This publication is funded through Cure CMD, an Association Contre Les Myopathies (AFM) grant (project 16297), and by the National Institutes of Health (grant numbers K08 AR059750, L40 AR057721 and 2R01NS047727).

NOTE: Protocoles pour la collecte de tissus humains doivent être examinés et approuvés par le comité institutionnel de la CISR. Collection de rebut, les tissus humains de-identifié a été approuvé par l&#39;Hôpital pour enfants de Boston et des comités hôpital Brigham and Women de la CISR. Les méthodes décrites ci-dessous ont été appliquées pour l&#39;isolement de cellules myogéniques de dépersonnalisés, jetés tissus. Les méthodes décrites sont applicables aux tissus prélevés à partir de matériaux patients consenti. 1. Isolement cellulaire <ol><li> La dissociation de la biopsie musculaire et la purification de cellules myogéniques <ol><li> Pré-peser un tissu plaque de culture de 10 cm dans une culture tissulaire biosécurité capot, puis re-peser la plaque contenant la biopsie musculaire pour calculer la quantité de tissu à être dissocié. </li><li> Utilisation de scalpels stériles, tissus mince musculaire finement et ajoutez quelques gouttes de HBSS stérile 1x à empêcher le tissu de se dessécher. </li><li> Ajouter 3,5 ml chacune de dispase II et de la collagénase D par gramme de tissu musculaire à être digérés. Pipeter le tissu haché et une solution d&#39;enzyme à travers une pipette de 25 ml stérile à plusieurs reprises. Incuber la plaque dans un incubateur de culture de tissu à 37 ° C avec 5% de CO 2 pendant 15 minutes et à digérer le tissu jusqu&#39;à ce que la suspension passe facilement si une pipette stérile 5 ml. REMARQUE: la dissociation des tissus est habituellement obtenue par digestion enzymatique dans les 45 à 90 min. Se il vous plaît se référer à la section &#39;Les résultats représentatifs de pour plus de détails. </li><li> Ajouter deux volumes de milieu de croissance stérile à un tissu dissocié, filtre à travers un tamis de 100 um de la cellule sur une 50 ml à tubes coniques et granulés pendant 10 min à 329 x g (~ 1100 tours par minute), la température ambiante. Se il vous plaît se référer à la table des matières pour la composition des médias. </li><li> Remettre en suspension le culot dans 1 volume de milieu de croissance stérile et ajouter sept volumes solution rouge sang de lyse cellulaire. Filtrer la solution à travers un tamis cellulaire 40 um, puis sédimenter tes cellules pendant 10 min à 329 xg, la température ambiante. </li><li> Compter les cellules dans un hémocytomètre et remettre en suspension les cellules dans du HBSS 1x 0,5% de BSA à une concentration de 1 x 10 6 cellules / 100 ul. Mettez de côté ~ 250 000 cellules dans un seul tube qui seront utilisés comme un contrôle négatif (non colorées). Régler une seule couleur colorées tubes de contrôle de côté supplémentaires pour l&#39;iodure de propidium et de CD56, qui sont nécessaires pour le bon déclenchement de cellules positives CD56 par tri FACS. Se il vous plaît se référer à la cellule manuels de tri ou de consulter FACS tri experts des installations de base pour assurer des contrôles appropriés sont inclus. </li><li> Colorer les cellules à trier avec 5 pi / 10 6 cellules d&#39;anticorps anti-CD56. Incuber tous les échantillons (y compris les contrôles) sur la glace pendant 30 min. </li><li> Laver les échantillons dans 10 ml 1x HBSS et un culot de cellules pendant 10 min à 329 xg (~ 1100 rpm) dans une centrifugeuse réfrigérée, 4 ° C la température. </li><li> Ajouter l&#39;iodure de propidium à une concentration finale de 1 pg / ml de l&#39;échantillon à être sorTed d&#39;exclusion des cellules mortes. On purifie des cellules positives CD56 de cellules myogéniques non myogéniques en utilisant le trieur de cellules activé par fluorescence. </li></ol></li><li> La dissociation de la biopsie de la peau NOTE: fibroblastes dermiques peuvent être isolés à partir d&#39;un coup de poing de la peau des patients lors des biopsies musculaires ne sont pas disponibles. Les fibroblastes dermiques peuvent être utilisés comme biomatériau pour de nombreuses études, y compris la transduction avec MyoD pour générer des cellules myogéniques. En outre, les fibroblastes dermiques peuvent être utilisés pour générer des cellules iPS, qui peuvent être différenciées en divers types de cellules pour une étude plus approfondie. <ol><li> Transports la biopsie de la peau au laboratoire dans un milieu de transport. Une fois que l&#39;échantillon est reçu, effectuer la culture primaire dès que possible. Si la culture primaire ne peut être établie le même jour, stocker l&#39;échantillon à température ambiante jusqu&#39;au lendemain. </li><li> Transférer la biopsie de la peau à une boîte de Pétri de 35 mm dans la hotte stérile à flux laminaire. </li><li> Rincer la biopsie de la peau dans une boîte de Pétri avec une stérilex PBS pour éliminer le sang et les débris. Retirer le tissu adipeux avec un scalpel stérile. </li><li> Ajouter une solution de collagénase de 2 ml et hacher le tissu avec un scalpel, incuber à 37 ° C pendant 45 min à 1 h, en fonction de la taille du tissu. </li><li> Transférer le tissu digéré dans un tube conique de 15 ml, rincer la boîte de Pétri avec 2 ml de milieu de culture de fibroblastes de deux fois, et de recueillir le milieu dans le même tube. </li><li> Pellet les cellules à 200 xg pendant 5 min à température ambiante. </li><li> Jeter le surnageant et laver le culot avec 3 ml de milieu de fibroblastes de retirer la collagénase, puis les cellules granules nouveau. Se il vous plaît se référer à la table des matières pour la composition des médias. </li><li> Répétez l&#39;étape 1.2.7 une fois de plus. </li><li> Remettre en suspension le culot dans des cellules de fibroblastes de taille et de la plaque 5 ml sur un T25 culture tissulaire stérile ballon. Incuber le flacon à 37 ° C avec 5% de CO 2. </li><li> Évaluer la culture pour la fixation des fibroblastes et la croissance au cours des 1-3 prochains jours. &lt;br /&gt; NOTE: Certains petits morceaux de tissus peuvent également se attacher à la plaque et les fibroblastes migrer hors de ces morceaux de tissu. </li><li> Maintenir la culture dans les mêmes conditions jusqu&#39;à ce que les fibroblastes sont cultivés jusqu&#39;à confluence d&#39;environ 80%. </li><li> Recueillir fibroblastes par trypsinisation et transfert sur des flacons de culture frais d&#39;expansion supplémentaire. Effectuer trypsinisation par lavage des cultures 3 fois en PBS 1x sans Ca ++ et Mg ++. Ajouter la trypsine-EDTA (voir Matériaux Table) pour les cellules (2 ml / fiole T25) pendant 2 minutes à 37 ° C. </li><li> Diviser les cellules individuelles dans des flacons supplémentaires. Si des morceaux de tissu restent attachés aux flacons T25 originaux, ajouter 5 ml de milieu de culture frais à ce ballon en plus de fibroblastes migrent hors continuellement. NOTE: La culture de fibroblastes élargi peut être congelé bas que P1 et stocké dans l&#39;azote liquide pour des expériences futures. </li></ol></li></ol> 2. L&#39;immortalisation des cellules myogéniques <ol><li&gt; cellules Plate 5000000 Phoenix écotrope emballage (PE) la nuit dans une plaque de culture de tissu de 10 cm stérile dans DMEM et moyennes 199 dans un rapport de 4: 1, complété avec du sérum de veau à 10%. </li><li> les cellules d&#39;alimentation 30 min avant la transfection avec 5 ml de milieu frais supplémenté avec 10 mM de caféine. </li><li> Homogénéiser un mélange de 2 pg d&#39;ADN de plasmide à partir d&#39;une préparation midi (CDK4 ou plasmide hTERT) et Polyjet et incuber à température ambiante pendant 15 min, tel que recommandé par le fabricant. </li><li> Ajouter le mélange plasmide / Polyjet aux cellules PE nuit. </li><li> Cellules nourrir avec du milieu frais (DMEM et moyennes 199 dans un rapport de 4: 1, complété avec du sérum de veau à 10%). Douze heures plus tard, recueillir le surnageant contenant le virus et la filtrer à travers un filtre de taille de pore de 0,45 um. </li><li> Utiliser 1 ml de surnageant recueilli pendant une nuit à infecter la cellule d&#39;encapsidation amphotrope PA317 ligne 5 et à obtenir une lignée de cellules productrices de virus stables après sélection avec soit 0,5 mg / ml de néomycine (G418) pour CDK4 ou 0,5 mg / ml d&#39;hygromycine pour hTERT. </li><li> Préparer travailler surnageants viraux par la croissance des cellules d&#39;emballage stables à près de confluence, puis en récoltant le surnageant chaque matin, soir et matin pour trois récoltes. </li><li> Filtrer les surnageants viraux et soit utiliser directement ou de les diviser en aliquots de 1 ml et conserver à -80 ° C pour une utilisation ultérieure. Notez que surnageant viral perd 50% d&#39;efficacité de l&#39;infection par chacun de congélation-décongélation. Ne oubliez pas de blanchir de lavage avant de jeter tout ce qui a touché les particules virales. NOTE: La lignée cellulaire PA317 virus produisant stable peut être congelé et maintenu à -150 ° C pour le stockage permanent (milieu de congélation est de 10% de DMSO; 90% de sérum). </li><li> Plate-purifiée FACS des cellules myogéniques (décrite dans les étapes 1.1 à 1.9) à une densité de 5 x 10 4 cellules / puits dans des plaques à 6 puits revêtues de gélatine à 0,1%. Se assurer que les cellules sont fixées à la plaque avant de procéder à l&#39;infection virale. </li><li> Ajouter 400 ul filtré, freshly produit surnageant viral ou aliquotes congelées à chaque plaque à six puits nuit (garder deux puits comme témoins). </li><li> Changer moyen en alimentant les cellules avec 2,5 ml / puits de milieu frais musculaire (4: 1 de milieu de Eagle modifié par Dulbecco (DMEM) et le milieu 199 supplémenté avec du sérum bovin fœtal à 15%; 0,02 M tampon HEPES; 1,4 mg / L de vitamine B12; 0,03 mg / L ZnSO4, 0,055 mg / L de dexaméthasone, 2,5 ug / l facteur de croissance des hépatocytes et 10 ug / L fibroblastes basique facteur de croissance). Jeter les médias et les pipettes contenant les particules virales dans un récipient de blanchiment. Laissez cellules dans le même milieu pendant 3 jours pour se remettre de l&#39;infection, puis traitent pour la sélection en utilisant soit 400 pg / ml de néomycine (infection de CDK4) ou 300 pg / ml d&#39;hygromycine (infection de hTERT). </li><li> Maintenir cellules dans la sélection des médicaments jusqu&#39;à ce que les cellules dans le plat de commande filière (1-2 semaines). </li><li> Passage cellules avant qu&#39;elles ne deviennent confluentes (60-80% de confluence; utilisant 0,05% de trypsine EDTA), même pendant la selectisur la période. Réensemencement cellules dans plusieurs plats 10cm avec un milieu de myoblastes frais (comme décrit dans 2.11) complétés avec le médicament de sélection. Maintenir des cellules immortalisées sélectionnées comme étant une population hétérogène ou cloner d&#39;obtenir un fond génétique complètement homogène (même de l&#39;insertion du transgène dans chaque cellule). </li><li> Effectuer la sélection clonale utilisant les étapes suivantes: <ol><li> Ensemencer les cellules à une faible densité (par exemple 300 à 500 cellules dans des plats de 10 cm) et les maintenir pendant environ deux semaines, jusqu&#39;à ce que de petites colonies sont formées (10 à 20 cellules). </li><li> Enlever la plupart du milieu à ce point, laissant seulement un film mince pour empêcher les cellules de se dessécher. </li><li> Placez un anneau de clonage (avec une fin trempé dans la graisse à vide de silicone) sur chaque clone désiré et ajouter quelques gouttes de trypsine / EDTA. </li><li> cellules de récolte une fois qu&#39;ils sont arrondis en aspirant soigneusement les cellules en utilisant une pointe de 1 ml ou une pipette Pasteur et de les transférer à la plus petite pla multipuits disponibleste (96 ou 48, ou 24 plaques de 12 puits). </li><li> Développer clones comme nécessaire pour éviter la confluence locale. </li></ol></li></ol>

<ol>
	<li>Flanigan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+muscular+dystrophies.">The muscular dystrophies.</a> Semin Neurol. 32, 255-263 (2012).</li><li>Mercuri, E., Muntoni, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscular+dystrophies.">Muscular dystrophies.</a> Lancet. 381, 845-860 (2013).</li><li>Sharples, A. P., Stewart, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myoblast+models+of+skeletal+muscle+hypertrophy+and+atrophy.">Myoblast models of skeletal muscle hypertrophy and atrophy.</a> Curr Opin Clin Nutr Metab Care. 14, 230-236 (2011).</li><li>Tran, T., Andersen, R., Sherman, S. P., Pyle, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+skeletal+muscle+development+and+applications+in+regenerative+medicine.">Insights into skeletal muscle development and applications in regenerative medicine.</a> Int Rev Cell Mol Biol. 300, 51-83 (2013).</li><li>Miller, A. D., Buttimore, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redesign+of+retrovirus+packaging+cell+lines+to+avoid+recombination+leading+to+helper+virus+production.">Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.</a> Mol Cell Biol. 6, 2895-2902 (1986).</li><li>Schubert, W., Zimmermann, K., Cramer, M., Starzinski-Powitz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+antigen+Leu-19+as+a+molecular+marker+of+regeneration+in+human+skeletal+muscle.">Lymphocyte antigen Leu-19 as a molecular marker of regeneration in human skeletal muscle.</a> Proc Natl Acad Sci U S A. 86, 307-311 (1989).</li><li>Mechtersheimer, G., Staudter, M., Moller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+natural+killer+(NK)+cell-associated+antigen+CD56(Leu-19),+which+is+identical+to+the+140-kDa+isoform+of+N-CAM,+in+neural+and+skeletal+muscle+cells+and+tumors+derived+therefrom.">Expression of the natural killer (NK) cell-associated antigen CD56(Leu-19), which is identical to the 140-kDa isoform of N-CAM, in neural and skeletal muscle cells and tumors derived therefrom.</a> Ann N Y Acad Sci. 650, 311-316 (1992).</li><li>Pavlath, G. K., Gussoni, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+myoblasts+and+muscle-derived+SP+cells.">Human myoblasts and muscle-derived SP cells.</a> Methods Mol Med. 107, 97-110 (2005).</li><li>Boldrin, L., Muntoni, F., Morgan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+human+and+mouse+satellite+cells+really+the+same.">Are human and mouse satellite cells really the same.</a> J Histochem Cytochem. 58, 941-955 (2010).</li><li>Belles-Isles, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+selection+of+donor+myoblast+clones+for+muscular+dystrophy+therapy+using+cell+surface+expression+of+NCAM.">Rapid selection of donor myoblast clones for muscular dystrophy therapy using cell surface expression of NCAM.</a> Eur J Histochem. 37, 375-380 (1993).</li><li>Meng, J., Adkin, C. F., Xu, S. W., Muntoni, F., Morgan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+human+muscle-derived+cells+to+skeletal+muscle+regeneration+in+dystrophic+host+mice.">Contribution of human muscle-derived cells to skeletal muscle regeneration in dystrophic host mice.</a> PLoS One. 6, e17454 (2011).</li><li>Zhu, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence+in+human+myoblasts+is+overcome+by+human+telomerase+reverse+transcriptase+and+cyclin-dependent+kinase+4:+consequences+in+aging+muscle+and+therapeutic+strategies+for+muscular+dystrophies.">Cellular senescence in human myoblasts is overcome by human telomerase reverse transcriptase and cyclin-dependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies.</a> Aging Cell. 6, 515-523 (2007).</li><li>Mamchaoui, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+pathological+human+myoblasts:+towards+a+universal+tool+for+the+study+of+neuromuscular+disorders.">Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders.</a> Skelet Muscle. 1, 34 (2011).</li><li>Sigmund, C. D., Stec, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetic Manipulation of the Renin-Angiotensin System Using Cre-loxP-Recombinase. Methods Mol Med. 51, 53-65 (2001).</li><li>Ludlow, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+telomerase+enzyme+activity+determination+using+droplet+digital+PCR+with+single+cell+resolution.">Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution.</a> Nucleic Acids Res. 42, e104 (2014).</li><li>Jankowski, R. J., Haluszczak, C., Trucco, M., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+characterization+of+myogenic+cell+populations+obtained+via+the+preplate+technique:+potential+for+rapid+isolation+of+muscle-derived+stem+cells.">Flow cytometric characterization of myogenic cell populations obtained via the preplate technique: potential for rapid isolation of muscle-derived stem cells.</a> Hum Gene Ther. 12, 619-628 (2001).</li><li>Gharaibeh, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li><li>Qu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+approaches+to+improve+cell+survival+in+myoblast+transfer+therapy.">Development of approaches to improve cell survival in myoblast transfer therapy.</a> J Cell Biol. 142, 1257-1267 (1998).</li><li>Choi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MyoD+converts+primary+dermal+fibroblasts,+chondroblasts,+smooth+muscle,+and+retinal+pigmented+epithelial+cells+into+striated+mononucleated+myoblasts+and+multinucleated+myotubes.">MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes.</a> Proc Natl Acad Sci U S A. 87, 7988-7992 (1990).</li><li>Huard, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+dermal+fibroblasts+expressing+MyoD1+in+mouse+muscles.">Transplantation of dermal fibroblasts expressing MyoD1 in mouse muscles.</a> Biochem Biophys Res Commun. 248, 648-654 (1998).</li><li>Lattanzi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+myogenic+conversion+of+human+fibroblasts+by+adenoviral+vector-mediated+MyoD+gene+transfer.+An+alternative+strategy+for+ex+vivo+gene+therapy+of+primary+myopathies.">High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.</a> J Clin Invest. 101, 2119-2128 (1998).</li><li>Park, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+modulates+Wnt+signalling+by+association+with+target+gene+chromatin.">Telomerase modulates Wnt signalling by association with target gene chromatin.</a> Nature. 460, 66-72 (2009).</li></ol>

The authors have nothing to disclose.

Cellules comme une ressource utile L&#39;isolement et la culture de populations de cellules myogéniques est extrêmement utile lors de l&#39;établissement des phénotypes de la maladie ou dans des modèles in vitro de la maladie. La procédure d&#39;isolement des cellules myogéniques décrite ici permet l&#39;isolement des myoblastes et fibroblastes à partir d&#39;échantillons de muscle squelettique, qui peuvent ensuite être propagées, différenciée, ou immédiateme...

A correction was made to the authors section in: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling
One of the authors names was corrected from:
Stacy C. Cossette
to:
Stacy A. Cossette

A correction was made to the authors section in: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

Erratum: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

Molecular diagnostics has dramatically evolved in the past 20 years. Genomic DNA is now routinely isolated from sputum or cheek swab, while in the past it required a blood draw. With the current fast turnaround time and ease of gene sequencing, many disease mutations are routinely identified with no need of additional testing. In the case of muscle disease diagnostics, identification of dozens of new genes in the past decade responsible for either muscular dystrophy or myopathy have dramatically changed the ways these diseases are diagnosed 1,2. Currently, there are dozens of genes that have been identified as causes of muscular dystrophy and congenital myopathy, although the mechanisms by which many of these genes produce disease remain unclear. In particular, rare diseases constitute a challenge due to the small size of the patient populations. For these cases, as well as for more common diseases, the generation of stable tools that facilitate studies on the mechanism of pathogenesis and screening of therapeutic drugs is highly desirable. 
Despite dramatic progress in DNA diagnostics, muscle biopsies are still performed to establish the primary diagnosis in many patients in whom primary metabolic or muscle disease is suspected. When a muscle biopsy is necessary, it offers the opportunity for additional diagnostic and research tissue collection with minimal additional morbidity risk for the patient. As there are a number of uses for each tissue specimen, it is highly desirable to establish techniques for primary cell culture using surgical tissue that are straightforward, efficient, and require minimal amounts of tissue. Proper triage of muscle or skin biopsies is required to maximize isolation of primary cells from tissue and long-term storage of live material. Additionally, stem cell research and drug screening holds great promise for developing therapies for many diseases using cell-based assays 3,4.
We herein describe methods for primary cell isolation from human muscle or skin biopsies. Additionally, we include a protocol for immortalization of myogenic cells, which is useful for generating large numbers of cells from an individual. These cells can be used for downstream applications, such as custom drug-screenings, which are otherwise unachievable with the overall low number of cells obtained from primary tissue.

<table border="0" cellpadding="0" cellspacing="0" width="1197">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;"><a name="RANGE!A1:D52">Name of Material/ Equipment</a></td>
			<td style="width:187px;">Company</td>
			<td style="width:207px;">Catalog Number</td>
			<td style="width:484px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Equipment</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Tissue&#160;culture biosafety hood</td>
			<td style="width:187px;">Baker Company, Inc.</td>
			<td style="width:207px;">Model SterilGuard Hood</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:320px;">Benchtop centrifuge, such as Beckman Model&#160;</td>
			<td style="width:187px;">Beckman Coulter</td>
			<td style="width:207px;">Model Allegra 6R</td>
			<td>If cell sorting is performed, a centrifuge with refrigeration is preferred</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Microscope</td>
			<td style="width:187px;">Nikon</td>
			<td style="width:207px;">Model Eclipse TS100</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:320px;">Tissue culture incubator, connected to a CO2 source</td>
			<td style="width:187px;">Forma Scientific&#160;</td>
			<td style="width:207px;">Series II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Fluorescence-activated cell sorter (FACS)</td>
			<td style="width:187px;">Becton Dickinson</td>
			<td style="width:207px;">Model Aria</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:320px;">Reagents for isolation of primary myoblasts from tissue</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dispase II</td>
			<td style="width:187px;">Roche Applied Science</td>
			<td style="width:207px;">#04942078001</td>
			<td>Prepare a sock solution of 2.4U/ml in DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase D&#160;</td>
			<td style="width:187px;">Roche Applied Science</td>
			<td style="width:207px;">#088882001</td>
			<td>Prepare a stock solution of 10 mg/ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">1x Sterile Hank&#8217;s Balanced Saline Solution (HBSS), calcium and magnesium free</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">#14185-052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin, fraction V</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">#05470</td>
			<td>Prepare a sterile&#160; solution of 1X HBSS 0.5% BSA for FACS sorting</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Sterile growth medium for myoblasts: Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) with high glucose (4.5g) supplemented with 30% fetal bovine serum</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">11965-092</td>
			<td>Contains L- glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">RBC lysis solution</td>
			<td style="width:187px;">Qiagen</td>
			<td style="width:207px;">158904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Propidium Iodide stock 10mg/mL</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">P4170</td>
			<td>Prepare the stock solution by diluting the powder in sterile distilled water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Anti-human CD56 antibody for flow cytometry</td>
			<td style="width:187px;">Biolegend</td>
			<td>318310</td>
			<td>APC-conjugated antibody, other labels are avaialable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Reagents for immortalization of primary myoblasts</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ecotropic packaging cell line PE</td>
			<td style="width:187px;">Cell Biolabs</td>
			<td style="width:207px;">RV-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Amphotropic packaging cell line PA3174</td>
			<td style="width:187px;">Cell Biolabs</td>
			<td style="width:207px;">RV-102</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Pig skin gelatin</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:207px;">G1890-500g</td>
			<td style="width:484px;">Prepare a stock solution of 0.1% gelatin in water. Coat the dish with the solution at 37&#176;C for one hour. Remove the solution and add medium.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">PolyJet&#160;</td>
			<td style="width:187px;">Signagen</td>
			<td>SL100688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">G418</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:207px;">345812</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hygromycin</td>
			<td style="width:187px;">EMD Biosciences</td>
			<td style="width:207px;">400051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">pBabe plasmids containing mCDK4 and hTERT</td>
			<td style="width:187px;">not commercially available</td>
			<td style="width:207px;"></td>
			<td>Stadler et al, 2013</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Qiagen plasmid midiprep kit</td>
			<td style="width:187px;">Qiagen</td>
			<td>12143</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Medium 199</td>
			<td style="width:187px;">Life Technologies&#160;</td>
			<td>Medium 199 (31150022)</td>
			<td style="width:484px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dulbecco&#8217;s modified Eagle medium (DMEM)</td>
			<td style="width:187px;">Life Technologies&#160;</td>
			<td>DMEM (11965-092)</td>
			<td style="width:484px;">Mix 4:1 DMEM:199</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:320px;">Myoblast growth medium: 4:1 Dulbecco&#8217;s modified Eagle medium (DMEM) and Medium 199 supplemented with 15% fetal bovine serum; 0.02 M HEPES buffer; 1.4 mg/l vitamin B12; 0.03 mg/l ZnSO4, 0.055 mg/l dexamethasone, 2.5 &#956;g/l hepatocyte growth factor and 10 &#956;g/l beta fibroblast growth factor.</td>
			<td style="width:187px;">Life technologie (DMEM, F199); Atlanta Biological (FBS); Invitrogen (Hepes); Fisher (ZincSulfate); Sigma (Vit.B12, Dexamethasone); Chemicon international (HGF); Biopioneer (betaFGF)&#160;</td>
			<td style="width:207px;">#15630-080 (Hepes); #Z68-500 (ZnSO); #V2876.#D4902 (Vit.B12, Dex); GF116 (HGF); HRP-0011 (bFGF).&#160;</td>
			<td style="width:484px;">Prepare media and stock solution Vit. B12 (20mg/ml); ZincSulfate (60&#181;g/ml); Dex (55&#181;g/&#181;l) separately for easier use. HGF stock solution (5&#181;g/ml) and FGF (20&#181;g/ml) should be added freshly every week at the final working concentration.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">TrypLE express</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">12605-010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Myosin heavy chain antibody for immunostaining</td>
			<td style="width:187px;">Developmental Hybridoma Bank</td>
			<td style="width:207px;">MF20</td>
			<td>Clone MF20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Desmin Antibody for immunostaining</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td>MS-376-S0</td>
			<td>Clone D33</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Horse serum</td>
			<td style="width:187px;">Invitrogen</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Reagents for primary skin fibroblast isolation</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Transport medium: RPMI 1640 supplemented with 10% fetal bovine serum and 0.2% penicillin/streptomycin</td>
			<td style="width:187px;">RPMI medium 1640: GIBCO Life Technologies</td>
			<td style="width:207px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Human primary fibroblast culture medium: RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin</td>
			<td style="width:187px;">Fetal bovine serum: Thermo Scientific</td>
			<td style="width:207px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:320px;">Collagenase solution: Collagenase type 2 (100mg) resuspended in 12.5 ml of fibroblast culture medium and filter-sterilized</td>
			<td style="width:187px;">Worthington</td>
			<td style="width:207px;">4176</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Sterile 1X Phosphate Buffer Saline (PBS) calcium and magnesium free</td>
			<td style="width:187px;">Lonza</td>
			<td style="width:207px;">17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Materials for isolation of primary myoblasts</td>
			<td style="width:187px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">50ml and 15ml sterile conical tubes</td>
			<td style="width:187px;">GeneMate/Bioexpress</td>
			<td style="width:207px;">C-3394-4 (50ml) ; C3394-1 (15ml))</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile scalpels</td>
			<td style="width:187px;">Aspen Surgical</td>
			<td style="width:207px;">372610</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td style="width:187px;">Hausser Scientific</td>
			<td style="width:207px;">1492</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 5, 10 and 25 ml pipettes</td>
			<td>Bellco glass</td>
			<td>1226-05010 (5ml); 1200-10010 (10ml) ;1228-25050 (25ml)</td>
			<td>Reusable pipettes are washed, cotton plugged and autoclaved before use</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile tissue culture-treated plastic dishes (10cm)</td>
			<td style="width:187px;">BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile nylon cell strainers (100&#181;m and 40&#181;m size)</td>
			<td style="width:187px;">BD Falcon</td>
			<td colspan="2">352340 (40&#181;m); 352360 (100&#181;m)</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">&#160;Materials for myoblast immortalization</td>
			<td style="width:187px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 0.45&#181;m filters</td>
			<td>Millipore</td>
			<td>SLHV013SL</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Cloning rings</td>
			<td style="width:187px;">Corning</td>
			<td>#3166-8</td>
			<td>To be cleaned and autoclaved before and after use</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Materials for fibroblast cell lines&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile 35mm tissue culture-treated plastic dishes</td>
			<td>Greiner bio-one</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile scalpels</td>
			<td>DeRoyal</td>
			<td>D4510A</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Sterile T25 tissue culture flasks</td>
			<td>Techno Plastic Product, TPP</td>
			<td>90026</td>
			<td>sold in the USA by MIDSCI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">TrypLE express</td>
			<td style="width:187px;">GIBCO Life Technologies</td>
			<td style="width:207px;">12605-010</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and immortalization of patient-derived cell lines from muscle biopsy for disease modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

La figure 1 illustre certaines des étapes clés impliquées dans la dissociation de tissu primaire: le montant exact du tissu est pesé dans un plat culture de tissus de Pétri stérile (Figure 1A, B). Le tissu est ensuite finement hachée à l&#39;aide de scalpels stériles, jusqu&#39;à ce qu&#39;une suspension de tissu est obtenu (figure 1C, D). Après l&#39;addition des enzymes de la digestion, les muscles primaire dissociation tissulaire est habituellement obtenue...

Watch this Scientific Journal Video about Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling at JoVE.com

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolement et immortalisation de lignées cellulaires de patients dérivé de la biopsie musculaire pour la modélisation des maladies

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

isolation-immortalization-patient-derived-cell-lines-from-muscle

Research

JoVE Journal

Medicine

16.4K vues.  UT Southwestern Medical Center. This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Vidéo: Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modélisation et Imagerie en 3 dimensions l&#39;invasion des cellules collectives

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Recherche

Médecine

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Skeletal Muscle dimorphisme des sexes de la protéomique

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Isolement des noyaux de cardiomyocytes post-mortem des tissus

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Différenciation isolement, caractérisation et comparative de l&#39;homme dentaires cellules souches de pulpe Dérivé de dents permanentes à l&#39;aide de deux méthodes différentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Patient Culture cellulaire dérivée et isolement de CD133<sup&gt; +</sup&gt; Putatifs cellules souches du cancer du mélanome

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Procédures Muscle squelettique humain biopsie aide de la technique de modification Bergström

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Enrichissement pour chimiorésistantes cancer de l&#39;ovaire des cellules souches à partir de lignées cellulaires humaines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Préparation et respirométrique évaluation des mitochondries isolées de tissu musculaire squelettique Obtenu par biopsie percutanée aiguille

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative Imagerie par résonance magnétique de la maladie du muscle squelettique

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Mise en place d&#39;épithéliales humaines Enteroids et Colonoids de tout le tissu et la biopsie

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...