This work was supported by the Swiss National Foundation Grant CR3I3_140717/1 and the Swiss Paraplegic Foundation.

REMARQUE: ponctions de moelle osseuse humaines de la crête iliaque ont été recueillies de consentir donateurs avec l&#39;approbation du comité d&#39;éthique du canton de Lucerne. Aspirats de moelle osseuse Canine de la crête iliaque ont été recueillies avec consent.Human de propriétaire de chien (env. 20 ml) et canin (env. 10 ml) aspirats de moelle osseuse étaient anti-coagulé par addition de 15 ml de citrate de sodium à 3,8% immédiatement après le retrait dans la salle d&#39;opération. Les échantillons ont été transférés à l&#39;environnement du laboratoire de traitement du même jour comme retirée. 1. Préparation de Urokinase (Avant le 1 er Utilisations) <ol><li> Reconstituer l&#39;urokinase en utilisant une solution saline stérile tamponnée au phosphate (PBS) selon les instructions du fabricant: Ajouter 10 ml de PBS dans le ballon de septum d&#39;entrée en utilisant une seringue de 10 ml. Dissoudre la substance sèche en agitant pour obtenir 50 000 unités d&#39;injection (U) par ml. </li><li> Préparer des aliquots de 500 ul (25,000) dans chaque U sterile tubes. aliquotes de magasins -20 ° C et l&#39;utiliser en cas de besoin pendant au moins six mois. </li></ol> 2. préparatives étapes (Avant chaque os traitement moelle) <ol><li> Décongeler une aliquote de l&#39;urokinase (25.000 U) coagulé par aspiration de moelle osseuse (volumes aspirés jusqu&#39;à 25 ml ont été traités avec succès en utilisant une seule portion aliquote de 25 000 U). </li><li> Préchauffer un bain d&#39;eau ou incubateur agitateur à 37 ° C. </li></ol> 3. digestion enzymatique du Clot <ol><li> Effectuer tous les travaux dans un biosécurité de flux laminaire armoire pour éviter la contamination microbienne des échantillons de moelle osseuse au cours du traitement. Lorsque vous travaillez avec des matières potentiellement infectieuses humaine, l&#39;utilisation de gants de protection ainsi que manipulation prudente est conseillée. </li><li> Placer un tamis de 100 um de la cellule au-dessus d&#39;un tube de 50 ml stérile. Versez délicatement la ponction de moelle osseuse à travers le tamis de la cellule, en inclinant la crépine ou déplacer le matériel d&#39;un caillot. Utiliser un embout de pipette stérile pourune meilleure circulation à travers les mailles du filtre. REMARQUE: éviter toute dilution du caillot, par exemple, en lavant le caillot avec du PBS. Urokinase agit indirectement et nécessite des composants du sérum (ie., Plasminogène) de la biopsie pour condensé efficace. Tout en continuant l&#39;opération avec le matériau de caillot, le filtrat peut être conservé à température ambiante jusqu&#39;à utilisation ultérieure dans l&#39;étape 3.6. </li><li> Transférer le matériel de caillots de moelle osseuse dans une boîte de culture de cellule vide aide de pinces stériles. Couper les débris en petits morceaux d&#39;environ 2 mm 3 à l&#39;aide d&#39;un scalpel stérile. </li><li> Transférer les petits morceaux de caillot dans un tube à réaction de 50 ml à l&#39;aide des pinces stériles. Veiller à ce que le caillot hachée a un aspect mouillé. Se il semble être sec, utiliser une partie du filtrat de l&#39;étape 3.1 pour humidifier. </li><li> Triturer le caillot par aspiration et pendant 5 fois en utilisant 5 ml microtitrage pipette. Ajouter une fraction aliquote de l&#39;échantillon d&#39;urokinase. Incuber pendant 30 min à 37 ° C soit dans un bain d&#39;eau ou dansune agitation (doucement) incubateur. </li><li> Triturer par pipetage de haut en bas pour les cinq fois à l&#39;aide d&#39;une pipette 5 ml. Effectuez cette étape dans une enceinte de sécurité biologique. Incuber pendant 30 minutes à 37 ° C. Après l&#39;incubation, triturer encore 5 fois à l&#39;aide d&#39;une pipette de 5 ml. Passez votre souris sur un nouveau 100 um tamis cellulaire de mettre en commun avec le filtrat de l&#39;étape 3.2. </li><li> Centrifuger la suspension de cellules à température ambiante pendant 10 min à 500 x g. Jeter le surnageant. Remettre en suspension le culot cellulaire dans un milieu tel que décrit ci-dessous ou selon le mode opératoire standard de votre laboratoire pour la culture d&#39;extension MSC. </li></ol> 4. Le classement pour Expansion Culture cellulaire et UFC Plaques <ol><li> Remettre en suspension les cellules (comprenant des érythrocytes et des cellules mononucléées) dans 50 ml de milieu de base: Alpha-MEM supplémenté avec 10% de sérum bovin fœtal (FBS), 100 U / ml de pénicilline, 100 mg / ml de streptomycine et 2,5 mg / ml d&#39;amphotéricine B . Compter les cellules 01h10 dilué dans une solution bleu Trypan utilisant une chambe Neubauerr. </li><li> Plaque les cellules dans des flacons de culture de cellules à une densité de 5 x 10 7 cellules / cm 2 et incuber dans un incubateur humidifié à 37 ° C. Si possible, utilisez hypoxiques (5% d&#39;oxygène). Pour le contrôle de dosage CFU, plaque 10 9 cellules dans une boîte de culture cellulaire de 10 cm de diamètre. </li><li> Après 3 jours de culture, les cellules souches mésenchymateuses sont attachés à la boîte de culture de cellules alors que les autres cellules restent en suspension. Changer les médias avec le milieu de base supplémenté avec 5 ng / ml de facteur de croissance des fibroblastes de base (bFGF). Pour le contrôle de dosage CFU, traiter la boîte de culture cellulaire de même. </li><li> Continuer culture cellulaire pour un total de deux semaines, l&#39;échange de médias trois fois par semaine. Si les cellules dépassent 80% de confluence, diviser par trypsinisation. Pour le contrôle de dosage CFU, échanger les médias de même, mais ne pas diviser les cellules pour le cours des deux semaines. </li></ol> 5. Giemsa pour le dosage CFU <ol><li> Préparer 10 ml de solution par Giemsa-plat: dilute la solution mère (7,6 g / l dans le glycérol Giemsa: méthanol) 1h10 dans l&#39;eau stérile (toujours préparer une dilution de travail frais). </li><li> Retirez le support de la boîte de culture cellulaire et laver les cellules avec du PBS. Soyez très précautionneux lors de l&#39;application liquide à la boîte de Pétri et éviter de laver hors des cellules. </li><li> Fixer les cellules dans du méthanol pur pendant 5 min à température ambiante. Jeter le méthanol. Ajouter la solution Giemsa et incuber pendant 60 min dans un incubateur humidifié à 37 ° C. </li><li> Laver deux fois avec du PBS. Air sécher la tête de la première plaque sur une serviette en papier. Compter les colonies manuellement. Ceci se fait à l&#39;aide d&#39;un marqueur sur le dos de la plaque. </li></ol> 6. MSC Différenciation <ol><li> La différenciation des cellules souches mésenchymateuses Canine REMARQUE: MSC Canine ont été différenciées en chondrogène, ostéogénique et lignées adipogéniques par stimulation avec le support approprié pour quatre semaines. </li><li> Pour La différenciation adipocytaire, MSC de culture en monocouche à 4 x 10 5 CELLS / cm 2 en alternance deux conditions de culture différentes de la manière suivante; <ol><li> Culture dans un milieu d&#39;entretien contenant de l&#39;adipogenèse DMEM + GlutaMAX, 3% de FBS, 100 unités / ml de pénicilline, 100 mg / ml de streptomycine, 2,5 pg / ml d&#39;amphotéricine B et 170 mM d&#39;insuline. </li><li> Culture dans l&#39;adipogenèse médias induire avec du milieu de maintenance additionné de 3% de FBS, sérum de lapin à 5%, 1 uM de dexaméthasone, 500 uM de 3-isobutyl-1-méthylxanthine, 33 uM de biotine, 5 uM et 17 uM de rosiglitazone 16 pantothenate. </li><li> Revel les gouttelettes lipidiques par coloration avec Oil Red-O. En bref, fixer les cellules avec 10% de formaldéhyde, laver avec du PBS et tacher avec O rouge 0,35% d&#39;huile dans de l&#39;isopropanol. </li></ol></li><li> Pour la culture de la différenciation ostéogénique MSC en monocouche à 7 x 10 3 cellules / cm 2 et de stimuler dans ce qui suit: <ol><li> Avancée DMEM (GIBCO) + GlutaMAX, 5% de FBS, 100 unités / ml de pénicilline, 100 mg / ml de streptomycine, 2,5 pg / ml d&#39;amphotéricine B, 501; M d&#39;acide L-ascorbique-2-phosphate, 10 mM de ß-glycerophosphate et 100 nM de dexaméthasone. </li><li> Identifier les dépôts de minéralisation de Von Kossa tache (5% AgNO 3). En bref, fixer les cellules avec 10% de formaldéhyde, laver avec du PBS et tache avec 5% AgNO 3 dans l&#39;eau distillée. </li></ol></li><li> Différenciation chondrogénique <ol><li> cubes coupées (3 mm de chaque côté) à partir d&#39;un dispositif médical en forme d&#39;éponge, constituées à partir lyophilisée de collagène de type I, et utilisés comme matériau d&#39;échafaudage pour soutenir 17 cellules. </li><li> MSC de semences sur le dessus des cubes à la concentration de 4 x 10 6 cellules / ml (~ 70 000 cellules / cube). </li><li> Avant l&#39;addition de médias, permettent aux cellules d&#39;adhérer aux cubes pendant 30 min. </li><li> Maintenir constructions MSC-collagène dans des milieux chondrogénique constitué de DMEM / F12 + GlutaMAX, 2,5% de FBS, 100 unités / ml de pénicilline, 100 mg / ml de streptomycine, 2,5 pg / ml d&#39;amphotéricine B, 40 ng / dexaméthasone ml, 50 pg / ascorbique ml acide 2-phosphate,50 ug / ml de L-proline, 1x insuline-transferrine-sélénium X, et 10 ng / ml de facteur de croissance transformant-β1. </li><li> Utilisez coloration au bleu alcian pour visualiser l&#39;accumulation des protéoglycanes dans les sections de constructions. Brièvement, tacher les sections O / N avec 0,4% de bleu alcian dissous dans 0,01% de H 2 SO 4 et 0,5 M de chlorhydrate de guanidine. Ensuite, laver les coupes ont été lavées pendant 30 min dans 40% de DMSO et 0,05 M MgCl 2. Les cellules ont été contre-rouge nucléaire rapide. </li></ol></li></ol>

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The authors have nothing to disclose.

Régulièrement on échantillonne moelle osseuse tandis que le patient subit une intervention chirurgicale (dans notre cas de chirurgie principalement la colonne vertébrale), avec l&#39;avantage que seul le travail peu supplémentaire doit être effectué par le personnel de la salle d&#39;opération. Même si les échantillons sont mélangés avec du citrate de sodium immédiatement après le retrait, de nombreux échantillons ont été partiellement coagulé quand ils sont arrivés dans le laboratoire pour le traitem...

Les cellules souches mésenchymateuses (CSM) jouent un rôle majeur dans la médecine régénérative et de l&#39;ingénierie tissulaire. Ils peuvent migrer, se différencier en différents types de cellules et une greffer, ce qui les rend idéales pour les candidats thérapies autologues 2,3. Dernièrement, des essais cliniques utilisant MSC pour les os et la réparation du cartilage, maladie du greffon contre l&#39;hôte ou une maladie cardiaque ont été lancés 4. Ces cellules souches mésenchymateuses peuvent être récoltées à partir du tissu du cordon ombilical ou adipeux, mais les résultats les plus prometteurs ont été obtenus à partir de la moelle osseuse les cellules souches dérivées 5. La crête iliaque permet de recueillir une quantité considérable de la moelle osseuse et sert donc de site principal d&#39;aspiration 6. Cependant, la qualité de l&#39;aspiration diminue avec l&#39;augmentation du volume de la moelle osseuse retirée. Alors que les cinq premiers ml de ponction de la moelle osseuse contiennent MSC de haute qualité, le retrait de volumes plus importants conduit à une dilution de l&#39;aspiration avec sang périphérique fepuis l&#39;os très vascularisé 7. En raison de la présente des mégacaryocytes et des plaquettes, des aspirats de moelle osseuse sont sujettes à la coagulation, à moins que les anticoagulants sont utilisés. Mais même avec des anticoagulants, des caillots peuvent se produire. Dans la moelle osseuse, cellules souches mésenchymateuses ne représentent qu&#39;une faible proportion de la piscine total de cellules 8 et doivent être multipliées en culture pour la plupart ingénierie tissulaire ou quatre applications thérapeutiques. La qualité d&#39;une telle culture dépend largement de la piscine de la cellule initiale, ce est à dire., La diversité et une grande départ numéro 9. De faibles effectifs d&#39;MSC de retraits peuvent se expliquer en partie par la variabilité des bailleurs de fonds. D&#39;autre part, les CSM à partir d&#39;échantillons de faible qualité nécessitent plus de temps dans la culture et repiquage prolongé pour atteindre le nombre souhaité de cellules. Dans les deux cas, des passages est une source étendue de la sénescence cellulaire et peut conduire à la perte de potentiel de 10 différenciation. Par conséquent, les protocoles qui peuvent maximiser cellulaire y optimiséield et prévenir des effets néfastes doivent être développés 11,12. Lorsque nous avons commencé à travailler avec MSC canines, nous avons été étonnés de voir que les échantillons de moelle osseuse canines environ trois sur quatre contenaient des caillots, tandis que les échantillons humains heureusement coagulé (un sur dix) ont été moins fréquentes. D&#39;autre part, il ne est pas surprenant, que nous avons observé des rendements beaucoup plus faibles de MSC à partir d&#39;échantillons coagulés. Pour résoudre le problème récurrent des échantillons coagulés, nous avons développé le protocole utilisant l&#39;urokinase de drogue thrombolytique lieu de rééchantillonnage. Thérapies thrombolytiques peuvent contrecarrer les situations de la vie en danger, comme l&#39;occlusion des vaisseaux sanguins provoquant une crise cardiaque, accident vasculaire cérébral ou embolie raison de coagulation non désirée. Ils fonctionnent par la dégradation des caillots par clivage enzymatique par la plasmine de la fibrine et du plasminogène activateurs enzymatiques. Malgré la large utilisation pour le traitement des patients, que très peu de publications existent que les activités thrombolytiques utiliséspour les applications de laboratoire pour sauver échantillons coagulés, en se concentrant principalement sur les lymphocytes. En 1987, Niku et al. décrit l&#39;utilisation de streptokinase pour dissoudre les caillots sanguins dans les lymphocytes fonctionnels résultant 13 et quatre ans plus tard, De Vis et al. étendu l&#39;utilisation de la streptokinase pour isoler les cellules leucémiques dans le sang et la moelle osseuse pour des applications de cytométrie en flux 14. Une publication plus récente suggère l&#39;utilisation de Alteplase pour le diagnostic de cancer 15. Tout en utilisant la même approche enzymatique, notre protocole met l&#39;accent sur l&#39;isolement des cellules souches mésenchymateuses multipotentes forme la moelle osseuse de fournir un outil pour les chercheurs dans le domaine des cellules souches.

<table border="0" cellpadding="0" cellspacing="0" width="674">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Basal Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PenStrep 100X</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">15140122</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human FGF-basic</td>
			<td style="width:227px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">MEM Alpha w/ Nucleoside, w/ stable Glutamine</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">1-23S50-I</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:328px;">FBS Heat Inactivated</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">2-01F36-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Amphotericin B</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1907</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Adipogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DMEM-HAM F12 + GlutaMAX</td>
			<td style="width:227px;">Amimed</td>
			<td style="width:119px;">1-26F09-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Insulin&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">I5500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rabbit serum&#160;</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">16120099</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Dexamethasone</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">D4902</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">3-Isobutyl-1-methylxanthine</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">I5879</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Biotin&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">B4639</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Rosiglitazone&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">R2408</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Pantothenate&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">P5155</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Oil Red-O&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">O0625</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Osteogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">L-ascorbic acid 2-phosphate</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">A8960</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">&#223;-glycerophosphate</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">G9422</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:328px;">Silver nitrate (AgNO3)</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">S6506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Chondrogenic Medium Components</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Biopad - sponge shaped medical device&#160;</td>
			<td style="width:227px;">Euroresearch</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">L-proline&#160;</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">P5607</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Insulin-Transferrin-Selenium X</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">51500056</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Human transforming growth factor-&#946;1&#160;</td>
			<td style="width:227px;">Peprotech</td>
			<td style="width:119px;">100-21</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Alcian Blue 8GX</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">A3157</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Nuclear fast red</td>
			<td style="width:227px;">Sigma</td>
			<td style="width:119px;">N8002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Generic</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tri-Sodium citrate dihydrate</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A3901</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">PBS</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">964.9100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Urokinase</td>
			<td style="width:227px;">Medac</td>
			<td style="width:119px;">1976826</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">0.5% Trypsin-EDTA</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:119px;">15400054</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Giemsa stain</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0885</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0877</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sulfuric acid (H2SO4)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A0655</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1584</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride (MgCl2)</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A3618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidine hydrochloride</td>
			<td style="width:227px;">Applichem</td>
			<td style="width:119px;">A1499</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Consumables</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">50 mL reaction tube</td>
			<td style="width:227px;">Axygen</td>
			<td style="width:119px;">SCT-50ML-25-S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">10 mL syringe</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">4606108V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Sterican needle (22G)</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">4657624</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">1.7 mL Microtubes</td>
			<td style="width:227px;">Brunschwig</td>
			<td style="width:119px;">MCT-175-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">100 &#956;m cell strainer</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:119px;">6.05935</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">sterile forceps</td>
			<td style="width:227px;">Bastos Viegas, SA</td>
			<td style="width:119px;">489-001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">sterile scalpel</td>
			<td style="width:227px;">Braun</td>
			<td style="width:119px;">5518059</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Primaria cell cuture dish</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:119px;">353803</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">C-Chip Neubauer Improved</td>
			<td style="width:227px;">Bioswisstech</td>
			<td style="width:119px;">505050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">cell culture flask - Flask T300</td>
			<td style="width:227px;">TPP</td>
			<td style="width:119px;">90301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;"></td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Equipment</td>
			<td style="width:227px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Microbiological biosafety cabinet class II</td>
			<td style="width:227px;">Skan</td>
			<td style="width:119px;">82011500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">water bath</td>
			<td style="width:227px;">Memmert</td>
			<td style="width:119px;">1305.0377</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Stripettes Serological Pipette 5ml</td>
			<td style="width:227px;">Corning</td>
			<td>4487-200ea</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">microscope</td>
			<td style="width:227px;">Olympus</td>
			<td style="width:119px;">CKX41</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">humidified incubator Heracells 240</td>
			<td style="width:227px;">Thermo scientific</td>
			<td>51026331</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Heraeus Multifuge 1S-R</td>
			<td style="width:227px;">Thermo scientific</td>
			<td style="width:119px;">75004331</td>
		</tr>
	</tbody>
</table>

an enzymatic method to rescue mesenchymal stem cells from clotted bone marrow samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Les faits que 74% des échantillons de moelle osseuse canins (n = 54) contenait des caillots quand ils sont arrivés dans notre laboratoire (figure 1A) ainsi diminué les rendements de MSC partir de ces échantillons, nous a fait croire qu&#39;un nombre considérable de MSC a été piégé dans les caillots . En effet, une simple DAPI-taches de matériau de caillot coupe confirme la présence de cellules nucléées à haute densité (figure 1B). Cela conduit finalement à un faible nomb...

Watch this Scientific Journal Video about An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples at JoVE.com

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Une méthode enzymatique pour secourir Cellules souches mésenchymateuses à partir d&#39;échantillons coagulés moelle osseuse

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

an-enzymatic-method-to-rescue-mesenchymal-stem-cells-from-clotted

Research

JoVE Journal

Developmental Biology

9.1K vues.  Swiss Paraplegic Research. Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Vidéo: An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Isolement de mésenchymateuses humaines cellules souches et de leur culture sur la matrice osseuse poreuse

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Recherche

Biologie du développement

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Conversion épigénétique comme une méthode sûre et simple pour obtenir des cellules sécrétrices d&#39;insuline à partir des adultes fibroblastes cutanés

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Isolement et expansion de mésenchymateuses cellules souches / stromales dérivées de placenta humain Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

Isoler cellules progénitrices Mesangiogenic (PPM) de Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Commande temporelle des données d&#39;expression dynamique d&#39;expression spatiale détaillée Plans

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

Isolement et caractérisation de cellules mésenchymateuses stromales du cordon ombilical humain et fœtale Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 &#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

Préparation de vésicules membranaires plasmatiques à partir de cellules souches mésenchymateuses de moelle osseuse pour une thérapie potentielle de remplacement du cytoplasme

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Bioingénierie des microenvironnements humanisés de la moelle osseuse dans la souris et leur visualisation par imagerie en direct

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Isolement, Culture et différenciation des cellules stromales de la moelle osseuse et les ostéoclastes progéniteurs de souris

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Isolation and Characterization of Cardiac Mesenchymal Stromal Cells from Endomyocardial Bioptic Samples of Arrhythmogenic Cardiomyopathy Patients

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The isolation of these cells offers the possibility of studying their involvement in cardiac diseases, and, in addition, provides a useful primary cell model to investigate biological mechanisms.
Here, the method for the isolation of C-MSC from arrhythmogenic cardiomyopathy patients&#39; bioptic samples is described. The endomyocardial biopsy sampling is guided in the right ventricular areas adjacent to the scar visualized by electro-anatomical mapping. The digestion of the biopsies in collagenase and their plating on a plastic dish in culture medium to allow C-MSC growth is described. The isolated cells can be expanded in culture for several passages. To confirm their mesenchymal phenotype, the description of immuno-phenotypical characterization is provided. C-MSC are able to differentiate into several cell types like adipocytes, chondrocytes, and osteoblasts: in the context of ACM, characterized by adipocyte deposits in patients&#39; hearts, the protocols for the adipogenic differentiation of C-MSC and the characterization of lipid droplet accumulation are described.

In this article, a method to isolate cardiac mesenchymal stromal cells from endomyocardial bioptic samples of arrhythmogenic cardiomyopathy patients is provided. Their characterization and the protocol to boost their adipogenic differentiation are described.

Isolement et caractérisation de cardiaques cellules stromales mésenchymateuses d’endomyocardique Bioptic échantillons de Patients de la cardiomyopathie arythmogène

A normal adult heart is composed of several different cell types, among which cardiac mesenchymal stromal cells represent an abundant population. The ...