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

Vorremmo ringraziare Robin Nye (Tufts Medical Center), Tomoya Uchimura e Dana Cairns (Tufts University) per aver fornito aiuto di stoccaggio liquido sinoviale e centrifugazione. Questo lavoro è stato finanziato dal NIH (1R01AR059106-01A1) per LZ

Un sistema 3D per condrociti articolari umani in coltura liquido sinoviale In questo lavoro, abbiamo incapsulato condrociti articolari umani in perle di alginato utilizzando una versione modificata produzione-ha suggerito protocollo di incapsulamento (Lonza, e 31). Utilizzando questi costrutti 3D, abbiamo sviluppato un sistema per la coltura delle cellule in un terreno di coltura contenente varie percentuali di liquido sinoviale e umane hanno valutato questi costrutti 3D per l&#39...

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In questo rapporto, abbiamo sviluppato un metodo che permette per la cultura dei condrociti articolari umani in un ambiente 3D in mezzo che contiene alte concentrazioni di liquido sinoviale umano. Liquido sinoviale è uno dei principali componenti che costituiscono l&#39;ambiente naturale nella cavità articolare, dove risiedono condrociti articolari. Tuttavia, la viscosità del liquido sinoviale è stata una grande sfida per tridimensionali a lungo termine coltura di condrociti. Per superare la sfida di mantenere la di...

Tabella di reagenti e attrezzature specifiche: <table border="1"><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Alginato (acido alginico sale di sodio) </td><td> Sigma </td><td> A2158-250G </td><td> 2,4% soluzione conservata a 40 ° C </td></tr><tr><td> Cloruro di calcio diidrato, granulare </td><td> JT Baker </td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" > La crescita dei media condrogenico </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 (supplemento) </td></tr><tr><td width="239" rowspan="2" > Differenziazione media condrogenico </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 (supplemento) </td></tr><tr><td> Condrociti articolari umani </td><td> Lonza </td><td > CC-2550 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-Diamidino-2-fenilindolo dicloridrato) </td><td> Sigma-Aldrich </td><td > D9542 </td><td></td></tr><tr><td> RNeasy mini kit (per l&#39;estrazione di RNA) </td><td> Qiagen </td><td > 74104 </td><td></td></tr><tr><td> Reagenti PCR: SYBR-green </td><td> Quanta </td><td > 95053-500 </td><td></td></tr><tr><td> Siringa da 12 ml </td><td> Tyco-Kendall-Monoject </td><td > 512852 </td><td></td></tr><tr><td> 22-Gague aghi ipodermici </td><td> Tyco Kendall-Monoject </td><td > 8881 </td><td></td></tr><tr><td> Microscopio </td><td> Olimpo </td><td > IX71 </td><td></td></tr><tr><td> Piattaforma rocker </td><td> Thermolyne Thermoscientific </td><td > Vari-mix </td><td></td></tr><tr><td></td><td></td><td ></td><td></td></tr><tr><td> Primer sequenze </td><td></td><td ></td><td></td></tr><tr><td> Collagene IIa-forward </td><td> 5&#39;-ATC CCA CCC TTC TCT CAC AGT-3 &#39; </td><td ></td><td></td></tr><tr><td> Collagene IIa-reverse </td><td> CCTCTGCCTTGACCCGAA-5&#39;-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13-forward </td><td> 5&#39;-GCC TGT CTT CTT ACA CAC GAC ACT-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13-reverse </td><td> 5&#39;-GAG AGC AGA TGA CTT GTC ATT GCC-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspasi 3-forward </td><td> 5&#39;-TCA TTA TTC AGG CCT GCC GTG GTA-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspasi 3-reverse </td><td> 5&#39;-TGG ATG AAC CAG GAG CCA CCT TTT -3 &#39; </td><td ></td><td></td></tr></table>

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

Distruzione della cartilagine è un elemento centrale patologica di osteoartrite, una delle principali cause di disabilità negli Stati Uniti. La cartilagine nell&#39;adulto non si rigenera in modo molto efficiente in vivo, e, di conseguenza, l&#39;artrosi conduce alla irreversibile perdita della cartilagine ed è accompagnata da dolore cronico e 1,2 immobilità. Ingegneria dei tessuti cartilagine offre un potenziale promettente per rigenerare e ripristinare la funzionalità dei tessuti. Questa tecnologia in genere implica condrociti semina in ponteggi naturale o sintetica e la coltura del 3D che dà luogo a costruire un mezzo equilibrato per un periodo di tempo con l&#39;obiettivo di un tessuto di ingegneria biochimica e biomeccanica mature che possono essere trapiantate in un sito difetto in vivo 3-6 . Raggiungere una condizione ottimale per la crescita dei condrociti e la deposizione della matrice è essenziale per il successo di ingegneria dei tessuti cartilaginei. Nella nativa comune cavità, cartilagine al articcolare superficie dell&#39;osso è immersa nel liquido sinoviale. Questo liquido chiaro e viscoso fornisce sostanze nutritive per la cartilagine articolare avascolare e contiene fattori di crescita, citochine e gli enzimi che sono importanti per il metabolismo dei condrociti 7,8. Inoltre, il liquido sinoviale facilita il movimento a basso attrito tra le superfici cartilaginee che secernono principalmente attraverso due componenti chiave, acido ialuronico e lubricin 9 10. Al contrario, la cartilagine di ingegneria tessutale è spesso colto in mezzi artificiali. Mentre questi media sono probabilmente in grado di offrire condizioni più definite per lo studio del metabolismo dei condrociti, liquido sinoviale più accuratamente riflette l&#39;ambiente naturale di cui condrociti articolari risiedono in Infatti, il liquido sinoviale ha il vantaggio di essere facile da ottenere e conservare, e che spesso può essere regolarmente rifornito dal corpo. Diversi gruppi hanno completato il terreno di coltura con liquido sinoviale in crescita umano, bovino, coniglio e cane chondrocytes, ma soprattutto utilizzato solo bassi livelli di liquido sinoviale (inferiore al 20%) 11-25. Mentre condrociti pollo, cavallo e umani sono stati colti nel mezzo con una maggiore percentuale di liquido sinoviale, questi sistemi di coltura erano bidimensionali 26-28. Qui vi presentiamo il nostro metodo di coltura di condrociti articolari umani in un sistema 3D con un alta percentuale di liquido sinoviale (fino al 100%) per un periodo di 21 giorni. Nel fare ciò, abbiamo superato un grande ostacolo presentato dalla viscosità del liquido sinoviale. Questo sistema offre la possibilità di studiare condrociti umani nel liquido sinoviale in un ambiente 3D, che può essere ulteriormente raggruppati con altri due importanti fattori (tensione di ossigeno e carico meccanico), 29,30 che costituiscono l&#39;ambiente naturale per la cartilagine di imitare l&#39;ambiente naturale per cartilagine di crescita. Inoltre, questo sistema può essere utilizzato anche per saggiare l&#39;attività del liquido sinoviale sui condrociti e fornire una piattaforma per lo sviluppotecnologie di rigenerazione della cartilagine e le opzioni terapeutiche per l&#39;artrite.

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 sistema 3D di coltura di condrociti articolari umani in alti livelli di liquido sinoviale è descritto. Liquido sinoviale riflette il microambiente più naturale per la cartilagine articolare, e può essere facilmente ottenuti e memorizzati. Questo sistema quindi può essere utilizzato per studiare la rigenerazione della cartilagine e per terapie di screening per il trattamento di artrite.

Un sistema 3D per condrociti articolari umani in coltura liquido sinoviale

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Biologia

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

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

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

Un sistema per<em&gt; Ex vivo</emColtura&gt; del pancreas embrionale

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

Analisi della funzione del gene e visualizzazione di Cilia-Generated flusso di fluido in vescicole di 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

Coltura di cellule umane nasale epiteliali presso l&#39;Air Interface Liquid

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

Un metodo per la coltura embrionale<em&gt; C. elegans</em&gt; Celle

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

Spazi di giunzione intercellulare di comunicazione: un biomarker funzionale per valutare gli effetti avversi dei tossici e tossine, e benefici per la salute di prodotti naturali

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

L&#39;Assemblea e l&#39;applicazione di &quot;Shear Anelli &#39;: A Novel endoteliale modello per Orbital, unidirezionale e periodica del fluido di flusso e lo stress di taglio

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

Magnetico-Activated Cell Sorting strategie per isolare e purificare Synovial cellule staminali mesenchimali liquido derivato da un modello di coniglio

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