développer durant la culture in vitro. <ol><li> L&#39;animal a été mis dans une chambre de l&#39;anesthésie et anesthésiés pendant environ cinq minutes. Pendant l&#39;anesthésie, d&#39;observer le taux de clignoter, la respiration et l&#39;activité motrice. Retirez les animaux de la chambre immédiatement après il arrête l&#39;activité motrice et le taux de clignotement est devenu rare. </li><li> Lay de l&#39;animal vers le bas sur une station de fonctionnement et de tuer l&#39;animal par dislocation cervicale. Coupez les fémurs et des tibias du membres arrière, enlever la peau et les muscles. </li><li> Mettez les fémurs et des tibias disséqué dans 70% d&#39;isopropanol pendant quelques secondes et le transfert à D-1X PBS. </li><li> Dans une enceinte de sécurité biologique, les fémurs et tibias ont été transférés à un plat de 10cm contenant du DMEM. Chaque os a ensuite eu lieu avec des pincettes et les deux extrémités ont été coupés avec des ciseaux ouverts. Fixer une aiguille 22G sur une seringue de 3 ml et remplissez-le avec du DMEM, puis rincer la moelle dans un tube de 50ml en insérant l&#39;aiguille à une extrémité ouverte de l&#39;os. Répétez 2 à 3 fois pour chaque os. Lorsque tous les courges ont été obtenus, remettre les cellules et passer la suspension de cellules à travers un tamis cellulaire 70μm pour enlever les débris osseux et des agrégats de sang. </li><li> Isoler les cellules à 200 g, 4 ° C pendant 5 minutes et retirer le surnageant par aspiration. Resuspendre les cellules dans 25ml MSC (DMEM contenant 10% de FBS et 1% de Pen-Strep). Suspension cellulaire 10ml a été ensemencées dans chaque boîte de culture 10cm pour un total de deux plats. Gardez les boîtes de culture dans un 37 ° C et 5% de CO 2 incubateur pour 1 ~ 2 semaines. Le milieu est changé tous les 2 ~ 3 jours. </li></ol> 2. Enrichissement des cellules souches mésenchymateuses Rat Un certain nombre de protéines de surface ont été utilisés pour enrichir les MSC, dont CD54, CD90, CD73, CD105 et CD271 3-5. Dans notre étude, nous avons utilisé CD54 et le CD90 en tant que marqueurs pour enrichir MSC par tri cellulaire magnétique. <ol><li> Lorsque les cellules ont atteint environ 80% de confluence, aspirer le moyen et ajouter les 4 ~ 5 ml de trypsine-EDTA à chaque plat. Mettez les plats de retour à l&#39;incubateur et incuber pendant environ 5 minutes afin de permettre le détachement des cellules. Une fois que les cellules ont été détachées, ajouter la même quantité de milieu de culture pour inactiver la trypsine. Recueillir la suspension cellulaire dans un tube de 15ml et les cellules spin down au 200g, 4 ° C pendant 5 minutes. </li><li> Les étapes suivantes décrivent comment enrichir MSC par deux marqueurs de surface CD54 et CD90 selon le manuel pour la séparation cellulaire utilisant BD IMagnet. Culot cellulaire a été resuspendu dans un tampon de coloration des cellules (FBS 3% de la chaleur inactivé dans D-1X PBS) à 20 millions de cellules / ml. Biotinylé CD54 anticorps (0.25μg par million de cellules) et CD90 biotinylé anticorps (0.15μg par million de cellules) a été ajoutée et mélangée doucement avec la suspension cellulaire. Après incubation sur la glace pendant 15 minutes, les cellules marquées ont été lavées avec un volume de plus de 1X BD Imag tampon. Les cellules marquées ont été centrifugés à 200g, 4 ° C pendant 5 minutes. </li><li> Vortex, le BD Imag particules streptavidine fond, on ajoute des particules de 40μl pour 10 millions de cellules. Mélanger la particule avec les cellules marquées soigneusement et incuber les cellules à 6 ~ 12 ° C pendant 30 minutes. Cela permet à la streptavidine particules de se lier à l&#39;biotinylé anti-CD54 et anti-CD90, ce qui est lié à des protéines de surface CD54 et CD90, respectivement. </li><li> Pendant le temps d&#39;incubation, l&#39;étiquette un tube à essai à fond rond pour recueillir la fraction positive. Après incubation, porter le volume d&#39;étiquetage pour 20 millions de cellules / ml avec du tampon 1X BD Imag et le transfert des cellules marquées sur le tube de collecte positive de fraction. Placez le tube de la fraction positive sur le IMagnet BD et laissez-le rester pendant 6 minutes, puis enlever le surnageant avec une pipette Pasteur en verre avec un tube de la fraction positive toujours sur la IMagnet BD. </li><li> Retirer le tube fraction positive-du IMagnet BD et le placer sur la glace. Ajouter 1ml glacée 1X BD Imag tampon et remettre les cellules en mélangeant doucement. Placer le tube de retour sur le IMagnet BD et laissez-le séjour pour 2 ~ 4 minutes. Enlever le surnageant avec une nouvelle pipette Pasteur en verre. Répétez le lavage comme décrit dans 2.5 une fois de plus. </li><li> Enlevez le tube de l&#39;IMagnet BD et remettre les cellules en milieu de culture, semences une 75cm 2 flacon pour maintenir les cellules et un plat de 10cm pour la cytométrie en flux. </li></ol> 3. Vérification de l&#39;expression du marqueur de surface par cytométrie en flux L&#39;analyse par cytométrie en flux a été réalisée afin de vérifier les cellules, nous avons obtenu d&#39;exprimer CD54 et CD90. Le marqueur CD45 HSC a été utilisée pour confirmer que les cellules souches mésenchymateuses ont été épuisés de CSH. <ol><li> Lorsque les cellules deviennent confluentes ~ 80%, trypsiniser cellules à la trypsine-EDTA et prélever des cellules dans un tube de 15ml. Centrifuger à 200 g, 4 ° C pendant 5 minutes pour collecter les cellules. </li><li> Aspirer le surnageant et a étécellules h avec D-1X PBS fois. Resuspendre les cellules dans un tampon de coloration des cellules (D-1X PBS contenant 2% de FBS et de l&#39;azide de sodium à 0,05%) à une concentration finale de 5 ~ 10 millions de cellules / ml et garder les cellules sur la glace. Suspension cellulaire Aliquoter 100 ul dans les six tubes étiquetés (1 cellules seulement; 2. Isotype contrôle IgG2a;.. 3 contrôle isotypique IgG1; 4 CD45; 5. CD54;.. 6 CD90). </li><li> Ajouter des contrôles isotype et anticorps primaires à des concentrations appropriées (IgG2a et IgG1: 20 pi par million de cellules; CD45: 0.5μg par million de cellules; CD54: 0.25μg par million de cellules; CD90: 0.15μg par million de cellules) et incuber à 4 ° C pour 30 minutes. </li><li> Laver les cellules avec du D-PBS 1X à deux reprises et ensuite remettre les cellules dans un tampon de coloration cellulaire 100 ul. Ajouter SA-PE (streptavidine-phycoérythrine, utilisez 0.15μg par million de cellules) et incuber à 4 ° C pendant 30 minutes dans l&#39;obscurité. </li><li> Laver les cellules marquées avec les D-1X PBS deux fois. Resuspendre les cellules dans un tampon de coloration cellulaire 400μl et transférer dans un tube falcon pour l&#39;analyse de cytométrie en flux. </li></ol> 4. Séparation des dérivés mono-colonie MSC MSC est une population hétérogène composée de différentes sous-populations de cellules de forme différente, le taux de croissance ainsi que la capacité de différenciation 6. Cependant, tous les sous-populations expriment les marqueurs CSM connue et donc il n&#39;est pas possible d&#39;utiliser des marqueurs pour séparer ces sous-populations. Par conséquent, nous avons appliqué le clonage cylindres de séparer les différentes sous-populations, qui sont des colonies formées par des cellules individuelles. <ol><li> Cellules de la plaque à environ 50 ~ 100 cellules par boîte de 10 cm. Incuber dans un 37 ° C et 5% de CO 2 incubateur pour 1 ~ 2 semaines. Durant cette période, d&#39;examiner des colonies bien isolées avec un microscope inversé. Une fois que les colonies ont atteint assez grande taille (plus de plus de 100 cellules dans chaque colonie), marquer les colonies avec un Sharpie au fond du plat. Lorsque prélever la colonie à être marqué, assurez-vous qu&#39;il n&#39;y a pas de colonies environnantes à proximité de celui choisi. </li><li> Aspirer moyennes et laver la vaisselle une fois avec D-1X PBS. Ramassez une bouteille de clonage stériles avec une pince stérile et doucement le placer autour de la colonie marquée. Répétez jusqu&#39;à ce que toutes les colonies marquée a un cylindre placé sur le clonage. Les colonies doivent être cueillies loin les uns des autres tels que chaque bouteille de clonage ne contient qu&#39;une seule colonie. </li><li> Ajouter 100 ul de trypsine-EDTA à chaque cylindre de clonage et de mettre le plat de retour à l&#39;incubateur pour ~ 5 minutes. Après 5 minutes, vérifier les cellules sous le microscope pour voir si elles sont arrondissant. Lorsque les cellules ont levé, ajouter la même quantité de milieu de culture pour inactiver la trypsine. Mélanger la suspension cellulaire avec une micropipette 200 pl et le transfert de la suspension cellulaire dans un plat contenant 3ml 60mm milieu de culture préchauffé. Étiqueter les plats correctement et remettez-les dans l&#39;incubateur. Suivi des changements morphologiques au cours des prochains jours. </li></ol> 5. Les résultats représentatifs Selon le protocole décrit dans la partie pour le rat MSC isolement, en plastique MSC adhérente doit être visible dès le lendemain après le placage. Comme les cellules continuent de proliférer, les cellules confluentes devrait ressembler à des cellules de la figure 1A. Lorsque les cellules atteignent environ 80% de confluence (figure 1B), repiquage peut être effectué. Pendant subculture, la trypsine-EDTA a été utilisé pour détacher les cellules et les cellules levées sont petites et rondes comme le montre la figure 1C. <img src="/files/ftp_upload/1852/1852fig1.jpg" alt="Figure 1" /> Figure 1. Des images à contraste de phase des cellules souches mésenchymateuses de rats. (A) MSC Confluent. La majorité des cellules sont de broche-like ou en étoile. (B) MSC environ 80% de confluence. (C) des cellules levée après trypsinisation sont petits et ronds. Une fois que les MSC sont enrichies par le tri magnétique de cellules, l&#39;analyse par cytométrie en flux est effectué pour vérifier les expressions marqueur de surface. Si l&#39;enrichissement est bonne, les cellules doivent montrer coloration positive contre des marqueurs CD54 et CD90 MSC, mais négatif par rapport au marqueur CD45 HSC (figure 2). Contrôle isotypique IgG2a et IgG1 sont utilisés comme témoins négatifs. <img src="/files/ftp_upload/1852/1852fig2.jpg" alt="Figure 2" /> Figure 2. L&#39;analyse par cytométrie en flux des cellules souches mésenchymateuses pour les marqueurs de surface. MSC ont été marquées avec des anticorps contre IgG2a (isotype contrôle 1), IgG1 (isotype contrôle 2), CD45, CD54 et CD90. MSC a exprimé CD54 et CD90 mais pas CD45. Lorsque les cellules sont ensemencées à densité clonale appropriée, les colonies devraient passer de cellules individuelles. La figure 3A représente une colonie formée par une seule cellule. Cylindres de clonage peuvent ensuite être utilisées pour séparer les colonies et les cellules provenant de colonies peuvent être cultivées séparément. Figure 3B et 3C représente cellules provenant de deux colonies individuelles. Les cellules provenant de la colonie sont une broche comme (figure 3B) alors que les cellules dérivées de la colonie 2 sont ronds (figure 3C). <pclass = &quot;jove_content&quot;&gt; <img src="/files/ftp_upload/1852/1852fig3.jpg" alt="Figure 3" /> Figure 3. Formation de colonies par MSC et unique colonie de cellules dérivées. MSC en culture à la densité des colonies clonales individuels formulaire. Ces colonies peuvent être séparés par des cylindres de clonage et les cellules provenant de colonies différentes peuvent être cultivées séparément. (A) Une colonie représentative formé par MSC lors plaquées à une densité clonale. (B) Spindle-like des cellules dérivées d&#39;une colonie. (C) cellules rondes dérivées d&#39;une autre colonie.

<ol>
	<li>de Hemptinne, I., Vermeiren, C., Maloteaux, J. M., Hermans, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+glial+glutamate+transporters+in+adult+mesenchymal+stem+cells.">Induction of glial glutamate transporters in adult mesenchymal stem cells.</a> J Neurochem. 91, 155-166 (2004).</li><li>Porter, R. M., Huckle, W. R., Goldstein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+dexamethasone+withdrawal+on+osteoblastic+differentiation+of+bone+marrow+stromal+cells.">Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells.</a> J Cell Biochem. 90, 13-22 (2003).</li><li>Chamberlain, G., Fox, J., Ashton, B., Middleton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+mesenchymal+stem+cells:+their+phenotype,+differentiation+capacity,+immunological+features,+and+potential+for+homing.">Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.</a> Stem Cells. 25, 2739-2749 (2007).</li><li>Abdallah, B. M., Kassem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells:+from+basic+biology+to+clinical+applications.">Human mesenchymal stem cells: from basic biology to clinical applications.</a> Gene Ther. 15, 109-116 (2008).</li><li>Erica, L., Herzog, L. C., Diane, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Krause+Plasticity+of+marrow-derived+stem+cells.">Krause Plasticity of marrow-derived stem cells.</a> Blood. 102, 3483-3493 (2003).</li><li>Colter, D. C., Sekiya, I., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+subpopulation+of+rapidly+self-renewing+and+multipotential+adult+stem+cells+in+colonies+of+human+marrow+stromal+cells.">Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells.</a> Proc Natl Acad Sci U S A. 98, 7841-7845 (2001).</li></ol>

Ce protocole décrit comment isoler et d&#39;enrichir les MSC. Une méthode pour séparer les cellules mono-colonie dérivée est également intégré. Il ya plusieurs étapes qui sont importantes pour une séparation réussie l&#39;isolement, l&#39;enrichissement et la colonie. Tout en faisant l&#39;isolement cellulaire de rat, il est recommandé de filtrer à travers une passoire cellule ou une maille de nylon stérile de taille similaire pour se débarrasser des caillots de sang et de débris osseux. Après étalemen...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>1X D-PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14040-133</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11885-084</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26140-079</td>
<td>&nbsp;</td>
<tr>
<td>Pen-Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>BD Imagnet</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>552311</td>
<td>&nbsp;</td>
<tr>
<td>Biotin mouse IgG2a</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>555572</td>
<td>&nbsp;</td>
<tr>
<td>Biotin mouse IgG1</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>555747</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD54</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL054B</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD90</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL005B</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD45</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL001B</td>
<td>&nbsp;</td>
<tr>
<td>BD Imag buffer</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>552362</td>
<td>&nbsp;</td>
<tr>
<td>Round-bottom tube</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352063</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin particles</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>557812</td>
<td>&nbsp;</td>
<tr>
<td>SA-PE</td>
<td>&nbsp;</td>
<td>R&amp;D systems</td>
<td>F0040</td>
<td>&nbsp;</td>
<tr>
<td>Cloning cylinder</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2059</td>
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isolation and enrichment of rat mesenchymal stem cells (mscs) and separation of single-colony derived mscs

MSC sont une population de cellules souches adultes qui est une source prometteuse pour des applications thérapeutiques. Ces cellules peuvent être isolées de la moelle osseuse et peut facilement être séparé des cellules souches hématopoïétiques (CSH) en raison de leur adhésion en plastique. Ce protocole décrit comment isoler des MSC à partir des fémurs et des tibias de rats. Les cellules isolées ont été encore enrichie contre deux marqueurs de surface CD54 et CD90 MSC par tri cellulaire magnétique. L&#39;expression de marqueurs de surface CD54 et CD90 ont ensuite été confirmés par une analyse de cytométrie en flux. HSC marqueur CD45 a également été inclus pour vérifier si les cellules souches mésenchymateuses ont été triés appauvri de CSH. MSC sont naturellement très hétérogènes. Il ya des sous-populations de cellules qui ont des formes différentes, la prolifération et les capacités de différenciation. Ces sous-populations expriment tous les marqueurs connus MSC et aucun marqueur unique n&#39;a pas encore été identifiés pour la sous-populations différentes. Par conséquent, une approche alternative pour séparer les différentes sous-populations est d&#39;utiliser le clonage cylindres de séparer une seule colonie de cellules dérivées. Les cellules provenant de colonies unique peut alors être cultivées et évaluées séparément.

Watch this Scientific Journal Video about Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs at JoVE.com

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSC ont été isolés à partir des fémurs et tibias et ensuite enrichi par tri cellulaire magnétique. Cellules triées ont été confirmés pour l&#39;expression des marqueurs de surface par cytométrie en flux. Ces cellules ont également été cultivées à une densité clonale pour former des colonies isolées et ensuite ces colonies ont été séparés par des cylindres de clonage.

L&#39;isolement et l&#39;enrichissement des cellules de rat souches mésenchymateuses (CSM) et de séparation des mono-colonie Dérivé MSC

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

isolation-enrichment-rat-mesenchymal-stem-cells-mscs-separation

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Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

28.3K vues.  City of Hope Cancer Center. Rat MSC ont été isolés à partir des fémurs et tibias et ensuite enrichi par tri cellulaire magnétique. Cellules triées ont été confirmés pour l'expression des marqueurs de surface par cytométrie en flux. Ces cellules ont également été cultivées à une densité clonale pour former des colonies isolées et ensuite ces colonies ont été séparés par des cylindres de clonage.

Vidéo: Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

L&#39;isolement et la transplantation de cellules souches hématopoïétiques (CSH)

Recherche

Biologie

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolement et animale expansion sans sérum du cordon ombilical humain dérivé cellules stromales mésenchymateuses (CSM) et des cellules endothéliales progénitrices Colony Forming (CPEF)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Isolement et caractérisation de Hoechst Faible CD45 Négatifs Souris Cellules souches mésenchymateuses du poumon

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induction et analyse des épithéliale à mésenchymateuses transition

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Isolation Manuel des cellules souches du tissu adipeux de Lipoaspirates humaines

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolement et culture des cellules souches épithéliales dentaire de l&#39;adulte de souris Incisive

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Isolement de CD146<sup&gt; +</sup&gt; Cellules Resident pulmonaire mésenchymateuses stromales de Rat Poumons

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Isolement, prolifération et différenciation des cellules souches adipeuses dérivées du macaque rhésus

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Isolement, caractérisation et application thérapeutique de vésicules extracellulaires à partir de cellules souches mésenchymateuses humaines en culture

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Isolement et caractérisation de cellules souches dérivées de tissu adipeux

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Pleins feux sur l’auteur : Isolement et identification de cellules souches mésenchymateuses dérivées du tissu adipeux de rats Sprague Dawley  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Isolement et identification de cellules souches mésenchymateuses dérivées du tissu adipeux de rats Sprague Dawley

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...