Nous tenons à remercier Robin Nye (Tufts Medical Center), Tomoya Uchimura et Dana Cairns (Tufts University) pour fournir une aide au stockage du liquide synovial et la centrifugation. Ce travail a été financé par le NIH (1R01AR059106-01A1) pour LZ

Un système 3D pour les chondrocytes articulaires humains en culture dans le liquide synovial Dans ce travail, nous avons encapsulé chondrocytes articulaires humains en billes d&#39;alginate en utilisant une modification de fabrication-a suggéré d&#39;encapsulation de protocole (Lonza, et 31). En utilisant ces constructions 3D, nous avons développé un système de culture de cellules dans un milieu de culture contenant des pourcentages variés de liquide synovial humain et ont évalué ces constructions 3D pour l&#39;expression des gènes du cartilage. 1. Préparer chondrocytes articulaires humains (HAC) pour trois dimensions (3D) d&#39;encapsulation <ol><li> Décongelez un flacon (1 ml) de chondrocytes articulaires humains (HAC) (Lonza) (passage 2) dans 37 ° C l&#39;eau de bain pendant 1 min. </li><li> Mélanger avec 1ml de milieu de croissance de chondrocytes (Lonza) et centrifuger à 3000 rpm pendant 3-5 min pour recueillir les cellules. Jeter le surnageant. </li><li> Resuspendre le culot cellulaire dans les milieux de croissance de chondrocytes (Lonza). </li><li> Cellules de la plaque dans un tissu de 10 cmplaque de culture (BD Biosciences), et la culture dans les milieux de croissance de chondrocytes (Lonza) jusqu&#39;à ce que les cellules sont confluentes. HAC devrait être utilisé à un nombre de passage ne dépasse pas 3 ou 4 (P3 ou P4). </li><li> Lavez CAH sur la plaque avec 155mm de NaCl au lieu de la PBS standard, suivie par trypsinzation pour recueillir des cellules pour l&#39;encapsulation d&#39;alginate de perles. Une fois que les cellules sont détachées, ralentit les cellules à 3000rpm pendant 5 min. Les cellules sont maintenant prêts pour l&#39;encapsulation 3D. </li></ol> 2. Encapsuler HAC en perles 3D Reprendre le HAC dans une solution d&#39;alginate de 1,2% (Sigma) à une densité de 8x10 5 cellules / ml. Les nombres de cellules ont été déterminés avant encapsulation, utilisant un compteur de cellules standard. Il est très important de bien mélanger afin d&#39;assurer une répartition homogène des cellules dans les billes. <ol><li> Pipeter le HAC / mélange de solution d&#39;alginate dans une seringue de 12 ml attachée à une aiguille de calibre 22 (Tyco Healthcare, Inc.) </li><li> Pendant ce temps, préparer un bécher de 50 mL avec 102 mM CaCl 2 à 5 fois le volume que de la solution HAC / aginate. Placer un barreau aimanté à l&#39;intérieur du bécher et agiter le 102 mM de CaCl2 solution lentement (à environ 150 rpm). </li><li> Tenez le bout de la seringue d&#39;environ 6 pouces au-dessus de la surface de la solution de CaCl 2 et ajouter le mélange HAC / alginate dans le CaCl 2 goutte à goutte une solution. En général, les billes d&#39;alginate qui en résultent sont de 2mm de diamètre avec une densité d&#39;encapsulation d&#39;environ 10 4 cellules / perle. Note: La hauteur de la seringue sur la solution de CaCl 2 est important. Courte distance se traduira par la déchirure en forme de goutte au lieu de perles sphériques en forme, causant inégale intégrité mécanique et l&#39;encapsulation cellulaire. </li><li> Laissez les perles remous dans le solution de CaCl 2 pendant 20-25 min. Alors que d&#39;autres protocoles d&#39;encapsulation d&#39;alginate indiquent un temps d&#39;incubation beaucoup plus courte de 10min, nous avons constaté que le temps d&#39;incubation plus longue avec la solution de CaCl 2 amélioré l&#39;intégrité de l&#39;êtreannonces. </li><li> Retirer la solution de CaCl 2, lavez-les perles 2-3 fois en 2-3 volumes de NaCl, puis une fois dans le milieu de différenciation des chondrocytes (Lonza). Alors que d&#39;autres protocoles d&#39;encapsulation perles d&#39;alginate impliquent l&#39;utilisation d&#39;un filtre pour le lavage des procédures ci-dessus, nous avons constaté que les billes d&#39;alginate deviennent souvent piégés dans le filtre et sèche, réduisant ainsi l&#39;efficacité de l&#39;encapsulation. Nous avons trouvé le plus efficace pour permettre aux billes de se déposer au fond du bécher avant de jeter la solution. </li><li> Utilisez une spatule standards pour transférer les billes dans des boîtes de culture. En général, nous la culture 12 perles par cm 3 ainsi pendant 2 jours dans un milieu de croissance de chondrocytes pour permettre de s&#39;adapter à des chondrocytes du processus d&#39;encapsulation dans la moyenne, ils ont été cultivés en avant de passer au milieu de différenciation de chondrocytes (Lonza) avec le liquide synovial. </li></ol> 3. Chondrocytes culture en milieu liquide synovial de la culture <ol><li> Frais liquide synovial peut être obtenu à partir d&#39;un oClinique utpatient (Nous avons obtenu le liquide synovial de la Tufts Medical Center). Le liquide synovial peuvent être transférées dans des tubes Falcon de 15 ml au laboratoire, et immédiatement centrifugé à 3000 rpm pendant 15min pour enlever les débris cellulaires. Aliquoter acellulaire du liquide synovial dans 1,5 ml microtubes, pour éviter répétés de congélation-décongélation. Surnageant peut être conservé à -80 ° C jusqu&#39;à utilisation. </li><li> Avant de se cultiver, mélanger le liquide synovial avec un milieu de différenciation des chondrocytes (MDP, Lonza) à différents rapports volumétriques, avec une concentration constante de l&#39;acide ascorbique 100mm et 9 mM CaCl 2 (ce montant trace de CaCl 2 est de garantir l&#39;intégrité de l&#39;alginate perles). </li><li> Conservez la plaque sur une plateforme à bascule dans un incubateur à 37 ° C (fréquence bascule: environ 75 fois / min) pour aider à la distribution des nutriments dans les billes d&#39;alginate, et pour réduire l&#39;agglutination des billes. Ceci est particulièrement important que nous la culture de ces chondrocytes pour une période de temps prolongée (jusqu&#39;à 4 semaines). Remarque: Alors que la croissance des chondrocytes et des médias de différenciation (Lonza) contiennent deux antibiotiques couramment utilisés gentamycine et amphotéricine, nous n&#39;avons pas compléter toute antibiotiques lorsque nous avons utilisé des pourcentages différents du liquide synovial pour la culture des chondrocytes. Aucune contamination n&#39;a jamais été observé. </li><li> Changer mélanges des médias tous les 2-3 jours. La viscosité du liquide synovial a été un obstacle majeur dans les chondrocytes en culture encapsulés dans des billes d&#39;alginate lors de cultures à long terme. Nous avons trouvé le plus efficace pour diluer le liquide synovial, complétés avec des moyennes de 102 mm de CaCl 2 (50% V / V), ce qui renforce également l&#39;intégrité des billes d&#39;alginate. De cette façon, le milieu peut être retiré lentement avec une pipette, sans endommager les perles. </li></ol> 4. HAC récolte dans des billes d&#39;alginate pour l&#39;analyse de l&#39;expression des gènes Des précautions particulières doivent être prises lors de la récolte de la CAH à partir des billes d&#39;alginate pour l&#39;analyse de l&#39;expression génique. <ol><li> Laver les billes d&#39;alginate 3 tiMES dans 102 mM de CaCl 2 pendant environ 5 min à chaque fois. </li><li> Récupérer les chondrocytes en ajoutant 55 mM NaCitrate à un volume 4-5 fois que des perles. Il est important que les billes d&#39;alginate sont totalement immergés dans la solution NaCitrate. Agiter ou de roche pour 20-30 min. </li><li> Isoler les cellules et jeter le surnageant. </li><li> Pour l&#39;analyse par RT-PCR, remettre en suspension les cellules dans un tampon de lyse cellulaire culot (Qiagen ARN kit d&#39;isolement), et de procéder à la purification d&#39;ARN. </li></ol> 5. Fix HAC en billes d&#39;alginate pour l&#39;analyse histologique Un soin particulier doit être pris à la récolte de la CAH à partir des billes en 3D pour l&#39;analyse histologique. <ol><li> Laver les billes d&#39;alginate 3 fois dans 102 mM de CaCl 2 pendant environ 5 min à chaque fois. Pour complètement laver le fluide visqueux synoviale, nous avons trouvé le plus efficace de se laver avec un milieu de différenciation des chondrocytes (Lonza) pendant la nuit, bascule dans un incubateur à 37 ° C (fréquence bascule: environ 75 foiss / min). Le milieu de croissance des chondrocytes a été utilisé pour assurer la survie de chondrocytes dans cette lave et prolongée pour permettre une pénétration maximale de l&#39;agent de fixation. </li><li> Fixer les perles dans de l&#39;éthanol 70% pendant la nuit, et procéder à une analyse histologique. Pour effectuer coloration DAPI, incuber avec des billes d&#39;alginate Dapi solution (500ng/ml) pendant 1 heure sous doux balancement, et laver 3 fois avec 102mm de CaCl 2 pendant 15 min à chaque fois. Dapi images peuvent ensuite être visualisées sous un microscope à fluorescence. Les billes peuvent également être sectionnés pour bleu alcian et coloration H &amp; E. </li></ol> 6. Les résultats représentatifs Notre méthode de culture en 3D pour des chondrocytes humains dans des pourcentages élevés de liquide synovial est représentée dans le schéma indiqué dans la Fig.1. Après chondrocytes humains ont été encapsulés dans des billes d&#39;alginate, ils ont été autorisés à croître en milieu supplémenté avec différents ratios de liquide synovial. En raison de la viscosité du liquide synovial, il est essentiel dechondrocytes culture dans des conditions à bascule constamment pour empêcher l&#39;agglutination des constructions de cartilage et d&#39;assurer une répartition homogène des nutriments. Il est également essentiel de se laver les billes d&#39;alginate longuement avant de récupérer les chondrocytes, de sorte que la fixation ou du tampon de lyse peuvent pénétrer les perles (Fig.1). Lumineux terrain (BF) des images de chondrocytes dans les billes d&#39;alginate sont présentés sur la Fig.2. Coloration DAPI a été réalisée pour confirmer la distribution usniform des cellules dans les billes (Fig. 2). A la fin de la période de culture de 21 jours, l&#39;analyse d&#39;expression génique a été réalisée par qRT-PCR. La référence gène GAPDH a été utilisée pour la normalisation de tous les rapports d&#39;achèvement, comme il a été déterminé à être l&#39;un des gènes de référence les plus fiables pour l&#39;analyse qPCR sur les chondrocytes 32. Un exemple est montré dans la Fig. 3, où nous avons analysé les résultats de chondrocytes articulaires humains en culture dans le liquide synovial poolées de six patients souffrant d&#39;arthrose. Cohérent avec le fait que les chondrocytes à partirLonza ont été élargis dans les cultures en 2D, ce qui conduirait inévitablement à la dédifférenciation 33, nous avons constaté que le jour 0 chondrocytes ont exprimé un niveau minimal de gènes matrice cartilagineuse (Fig. 3). Culture 3D de chondrocytes avec un milieu de différenciation des chondrocytes (Lonza) ou milieu supplémenté avec du liquide synovial augmenté de façon significative l&#39;expression du gène du collagène du cartilage, aggrécane et MMP13, qui indique chondrocytes re-différenciation par Jour 21 (Fig.3) 34. Augmenter le pourcentage de liquide synovial dans les médias a entraîné des niveaux comparables de marqueurs matrice cartilagineuse II de collagène et d&#39;expression de l&#39;ARNm aggrécane (Fig.3A et 3B). Par ailleurs, les chondrocytes cultivés dans 100% de liquide synovial, même présentaient une diminution dans le cartilage d&#39;enzymes dégradant l&#39;expression des ARNm MMP13 par rapport à celles cultivées en milieu seul (Fig.3C). Fait intéressant, le niveau d&#39;expression de la mort cellulaire indicateur de la caspase 3 a progressivement diminué avec l&#39;augmentation des ratios de liquide synovial, ce qui suggère que sculture du liquide ynovial a conduit à des niveaux diminution de l&#39;apoptose (Fig. 3D). Par conséquent, nos résultats montrent que les chondrocytes articulaires humains en culture dans les niveaux élevés de liquide synovial, dans un cadre 3D est une technologie réalisable. Tableaux et figures <img alt="Figure 1." src="/files/ftp_upload/3587/3587fig1.jpg" /> Figure 1. Schéma de la méthode à la culture de chondrocytes articulaires humains en pourcentages élevés de liquide synovial dans des billes d&#39;alginate 3D. Premièrement, les chondrocytes et la solution d&#39;alginate sont mélangés. Lorsqu&#39;il est appliqué goutte à goutte à la solution de CaCl 2, les chondrocytes sont immobilisées dans des billes d&#39;hydrogel réticulé Ca-alginate. Ces constructions en 3D sont ensuite cultivées dans le milieu de différenciation de chondrocytes (Lonza) avec différents ratios de liquide synovial humain. Après 21 jours de culture dans une condition à bascule, des billes d&#39;alginate contenant des cellules sont lavées abondamment après quoi les cellules sont récupérées pour le gèneanalyse de l&#39;expression. <img alt="Figure 2." src="/files/ftp_upload/3587/3587fig2.jpg" /> Figure 2. Fond clair (BF) et des images Dapi des chondrocytes articulaires humains cultures encapsulées avec 0%, 30%, 50%, 70% et 100% liquide synovial. Les chondrocytes ont été de forme sphérique dans toutes les conditions de culture. Images du champ clair (BF) et Dapi ont été superposées pour confirmer l&#39;emplacement et la distribution des chondrocytes. EISN, des images agrandies. Pointes de flèches, colocalisation des chondrocytes au BF et images coloration DAPI. <img alt="Figure 3." src="/files/ftp_upload/3587/3587fig3.jpg" /> Figure 3. QRT PCR analyse des encapsulée chondrocytes articulaires humains au jour 0 (J0) une après 21 jours de culture (D21) en milieu supplémenté avec des proportions variables de liquide synovial (SF) (0%, 30%, 50%, 70 % et 100%). Les résultats de quatre échantillons indépendants sont montrés ici. GAPDH a été utilisé comme référence interne pour toutes les PCR. <strong&gt; A. L&#39;expression du collagène ARNm II. B. Expression de l&#39;ARNm aggrécane. C. MMP13 expression de l&#39;ARNm. D. L&#39;expression d&#39;ARNm Caspase 3. La signification statistique a été évaluée pour la Journée des échantillons et 0 Jour 21 échantillons de 0% et 100% liquide synovial (SF) en culture, en utilisant le logiciel INSTAT. * Indique P &lt;0,05.

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synovial+fluid+and+synovial+fluid+cells+on+chondrocyte+metabolism+in+short+term+tissue+culture.">Effects of synovial fluid and synovial fluid cells on chondrocyte metabolism in short term tissue culture.</a> J. Rheumatol. 15, 210-216 (1988).</li><li>Beekhuizen, M., Bastiaansen-Jenniskens, Y. M., Koevoet, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+tissue+inhibits+proteoglycan+production+in+human+osteoarthritic+cartilage;+Establishment+and+characterisation+of+a+long-term+coculture.">Osteoarthritic synovial tissue inhibits proteoglycan production in human osteoarthritic cartilage; Establishment and characterisation of a long-term coculture.</a> Arthritis Rheum. , (2011).</li><li>Rodrigo, J. J., Steadman, J. R., Syftestad, G., Benton, H., Silliman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+human+knee+synovial+fluid+on+chondrogenesis+in+vitro.">Effects of human knee synovial fluid on chondrogenesis in vitro.</a> Am. J. Knee. Surg. 8, 124-129 (1995).</li><li>van den Hoogen, B. M., van de Lest, C. H., van Weeren, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loading-induced+changes+in+synovial+fluid+affect+cartilage+metabolism.">Loading-induced changes in synovial fluid affect cartilage metabolism.</a> Br. J. Rheumatol. 37, 671-676 (1998).</li><li>Webb, G. R., Westacott, C. I., Elson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+fluid+and+synovium+supernatants+up-regulate+tumor+necrosis+factor+receptors+on+human+articular+chondrocytes.">Osteoarthritic synovial fluid and synovium supernatants up-regulate tumor necrosis factor receptors on human articular chondrocytes.</a> Osteoarthritis Cartilage. 6, 167-176 (1998).</li><li>Kook, S. H., Son, Y. O., Lee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+affects+positively+the+proliferation+of+bovine+satellite+cells+and+their+myogenic+differentiation+through+up-regulation+of+MyoD.">Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD.</a> Cell. Biol. Int. 32, 871-878 (2008).</li><li>Knobloch, T. J., Madhavan, S., Nam, J., Agarwal, S., Agarwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chondrocytic+gene+expression+by+biomechanical+signals.">Regulation of chondrocytic gene expression by biomechanical signals.</a> Crit. Rev. Eukaryot. Gene. Expr. 18, 139-150 (2008).</li><li>Guo, J. F., Jourdian, G. W., MacCallum, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+growth+characteristics+of+chondrocytes+encapsulated+in+alginate+beads.">Culture and growth characteristics of chondrocytes encapsulated in alginate beads.</a> Connect. Tissue. Res. 19, 277-297 (1989).</li><li>Toegel, S., Huang, W., Piana, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+reliable+reference+genes+for+qPCR+studies+on+chondroprotective+action.">Selection of reliable reference genes for qPCR studies on chondroprotective action.</a> BMC. Mol. Biol. 8, 13-13 (2007).</li><li>Lin, Z., Fitzgerald, J. B., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+human+chondrocytes+during+passaged+monolayer+cultivation.">Gene expression profiles of human chondrocytes during passaged monolayer cultivation.</a> J. Orthop. Res. 26, 1230-1237 (2008).</li><li>Goessler, U. R., Bieback, K., Bugert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chondrocytes+differentially+express+matrix+modulators+during+in+vitro+expansion+for+tissue+engineering.">Human chondrocytes differentially express matrix modulators during in vitro expansion for tissue engineering.</a> Int. J. Mol. Med. 16, 509-515 (2005).</li><li>Anat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 121, 107-118 (1976).</li></ol>

Nous n&#39;avons rien à révéler.

Dans ce rapport, nous avons développé une méthode qui permet la culture de chondrocytes articulaires humains dans un environnement 3D en milieu contenant des concentrations élevées de liquide synovial humain. Le liquide synovial est l&#39;un des principaux composants qui constituent l&#39;environnement naturel dans la cavité articulaire, où résident les chondrocytes articulaires. Cependant, la viscosité du liquide synovial a été un défi majeur pour trois dimensions à long terme de culture des chondrocytes. ...

Table des réactifs spécifiques et des équipements: <table border="1"><tr><th> Le nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> Alginate (sel de sodium d&#39;acide alginique) </td><td> Sigma </td><td> A2158-250G </td><td> Solution de 2,4% stockés à 40 ° C </td></tr><tr><td> Chlorure de calcium granulaire </td><td> JT Baker </td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" > Les milieux de croissance chondrocytaire </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3156 (base des médias) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4409 (supplément) </td></tr><tr><td width="239" rowspan="2" > Médias différenciation chondrocytaire </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3226 (base des médias) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4408 (supplément) </td></tr><tr><td> Chondrocytes articulaires humains </td><td> Lonza </td><td > CC-2550 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-diamidino-2-phénylindole dichlorhydrate) </td><td> Sigma-Aldrich </td><td > D9542 </td><td></td></tr><tr><td> RNeasy mini kit (pour l&#39;extraction de l&#39;ARN) </td><td> Qiagen </td><td > 74104 </td><td></td></tr><tr><td> Réactifs de PCR: SYBR-vert </td><td> Quanta </td><td > 95053-500 </td><td></td></tr><tr><td> Seringue 12 ml </td><td> Tyco-Kendall-Monoject </td><td > 512852 </td><td></td></tr><tr><td> Aiguille hypodermique de 22 Gague </td><td> Tyco-KendalL-Monoject </td><td > 8881 </td><td></td></tr><tr><td> Microscope </td><td> Olympus </td><td > IX71 </td><td></td></tr><tr><td> Rocker plate-forme </td><td> Thermolyne Thermoscientific </td><td > Vari-mix </td><td></td></tr><tr><td></td><td></td><td ></td><td></td></tr><tr><td> Séquences amorces </td><td></td><td ></td><td></td></tr><tr><td> Collagène IIa-forward </td><td> 5&#39;-TTC ATC CCA CCC TCT CAC AGT-3 &#39; </td><td ></td><td></td></tr><tr><td> Collagène IIa-reverse </td><td> 5&#39;-CCTCTGCCTTGACCCGAA-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13-forward </td><td> 5&#39;-TGT GCC CTT CTT CAC GAC ACA ACT-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13-reverse </td><td> 5&#39;-GAG CAG AGA CTT TGA GTC ATT GCC-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspase 3-forward </td><td> 5&#39;-TCA TTA TTC AGG CCT GCC GTG GTA-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspase 3-reverse </td><td> 5&#39;-TGG ATG CAA ACG GAG CCA TCC TTT -3 &#39; </td><td ></td><td></td></tr></table>

a 3d system for culturing human articular chondrocytes in synovial fluid

Cartilage de destruction est un élément central pathologiques de l&#39;arthrose, une des principales causes d&#39;invalidité dans les Etats-Unis. Cartilage de l&#39;adulte ne se régénère pas très efficace in vivo, et par conséquent, l&#39;arthrose conduit à une perte irréversible du cartilage et est accompagné par la douleur chronique et 1,2 immobilité. Génie tissulaire du cartilage offre un potentiel prometteur pour régénérer et restaurer la fonction des tissus. Cette technologie implique généralement chondrocytes semis dans des échafaudages naturel ou synthétique et la culture de la 3D résultant de construire dans un milieu équilibré sur une période de temps avec un but de l&#39;ingénierie biochimique et un tissu biomécaniquement matures qui peuvent être transplantées dans un site de la lésion in vivo 3-6 . Atteindre une condition optimale pour la croissance des chondrocytes et dépôt de matrice est essentiel pour la réussite de l&#39;ingénierie tissulaire du cartilage. Dans la cavité natale, conjointe du cartilage à l&#39;articla surface de l&#39;os EXCLUES baigne dans le liquide synovial. Ce liquide clair et visqueux fournit des nutriments au cartilage articulaire avasculaire et contient des facteurs de croissance, des cytokines et des enzymes qui sont importants pour le métabolisme des chondrocytes 7,8. Par ailleurs, le liquide synovial facilite faible coefficient de frottement entre les surfaces cartilagineuses mouvement principalement par sécrétant deux éléments clés, le hyaluronane et Lubricin 9 10. En revanche, l&#39;ingénierie tissulaire du cartilage est le plus souvent cultivés en milieu artificiel. Bien que ces médias sont vraisemblablement en mesure d&#39;offrir des conditions plus définies pour étudier le métabolisme des chondrocytes, le liquide synovial reflète le plus fidèlement l&#39;environnement naturel, dont les chondrocytes articulaires résident po En effet, le liquide synovial a l&#39;avantage d&#39;être facile à obtenir et à stocker, et peut souvent être régulièrement réapprovisionné par le corps. Plusieurs groupes ont complété le milieu de culture avec du liquide synovial dans la croissance humaine, bovine, lapin et chien chondrocytes, mais surtout utilisé seulement de faibles niveaux de liquide synovial (inférieur à 20%) 11-25. Alors que les chondrocytes de poulet, de cheval et humaines ont été cultivées dans le milieu avec des pourcentage plus élevé de liquide synovial, ces systèmes de culture ont été à deux dimensions 26-28. Ici, nous présentons notre méthode de culture de chondrocytes articulaires humains dans un système 3D avec un pourcentage élevé de liquide synovial (jusqu&#39;à 100%) sur une période de 21 jours. Ce faisant, nous avons surmonté un obstacle majeur présenté par la haute viscosité du liquide synovial. Ce système offre la possibilité d&#39;étudier des chondrocytes humains dans le liquide synovial, dans un cadre 3D, qui peut être combiné avec deux autres facteurs importants (la tension d&#39;oxygène et le chargement mécanique) 29,30 qui constituent l&#39;environnement naturel pour le cartilage d&#39;imiter le milieu naturel pour la croissance du cartilage. Par ailleurs, ce système peut également être utilisé pour le dosage de l&#39;activité du liquide synovial sur les chondrocytes et de fournir une plateforme pour le développementcartilage de technologies de régénération et les options thérapeutiques pour l&#39;arthrite.

Watch this Scientific Journal Video about A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid at JoVE.com

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Un système 3D de chondrocytes articulaires humains en culture dans les niveaux élevés du liquide synovial est décrite. Le liquide synovial reflète le microenvironnement plus naturelle pour le cartilage articulaire, et peut être facilement obtenu et stocké. Ce système peut donc être utilisée pour étudier la régénération du cartilage et de dépistage pour les thérapies pour traiter l&#39;arthrite.

Un système 3D pour les chondrocytes articulaires humains Culture dans le liquide synovial

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

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20.7K vues.  Tufts University School of Medicine. Un système 3D de chondrocytes articulaires humains en culture dans les niveaux élevés du liquide synovial est décrite. Le liquide synovial reflète le microenvironnement plus naturelle pour le cartilage articulaire, et peut être facilement obtenu et stocké. Ce système peut donc être utilisée pour étudier la régénération du cartilage et de dépistage pour les thérapies pour traiter l'arthrite.

Vidéo: A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Un système de culture de cellules épithéliales de l&#39;iris Pigment étudier la régénération Lens Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

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Biologie

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Dérivation efficace des ressources humaines précurseurs cardiaques et les cardiomyocytes à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un système d&#39;<em&gt; Ex vivo</emLa culture&gt; de pancréas embryonnaire

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

L&#39;analyse de la fonction des gènes et visualisation de Cilia généré par l&#39;écoulement des fluides dans des vésicules de Kupffer

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Culture de cellules humaines épithéliales nasales à l&#39;interface Air Liquide

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap Junctional intercellulaire Communication: Un biomarqueur fonctionnelle pour évaluer les effets néfastes des substances toxiques et les toxines, et bienfaits pour la santé des produits naturels

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

L&#39;Assemblée et l&#39;application des «Shear Anneaux»: A Novel Endothelial Modèle pour Orbital, unidirectionnel et périodique Fluid débit et contrainte de cisaillement

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Cellule magnétique activé tri des stratégies visant à isoler et purifier Synovial fluide mésenchymateuses cellules souches dérivées d’un modèle de lapin

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Culture 3D d’organoïdes de l’épithélium intestinal des villosités en cours de dédifférenciation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...