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 ønsker å takke Robin Nye (Tufts Medical Center), Tomoya Uchimura og Dana Cairns (Tufts University) for å gi hjelp til synovialvæsken lagring og sentrifugering. Dette arbeidet ble finansiert av NIH (1R01AR059106-01A1) for LZ

En 3D-system for dyrking human artikulære chondrocytes i synovialvæsken I dette arbeidet, innkapslet vi menneskelige artikulære chondrocytes i alginat perler ved hjelp av en modifisert produksjon-foreslo innkapsling protokoll (Lonza, og 31). Ved hjelp av disse 3D-konstruksjoner, har vi utviklet et system for dyrking celler i en kultur medium som inneholder varierte prosenter av menneskelig synovialvæsken og har vurdert disse 3D konstruksjoner for brusk genuttrykk. <p class...

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I denne rapporten har vi utviklet en metode som gjør det mulig for kulturen av menneskelig artikulære chondrocytes i et 3D miljø i medium som inneholder høye konsentrasjoner av menneskelig leddvæske. Synovialvæsken er en av de viktigste komponentene som utgjør det naturlige miljøet i det felles hulrom, hvor artikulær chondrocytes bor. Imidlertid har viskositet synovialvæsken vært en stor utfordring for tredimensjonale langsiktig dyrking av chondrocytes. For å overvinne utfordringen med å opprettholde selv n...

Tabell over spesifikke reagenser og utstyr: <table border="1"><tr><th> Navn på Reagens </th><th> Firma </th><th> Katalog nummer </th><th> Kommentarer </th></tr><tr><td> Alginat (alginsyre natrium salt) </td><td> Sigma </td><td> A2158-250G </td><td> 2,4% løsning oppbevares ved 40 ° C </td></tr><tr><td> Kalsiumklorid dihydrat, Granular </td><td> JT Baker </td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" > Chondrogenic Vekst media </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3156 (base media) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4409 (supplement) </td></tr><tr><td width="239" rowspan="2" > Chondrogenic Differensiering Media </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3226 (base media) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4408 (supplement) </td></tr><tr><td> Menneskelig artikulære chondrocytes </td><td> Lonza </td><td > CC-2550 </td><td></td></tr><tr><td> Dapi (4 &#39;,6-Diamidino-2-phenylindole dihydrochloride) </td><td> Sigma-Aldrich </td><td > D9542 </td><td></td></tr><tr><td> RNeasy mini kit (for RNA ekstraksjon) </td><td> Qiagen </td><td > 74104 </td><td></td></tr><tr><td> PCR reagenser: SYBR-grønn </td><td> Quanta </td><td > 95053-500 </td><td></td></tr><tr><td> 12 ml sprøyte </td><td> Tyco-Kendall-Monoject </td><td > 512852 </td><td></td></tr><tr><td> 22-Gague Hypodermic Needle </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> Plattform 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> Collagen IIa frem </td><td> 5&#39;-TTC ATC CCA CCC TCT CAC AGT-3 &#39; </td><td ></td><td></td></tr><tr><td> Collagen IIa-revers </td><td> 5&#39;-CCTCTGCCTTGACCCGAA-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13 frem </td><td> 5&#39;-TGT GCC CTT CTT CAC ACA GAC ACT-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13-revers </td><td> 5&#39;-GAG AGC AGA CTT TGA GTC ATT GCC-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspase 3-framover </td><td> 5&#39;-TCA TTA TTC AGG CCT GCC GTG GTA-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspase 3-revers </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

Brusk ødeleggelse er et sentralt patologisk trekk ved slitasjegikt, en ledende årsak til uførhet i USA. Brusk i den voksne ikke regenerere ikke veldig effektivt in vivo, og som et resultat fører slitasjegikt til irreversible brusk tap og er ledsaget av kroniske smerter og immobilitet 1,2. Brusk tissue engineering tilbyr lovende potensial til å fornye og gjenopprette vev funksjon. Denne teknologien innebærer vanligvis seeding chondrocytes i naturlige eller syntetiske stillaser og dyrking den resulterende 3D ​​konstruere en balansert medium over en tidsperiode med et mål av engineering en biokjemisk og biomekanisk modne vev som kan transplanteres inn i en defekt område in vivo 3-6 . Oppnå en optimal forutsetning for chondrocyttransplantasjon vekst og matrise deponering er avgjørende for å lykkes med brusk tissue engineering. I de innfødte felles cavity, brusk på arktiskeular overflaten av benet er badet i synovialvæsken. Dette klar og tyktflytende væske gir næringsstoffer til avascular leddbrusk og inneholder vekstfaktorer, cytokiner og enzymer som er viktige for chondrocyttransplantasjon metabolisme 7,8. Videre letter synovialvæsken lav friksjon bevegelse mellom cartilaginous flater hovedsakelig gjennom sekresjon to viktige komponenter, hyaluronan og lubricin 9 10. I kontrast er vevet utviklet brusk oftest dyrket i kunstige medier. Mens disse mediene er trolig i stand til å gi mer definerte vilkår for å studere chondrocyttransplantasjon stoffskifte, synovialvæsken mest presist reflekterer naturen som artikulære chondrocytes bor i. Har faktisk leddvæsken fordelen av å være lett å få tak og lagre, og kan ofte fornyes jevnlig av kroppen. Flere grupper har supplert den kulturen medium med synovialvæsken i voksende menneske, storfe, kanin og hund chondrocytes, men for det meste brukes kun lave nivåer av leddvæske (under 20%) 11-25. Mens kylling, hest og menneske chondrocytes har vært dyrket i medium med høyere prosentandel av synovialvæsken, ble disse kultur systemene todimensjonal 26-28. Her presenterer vi vår metode for dyrking menneskelig artikulære chondrocytes i et 3D-system med en høy andel av leddvæske (opp til 100%) over en periode på 21 dager. Ved å gjøre det, overvant vi et stort hinder presentert av den høye viskositeten på synovialvæsken. Dette systemet gir mulighet for å studere menneskelig chondrocytes i synovialvæsken i et 3D miljø, som kan videre kombineres med to andre viktige faktorer (oksygen spenninger og mekanisk belastning) 29,30 som utgjør det naturlige miljø for brusk å etterligne de naturlige miljø for brusk vekst. Videre kan dette systemet også brukes for å analysere synovialvæsken aktivitet på chondrocytes og gi en plattform for utviklingbrusk regenerering teknologier og behandlingsalternativer for leddgikt.

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

En 3D-system for dyrking menneskelig artikulære chondrocytes i høye nivåer av synovial væske er beskrevet. Leddvæsken reflekterer den mest naturlige mikromiljøet for leddbrusk, og kan lett innhentes og lagres. Dette systemet kan dermed brukes til å studere brusk regenerering og for screening legemiddelselskap for behandling av leddgikt.

En 3D System for dyrking Menneskelig artikulære chondrocytes i synovialvæsken

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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 for<em&gt; Ex vivo</em&gt; Dyrking av 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 ...

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

Analyse av gen-funksjon og visualisering av Cilia-generert Væskestrøm i Kupffer sin Vesikkel

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

Dyrking av menneskelig Nasal Epitelceller på Air Flytende Interface

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 metode for dyrking Embryonic<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 junctional interkommunikasjons: En funksjonell biomarkør for å vurdere skadevirkninger av giftstoffer og giftstoffer, og helsemessige fordeler av 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

Forsamlingen og anvendelse av &#39;Skjær Rings&#39;: A Novel endotel modell for Orbital, Enveis og Periodisk Fluid Flow og skjærspenning

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-aktivert celle sortering strategier for å isolere og rense Synovial Fluid-avledet Mesenchymal stamceller fra en kanin 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 ...