Name: a 3d system for culturing human articular chondrocytes in synovial fluid
Uploaded: 2012-01-31T13:00:00
Description: Watch this Scientific Journal Video about A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid at JoVE.com

Vi vil gerne takke Robin Nye (Tufts Medical Center), Tomoya Uchimura og Dana Cairns (Tufts University) for at yde hjælp til synovialvæske opbevaring og centrifugering. Dette arbejde blev finansieret af NIH (1R01AR059106-01A1) til LZ

En 3D-system til dyrkning menneskelige artikulær chondrocytter i synovialvæske I dette arbejde, indkapslet vi menneskelige artikulær chondrocytter i alginat perler ved hjælp af en modificeret fremstilling-foreslået indkapsling protokol (Lonza, og 31). Ved hjælp af disse 3D-konstruktioner, har vi udviklet et system til dyrkning af celler i en kultur, medium, der indeholder forskellige procenter af menneskelige synovialvæske og har vurderet disse 3D-konstruktioner for brusk g...

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I denne rapport har vi udviklet en metode, der giver mulighed for dyrkning af humane artikulær chondrocytter i et 3D-miljø i medium, der indeholder høje koncentrationer af menneskelig synovialvæske. Synovialvæsken er en af ​​de vigtigste komponenter, der udgør det naturlige miljø i den fælles hulrum, hvor artikulær chondrocytter bor. Har imidlertid viskositet synovialvæsken været en stor udfordring for tre-dimensionelle langsigtet dyrkning af chondrocytter. For at overvinde den udfordring at fastholde sel...

Tabel over specifikke reagenser og udstyr: <table border="1"><tr><th> Navn på Reagens </th><th> Firma </th><th> Katalognummer </th><th> Kommentarer </th></tr><tr><td> Alginat (alginsyre natriumsalt) </td><td> Sigma </td><td> A2158-250G </td><td> 2,4% opløsning opbevares ved 40 ° C </td></tr><tr><td> Calciumchlorid dihydrat, Granular </td><td> JT Baker </td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" > Chondrogenic Vækst medier </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3156 (base medier) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4409 (tillæg) </td></tr><tr><td width="239" rowspan="2" > Chondrogenic Differentiering Medier </td><td width="254" rowspan="2" > LOnza </td><td width="182" > CC-3226 (base medier) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4408 (tillæg) </td></tr><tr><td> Menneskelige artikulær chondrocytter </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 (for RNA ekstraktion) </td><td> Qiagen </td><td > 74104 </td><td></td></tr><tr><td> PCR reagenser: SYBR-grøn </td><td> Quanta </td><td > 95053-500 </td><td></td></tr><tr><td> 12 ml sprøjte </td><td> Tyco-Kendall-Monoject </td><td > 512852 </td><td></td></tr><tr><td> 22-Gague kanyle </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> Platform rocker </td><td> Thermoscientific thermolyne </td><td > Vari-mix </td><td></td></tr><tr><td></td><td></td><td ></td><td></td></tr><tr><td> Primere sekvenser </td><td></td><td ></td><td></td></tr><tr><td> Kollagen IIa-forward </td><td> 5&#39;-TTC ATC CCA CCC TCT CAC AGT-3 &#39; </td><td ></td><td></td></tr><tr><td> Kollagen 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-frem </td><td> 5&#39;-TCA TTA TTC AGG FTT GCC GTG GTA-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspase 3-reverse </td><td> 5&#39;-TGG ATG AAC KAG GAG CCA TCC TTT -3 &#39; </td><td ></td><td></td></tr></table>

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

Bruskdestruktionen er et centralt patologisk træk ved slidgigt, en førende årsag til invaliditet i USA. Brusk i den voksne ikke regenerere meget effektivt in vivo, og som et resultat, fører slidgigt til uoprettelige brusk tab og er ledsaget af kroniske smerter og immobilitet 1,2. Brusk tissue engineering tilbyder lovende potentiale til at regenerere og genoprette væv funktion. Denne teknologi involverer typisk såning chondrocytter i naturlige eller syntetiske stilladser og dyrkning den resulterende 3D ​​konstruere på en afbalanceret medium over en periode på tiden med et mål om Engineering A biokemisk og biomekanisk modne væv, der kan transplanteres ind i en defekt websted in vivo 3-6 . At opnå en optimal betingelse for chondrocytter vækst og matrix deposition er afgørende for succesen af ​​brusk tissue engineering. I den indfødte fælles hulrum, brusken på Articular overfladen af ​​knoglen er badet i synovialvæske. Denne klare og tyktflydende væske giver næringsstoffer til avascular ledbrusken og indeholder vækstfaktorer, cytokiner og enzymer, der er vigtige for chondrocytter stofskifte 7,8. Desuden synovialvæske letter lav friktion bevægelse mellem bruskspidserne overflader hovedsagelig gennem secernerende to centrale komponenter, hyaluronan og lubricin 9 10. I modsætning hertil er manipuleret væv brusk oftest dyrket i kunstige medier. Mens disse medier er sandsynligvis i stand til at yde mere definerede betingelser for at studere chondrocytter metabolisme, synovialvæske mest præcist afspejler den naturlige miljø, som artikulær chondrocytter bor i. Faktisk synovialvæske har den fordel af at være let at indhente og opbevare, og kan ofte regelmæssigt fornys af kroppen. Flere grupper har suppleret dyrkningsmediet med ledvæske i voksende menneskelige, kvæg, kaniner og hunde chondrocytes, men mest bruges kun lave niveauer af synovialvæske (under 20%) 25/11. Mens kylling, hest og menneske chondrocytter har været dyrket i medium med højere procentdel af synovialvæske, disse kultur systemer var to-dimensionelle 26-28. Her kan vi præsentere vores metode til dyrkning menneskelige artikulær chondrocytter i et 3D-system med en høj procentdel af synovialvæske (op til 100%) over en periode på 21 dage. Dermed overvandt vi en stor forhindring forelagt af høj viskositet synovialvæsken. Dette system giver mulighed for at studere menneskets chondrocytter i synovialvæske i et 3D-indstilling, som kan yderligere kombineres med to andre vigtige faktorer (oxygenspændingen og mekaniske belastning) 29,30, som udgør det naturlige miljø for brusk til at efterligne det naturlige miljø for brusk vækst. Desuden kan dette system også anvendes til analysering synovialvæske aktivitet på chondrocytter og skabe en platform for udviklingbrusk regeneration teknologier og terapeutiske muligheder for gigt.

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

Et 3D-system med dyrkning menneskelige artikulær chondrocytter i høje niveauer af synovialvæsken er beskrevet. Synovialvæske afspejler den mest naturlige mikromiljø for ledbrusken, og kan let udtages og opbevares. Dette system kan derfor bruges til at studere brusk regenerering og til screening af lægemidler til behandling af gigt.

En 3D-system til dyrkning Menneskelige artikulær chondrocytter i synovialvæske

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

Effektiv Udledning af menneskelige Hjerte prækursorer og cardiomyocytter fra Pluripotente humane embryonale stamceller med småmolekyle Induktion

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

A System for ex vivo Culturing of Embryonic Pancreas

Et system til<em&gt; Ex vivo</em&gt; Dyrkning af embryonale Pancreas

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

Analyse af genfunktioner og Visualisering af Cilia er genereret Fluid Flow i Kupffer s vesikel

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

Dyrkning af human Nasal epithelialceller på Air Liquid grænseflade

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

A Method for Culturing Embryonic C. elegans Cells

A Method for Culturing Embryonic C. elegans Cells

En fremgangsmåde til dyrkning Embryonale<em&gt; C. elegans</em&gt; Celler

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

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

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 ...

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

Gap junktion intercellulær kommunikation: En funktionel biomarkør at vurdere negative virkninger af giftstoffer og toksiner, og Sundhedsmæssige fordele af Naturprodukter

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

Forsamlingen og anvendelse af &#39;Shear Rings &quot;: A Novel Endothelial Model for Orbital, ensrettet og periodisk Fluid Flow og Shear stress

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

Magnetisk aktiveret celle sortering strategier for at isolere og rense Synovial væske-afledte mesenkymale stamceller fra en kanin 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 ...