Die Arbeit wurde teilweise durch National Institute of Health (R01GM079688, R21RR024439 und R21GM075838), National Science Foundation (CBET 0941055) und der MSU-Stiftung unterstützt.

1. Isolation der Rat MSCs Mesenchymale Stammzellen wurden aus 6-8 Wochen alten Sprague-Dawley weiblichen Ratten wie oben beschrieben 1, 2 isoliert. Isolierte MSCs zu einer plastischen Oberfläche haften und leicht während in vitro-Kultur zu erweitern. <ol><li> Das Tier war in eine Narkose Kammer gestellt und betäubt für rund fünf Minuten. Während der Narkose, beobachten die Rate der blinkt, Atmung und die motorische Aktivität. Entfernen Sie das Tier aus der Kammer sofort nach motorischer Aktivität stoppt und die blinkende Rate wurde selten. </li><li> Legen Sie das Tier sich auf eine Operation entfernt und tötet das Tier durch Genickbruch. Schneiden Sie die Oberschenkel-und Unterschenkelknochen von hinten Gliedmaßen, entfernen Sie die Haut und Muskeln. </li><li> Setzen Sie den sezierten Oberschenkel-und Unterschenkelknochen in 70% Isopropanol für ein paar Sekunden und Transfer zum 1X D-PBS. </li><li> In einem Biosicherheitswerkbank wurden die Oberschenkel-und Unterschenkelknochen eine 10cm Schüssel mit DMEM übertragen. Jeder Knochen wurde dann mit einer Pinzette gehalten und die beiden Enden offen waren mit einer Schere abgeschnitten. Bringen Sie eine 22G-Nadel auf eine 3ml Spritze und füllen Sie ihn mit DMEM, dann spülen Sie die Mark in eine 50 ml Tube, indem Sie die Nadel in einem offenen Ende des Knochens. Wiederholen Sie 2 bis 3 mal pro Knochen. Wenn alle Zucchini erhalten wurden, resuspendieren und übergeben Sie die Zellsuspension durch ein 70μm Zellsieb bis auf die Knochen Schmutz und Blut Aggregate zu entfernen. </li><li> Spin down Zellen bei 200g, 4 ° C für 5 Minuten und entfernen Sie die Überstände durch Absaugen. Die Zellen in 25ml MSC-Medium (DMEM mit 10% FBS und 1% Pen-Strep). 10ml Zellsuspension wurde in jedem 10cm Kulturschale für insgesamt zwei Schalen ausgesät. Halten Sie den Kulturschalen in einem 37 ° C und 5% CO 2-Inkubator für 1 bis 2 Wochen. Medium wurde alle 2 bis 3 Tagen. </li></ol> 2. Anreicherung von Rat MSCs Eine Reihe von Oberflächenproteinen wurden verwendet, um MSCs zu bereichern, einschließlich CD54, CD90, CD73, CD105 und CD271 3-5. In unserer Studie haben wir CD54 und CD90 als Marker für MSCs durch magnetische Zellsortierung bereichern. <ol><li> Wenn die Zellen etwa 80% Konfluenz erreicht, Saugen Sie das Medium und fügen 4 ~ 5 ml Trypsin-EDTA zu jedem Gericht. Legen Sie das Geschirr wieder in den Inkubator und inkubieren für rund 5 Minuten zum Ablösen der Zellen zu ermöglichen. Sobald Zellen wurden abgenommen, fügen Sie die gleiche Menge Kulturmedium zu Trypsin zu inaktivieren. Sammeln Zellsuspension in ein 15ml Tube und Spin Zellen nach unten auf 200g, 4 ° C für 5 Minuten. </li><li> Die nächsten Schritte beschreiben, wie MSCs durch zwei Oberflächenmarker CD54 und CD90 bereichern gemäß dem Handbuch für die Zellseparation mittels BD IMagnet. Zellpellet wurde in Zellfärbung Puffer (3% hitzeinaktiviertem FBS in 1X D-PBS) bei 20 Millionen Zellen / ml resuspendiert. Biotinylierten CD54-Antikörper (0.25μg pro Million Zellen) und biotinylierten CD90-Antikörper (0.15μg pro Million Zellen) zugegeben und vorsichtig mit der Zellsuspension gemischt. Nach der Inkubation auf Eis für 15 Minuten wurden die markierten Zellen mit einem Überschuss Volumen von 1X BD Imag gewaschen. Die markierten Zellen wurden bei 200g gesponnen, 4 ° C für 5 Minuten. </li><li> Vortex die BD Imag Streptavidin Partikel gründlich, fügen Sie 40 ul Partikel für alle 10 Millionen Zellen. Mix der Partikel mit der markierten Zellen gründlich und inkubieren Sie die Zellen bei 6 ~ 12 ° C für 30 Minuten. Dies ermöglicht dem Streptavidin Partikel zu binden die biotinylierten anti-CD54 und anti-CD90, das auf der Oberfläche Proteine ​​CD54 und CD90 gebunden ist jeweils. </li><li> Während der Inkubationszeit Label ein Rundboden-Röhrchen, die positive Fraktion zu sammeln. Nach der Inkubation bringen die Kennzeichnung Volumen bis 20 Millionen Zellen / ml mit 1X BD Imag Puffer und übertragen Sie die markierten Zellen, die positiv-Fraktion Sammelrohr. Legen Sie die positive-Fraktion Tube auf den BD IMagnet und lassen Sie es bleiben für 6 Minuten, dann entfernen Sie den Überstand mit einem Glas Pasteurpipette mit der Positiv-Fraktion Röhre immer noch auf der BD IMagnet. </li><li> Entfernen Sie die positive-Fraktion Rohr aus dem BD IMagnet und legen Sie sie auf Eis. 1ml eiskaltem 1X BD Imag Puffer und die Zellen durch vorsichtiges Mischen. Das Röhrchen wieder auf die BD IMagnet und lassen Sie es für 2 ~ 4 Minuten zu bleiben. Entfernen Sie den Überstand mit einem neuen Glas-Pasteur-Pipette. Wiederholen Sie die Wäsche wie in 2.5 noch einmal beschrieben. </li><li> Entfernen Sie den Schlauch aus dem BD IMagnet und die Zellen in Kulturmedium, Saatgut ein 75cm 2 Flasche für die Erhaltung der Zellen und eine 10cm Schüssel für die Durchflusszytometrie. </li></ol> 3. Überprüfung der Surface Marker Expression mittels Durchflusszytometrie Durchflusszytometrie-Analyse wurde durchgeführt, um die Zellen erhielten wir ausdrücken CD54 und CD90 zu überprüfen. Die HSC-Marker CD45 verwendet wurde, um zu bestätigen, dass die MSCs von HSK waren erschöpft. <ol><li> Wenn Zellen ~ 80% konfluent geworden, trypsinize Zellen mit Trypsin-EDTA und sammeln Zellen in ein 15ml Tube. Zentrifugation bei 200g, 4 ° C für 5 Minuten, um Zellen zu sammeln. </li><li> Saugen Sie den Überstand und wurdeh Zellen mit 1x D-PBS einmal. Die Zellen in Zellfärbung Puffer (1X D-PBS mit 2% FBS und 0,05% Natriumazid) auf eine Endkonzentration von 5 ~ 10 Mio. Zellen / ml und halten Zellen auf Eis. Aliquot 100 &amp;mgr; Zellsuspension in den sechs beschrifteten Röhrchen (1 Zellen nur;. 2 Isotypkontrolle IgG2a;.. 3 Isotypkontrolle IgG1, 4 CD45;. 5 CD54;.. 6 CD90). </li><li> Add Isotypkontrollen und primären Antikörpern bei entsprechenden Konzentrationen (IgG2a und IgG1: 20 &amp;mgr; l pro Million Zellen; CD45: 0.5μg pro Million Zellen; CD54: 0.25μg pro Million Zellen; CD90: 0.15μg pro Million Zellen) und inkubieren Sie bei 4 ° C für 30 Minuten. </li><li> Wash-Zellen mit 1x D-PBS zweimal und dann die Zellen in 100 &amp;mgr; Zellfärbung Puffer. Fügen Sie SA-PE (Streptavidin-Phycoerythrin, verwenden 0.15μg pro Million Zellen) und inkubieren Sie bei 4 ° C für 30 Minuten im Dunkeln. </li><li> Wash markierten Zellen mit 1x D-PBS zweimal. Die Zellen in 400μl Zelle Färbepuffer und in ein Falcon-Röhrchen für die Durchflusszytometrie-Analyse. </li></ol> 4. Trennung von Einzel-Kolonie Derived MSCs MSCs ist eine heterogene Population von verschiedenen Subpopulationen mit verschiedenen Zell-Form, Wachstum und die Differenzierung Fähigkeit 6 zusammen. Allerdings äußern alle Subpopulationen der bekannten MSC Marker und daher ist es nicht möglich, Marker verwenden zu trennen diese Subpopulationen. Deshalb wandten wir Klonzylinder zur Abtrennung der verschiedenen Bevölkerungsgruppen, die Kolonien, die von einzelnen Zellen gebildet werden. <ol><li> Platte Zellen bei etwa 50 ~ 100 Zellen pro 10cm Schüssel. Inkubieren bei 37 ° C und 5% CO 2-Inkubator für 1 bis 2 Wochen. Während dieser Zeit prüfen, gut isolierte Kolonien mit einem inversen Mikroskop. Nachdem die Kolonien groß genug Größe (besser mehr als 100 Zellen in jeder Kolonie) erreicht haben, markieren Sie die Kolonien mit einem sharpie am unteren Rand der Schale. Wenn die Kolonie markiert werden, stellen Sie sicher, es gibt keine umliegenden Kolonien in der Nähe des abgeholt eins. </li><li> Saugen Sie mittel-und waschen Sie die Schale einmal mit 1x D-PBS. Pick-up einer sterilen Klonen Zylinder mit einer sterilen Pinzette und legen Sie sie vorsichtig um die markierte Kolonie. Wiederholen Sie diesen Vorgang, bis alle markierten Kolonien hat ein Klonen Zylinder angeordnet vorbei. Die abgeholt Kolonien weit entfernt von einander, so dass jeder das Klonen Zylinder enthält nur eine Kolonie. </li><li> Add 100 &amp;mgr; Trypsin-EDTA zu jedem Klonen Zylinder und die Schüssel wieder in den Inkubator für ~ 5 Minuten. Nach 5 Minuten überprüfen Sie die Zellen unter dem Mikroskop zu sehen, ob sie Rundung auf. Wenn die Zellen hob, fügen Sie die gleiche Menge Kulturmedium zu Trypsin zu inaktivieren. Mischen Sie die Zellsuspension mit einer 200 ul Mikropipettierer und übertragen Sie die Zellsuspension auf eine 60mm Schale mit 3 ml vorgewärmtem Kulturmedium. Beschriften Sie die Speisen richtig und lege sie wieder in den Inkubator. Tracking-morphologische Veränderungen im Laufe der nächsten Tage. </li></ol> 5. Repräsentative Ergebnisse Nach dem Protokoll in dem Teil für Ratten MSCs Isolation beschrieben, sollten Kunststoff-adhärenten MSCs sichtbar am nächsten Tag nach dem Ausplattieren. Wie Zellen sich zu vermehren fortzusetzen, sollte die konfluenten Zellen wie die Zellen in Abbildung 1A dargestellt aussehen. Wenn die Zellen zu erreichen ~ 80% Konfluenz (Abbildung 1B), kann Subkultivierung durchgeführt werden. Während Subkultur wurde Trypsin-EDTA verwendet, um Zellen zu lösen und hob Zellen sind klein und rund, wie in Abbildung 1C gezeigt. <img src="/files/ftp_upload/1852/1852fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Phasenkontrast-Bilder von Ratten MSCs. (A) Confluent MSCs. Die Mehrzahl der Zellen sind spindelförmige oder sternförmige. (B) MSCs rund 80% Konfluenz. (C) Lifted Zellen nach Trypsinierung sind klein und rund. Sobald MSCs durch magnetische Zellsortierung angereichert sind, ist die Durchflusszytometrie-Analyse durchgeführt, um die Oberfläche Marker Ausdrücke zu überprüfen. Wenn die Anreicherung gut ist, sollten die Zellen positive Färbung gegen MSC Marker CD54 und CD90, aber negativ gegen die HSC-Marker CD45 (Abbildung 2) zeigen. Isotypkontrollen IgG2a und IgG1 werden als negative Kontrollen verwendet. <img src="/files/ftp_upload/1852/1852fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Durchflusszytometrie Analyse von MSCs für Oberflächen-Marker. MSCs wurden mit Antikörpern gegen IgG2a (Isotyp-Kontrolle 1), IgG1 (Isotyp-Kontrolle 2), CD45, CD54 und CD90 markiert. MSCs exprimiert CD54 und CD90, aber nicht CD45. Wenn die Zellen am richtigen klonaler Dichte ausgesät werden, sollten Kolonien aus einzelnen Zellen steigen. 3A ist eine Kolonie, die von einer einzigen Zelle gebildet. Cloning Zylinder kann dann zur Trennung der Kolonien und Zellen aus den Kolonien abgeleitet getrennt kultiviert werden. 3B und 3C stellt Zellen aus zwei einzelnen Kolonien stammen. Die Zellen aus Kolonie 1 abgeleitet sind Spindel wie (3B), während die Zellen aus Kolonie 2 abgeleitet rund sind (Abb. 3C). <pclass = &quot;jove_content&quot;&gt; <img src="/files/ftp_upload/1852/1852fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Colony Formation von MSCs und Single-Kolonie abgeleiteten Zellen. MSCs bei klonaler Dichte zu bilden einzelne Kolonien kultiviert. Diese Kolonien können durch Klonen Zylindern und Zellen aus verschiedenen Kolonien getrennt werden können getrennt kultiviert werden. (A) Ein Vertreter Kolonie von MSCs entsteht, wenn bei klonaler Dichte ausplattiert. (B) Spindel-ähnliche Zellen aus einer Kolonie abgeleitet. (C) Round-Zellen aus einer anderen Kolonie abgeleitet.

<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>

Dieses Protokoll beschreibt, wie zu isolieren und zu bereichern MSCs. Eine Methode, um die Single-Kolonie abgeleiteten Zellen trennen, ist ebenfalls integriert. Es gibt mehrere Schritte, die wichtig sind für eine erfolgreiche Isolierung, Anreicherung und Trennung Kolonie. Währenddessen Zelle isoliert von Ratten, empfiehlt es sich, durch eine Zelle Sieb oder eine sterile Nylon-Mesh von ähnlicher Größe, um loszuwerden, das Blut gerinnt und Knochendebris Filter. Nach Ausplattieren der Zellen über Nacht werden viele t...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and enrichment of rat mesenchymal stem cells (mscs) and separation of single-colony derived mscs

MSCs sind eine Population von adulten Stammzellen, die eine vielversprechende Quelle für therapeutische Anwendungen geeignet ist. Diese Zellen können aus dem Knochenmark isoliert werden und lässt sich leicht aus den hämatopoetischen Stammzellen (HSZ) aufgrund ihrer plastischen Einhaltung getrennt werden. Dieses Protokoll beschreibt, wie MSCs aus Ratten-Oberschenkel-und Unterschenkelknochen zu isolieren. Die isolierten Zellen wurden weiter gegen zwei MSCs Oberflächenmarker CD54 und CD90 durch magnetische Zellsortierung angereichert. Expression der Oberflächenmarker CD54 und CD90 wurden dann mittels Durchflusszytometrie-Analyse bestätigt. HSC CD45 wurde ebenfalls enthalten, um zu überprüfen, ob die sortierten MSCs von HSK aufgebraucht waren. MSCs sind natürlich sehr heterogen. Es gibt Subpopulationen von Zellen, die verschiedenen Formen, Proliferation und Differenzierung Fähigkeiten haben. Diese Subpopulationen alle exprimieren den bekannten MSC-Marker und keine eindeutige Marker noch für die verschiedenen Subpopulationen identifiziert worden. Daher ist ein alternativer Ansatz zur Abtrennung der verschiedenen Subpopulationen mit Klonzylinder zu trennen Single-Kolonie abgeleiteten Zellen. Die Zellen aus dem Single-Kolonien abgeleitet werden dann kultiviert und getrennt ausgewertet.

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 MSCs wurden aus Oberschenkel-und Unterschenkelknochen isoliert und dann durch magnetische Zellsortierung angereichert. Sortiert Zellen wurden für die Expression der Oberflächenmarker durchflusszytometrisch bestätigt. Diese Zellen wurden auch bei klonaler Dichte kultiviert werden, um einzelne Kolonien bilden, und dann dieser Kolonien wurden durch Klonen Zylinder getrennt.

Isolierung und Anreicherung von Rat mesenchymalen Stammzellen (MSCs) und Separation von Single-Kolonie Derived MSCs

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 ...

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

28.3K Aufrufe.  City of Hope Cancer Center. Rat MSCs wurden aus Oberschenkel-und Unterschenkelknochen isoliert und dann durch magnetische Zellsortierung angereichert. Sortiert Zellen wurden für die Expression der Oberflächenmarker durchflusszytometrisch bestätigt. Diese Zellen wurden auch bei klonaler Dichte kultiviert werden, um einzelne Kolonien bilden, und dann dieser Kolonien wurden durch Klonen Zylinder getrennt.

Serie: 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.

Isolierung und Transplantation von hämatopoetischen Stammzellen (HSZ)

Forschung

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.

Isolation und tierisches Serum Freie Expansion der menschlichen Nabelschnur Derived mesenchymale Stromazellen (MSCs) und Endothelial Colony Forming Progenitorzellen (ECFCs)

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

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolation und Ableitung von embryonalen Maus-Keimzellen

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 & 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.

Isolation und Charakterisierung von Hoechst Low CD45 Negative Mouse Lung mesenchymalen Stammzellen

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

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Isolierung und Primärkultur von Ratten Leberzellen

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

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.

Induktion und Analyse von epithelialen zu mesenchymalen 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.

Manuelle Isolierung von Fettgewebe gewonnene Stammzellen aus menschlichen Lipoaspirates

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

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Isolierung, Kultur und Transplantation von Muskel-Satelliten-Zellen

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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.

Isolation und Kultur der Dental epithelialen Stammzellen aus dem erwachsenen Maus Schneidezahn

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.

Isolierung von CD146<sup&gt; +</sup&gt; Resident Lung mesenchymalen Stromazellen aus Rattenlungen

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