Wir möchten Robin Nye (Tufts Medical Center), Tomoya Uchimura und Dana Cairns (Tufts University) für die Bereitstellung von Hilfe bei der Gelenkflüssigkeit Lagerung und Zentrifugation danken. Diese Arbeit wurde von den NIH (1R01AR059106-01A1) für LZ finanziert

Ein 3D-System zur Kultivierung von menschlichen Chondrozyten in der Synovialflüssigkeit In dieser Arbeit haben wir die menschliche Chondrozyten in Alginatkügelchen mit einem modifizierten Herstellung-schlug Encapsulation Protokoll (Lonza, und 31) gekapselt. Mit Hilfe dieser 3D-Konstrukte, haben wir ein System zur Kultivierung von Zellen in einem Kulturmedium, das abwechslungsreiche Prozentsätze der menschlichen Synovialflüssigkeit entwickelt und bewertet diese 3D-Konstrukte für die Knorpel-Genexpression. 1. Bereiten menschlichen Chondrozyten (HAC) für die dreidimensionale (3D)-Kapselung <ol><li> Tauwetter ein Fläschchen (1 ml) des menschlichen Chondrozyten (HAC) (Lonza) (Passage 2) in 37 ° C Wasserbad für 1 min. </li><li> Mix mit 1 ml Chondrozyten Nährmedien (Lonza) und Zentrifuge bei 3000 rpm für 3-5 min, um die Zellen zu sammeln. Überstand verwerfen. </li><li> Resuspendieren Zellpellet in Chondrozyten Nährmedien (Lonza). </li><li> Platte von Zellen in einer 10 cm GewebeKultur-Platte (BD Biosciences), und die Kultur in Chondrozyten Nährmedien (Lonza), bis die Zellen konfluent. HAC sollte in einem Durchgang Zahl nicht höher als 3 oder 4 (P3 oder P4) verwendet werden. </li><li> Wash HAC auf dem Teller mit 155mm NaCl anstelle des Standard-PBS, durch trypsinzation gefolgt, um Zellen für die Alginat-Bead-Kapselung zu sammeln. Sobald Zellen sind freistehend, Spin-down der Zellen bei 3000rpm für 5min. Die Zellen sind nun bereit für 3D-Kapselung. </li></ol> 2. Encapsulate HAC in 3D-Perlen Resuspendieren HAC in 1,2% Alginat-Lösung (Sigma) in einer Dichte von 8x10 5 Zellen / ml. Die Zellzahlen wurden vor der Einkapselung, bestimmt mit einem Standard-Zell-Zähler. Es ist sehr wichtig, gut zu mischen, um eine gleichmäßige Verteilung der Zellen in die Perlen zu sichern. <ol><li> Pipette die HAC / Alginat-Lösung Mischung in eine 12 ml Spritze an einer 22-Gauge-Nadel (Tyco Healthcare, Inc.). </li><li> Inzwischen bereiten ein 50 mL Becherglas mit 102 mM CaCl 2 bei einem Volumen 5 Mal, dass der HAC / aginate Lösung. Platzieren Sie einen Rührstab in das Becherglas und rühren die 102 mM CaCl 2-Lösung langsam (bei ​​ca. 150 rpm). </li><li> Halten Sie die Spritze Spitze ca. 6 cm über der Oberfläche der CaCl 2-Lösung und fügen Sie den HAC / Alginat-Gemisch in die CaCl 2-Lösung getropft. Im Allgemeinen sind die daraus resultierenden Alginatkügelchen 2mm im Durchmesser mit einer Verkapselung Dichte von etwa 10 4 Zellen / Perle. Hinweis: Die Höhe der Spritze über die CaCl 2-Lösung ist wichtig. Kürzere Strecke wird in Tropfenform statt kugelförmige Perlen geformt führen, wodurch ungleichmäßige mechanische Integrität und Verkapselung von Zellen. </li><li> Lassen Sie die Kugeln in die CaCl 2-Lösung für 20-25 min rühren. Während andere Alginat Kapselungsprotokolle eine viel kürzere Inkubationszeit von 10min zeigen, fanden wir, dass längere Inkubationszeit mit der CaCl 2-Lösung die Integrität der verbessert werdenAnzeigen. </li><li> Entfernen Sie die CaCl 2-Lösung, waschen Sie die Perlen 2-3 mal in 2-3 Volumina NaCl, und dann noch einmal in die Differenzierung der Chondrozyten Medium (Lonza). Während andere Alginat Perle Kapselung Protokolle mit einzubeziehen einen Filter für die oben genannten Waschverfahren, fanden wir, dass die Alginatkügelchen oft gefangen werden in den Filter und austrocknen, wodurch die Effizienz der Kapselung. Wir fanden es am effizientesten, damit die Perlen auf den Boden des Bechers vor der Entsorgung regeln die Lösung. </li><li> Verwenden Sie ein Standard-Spatel auf die Perlen in Kulturschalen übertragen. Wir in der Regel Kultur 12 Perlen pro 3 cm auch für 2 Tage in Chondrozyten Wachstumsmedium zu ermöglichen Chondrozyten auf die Verkapselung Prozess in dem Medium, den sie vor der Umstellung auf die Differenzierung der Chondrozyten Medium (Lonza) mit Gelenkflüssigkeit angebaut wurden anzupassen. </li></ol> 3. Kultur Chondrozyten in Synovialflüssigkeit Kulturmedium <ol><li> Frische Gelenkflüssigkeit kann aus einem o erhaltenutpatient Klinik (Wir erhalten Synovialflüssigkeit von der Tufts Medical Center). Die Gelenkflüssigkeit kann in 15ml Falcon-Röhrchen an das Labor übertragen werden, und sofort bei 3000 rpm für 15 Minuten zentrifugiert, um Zelltrümmer zu entfernen. Aliquot zellfreien Synovialflüssigkeit in 1,5 ml Reaktionsgefäße, um zu vermeiden wiederholtes Einfrieren und Auftauen. Überstand kann bei -80 ° C bis zur Verwendung gelagert werden. </li><li> Vor der Kultivierung Mix der Gelenkflüssigkeit mit Chondrozyten-Differenzierung Medium (CDM, Lonza) bei unterschiedlichen Volumenverhältnisse, mit einer konstanten Konzentration von 100 mM Ascorbinsäure und 9 mM CaCl 2 (diese Spur von CaCl 2 ist es, die Integrität der Alginat sorgen Perlen). </li><li> Halten Sie die Platte auf einem Schaukelstuhl-Plattform in einem 37 ° C Inkubator (rocking-Frequenz: etwa 75 mal / min), um Nährstoffe Verteilung innerhalb der Alginatkügelchen helfen, und zur Verringerung der Verklumpung der Perlen. Dies ist besonders wichtig, da wir diese Kultur Chondrozyten über einen längeren Zeitraum (bis zu 4 Wochen). Note: Während Chondrozyten Wachstum und die Differenzierung Medien (Lonza) zwei häufig verwendete Antibiotika Gentamycin und Amphotericin enthalten, haben wir nicht alle Antibiotika zu ergänzen, wenn wir unterschiedliche Anteil der Synovialflüssigkeit verwendet für Chondrozyten kultiviert. Keine Kontamination wurde niemals beobachtet. </li><li> Ändern Medien Mischungen alle 2-3 Tage. Die Viskosität der Gelenkflüssigkeit hat eine wichtige Hürde in Kultivierung Chondrozyten in Alginatkügelchen während der langfristigen Kulturen eingekapselt worden. Wir fanden es am effektivsten, Synovialflüssigkeit-ergänzt Medium mit 102mm CaCl 2 (50% V / V), die verstärkt auch die Integrität der Alginatkügelchen verdünnen. Auf diese Weise kann das Medium langsam mit einer Pipette entfernt werden, ohne Beschädigung der Perlen. </li></ol> 4. Ernte-HAC in Alginatkügelchen zur Genexpressionsanalyse Besondere Vorsicht ist bei der Ernte der HAC von der Alginatkügelchen zur Genexpressionsanalyse werden. <ol><li> Waschen Sie die Alginatkügelchen 3 Times in 102 mM CaCl 2 für ca. 5 min jeder Zeit. </li><li> Rufen Sie die Chondrozyten, indem 55 mM NaCitrate bei einem Volumen 4-5 mal, dass der Perlen. Es ist wichtig, dass die Alginatkügelchen total in die NaCitrate Lösung eingetaucht. Shake oder Rock für 20-30 min. </li><li> Spin down den Zellen und den Überstand verwerfen. </li><li> Für die RT-PCR-Analyse, Zellpellet in Zell-Lyse-Puffer (Qiagen RNA Isolation Kit), und fahren Sie mit RNA Aufreinigung. </li></ol> 5. Fix HAC in Alginatkügelchen für die histologische Analyse Besondere Sorgfalt ist darauf zu achten, die HAC aus dem 3D-Perlen für die histologische Analyse der Ernte werden. <ol><li> Waschen Sie die Alginatkügelchen 3 mal in 102 mM CaCl 2 für ca. 5 min jeder Zeit. Um das Gerät vollständig abzuwaschen das zähflüssige Gelenkschmiere, fanden wir es am effektivsten, mit Chondrozyten-Differenzierung Medium (Lonza) über Nacht zu waschen, rocking in einem 37 ° C Inkubator (rocking-Frequenz: etwa 75 Mals / min). Die Chondrozyten-Wachstumsmedium wurde verwendet, um das Überleben der Chondrozyten in dieser verlängerten waschen zu gewährleisten und maximale Durchdringung des Fixiermittels zu ermöglichen. </li><li> Fix die Perlen in 70% Ethanol über Nacht, und fahren Sie mit histologische Analyse. Um DAPI-Färbung, inkubieren Alginatkügelchen mit DAPI-Lösung (500ng/ml) für 1 Stunde unter leichtem Schütteln, und waschen für 3-mal mit 102mm CaCl 2 für je 15 min Zeit. Dapi Bilder können dann unter einem Fluoreszenzmikroskop betrachtet werden. Die Perlen können auch geschnitten für Alcianblau und H &amp; E Färbung. </li></ol> 6. Repräsentative Ergebnisse Unsere 3D-Kultivierung Methode für den menschlichen Chondrozyten in hohen Prozentsätzen der Gelenkflüssigkeit ist in der schematischen Darstellung in Abbildung 1 gezeigt, dargestellt. Nach humanen Chondrozyten in Alginatkügelchen verkapselt waren, durften sie in Medium mit unterschiedlichen Verhältnissen der Gelenkflüssigkeit ergänzt wachsen. Wegen der Viskosität der Gelenkflüssigkeit, ist es wichtig,Kultur Chondrozyten unter ständiger rocking Bedingungen verhindern das Verklumpen des Knorpels Konstrukte und um eine gleichmäßige Verteilung der Nährstoffe zu gewährleisten. Es ist auch wichtig, die Alginatkügelchen ausgiebig zu waschen, bevor das Abrufen der Chondrozyten, so dass die Festsetzung oder Lysepuffer können die Perlen zu durchdringen (Abb. 1). Hellfeld (BF) Bilder von Chondrozyten in der Alginat-Beads sind in Abb. 2 dargestellt. DAPI-Färbung wurde durchgeführt, um usniform Verteilung der Zellen innerhalb der Kügelchen (Abb. 2) zu bestätigen. Am Ende der 21-tägigen Kultur Periode wurde Genexpressionsanalyse mittels qRT-PCR durchgeführt. Die Referenz-Gen GAPDH wurde zur Normierung für alle PCRs verwendet, wie es war bestimmt einer der zuverlässigsten Hinweis Gene für qPCR-Analyse auf Chondrozyten 32 sein. Ein Beispiel ist in Abb. dargestellt. 3, wo wir analysierten die Ergebnisse von menschlichen Chondrozyten in der Synovialflüssigkeit von sechs Patienten mit Osteoarthritis zusammengefasst kultiviert. Im Einklang mit der Tatsache, dass die Chondrozyten ausLonza in 2D Kulturen, was zwangsläufig zu Entdifferenzierung 33 würde führen erweitert worden, fanden wir, dass Tag 0 Chondrozyten exprimiert minimal Ebenen der Knorpelmatrix Gene (Abb. 3). 3D-Kultivierung von Chondrozyten mit Chondrozyten-Differenzierung Medium (Lonza) oder Medium mit Gelenkflüssigkeit ergänzt deutlich erhöht Knorpel Genexpression von Kollagen, Aggrecan und MMP13, die Chondrozyten re-Differenzierung zeigt by Day 21 (Abb. 3) 34. Erhöhung des Anteils der Gelenkflüssigkeit in den Medien führte zu einem vergleichbaren Grad der Knorpelmatrix Marker Kollagen II und Aggrecan mRNA-Expression (Abb. 3a und 3b). Darüber hinaus Chondrozyten in 100% Synovialflüssigkeit kultivierten sogar zeigten eine Abnahme der Knorpel abbauende Enzym MMP13 mRNA-Expression als mit den kultivierten in Medium allein (Abb. 3c) verglichen. Interessanterweise ist die Expression von Zelltod Indikator Caspase 3 allmählich nahm mit zunehmendem Verhältnis von Gelenkflüssigkeit, was darauf hindeutet, dass synovial Flüssigkeit Kultivierung hat zu einer verminderten Apoptose Ebenen (Abb. 3D) geführt. Deshalb zeigen unsere Ergebnisse, dass Kultivierung menschlicher Chondrozyten in ein hohes Maß an Gelenkflüssigkeit in einer 3D-Umgebung eine praktikable Technologie ist. Tabellen und Abbildungen <img alt="Abbildung 1." src="/files/ftp_upload/3587/3587fig1.jpg" /> Abbildung 1. Schematische Darstellung des Verfahrens zur Kultur menschlichen Chondrozyten in hohen Prozentsätzen der Gelenkflüssigkeit in 3D Alginatkügelchen. Erstens sind Chondrozyten und Alginat-Lösung gemischt. Beim Auftragen tropfenweise zu der CaCl 2-Lösung werden Chondrozyten innerhalb der vernetzten Ca-Alginat-Hydrogel-Kügelchen immobilisiert. Diese 3D-Konstrukte werden dann in der Chondrozyten-Differenzierung Medium (Lonza) mit unterschiedlichen Verhältnissen des menschlichen Synovialflüssigkeit kultiviert. Nach 21 Tagen der Kultivierung unter einem Schaukelstuhl Zustand sind Alginatkügelchen enthaltenden Zellen intensiv, wonach die Zellen für die Gen abgerufen werden gewaschenExpressions-Analyse. <img alt="Abbildung 2." src="/files/ftp_upload/3587/3587fig2.jpg" /> Abbildung 2. Hellfeld (BF) und DAPI Bilder von menschlichen Chondrozyten gekapselt Kulturen mit 0%, 30%, 50%, 70% und 100% Synovialflüssigkeit. Chondrozyten wurden kugelförmig in alle Kulturbedingungen. Bilder von Hellfeld (BF) und DAPI wurden überlagert, um die Lage und Verteilung der Chondrozyten zu bestätigen. Intarsien, vergrößerten Bildern. Pfeilspitzen, Kolokalisation von Chondrozyten in BF und DAPI-Färbung Bilder. <img alt="Abbildung 3." src="/files/ftp_upload/3587/3587fig3.jpg" /> Abbildung 3. QRT-PCR-Analyse von verkapselten menschlichen Chondrozyten am Tag 0 (D0) ein nach 21 Tagen der Kultivierung (D21) in Medium mit unterschiedlichen Verhältnissen der Gelenkflüssigkeit (SF) (0%, 30%, 50%, 70 ergänzt % und 100%). Die Ergebnisse von vier unabhängigen Proben werden hier gezeigt. GAPDH wurde als interne Referenz für alle PCRs verwendet. <strong&gt; A. Collagen II mRNA-Expression. B. Aggrecan mRNA-Expression. C. MMP13 mRNA-Expression. D. Caspase 3-mRNA-Expression. Statistische Signifikanz wurde für Proben vom Tag 0 und Tag 21 Proben von 0% und 100% Synovialflüssigkeit (SF) Kultivieren, mit INSTAT Software beurteilt. * Bezeichnet 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>

Wir haben nichts zu offenbaren.

In diesem Bericht haben wir eine Methode, die für die Kultivierung humaner Chondrozyten ermöglicht in einer 3D-Umgebung in Medium, das hohe Konzentrationen von menschlichen Gelenkflüssigkeit enthält. Synovialflüssigkeit ist eine der wichtigsten Komponenten, die die natürliche Umwelt darstellen in der Gelenkpfanne, wo Chondrozyten befinden. Allerdings hat die Viskosität der Gelenkflüssigkeit eine große Herausforderung für die dreidimensionale langfristige Kultivierung von Chondrozyten wurde. Zur Überwindung de...

Tabelle der spezifischen Reagenzien und Geräte: <table border="1"><tr><th> Name des Reagens </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> Alginat (Alginsäure Natriumsalz) </td><td> Sigma </td><td> A2158-250G </td><td> 2,4%-Lösung bei 40 ° C gelagert </td></tr><tr><td> Calciumchlorid-Dihydrat, Granular </td><td> JT Baker </td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" > Chondrogene Nährmedien </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3156 (base Medien) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4409 (Ergänzung) </td></tr><tr><td width="239" rowspan="2" > Chondrogene Differenzierung Medien </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3226 (base Medien) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4408 (Ergänzung) </td></tr><tr><td> Menschliche Chondrozyten </td><td> Lonza </td><td > CC-2550 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-Diamidino-2-phenylindole dihydrochlorid) </td><td> Sigma-Aldrich </td><td > D9542 </td><td></td></tr><tr><td> RNeasy Mini Kit (für RNA-Extraktion) </td><td> Qiagen </td><td > 74104 </td><td></td></tr><tr><td> PCR-Reagenzien: SYBR-Grün </td><td> Quanta </td><td > 95053-500 </td><td></td></tr><tr><td> 12 ml Spritze </td><td> Tyco-Kendall-Monoject </td><td > 512852 </td><td></td></tr><tr><td> 22-Gague Injektionsnadel </td><td> Tyco-Kendall-Monoject </td><td > 8881 </td><td></td></tr><tr><td> Mikroskop </td><td> Olymp </td><td > IX71 </td><td></td></tr><tr><td> Platform Wippe </td><td> Thermoscientific Thermolyne </td><td > Vari-mix </td><td></td></tr><tr><td></td><td></td><td ></td><td></td></tr><tr><td> Primer-Sequenzen </td><td></td><td ></td><td></td></tr><tr><td> Collagen IIa-forward </td><td> 5&#39;-TTC ATC CCA CCC TCT CAC AGT-3 &#39; </td><td ></td><td></td></tr><tr><td> Collagen 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 ACA GAC ACT-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13-reverse </td><td> 5&#39;-GAG AGC AGA CTT TGA GTC ATT GCC-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspase 3-vorne </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 AAC CAG GAG CCA TCC TTT -3 &#39; </td><td ></td><td></td></tr></table>

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

Knorpelabbau ist ein zentrales pathologisches Merkmal der Arthrose, eine führende Ursache von Behinderungen in den USA. Knorpel in den Erwachsenen nicht sehr effizient regenerieren in vivo, und als Ergebnis führt Arthrose, Knorpel Verlust irreversibel und wird von chronischen Schmerzen und Unbeweglichkeit 1,2 begleitet. Cartilage Tissue Engineering bietet vielversprechende Möglichkeiten, sich zu regenerieren und wiederherstellen Gewebe-Funktion. Diese Technologie umfasst in der Regel Aussaat Chondrozyten in natürlichen oder synthetischen Gerüsten und Kultivieren der resultierenden 3D in einem ausgewogenen Medium bauen über einen Zeitraum von Zeit, mit dem Ziel der Technik ein biochemisch und biomechanisch ausgereifte Gewebe, das in eine Fehlstelle in vivo 3-6 können transplantiert werden . Das Erreichen einer optimalen Voraussetzung für Wachstum und Chondrozyten-Matrix Ablagerung ist für den Erfolg von Knorpel Tissue Engineering unverzichtbar. In der nativen Gelenkspalt, Knorpel an der articdere Oberfläche des Knochens ist in Gelenkflüssigkeit gebadet. Diese klare und viskose Flüssigkeit liefert Nährstoffe an die avaskuläre Gelenkknorpel und enthält Wachstumsfaktoren, Zytokine und Enzyme, die wichtig sind für Chondrozytenmetabolismus 7,8. Darüber hinaus erleichtert Synovialflüssigkeit reibungsarme Bewegung zwischen knorpeligen Oberflächen hauptsächlich durch Sekretion zwei Hauptkomponenten, Hyaluronsäure und lubricin 9 10. Im Gegensatz dazu ist Gewebezüchtung Knorpel meist in künstlichen Medien kultiviert. Während dieser Medien eher in der Lage sind, mehr definierten Bedingungen für das Studium Chondrozytenmetabolismus bieten, Synovialflüssigkeit am besten reflektiert die natürliche Umwelt, von denen Chondrozyten wohnen in. In der Tat hat Synovialflüssigkeit den Vorteil, leicht zu beschaffen und zu speichern, und können oft regelmäßig durch den Körper wieder aufgefüllt werden. Mehrere Gruppen haben das Kulturmedium mit Gelenkflüssigkeit ergänzt wachsende Mensch, Rind, Kaninchen und Hund chondrocytes, aber meist nur geringe Mengen von Gelenkflüssigkeit (unter 20%) 11-25 verwendet. Während Huhn, Pferd und Mensch Chondrozyten in das Medium mit höherer Prozentsatz der Gelenkflüssigkeit gezüchtet haben, wurden diese Kultur Systemen zweidimensionalen 26-28. Hier präsentieren wir unsere Methode der Züchtung menschlichen Chondrozyten in ein 3D-System mit einem hohen Anteil von Gelenkflüssigkeit (bis zu 100%) über einen Zeitraum von 21 Tagen. Dabei überwanden wir eine große Hürde durch die hohe Viskosität der Gelenkflüssigkeit vor. Dieses System bietet die Möglichkeit des Studiums humanen Chondrozyten in Synovialflüssigkeit in einer 3D-Umgebung, die weiter mit zwei weiteren wichtigen Faktoren (Sauerstoff-Partialdruck und mechanischer Belastung) 29,30, dass die natürliche Umwelt darstellen für Knorpel, die natürliche Milieu für imitieren können kombiniert werden Knorpel Wachstum. Darüber hinaus kann dieses System auch für die Bestimmung von Gelenkflüssigkeit Aktivität auf Chondrozyten verwendet werden und bieten eine Plattform für die EntwicklungKnorpelregeneration Technologien und therapeutische Optionen für Arthritis.

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

Ein 3D-System zur Kultivierung menschlichen Chondrozyten in ein hohes Maß an Gelenkflüssigkeit beschrieben. Synovialflüssigkeit spiegelt den natürlichen Mikroumgebung für Gelenkknorpel, und kann leicht erreicht werden und gespeichert werden. Dieses System kann daher für die Untersuchung Knorpelregeneration und zum Screening von Therapeutika zur Behandlung von Arthritis verwendet werden.

Ein 3D-System für Züchtung menschlichen Chondrozyten in der Synovialflüssigkeit

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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Research

JoVE Journal

Biology

20.7K Aufrufe.  Tufts University School of Medicine. Ein 3D-System zur Kultivierung menschlichen Chondrozyten in ein hohes Maß an Gelenkflüssigkeit beschrieben. Synovialflüssigkeit spiegelt den natürlichen Mikroumgebung für Gelenkknorpel, und kann leicht erreicht werden und gespeichert werden. Dieses System kann daher für die Untersuchung Knorpelregeneration und zum Screening von Therapeutika zur Behandlung von Arthritis verwendet werden.

Serie: 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.

Ein System zur Kultivierung Iris Pigmentepithelzellen nach Lens Regeneration in Newt Study

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, ...

Forschung

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.

Effiziente Ableitung von Human Cardiac Vorstufen und Kardiomyozyten aus pluripotenten humanen embryonalen Stammzellen mit 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 ...

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.

Ein System für<em&gt; Ex vivo</em&gt; Kultivierung von embryonalen Pankreas

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.

Analyse der Genfunktion und Visualisierung von Cilia-Generated Fluid Flow in Kupffer-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 ...

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.

Die Kultivierung der menschlichen Nasen Epithelzellen an der Air flüssig-Grenzfläche

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.

Interzelluläre Gap Junction-Kommunikation: Eine funktionelle Biomarker zur Beurteilung Unerwünschte Auswirkungen von Schadstoffen und Toxinen und gesundheitlichen Vorteile von Natural Products

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.

Die Montage und Anwendung der &quot;Shear Ringe &#39;: A Novel Endothelial Modell für Orbital, unidirektional und periodische Strömungs- und Scherspannung

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.

Magnetische aktiviert Zelle Sortieren Strategien zu isolieren und reinigen Synovial Flüssigkeit abgeleitet mesenchymalen Stammzellen aus einem Kaninchen-Modell

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.

3D-Kultivierung von Organoiden aus dem intestinalen Zottenepithel, das einer Dedifferenzierung unterzogen wird

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