Name: Isolatie van chondrocyten en chondroprogenitoren met behulp van fibronectine-adhesie en migratoire assay (Video) | JoVE Journal
Uploaded: 2024-10-04T14:30:00.000+00:00
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Description: Leer de isolatie van chondrocyten en chondroprogenitoren uit menselijk kraakbeen via fibronectine-adhesie en migratie-assays. Deze methode ondersteunt regeneratieve geneeskunde en kraakbeentransplantatie.

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<meta charset="utf8"></head><body>Chondroprogenitor en chondrocyten isolatie van menselijk gewrichtskraakbeen

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<li>Vinod, E., Padmaja, K., Ramasamy, B., Sathishkumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+review+of+articular+cartilage+derived+chondroprogenitors+for+cartilage+repair+in+animal+models%5BTitle%5D)+AND+%22J+Orthopaed%22%5BJournal%5D)">Systematic review of articular cartilage derived chondroprogenitors for cartilage repair in animal models.</a> J Orthopaed. 35, 43-53 (2022).</li>
</ol>

<table><tbody><tr><td>22-scalpel blade&nbsp;</td><td>GLASS VAN</td><td></td><td></td></tr><tr><td>6-well plate&nbsp;</td><td>CORNING</td><td>3516</td><td></td></tr><tr><td>Alcian Blue</td><td>THERMO SCIENTIFIC</td><td>J6012</td><td></td></tr><tr><td>Alizarin Red&nbsp;</td><td>SIGMA</td><td>130223</td><td></td></tr><tr><td>Amphotericin-B (2 &mu;g/mL).</td><td>GIBCO</td><td>15240062</td><td></td></tr><tr><td>Ascorbic acid (62 &mu;g/mL)</td><td>SIGMA ALDRICH</td><td>A4544-25G</td><td></td></tr><tr><td>BC CytoFLEX LX flow cytometer&nbsp;</td><td>BECKMAN COULTER</td><td>CYTExpert Software Version 2.5</td><td></td></tr><tr><td>CaCl2</td><td>SIGMA ALDRICH</td><td>&nbsp;C34006</td><td></td></tr><tr><td>CD105-FITC</td><td>BD BIOSCIENCE</td><td>561443</td><td></td></tr><tr><td>CD106-APC</td><td>BD BIOSCIENCE</td><td>551147</td><td></td></tr><tr><td>CD14-FITC</td><td>BD BIOSCIENCE</td><td>555397</td><td></td></tr><tr><td>CD29-APC</td><td>BD BIOSCIENCE</td><td>559883</td><td></td></tr><tr><td>CD34-PE</td><td>BD BIOSCIENCE</td><td>348057</td><td></td></tr><tr><td>CD45-FITC</td><td>BD BIOSCIENCE</td><td>347463</td><td></td></tr><tr><td>CD49b-FITC</td><td>MILTENYL BIOTEC</td><td>&nbsp;MACS 130/100337</td><td></td></tr><tr><td>CD49e-PE</td><td>BD BIOSCIENCE</td><td>555617</td><td></td></tr><tr><td>CD73-PE</td><td>BD BIOSCIENCE</td><td>550257</td><td></td></tr><tr><td>CD90-PE</td><td>BD BIOSCIENCE</td><td>561970</td><td></td></tr><tr><td>Cell counter</td><td>DE NOVIX</td><td>Cell Drop BF</td><td></td></tr><tr><td>Cell strainer</td><td>HIMEDIA</td><td>TCP024</td><td></td></tr><tr><td>Centrifuge</td><td>BECKMAN COULTER</td><td>Allegra X-30R</td><td></td></tr><tr><td>CO2 incubator&nbsp;</td><td>THERMO SCIENTIFIC</td><td>MODEL-371</td><td></td></tr><tr><td>Collagen type X (COL10A1),Runt-related transcription factor (RUNX2),SRY-Box Transcription Factor 9 (SOX-9), Aggrecan (ACAN), and Collagen type II (COL2A1)&nbsp;</td><td>EUROGENTEC, BELGIUM</td><td></td><td></td></tr><tr><td>Collagenase type II</td><td>WORTHINGTON</td><td>LS004176</td><td></td></tr><tr><td>DMEM F-12 (Dulbecco's Modified Eagle's Medium F-12)</td><td>SIGMA ALDRICH</td><td>D8900-1L</td><td></td></tr><tr><td>ELISA plate reader</td><td>MOLECULAR DEVICES</td><td>SpectraMax i3x Reader</td><td></td></tr><tr><td>Fast Green</td><td>FISHER SIENTIFIC</td><td>2353459</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO</td><td>10270106</td><td></td></tr><tr><td>FGF2</td><td>CLOUD CLONE CORP</td><td>APA551Hu01</td><td></td></tr><tr><td>Fibronectin (10 &micro;L/mL)</td><td>SIGMA ALDRICH</td><td>F1141</td><td></td></tr><tr><td>First-Strand synthesis system</td><td>TAKARA BIO</td><td>6110A</td><td></td></tr><tr><td>Glutamax</td><td>GIBCO</td><td>35050061</td><td></td></tr><tr><td>Hematoxylin</td><td>QUALIGENS</td><td>Q39411</td><td></td></tr><tr><td>MgCl2&nbsp;</td><td>SIGMA ALDRICH</td><td>M8787</td><td></td></tr><tr><td>Oil Red O</td><td>SIGMA</td><td>1320065</td><td></td></tr><tr><td>PBS (Phosphate Buffered Saline)</td><td>GIBCO</td><td>10010023</td><td></td></tr><tr><td>PCR thermocycler&nbsp;</td><td>APPLIED BIOSYSTEMS</td><td>Quantstudio 12K Flex thermocycler&nbsp;</td><td></td></tr><tr><td>Penicillin-streptomycin (100 IU/mL)</td><td>GIBCO</td><td>15240062</td><td></td></tr><tr><td>Picrosirius Red</td><td>ALFA AESAR</td><td>2610108</td><td></td></tr><tr><td>Primary antibody (mouse Collagen type II)</td><td>DSHB</td><td>DSHB II II6B3</td><td></td></tr><tr><td>Pronase</td><td>ROCHE</td><td>10165913103</td><td></td></tr><tr><td>Quant-iT Picogreen dsDNA reagent</td><td>THERMO SCIENTIFIC</td><td>P7589</td><td></td></tr><tr><td>Refrigerator</td><td>ELANPRO</td><td></td><td></td></tr><tr><td>RNeasy MiniKit</td><td>QIAGEN</td><td>74104</td><td></td></tr><tr><td>Safranin O</td><td>QUALIGENS</td><td>Q39962</td><td></td></tr><tr><td>Secondary antibody (Goat Anti-Mouse IgG Antibody, HRP conjugate)</td><td>THERMO SCIENTIFIC</td><td>31430</td><td></td></tr><tr><td>Shaking water bath&nbsp;</td><td>REMI</td><td></td><td></td></tr><tr><td>StemPro differentiating kits&nbsp;</td><td>GIBCO</td><td>1007201, A1007001, and A1007101</td><td></td></tr><tr><td>T-25 flask&nbsp;</td><td>CORNING</td><td>430639</td><td></td></tr><tr><td>TakyonTM Low Rox SYBR Master Mix dTTP Blue&nbsp;</td><td>EUROGENTEC</td><td>UF-LSMT-B0701</td><td></td></tr><tr><td>TGF&beta;</td><td>ABCAM</td><td>ab277760</td><td></td></tr><tr><td>Tissue Culture Petridish</td><td>TARSONS</td><td>960010</td><td></td></tr><tr><td>Toluidine Blue</td><td>QUALIGENS</td><td>2040</td><td></td></tr><tr><td>Tryphan blue&nbsp;</td><td>GIBCO</td><td>15250061</td><td></td></tr><tr><td>Trypsin EDTA</td><td>GIBCO</td><td>25200072</td><td></td></tr></tbody></table>

isolatie van chondrocyten en chondroprogenitoren met behulp van fibronectine-adhesie en migratoire assay

Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

Isolatie van chondrocyten en chondroprogenitoren met behulp van fibronectine-adhesie en migratoire assay

Chondroprogenitor cells (CPCs), recently identified as a distinct subpopulation, exhibit promise due to their mesenchymal properties, heightened ...

isolation-chondrocytes-chondroprogenitors-using-fibronectin-adhesion

Research

JoVE Journal

Biology

Biologie

Een 3D-systeem van het kweken van menselijke articulaire chondrocyten in hoge mate van gewrichtsvocht wordt beschreven. Gewrichtsvocht geeft de meest natuurlijke micro-omgeving voor het kraakbeen, en kan gemakkelijk worden verkregen en opgeslagen. Dit systeem kan dus gebruikt worden voor het bestuderen van het herstel van kraakbeen en voor het screenen van geneesmiddelen voor de behandeling van artritis.

Een 3D-systeem voor het kweken van menselijke articulaire chondrocyten in gewrichtsvloeistof

De biosynthese van kraakbeenachtige extracellulaire matrix door chondrocyten kan worden beïnvloed door toepassing van mechanische stimuli. Deze methode beschrijft de technische uitvoering van dynamische druk-stammen chondrocyten ingekapseld in 3D constructen en de evaluatie van geïnduceerde veranderingen in chondrocyte metabolisme.

Mechanische stimulatie van Chondrocyten-agarose Hydrogelen

Kraakbeenherstel vormt een onopgeloste medische uitdaging en cel-gebaseerde benaderingen om menselijk articulair kraakbeen te ontwikkelen zijn een veelbelovende oplossing. Hier beschrijven we driedimensionale (3D) biomimetische hydrogels als een ideaal hulpmiddel voor de uitbreiding en rijping van menselijke articulaire kraakbeencellen.

3D Hydrogel Stellingen voor Articulaire Chondrocyte Cultuur en Kraakbeen Generation

Wij presenteren een protocol voor het genereren van een chondrogeen afkomst van menselijk perifere bloed (PB) via geïnduceerde pluripotente stamcellen (iPSCs) met behulp van een integratievrije methode, waaronder embryoïde lichaamsvorming (EB), uitbreiding van fibroblastische cellen en chondrogeeninductie.

Differentiatie van chondrocyten van perifere bloed afgeleide humane geïnduceerde pluripotente stamcellen

Een werkwijze te halen Dendrimeer gebaseerde ongelijke nanopatterns waarmee de nanoschaal controle van lokale arginine-glycine-aspartic acid (RGD) oppervlakte dichtheid is beschreven en toegepast op de studie van celdifferentiatie hechting en chondrogenic.

Dendrimeer gebaseerde ongelijke Nanopatterns naar lokaal controle oppervlak hechting: een methode om directe Chondrogenic differentiatie

Het verkrijgen van een zuivere populatie van fibroblasten is cruciaal voor het bestuderen van hun rol in wondherstel en fibrose. Hier beschreven is een gedetailleerde methode om fibroblasten en myofibroblasten te isoleren van niet gewonde en gewonde muisharten, gevolgd door karakterisering van hun zuiverheid en functionaliteit door immunofluorescentie, RTPCR, fluorescentie-ondersteunde cel sorteren, en collageen gel contractie.

Isolatie en karakterisering van volwassen cardiale fibroblasten en myofibroblasten

Infrapatellaire vetkussen mesenchymale stamcellen (IFP-MSC's) kunnen gemakkelijk worden geïsoleerd uit de infrapatellaire vetschijf van het kniegewricht. Ze vermenigvuldigen zich goed in vitro, vormen CFU-F-kolonies en differentiëren in adipogene, chondrogene en osteogene afstammingslijnen. Hierin wordt de methodologie gegeven voor de isolatie, uitbreiding en differentiatie van IFP-MSC's van geitenstikselgewricht.

Isolatie, uitbreiding en differentiatie van mesenchymale stamcellen uit de infrapatellaire vetpad van de geitenstikkingsverbinding

Deze studie beschrijft een methode voor het produceren van chondrocytische sferoïden door cellen samen te voegen tot sferoïden onder omstandigheden met een lage adhesie met behulp van zwaartekracht, gevolgd door het kweken van de resulterende sferoïden in mini-bioreactoren.

Het genereren van sferoïden uit verschillende chondrocyten met behulp van weinig klevende omstandigheden onder zwaartekracht en zelfgemaakte mini-bioreactoren

Beschrijven we een kwalitatieve analyse voor gist hechting en agar invasie als een maat voor invasieve en pseudohyfale differentiatie. Deze eenvoudige test kan gebruikt worden om de invasieve fenotype van verschillende mutanten, alsmede de effecten omgevingsfactoren te beoordelen en signaalwegen op gist differentiatie.

Test voor Adhesie en Agar invasie in S. cerevisiae

Deze video toont de techniek die gebruikt wordt voor de voorbereiding van organisator en dier pole explantaten van Xenopus laevis embryo's, waaronder het gebruik van de wenkbrauw mes - een speciale tool gemaakt dissectie van je wenkbrauw. Het protocol voor het monteren van een adhesie-test wordt ook gegeven, die probes voor de aanwezigheid van belangrijke adhesiemoleculen op het oppervlak aanwezig organisator of dierlijke paal cellen die essentieel zijn voor een goede ontwikkeling.

Ontleding van Organisator en dierlijke Pole Explantaten van Xenopus laevis embryo&#39;s en montage van een Celadhesie Assay

Leer de isolatie van chondrocyten en chondroprogenitoren uit menselijk kraakbeen via fibronectine-adhesie en migratie-assays. Deze methode ondersteunt regeneratieve geneeskunde en kraakbeentransplantatie.

Isolatie van chondrocyten en chondroprogenitoren met behulp van fibronectine-adhesie en migratoire assay

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