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;espressione genica della cartilagine. 1. Preparare condrociti articolari umani (HAC) per tre dimensioni (3D) incapsulamento <ol><li> Scongelare una fiala (1 ml) di condrociti articolari umani (HAC) (Lonza) (Passaggio 2) a bagnomaria 37 ° C per 1 min. </li><li> Mescolare con 1 ml di terreni di crescita dei condrociti (Lonza) e centrifugare a 3000 rpm per 3-5 minuti per raccogliere le cellule. Scartare il surnatante. </li><li> Risospendere il pellet di cellule in terreno di coltura di condrociti (Lonza). </li><li> Le cellule in un tessuto piatto 10 centimetripiastra di coltura (BD Biosciences), e la cultura in terreni di coltura di condrociti (Lonza) fino a quando le cellule sono confluenti. HACs deve essere utilizzato in un numero passaggio non superiore a 3 o 4 (P3 e P4). </li><li> Lavare HACs sulla piastra con 155mm di NaCl al posto della PBS standard, seguita da trypsinzation per raccogliere le cellule per l&#39;incapsulamento alginato tallone. Una volta che le cellule si staccano, centrifugare le cellule a 3000rpm per 5min. Le cellule sono ora pronti per l&#39;incapsulamento 3D. </li></ol> 2. Incapsulare HACs in perline 3D Risospendere il HACs alginato in soluzione 1,2% (Sigma) ad una densità di 8x10 5 cellule / ml. Numero di cellule è stato determinato prima di incapsulamento, utilizzando un contatore cella standard. E &#39;molto importante mescolare bene per assicurare una distribuzione uniforme delle celle in perline. <ol><li> Pipettare l&#39;HAC / soluzione di alginato miscela in una siringa da 12 ml collegata ad un 22-gauge (Tyco Healthcare, Inc.). </li><li> Nel frattempo, preparare un bicchiere da 50 ml con 102 mM CaCl 2 in un volume di 5 volte quella della soluzione HAC / aginate. Inserire una ancoretta all&#39;interno del bicchiere e mescolare il 102 mm CaCl 2 soluzione lentamente (a circa 150 giri al minuto). </li><li> Tenere la punta della siringa di circa 6 centimetri al di sopra della superficie della soluzione di CaCl 2 e aggiungere l&#39;HAC / alginato composto nella soluzione di CaCl 2 goccia a goccia. In generale, le perle alginato risultanti sono 2 millimetri di diametro con una densità di incapsulamento di circa 10 4 cellule / tallone. Nota: L&#39;altezza della siringa sulla soluzione di CaCl 2 è importante. Distanza più breve si tradurrà in lacrima a forma sferica, invece di perle a forma, causando irregolare integrità meccanica e l&#39;incapsulamento delle celle. </li><li> Lasciate che i grani mescolare nella soluzione di CaCl 2 per 20-25 min. Mentre altri protocolli di incapsulamento alginato di indicare un tempo di incubazione molto più breve di 10 minuti, abbiamo trovato che il tempo di incubazione più lungo con il CaCl 2 soluzione migliore l&#39;integrità del essereannunci. </li><li> Rimuovere la soluzione di CaCl 2, lavare le perle 2-3 volte in 2-3 volumi di NaCl, e poi una volta in media di differenziazione dei condrociti (Lonza). Mentre altri protocolli di incapsulamento alginato tallone comportano l&#39;uso di un filtro per le suddette procedure di lavaggio, abbiamo scoperto che le perle alginato diventano spesso intrappolato nel filtro fuori e asciutto, riducendo così l&#39;efficienza di incapsulamento. Abbiamo trovato più efficiente per consentire le perle di depositarsi sul fondo del bicchiere prima di scartare la soluzione. </li><li> Utilizzare una spatola standard per trasferire le perline in piatti della cultura. Noi di solito la cultura 12 perle per ben 3 centimetri per 2 giorni nel terreno di coltura di condrociti condrociti per consentire di adeguarsi al processo di incapsulamento nel medio sono state coltivate a loro prima di passare alla media di differenziazione dei condrociti (Lonza) con liquido sinoviale. </li></ol> 3. Condrociti cultura nella coltura del liquido sinoviale medio <ol><li> Fresco liquido sinoviale può essere ottenuto da un outpatient clinica (Abbiamo ottenuto liquido sinoviale presso la Tufts Medical Center). Il liquido sinoviale può essere trasferito in tubi 15ml falco al laboratorio, e immediatamente centrifugato a 3000 rpm per 15 minuti per rimuovere i detriti cellulari. Aliquota cell-free liquido sinoviale in provette da 1,5 ml microcentrifuga, al fine di evitare ripetuti di congelamento-scongelamento. Surnatante può essere conservato a -80 ° C fino al momento dell&#39;uso. </li><li> Prima di coltura, mescolare il liquido sinoviale con il mezzo di differenziazione dei condrociti (CDM, Lonza) a diversi rapporti volumetrici, con una concentrazione costante di acido ascorbico 100mM e 9 mM CaCl 2 (tale importo traccia di CaCl 2 è quello di garantire l&#39;integrità del alginato perline). </li><li> Tenere la piastra su una piattaforma oscillante in un incubatore ° C 37 (frequenza a dondolo: circa 75 volte / min) per aiutare la distribuzione dei nutrienti all&#39;interno della perline alginato, e per ridurre aggregazione delle perline. Ciò è particolarmente importante in quanto abbiamo la cultura di questi condrociti per un lungo periodo di tempo (fino a 4 settimane). Nota: Mentre la crescita dei condrociti e dei media differenziazione (Lonza) contengono due comuni antibiotici gentamicina e amfotericina, non abbiamo alcun supplemento antibiotici quando abbiamo usato diverse percentuali di liquido sinoviale per la coltura di condrociti. Nessuna contaminazione è stata mai osservata. </li><li> Cambia miscele dei media ogni 2-3 giorni. La viscosità del liquido sinoviale è stato un grande ostacolo in condrociti coltura incapsulati in microsfere di alginato durante il lungo periodo culture. Abbiamo trovato più efficace per diluire il liquido sinoviale medio-integrato con 102mm CaCl 2 (50% v / v), che rafforza anche l&#39;integrità delle perline alginato. In questo modo, il mezzo può essere rimosso lentamente con una pipetta, senza danneggiare le perline. </li></ol> 4. HACs raccolto in perle di alginato per l&#39;analisi di espressione genica Particolare cura deve essere presa durante la raccolta del HACs dalle perle alginato per l&#39;analisi di espressione genica. <ol><li> Lavare le perline alginato 3 Times in 102 mM CaCl 2 per circa 5 minuti ogni volta. </li><li> Recuperare i condrociti con l&#39;aggiunta di 55 mM NaCitrate a un volume 4-5 volte quella delle perline. E &#39;importante che le perline alginato sono totalmente immerso nella soluzione NaCitrate. Agitare o roccia per 20-30 min. </li><li> Spin giù le cellule ed eliminare il surnatante. </li><li> Per RT-PCR, risospendere il pellet cellulare in un tampone di lisi cellulare (Qiagen RNA Kit di isolamento), e procedere con la purificazione di RNA. </li></ol> 5. Fix HACs in perline alginato per l&#39;analisi istologica Particolare cura deve essere presa per raccogliere il HACs dalle perle 3D per l&#39;analisi istologica. <ol><li> Lavare le perline alginato 3 volte in 102 mM CaCl 2 per circa 5 minuti ogni volta. Per lavare via tutto il liquido viscoso sinoviale, abbiamo trovato più efficace di lavare con mezzo di differenziazione dei condrociti (Lonza) durante la notte, oscillando in un incubatore ° C 37 (frequenza a dondolo: circa 75s / min). Il terreno di coltura di condrociti è stato utilizzato per assicurare la sopravvivenza di condrociti in questo lavaggio prolungato e per consentire la massima penetrazione dell&#39;agente di fissaggio. </li><li> Fissare le perline in etanolo al 70% durante la notte, e procedere con l&#39;analisi istologica. Per eseguire la colorazione DAPI, incubare perline alginato con DAPI soluzione (500ng/ml) per 1 ora in dolce dondolio, e lavare per 3 volte con 102 mm CaCl 2 per 15 minuti ogni volta. Dapi immagini possono poi essere visualizzate al microscopio a fluorescenza. Le sfere possono anche essere sezionate per alcian blu e colorazione H &amp; E. </li></ol> 6. Rappresentante Risultati Il nostro metodo di coltura 3D per condrociti umani in alte percentuali di liquido sinoviale è raffigurato nello schema elettrico mostrato in Fig.1. Dopo condrociti umani sono stati incapsulati in microsfere di alginato, sono stati lasciati crescere in media integrato con diversi rapporti di liquido sinoviale. A causa della viscosità del liquido sinoviale, è essenzialecondrociti cultura a dondolo in condizioni costanti per evitare l&#39;accumulo dei costrutti cartilagine e per garantire un&#39;equa distribuzione delle sostanze nutritive. E &#39;anche essenziale per lavare le sfere di alginato ampiamente prima di recuperare i condrociti, in modo che la fissazione o tampone di lisi può penetrare le perline (Fig.1). Luminoso campo (BF) immagini di condrociti nelle perline alginato sono mostrati in figura 2. Colorazione DAPI è stata eseguita per confermare usniform distribuzione delle cellule all&#39;interno della sfere (Fig. 2). Al termine dei 21 giorni di cultura, analisi di espressione genica è stata effettuata da qRT-PCR. Il riferimento gene GAPDH è stato utilizzato per la normalizzazione di tutti PCR, come è stato determinato da uno dei geni di riferimento più affidabili per l&#39;analisi qPCR sui condrociti 32. Un esempio è mostrato in fig. 3, dove abbiamo analizzato i risultati di condrociti articolari umani colti nel liquido sinoviale aggregati di sei pazienti con osteoartrosi. Coerente con il fatto che i condrociti daLonza sono stati estesi in culture 2D, che porterebbe inevitabilmente alla de-differenziazione 33, abbiamo scoperto che giorno 0 condrociti espresso livelli minimi di cartilagine geni della matrice (Fig. 3). 3D coltura di condrociti di media differenziazione dei condrociti (Lonza) o media integrato con liquido sinoviale significativo aumento di espressione genica cartilagine del collagene, aggrecano e MMP13, che indica condrociti ri-differenziazione per giorno 21 (Fig.3) 34. Aumentare la percentuale di liquido sinoviale nei media ha portato a livelli comparabili di matrice cartilaginea marcatori collagene II e l&#39;espressione di mRNA aggrecano (Fig.3a e 3B). Inoltre, condrociti coltivati ​​nel 100% del liquido sinoviale anche mostrato una diminuzione di enzimi degradanti cartilagine MMP13 espressione di mRNA rispetto a quelli coltivati ​​in media solo (Fig.3C). È interessante notare che il livello di espressione di indicatore delle cellule morte caspasi 3 gradualmente diminuita con rapporti sempre maggiore di liquido sinoviale, suggerendo che scoltura del liquido ynovial ha portato a livelli di apoptosi diminuzione (Fig. 3D). Pertanto, i nostri risultati mostrano che condrociti articolari umani in coltura alti livelli di liquido sinoviale in un ambiente 3D è una tecnologia fattibile. Tabelle e figure <img alt="Figura 1." src="/files/ftp_upload/3587/3587fig1.jpg" /> Figura 1. Schema del metodo di condrociti articolari cultura umana in alte percentuali di liquido sinoviale in perle di alginato 3D. In primo luogo, condrociti e la soluzione di alginato si mescolano. Quando viene applicato goccia a goccia alla soluzione di CaCl 2, condrociti sono immobilizzati all&#39;interno del reticolato di Ca-alginato di perline idrogel. Questi costrutti 3D vengono poi coltivate in mezzo differenziazione dei condrociti (Lonza) con diversi rapporti di umana liquido sinoviale. Dopo 21 giorni di coltura in una condizione a dondolo, perle di alginato contenenti le cellule sono lavate ampiamente trascorso il quale le cellule vengono recuperati per il geneanalisi di espressione. <img alt="Figura 2." src="/files/ftp_upload/3587/3587fig2.jpg" /> Figura 2. Campo chiaro (BF) e le immagini DAPI di condrociti articolari umani culture incapsulato con lo 0%, 30%, 50%, 70% e il 100% del liquido sinoviale. Condrociti erano di forma sferica in tutte le condizioni di coltura. Immagini del campo chiaro (BF) e DAPI sono stati sovrapposti per confermare la posizione e la distribuzione dei condrociti. Inserti, le immagini ingrandite. Punte di freccia, colocalizzazione dei condrociti in BF e immagini colorazione DAPI. <img alt="Figura 3." src="/files/ftp_upload/3587/3587fig3.jpg" /> Figura 3. QRT-PCR di incapsulato condrociti articolari umani al giorno 0 (D0) uno dopo 21 giorni di coltura (D21) in un mezzo integrato con rapporti variabili di liquido sinoviale (SF) (0%, 30%, 50%, 70 % e 100%). Risultati di quattro campioni indipendenti sono mostrati qui. GAPDH è stato utilizzato come riferimento interno per tutte le PCR. <strong&gt; A. Collagene II espressione di mRNA. B. Aggrecano espressione di mRNA. C. MMP13 espressione di mRNA. D. Caspasi 3 mRNA espressione. La significatività statistica è stata valutata per il Giorno 0 Giorno 21 campioni e campioni dello 0% e 100% del liquido sinoviale (SF) coltura, utilizzando il software INSTAT. * Denota 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>

Non abbiamo nulla da rivelare.

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

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 Views.  Tufts University School of Medicine. 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.

Video: 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.

Un sistema per la coltura cellule epiteliali del pigmento Iris a studio Rigenerazione Lens 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, ...

Ricerca

Biologia

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.

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

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.

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

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.

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

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.

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

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured 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

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.

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

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.

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

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.

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 ...