Ce projet a été soutenu par la Fondation nationale suisse (FNS # 310030-127586/1).

1. La récolte du disque intervertébral <ol><li> Whole queue bovins longueur est obtenue à partir d&#39;un abattoir local, si possible sans la peau car la présence de la peau augmente le risque de contamination (figure 2). </li><li> Préparer grande planche à découper et préparer la station de travail et des instruments stériles au sommet d&#39;une planche à découper (Fig. 2). </li><li> Préparer sous le capot de la gaze stérile à flux laminaire stérile humidifié avec du chlorure de sodium à 0,9% contenant du citrate de sodium 55mm et mis dans chaque puits de la plaque de 6 puits. </li><li> Préparer un bassin et diluer 1:100 solution de Betadine avec de l&#39;eau du robinet. </li><li> Plonger la queue entière bovine dans un bassin contenant une solution de Bétadine 1% pendant 5 min. </li><li> Brièvement la queue à sec avec une gaze stérile et le placer sur la station de travail stérile. </li><li> Retirez délicatement les muscles autour de la queue avec lame de bistouri n ° 22. </li><li> Hacher l&#39;écart des pièces de la queue qui ne sont pas nécessaires, généralement les deux extrémités de la queue, qui contiennent relativement grandes et très petites entreprisesIVD (Fig. 3). </li><li> Garniture davantage les muscles et les tendons autour de la DIV en utilisant lame de bistouri n ° 10. Attention à ne pas couper l&#39;anneau externe des disques. </li><li> Marquer la partie antérieure de l&#39;IVD avec un marqueur de peau chirurgical (cette étape est facultative, elle permet de déterminer le centre de rotation axiale dans notre bioréacteur). </li><li> Localisez le point de connexion IVD et vertèbres de la queue en se déplaçant doucement. Sentez-vous la frontière entre l&#39;IVD et d&#39;os avec le côté mat de la lame d&#39;un scalpel. Le site de clivage devrait être de 1-2 mm de distance de l&#39;IVD vers la vertèbre. Placez personnalisée fait le porte-lame industrielle sur le site de clivage (Fig. 4). </li><li> Cleave IVD et vertèbre par martelage sur le dessus du support de lame sur-mesure industrielle (Fig. 3). </li><li> Répétez de l&#39;autre côté de la connexion IVD vertèbre. </li><li> Enveloppez isolés DIV avec une gaze stérile humidifié avec du chlorure de sodium à 0,9% contenant du citrate de sodium 55mm. </li><li> Continuer l&#39;isolement des DIV à l&#39;échantillon numéro désiré (généralement autour de 6 peutêtre récoltés avec un diamètre de 10-20 mm). </li><li> Connectez le système de lavage à jet (Zimmer, Inc.) Avec une solution stérile de Ringer lactate, 5L sacs sont d&#39;une taille utile (alternativement PBS stérile ou 0,9% de solution saline). </li><li> Tenir la DIV avec une pince et le jet-lavage des deux côtés de la surface des plateaux vertébraux en utilisant Zimmer Pulsavac jet de lavage du système (Fig. 1C). Le pistolet à jet devrait être tourné sur la surface des plateaux vertébraux à un angle compris entre 30 - 60 ° pendant environ 30s de chaque côté. (Fig. 1 et 7) </li><li> Mettez la DIV de revenir à la plaque de 6 puits et envelopper avec une gaze humidifiée tout jet de lave un autre échantillon du disque. </li><li> DIV sont prêts pour la culture d&#39;organes et en aval des applications (par exemple la viabilité cellulaire, le chargement mécanique, la culture d&#39;organes) (Fig. 1D). </li></ol> 2. Les résultats représentatifs Résultat des mesures pour juger une préparation réussie du disque intervertébral peut être une expérience de diffusion où un colorant fluorescent de faible poids moléculaire (Procion par exemple rouge)est préparée comme une solution à 1% dans PBS 2. L&#39;IVD est alors maintenu avec au moins 100 ml de solution de colorant et le colorant est alors autorisé à diffuser passivement dans l&#39;IVD pour 24h dans le gonflement libre. L&#39;IVD est alors flash-congelés dans l&#39;azote liquide et ramené à température ambiante et déshydraté par une série de transferts d&#39;acétone (première pré-refroidi dans -80 ° C, puis à -20 ° C, puis 4 ° C et enfin à la température ambiante ). Le disque peut ensuite être intégrées dans le poly-méthyl-acrylate de méthyle (PMMA) et couper avec une lame tranchante pour produire 100 um d&#39;épaisseur des sections. Ces sections peuvent ensuite être consultés par n&#39;importe quel microscope fond clair standard, mais préférentiellement par l&#39;utilisation de la microscopie à fluorescence, depuis Procion Red émet une fluorescence rouge (fig. 5-6). Le trabeculi de la surface osseuse apparaissent très propre après l&#39;étape lavaging jet (Fig. 7). Paramètres pour une culture d&#39;organe réussie sont d&#39;abord l&#39;absence de contamination, la viabilité des cellules maintenues (fig. 8-9), la hauteur discale etglobale &quot;de l&#39;intégrité du disque&quot;, mesurée avec des coupes histologiques et de safranine O / vert rapide tache ou hématoxiline Meyer 3. Nous avons développé des protocoles et des macros publiés ailleurs 4,5 à tacher les tissus du disque en direct et de balayer la viabilité des cellules en 3D en utilisant un microscope confocal à balayage laser et le kit de coloration vivants / morts (Molecular Probes, Invitrogen, Bâle, Suisse). Nous avons également développé une macro et de routine à effectuer la cellule de comptage automatique pour estimer la viabilité des cellules dans les tissus 3D ou 3D transporteurs utilisant la plateforme NIH ImageJ 4-6. Protocoles de rechange ont été proposées pour déterminer la viabilité cellulaire des cellules du disque par digestion initiale à l&#39;aide la digestion séquentielle de la matrice extracellulaire en utilisant la pronase et la collagénase de type 2 et ensuite de colorer la suspension cellulaire 7. Une version antérieure du protocole impliquait une étape d&#39;incubation dans du PBS contenant dix fois plus concentrée de concentration de pénicilline / streptomycine pendant 10 minutes après la récolte des disques d&#39;orgue avant la cuLTURE. Avec l&#39;étape de lavage à jet cette étape devient obsolète puisque cette étape semble bénéfique à des fins de culture. <img alt="Figure 1" src="/files/ftp_upload/3490/3490fig1.jpg" /> Figure 1. Vue d&#39;ensemble des étapes méthodologiques pour préparer bovines coccygienne disques intervertébraux avec plateaux intact et une fine couche (1-2mm) de l&#39;os. Vue schématique A) du disque intervertébral (DIV) et ses voies nutritionnelles. B) Procédure à découper la DIV avec un personnalisées porte-lame et une lame industrielle forte. C) étape de lavage Jet pour assurer la viabilité des cellules de haute et de permettre l&#39;échange de nutriments gardant les plateaux et 1-2 mm d&#39;os attachée sur les deux côtés. D) Enfin, la DIV peut être cultivé dans la chambre de spécimens d&#39;un bioréacteur ou être utilisé pour toute application en aval. <img alt="Figure 2" src="/files/ftp_upload/3490/3490fig2.jpg" /> Figure 2 TOP:. Pleine longueur de la queue fraîches bovines (idéalement bénéficier du droit àINED dans les 2-3 heures post-mortem afin d&#39;assurer la viabilité cellulaire élevée. En bas: X-ray image d&#39;une queue de bovins âgés de 6 mois environ illustrant l&#39;existence des plaques de croissance (GP), centre secondaire d&#39;ossification, entre les plateaux osseux (PE) et les corps vertébral (VB). <img alt="Figure 3" src="/files/ftp_upload/3490/3490fig3.jpg" /> Figure 3. Un Tools) pour disséquer la queue bovins dans des conditions stériles. B) Customized porte-lame pour couper des segments-motionnelle de la queue de l&#39;espèce bovine dans l&#39;action tout en coupant le disque intervertébral. C) Vue latérale de l&#39;personnalisées porte-lame avec une lame standard industriel (Lutz, Allemagne). <img alt="Figure 4" src="/files/ftp_upload/3490/3490fig4.jpg" /> Figure 4. Dessin et de l&#39;image de la queue bovins après avoir enlevé le muscle environnant et du ligament illustrant les sites de clivage de couper les segments promotionnelle. Partiel vertèbre doit être &quot;craqué&quot; wie le porte-lame sur-mesure, idéalement 1-2 mm loin de les plateaux cartilagineux pour assurer surface osseuse complète. <img alt="Figure 5" src="/files/ftp_upload/3490/3490fig5.jpg" /> Figure 5. Démonstration de l&#39;efficacité du nettoyage de l&#39;étape de jet-lavage avant (A) et après (B). Avis des surfaces osseuses nettoyage des segments du disque intervertébral après la procédure de pulvérisation. <img alt="Figure 6" src="/files/ftp_upload/3490/3490fig6.jpg" /> Figure 6. Expérimentation de diffusion gratuite de gonflement du fraîchement préparés excisée disques intervertébraux queue bovin (DIV) laissé pendant 24 heures dans une solution de 1% Procion rouge, démontrant l&#39;effet de blocage par la plaque de croissance. Une radiographie numérique vue sagittale d&#39;un DIV contenant encore la plaque de croissance et une seconde couche de l&#39;image de microscopie à fluorescence de l&#39;os à travers la coupe sagittale explant coccygienne disque intervertébral bovins des plateaux vertébraux cartilagineux et osseux après deux4h de diffusion gratuite. GP: plaque de croissance, 2ndry CO: centre secondaire d&#39;ossification. <img alt="Figure 7" src="/files/ftp_upload/3490/3490fig7.jpg" /> Figure 7. Gratuit-gonflement expérience de fraîchement préparés excisée disques intervertébraux queue bovin (DIV) laissé pendant 24 heures dans une solution de 1% Procion rouge, démontrant de nettoyage à jet d&#39;effet du traitement de lavage. Sagittale sections épaisses (~ 100 mm) de diagnostic in vitro excisés bovins avec des plaques de ~ 1,5 mmthick fin osseuses qui ont subi un lavage à jet d&#39;un traitement (à gauche) contrôle sans traitement (droite) un lavage de jet-traitement. Inlet montre le jet, qui a été utilisé par le système plaies Zimmer débridement expériences 24h diffusion libre des bovins explants disque intervertébral en rouge Procion 1%. <img alt="Figure 8" src="/files/ftp_upload/3490/3490fig8.jpg" /> Figure 8. Images Live / Dead de projections de piles imagerie confocale (250μm) prélevés bovinsegments e disque intervertébral préparé avec cette méthode de lavage de jet-gardés sous-gonflement libre pour 0 et 14 jours respectivement pour le nucleus pulposus (NP), l&#39;intérieur l&#39;anneau fibreux (IA) et l&#39;anneau fibreux externe (OA). Vert = cellules des cellules vivantes souillé par calcéine AM (acétyl méthylique), les globules rouges = noyau de cellules mortes colorées par éthidium homodimère-1. <img alt="Figure 9" src="/files/ftp_upload/3490/3490fig9.jpg" /> Figure 9. Viabilité cellulaire du disque intervertébral par laser microscopie confocale à balayage sur les tissus vivants et de numérisation 3D pile. La viabilité des cellules du noyau pulpeux (NP) et l&#39;anneau de fibrose (AF) au cours des 21 jours-culture sous-gonflement sans condition. (Moyenne ± SEM, n = 6) Les différences statistiques ont été testés à l&#39;aide non-paramétrique de Kruskal-Wallis signé test de la somme parmi les groupes. Des différences significatives ont été trouvées entre 21 jours et tous les autres moments dans NP. Alors que dans les différences significatives ont été trouvées AF entre le jour de 7 àjour 0. (* P &lt;0,05, ** p &lt;0,01).

<ol>
	<li>Lee, C. R., Iatridis, J. C., Poveda, L., Alini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+organ+culture+of+the+bovine+intervertebral+disc:+effects+of+vertebral+endplate+and+potential+for+mechanobiology+studies.">In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies.</a> Spine (Phila Pa 1976). 31, 515-522 (1976).</li><li>Chan, S. C. W., Gantenbein-Ritter, B., Leung, V. Y., Chan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreserved+intervertebral+disc+with+injected+bone+marrow-derived+stromal+cells:+a+feasibility+study+using+organ+culture.">Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture.</a> Spine. J. 10 (6), 486-496 (2010).</li><li>Chan, S. C., Ferguson, S. J., Wuertz, K., Gantenbein-Ritter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Response+of+the+Intervertebral+Disc+to+Repetitive+Short+Term+Cyclic+Torsion.">Biological Response of the Intervertebral Disc to Repetitive Short Term Cyclic Torsion.</a> Spine (Phila Pa 1976). , (2011).</li><li>Gantenbein-Ritter, B., Sprecher, C. M., Chan, S., Illien-J&#252;nger, S., Grad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+protocols+for+live/dead+staining+in+three-dimensional+carriers.">Confocal imaging protocols for live/dead staining in three-dimensional carriers.</a> Methods Mol. Biol. 740, 127-140 (2011).</li><li>Gantenbein-Ritter, B., Potier, E., Zeiter, S., van der Werf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+three+techniques+to+determine+cell+viability+in+3D+tissues+or+scaffolds.">Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds.</a> Tissue Engineering Part C Methods. 14, 353-358 (2008).</li><li>Rasband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li><li>Haschtmann, D., Stoyanov, J. V., Ettinger, L., Nolte, L. P., Ferguson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+intervertebral+disc/endplate+culture+model:+analysis+of+an+ex+vivo+in+vitro+whole-organ+rabbit+culture+system.">Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system.</a> Spine. 31, 2918-2925 (2006).</li><li>Gantenbein, B., Gr&#252;nhagen, T., Lee, C. R., van Donkelaar, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+organ+culturing+system+for+intervertebral+disc+explants+with+vertebral+endplates:+a+feasibility+study+with+ovine+caudal+discs.">An in vitro organ culturing system for intervertebral disc explants with vertebral endplates: a feasibility study with ovine caudal discs.</a> Spine. 31, 2665-2673 (2006).</li><li>Korecki, C. L., Kuo, C. K., Tuan, R. S., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervertebral+disc+cell+response+to+dynamic+compression+is+age+and+frequency+dependent.">Intervertebral disc cell response to dynamic compression is age and frequency dependent.</a> J. Orthop. Res. 27, 800-806 (2009).</li><li>Korecki, C. L., MacLean, J. J., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+effects+on+intervertebral+disc+mechanics+and+biology.">Dynamic compression effects on intervertebral disc mechanics and biology.</a> Spine (Phila Pa 1976). 33, 1403-1409 (1976).</li><li>Jim, B., Steffen, T., Moir, J., Roughley, P., Haglund, L. <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+an+intact+intervertebral+disc+organ+culture+system+in+which+degeneration+can+be+induced+as+a+prelude+to+studying+repair+potential.">Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential.</a> European Spine Journal. , 1-11 (2011).</li></ol>

Nous n&#39;avons rien à révéler.

La première étape pour la culture d&#39;organe réussie est de faire en sorte que l&#39;explant ne doivent pas être contaminés. La queue doit être dépecé avant de commencer la procédure. Toute poils amené dans un laboratoire stérile pourrait être problématique en termes de contamination. La queue bovins devraient idéalement être aussi frais que possible (ce qui affecte la viabilité des cellules initiales). Par ailleurs, l&#39;étape de lavage bétadine est recommandée pour réduire le risque de contamina...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td colspan="4"> Frais tissus bovins disque intervertébral de queues de bovins, d&#39;abattoir local (idéalement dans les heures post-mortem et sans peau). </td></tr><tr><td> Pulsavac AC Plus Système </td><td> Zimmer Inc., Suisse </td><td> 00-5150-486-01 </td><td> Meilleure performance à la tête de pulvérisation et de hip-avec alimentation AC (celui avec le pack de 8 piles AA ne fonctionnera aussi mais c&#39;est moins pratique) </td></tr><tr><td> Haute capacité de ventilateur pulvérisation W / Splash Bouclier, longueur 12,7 cm </td><td> Zimmer Inc., Suisse </td><td> 00-5150-175-00 </td><td> Il ya plusieurs têtes de pulvérisation disponibles, nous avons testé celui-ci avec succès </td></tr><tr><td> Les lames de bistouri n ° 22 et n ° 10 </td><td> Swann-Mort, l&#39;Angleterre </td><td> # 10: 0201 # 22: 0208 </td><td></td></tr><tr><td> Porte-lame de scalpel # 3 et # 4 </td><td> Hausmann, Allemagne </td><td> # 3: 06.103.00 # 4: 06.104.00 </td><td></td></tr><tr><td> Lutz industriels lames </td><td> Lutz, de l&#39;Allemagne </td><td> 1022.0884 </td><td></td></tr><tr><td> Tampon phosphate salin (PBS) </td><td> Invitrogen, Suisse </td><td> 10010-023 </td><td></td></tr><tr><td> Eagle modifié par Dulbecco (DMEM) </td><td> Gibco, Suisse </td><td> 11960-044 </td><td></td></tr><tr><td> Solution de Ringer lactate (sans glucose) </td><td> Bichsel, Suisse </td><td> 133 0002 </td><td></td></tr><tr><td> 6-même plaque multi-puits </td><td> TPP, Suisse </td><td> 92006 </td><td></td></tr><tr><td> Solution de Betadine </td><td> Mundipharma, Suisse </td&gt; <td> 10055025 </td><td></td></tr><tr><td> Marqueur de la peau chirurgicale </td><td> Porex Surgical, Suisse </td><td> 9560 </td><td></td></tr><tr><td> Grande planche à découper </td><td></td><td></td><td> Toute marque est possible </td></tr></tbody></table>

preparation of intact bovine tail intervertebral discs for organ culture

Le disque intervertébral (DIV) est l&#39;articulation de la colonne vertébrale reliant vertèbre en vertèbre. Il fonctionne pour transmettre le chargement de la colonne vertébrale et donner de la flexibilité de la colonne vertébrale. Il compose de trois compartiments: le noyau pulpeux intime (NP) englobant l&#39;annulus fibrosus (AF), et deux plateaux cartilagineux reliant les NP et AF pour le corps vertébral, des deux côtés. Douleur d&#39;origine discale éventuellement causé par une maladie dégénérative du disque intervertébral (DDD) et les hernies discales a été identifié comme un problème majeur dans notre société moderne. Pour étudier les mécanismes possibles de la dégénérescence IVD, dans les systèmes de culture in vitro d&#39;organes avec cellules du disque live sont très attrayante. La culture in vitro de bovins intacts les DIV coccygienne a avancé à un système de modèle pertinent, qui permet l&#39;étude de la mécano-biologique des aspects dans un environnement bien contrôlé physiologiques et mécaniques. DIV de la queue bovine peut être obtenue assez facilement en plus grand nombre uneD sont très similaires aux humains les DIV lombaires à l&#39;égard de la densité cellulaire, la population de cellules et de dimensions. Toutefois, avant bovins caudale techniques de récolte DIV conservant plateaux cartilagineux et osseux plateaux échoué après 1-2 jours de culture depuis les voies de la nutrition étaient évidemment bloqué par du sang coagulé. DIV sont les plus grands organes avasculaire, par conséquent, les nutriments aux cellules de la NP dépendent uniquement sur la diffusion via les bourgeons capillaires du corps vertébraux adjacents. Présence de débris d&#39;os et de sang coagulé sur les surfaces des plateaux vertébraux peuvent entraver la diffusion des nutriments dans le centre du disque et la viabilité cellulaire compromis. Notre groupe a établi un protocole relativement rapide à &quot;craquer&quot; les DIV-le de la queue avec un faible risque de contamination. Nous sommes capables de perméabiliser les surfaces fraîchement coupées des plateaux vertébraux osseux en utilisant un système de jet de lavage chirurgical, ce qui élimine les caillots de sang et de débris de coupe et de façon très efficace rouvre la voie de la diffusion de nutritionau centre de la DIV. La présence de plaques de croissance des deux côtés de l&#39;os vertébral doit être évitée et d&#39;être enlevés avant la culture. Dans cette vidéo, nous décrivons les étapes cruciales lors de la préparation et de démontrer la clé d&#39;une culture d&#39;organe réussie maintien de la viabilité cellulaire élevée pendant 14 jours dans la culture gonflement libre. Le temps de la culture pourrait être prolongé le cas échéant environnement mécanique peut être maintenue en utilisant bioréacteur chargement mécanique. La technique démontrée ici peuvent être étendues à d&#39;autres espèces animales comme les porcins, ovins et lépreux isolement caudale ou lombaire DIV.

Watch this Scientific Journal Video about Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture at JoVE.com

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

Ce protocole illustre une technique de récolte pour les bovins coccygienne disques intervertébraux pour la culture d&#39;organes pour<em&gt; In vitro</emCulture d&#39;organe&gt;.

Préparation d&#39;Intact disques intervertébraux Queue bovine pour la culture d&#39;organe

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

preparation-intact-bovine-tail-intervertebral-discs-for-organ

Research

JoVE Journal

Bioengineering

14.4K vues.  University of Bern. Ce protocole illustre une technique de récolte pour les bovins coccygienne disques intervertébraux pour la culture d'organes pour In vitro

Vidéo: Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Les hydrogels d&#39;alginate de culture d&#39;organe en trois dimensions des ovaires et des trompes

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Recherche

Bio-ingénierie

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Préparation de la 3D pour les applications de fibrine Échafaudages CULTURE DE CELLULES SOUCHES

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Exemple Stratégies Préparation de Spectrométrie de Masse Imagerie de culture cellulaire 3D Modèles

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

L&#39;étude de la Stœchiométrie Epidermal Growth Factor Receptor dans des cellules intactes en utilisant la microscopie corrélative

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Un<em&gt; In vitro</em&gt; Orgue Culture Modèle de la murine disque intervertébral

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Fabrication et Validation d’un système d’orgue-on-chip avec électrodes intégrées à quantifier directement transendothéliale résistance électrique

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales &#8211; nano-, micro-, meso-, and macroscopic &#8211; fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

This study assesses the fracture toughness of bovine cortical bone at the sub-meso levels using microscopic scratch tests. This is an original, objective, rigorous, and reproducible method proposed to probe fracture toughness below the macroscopic scale. Potential applications are studying changes in bone fragility due to diseases like osteoporosis.

Évaluation de fragilité de l’OS Cortical Bovine à l’aide de Tests de Scratch

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Fabrication évolutive d’étirable, Dual Channel, puces microfluidiques orgue

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Plate-forme de culture d’organes du segment antérieur pour le suivi des blessures du globe ouvert et des performances thérapeutiques

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

Reconstitution de la cytoarchitecture et de la fonction des tissus épithéliaux humains sur une puce organique à sommet ouvert

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...