Questo lavoro è stato sostenuto dalla Washington University muscoloscheletrico Research Center (NIH P30 AR057235), Molecular Imaging Center (NIH P50 CA094056), mechanobiology Training Grant (NIH 5T32EB018266), NIH R21AR069804, e NIH K01AR069116. Gli autori desiderano ringraziare Patrick Wong per il suo contributo nella raccolta dei dati.

Tutti gli esperimenti sugli animali sono stati eseguiti in conformità alla Washington University di Comitato Animal Studies St. Louis. 1. Gli animali <ol><li> Ottenere due ceppi di topi: 10 settimane di età BALB / c (n = 6, BALB-M, BALB / cAnNTac) e 10 settimane kappa-B-luciferasi animali giornalista vecchi fattore nucleare (NF-κβ-luc) allevato in BALB / c sfondo (n = 6, BALB / c-Tg (Rela-luc) 31Xen). </li><li> Prima della dissezione, eutanasia animali con CO 2 sovradosaggio con un flusso di 2,5-3 l / min per 5 minuti seguita da un ulteriore 2 min di tempo di permanenza. </li><li> Disinfettare la regione esterna dell&#39;animale da bagno in un bagno di etanolo al 70% per 2 min prima della dissezione. </li></ol> 2. Dissection <ol><li> Eseguire un taglio verticale longitudinale sulla superficie dorsale del mouse utilizzando piccole forbici dissezione per esporre la cavità del corpo. </li><li> Fare due tagli verticali longitudinali su entrambi i lati della colonna vertebrale dell&#39;animale a partire dalla prima vertebra lombare (L1) alle vertebre caudali (C8). </li><li> Usando un bisturi, pinza sottile, e forbici dissezione fine, rimuovere accuratamente la colonna vertebrale da L1-C8 dalla cavità del corpo dell&#39;animale. </li><li> rimuovere accuratamente tessuti molli circostanti eccesso sulla colonna vertebrale, assicurando nel contempo a non graffiare o danneggiare l&#39;IVD sul lato ventrale. </li><li> Ulteriormente sezionare la colonna vertebrale in vertebre-disc-vertebre spinali unità funzionali (FSU) al L1 / L2, L3 / L4, L5 / L6, C3 / C4, C5 / C6, C7 e dischi / C8. </li><li> Sciacquare la FSU in soluzione salina bilanciata di Hank integrato con 1% di penicillina-streptomicina per 2 min. </li><li> Casualmente assegnare i FSUs in tre gruppi: uncultured e greggia FSU (fresca), coltivate ma greggia FSU (controllo), e coltivate e stab trattati FSU (Stab). </li><li> Snap congelare i campioni freschi FSU con azoto liquido immediatamente dopo la dissezione e conservare in un congelatore C -20 °. </li></ol> 3. Condizioni coltura d&#39;organo<ol><li> Preparare 500 ml di terreni di coltura sterile di 1: 1 di Dulbecco mezzo di Eagle modificato: Nutrient Mixture F12 (DMEM: F12) supplementato con siero fetale bovino 20% (FBS) e 1% di penicillina-streptomicina. </li><li> Utilizzando 10 ml pipette sierologiche, pipetta 2 ml di terreni di coltura in ciascun pozzetto di una piastra di coltura a 24 pozzetti. </li><li> Seguendo dissezione e risciacquare, collocare ogni FSU in un pozzo individuo nella piastra a 24 pozzetti media-riempita. </li><li> Incubare i campioni in un incubatore sterile che mantiene 37 ° C, 5% di CO 2, 20% O 2 e&gt; 90% di umidità. </li><li> Dopo un periodo di coltura iniziale di 24 h, danneggiare meccanicamente i campioni Stab gruppo tramite puntura dell&#39;anello fibroso utilizzando un ago calibro 27 sterile come mostrato nella Figura 1A. </li><li> Cambiare supporto ogni 48 h preparando un nuovo 24-pozzetti di coltura-riempite, e campioni di trasferimento dal vecchio piastra alle nuove usando pinzette sterili. Aspirare i vecchi media e dispose della vecchia piastra di coltura in un contenitore per rifiuti a rischio biologico. </li><li> L&#39;ultimo giorno di coltura, incubare tutti i campioni supporti contenenti 0,75 mg / ml di cloruro nitro blu tetrazolio per 24 h. </li><li> Successivamente bloccare tutto il FSUs con azoto liquido e conservare in un freezer a -20 ° C fino al momento prove meccaniche o istologia (Figura 1B). </li></ol> 4. Misure longitudinali NF-kB <ol><li> Immagine del FSU da animali NF-κβ-luciferasi a 1, 5, 13, e 19 giorni per valutare l&#39;espressione di NF-kB. </li><li> Aggiungere 10 microlitri di 1 mg / mL di soluzione luciferina ciascun pozzetto ed incubare in un incubatore sterile a 37 ° C per 10 min. </li><li> Immagine i campioni per bioluminescenza utilizzando il sistema di imaging con un tempo di esposizione 1 min e l&#39;impostazione bin 2. </li><li> Contemporaneamente, utilizzare la macchina per scattare una fotografia e sovrapporre l&#39;immagine è bioluminescenza con per determinare la posizione anatomica della luminescenza nel IVD. </li></ol><pclass = &quot;jove_title&quot;&gt; 5. valutazione meccanica <ol><li> Determinare il comportamento meccanico dei IVD mediante compressione dinamica spostamento controllato 9. </li><li> Utilizzando un microscopio dissezione, bisturi e pinzette, rimuovere i corpi vertebrali ossee del FSU da ciascun campione mantenendo intatto e attaccato al IVD le placche terminali cartilaginee. </li><li> Fissare le IVD isolato ad una piastra di alluminio 1 cm x 1 cm x 0,2 centimetri con colla di cianoacrilato. </li><li> Misurare l&#39;altezza del disco e larghezza considerando una media di tre misurazioni sull&#39;asse longitudinale di ciascun disco utilizzando un micrometro laser. Calcolare il rapporto di altezza del disco dividendo l&#39;altezza media disco per la larghezza massima del disco. </li><li> Utilizzare l&#39;altezza del disco misurata per calcolare i valori di deformazione ingresso utilizzati in prove meccaniche. Si noti che l&#39;altezza media disco era approssimativamente 690 um ± 39 um. </li><li> Porre i campioni in un tampone fosfato salino bagno sotto il compressisulla macchina e precaricare il disco da 0,02 N. </li><li> Ciclicamente comprimere il disco utilizzando una forma d&#39;onda sinusoidale a 1 Hz per 20 cicli a livello di deformazione 1% e livello di deformazione 5% per 3 prove ciascuno e registrare i valori di carico e spostamento della IVD. Attendere 10 min di tempo di riposo tra le prove per il disco per rilassarsi e per evitare lesioni. </li><li> Calcolare la media rigidità dalla fase di carico dell&#39;ultimo ciclo, e calcolare la tangente di perdita dall&#39;angolo di fase tra i dati di carico e di spostamento. </li></ol> 6. Proteoglycan e Collagene Quantificazione <ol><li> Seguendo prove meccaniche, misurare il peso umido disco posizionando l&#39;IVD isolato in una provetta da centrifuga pre-tarato e misurando il peso con una bilancia analitica. </li><li> Digerire i IVD isolati in 250 microlitri soluzione di digestione papaina (0,1 M di acetato di sodio, EDTA 0,01 M, 0,005 M cisteina HCl, pH 6,4) per una notte a 65 ° C utilizzando un riscaldatore blocco. </li><li> campioni di centrifugazione per 10 mina 2.000 xg e raccogliere il liquido surnatante. </li><li> Misurare il contenuto di proteoglicani della IVD utilizzando il saggio (DMMB) metilmetilene blu con standard condroitina solfato. </li><li> Preparare la soluzione DMMB (21 mg DMMB, 5 mL di etanolo assoluto, 2 g di formiato di sodio in 1 L DDH 2 O). </li><li> Pipettare 30 ml di standard e campioni in triplice copia in una piastra a 96 pozzetti, insieme a 250 ml di soluzione DMMB in ciascun pozzetto. </li><li> Leggere l&#39;assorbanza della piastra a 525 nm utilizzando uno spettrofotometro. </li><li> Misurare il contenuto di collagene utilizzando un kit di analisi idrossiprolina secondo le istruzioni del produttore. </li></ol> 7. Istologia <ol><li> Fissare un sottoinsieme di campioni in paraformaldeide al 4% per 24 h, de-calcify in acido formico al 5% per 48 ore, disidratare con etanolo, e incorporare in paraffina. </li><li> Utilizzando un microtomo, sezione sagittale i campioni a 10 micron di spessore e applicare le sezioni per vetrini. Macchiare le sezioni usando safranina-O / Fast Green e DAPI. </li><li> Utilizzando un microscopio ottico, immagine scorre a ingrandimento 10X. </li></ol> 8. Contrast-enhanced microComputed Tomografia (microCT) <ol><li> Scansione di un sottoinsieme di campioni al momento del dato 0, 2, 5, e 7 giorni utilizzando longitudinale ioversol contrasto enhancement 10, e terminale acido fosfomolibdico (PMA) contrasto enhancement al punto temporale di 7 giorni. </li><li> 4 h prima microCT tempo di scansione, aggiungere 300 ml di 350 mg / mL di soluzione di iodio ioversol ai terreni di coltura per una concentrazione finale di circa 50 mg / soluzione ioversol contenenti iodio ml e incubare in un incubatore sterile a 37 ° C per 4 h. </li><li> Dopo l&#39;incubazione, il campione avvolgere in garza sterile e posto in una provetta 1 ml. </li><li> I campioni del sistema microCT e scansione a 45 keVp, 177 uA, 10,5 um dimensione voxel, e 300 ms integrazione. </li><li> Esportare dati dal microCT come file DICOM e visualizzare con il software ( <em&gt; ad esempio, OsiriX). </li><li> Al punto di tempo di 7 giorni, fissare campioni utilizzando una soluzione di paraformaldeide al 4% per 24 ore, seguita da una soluzione PMA 5% per 3 giorni, e la scansione utilizzando le stesse impostazioni come quelli utilizzati in fase 8.4. </li></ol> 9. Tridimensionale di modellazione agli elementi finiti <ol><li> Segmento i file DICOM dei IVD utilizzando il software con della soglia manuale (per esempio, OsiriX). </li><li> Convertire i volumi NP e AF della IVD in mesh di elementi finiti voxel-based utilizzando MeshLab. </li><li> Combinare le microstrutture della NP e AF per formare un IVD completa ed applicare condizioni al contorno determinate sperimentalmente nel software elementi finiti. </li></ol>

<ol>
	<li>Dagenais, S., Caro, J., Haldeman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+low+back+pain+cost+of+illness+studies+in+the+United+States+and+internationally.">A systematic review of low back pain cost of illness studies in the United States and internationally.</a> Spine J. 8 (1), 8-20 (2008).</li><li>Urban, J., Roberts, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degeneration+of+the+intervertbral+disc.">Degeneration of the intervertbral disc.</a> Arthritis Res Ther. 5 (6), 1-48 (2003).</li><li>Mirza, S. K., White, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+intervertebral+disc+and+pathophysiology+of+herniated+disc+disease.">Anatomy of intervertebral disc and pathophysiology of herniated disc disease.</a> J Clin Laser Med Surg. 13 (3), 131-142 (1995).</li><li>Acaroglu, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degeneration+and+aging+affect+the+tensile+behavior+of+human+lumbar+anulus+fibrosus.">Degeneration and aging affect the tensile behavior of human lumbar anulus fibrosus.</a> Spine. 20 (24), 2690-2701 (1995).</li><li>Abraham, A. C., Liu, J. W., Tang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+changes+in+the+structure+and+inflammatory+response+of+the+intervertebral+disc+due+to+stab+injury+in+a+murine+organ+culture+model.">Longitudinal changes in the structure and inflammatory response of the intervertebral disc due to stab injury in a murine organ culture model.</a> J Orthop Res. 34 (8), 1431-1438 (2016).</li><li>Ohtori, S., Inoue, G., Miyagi, M., Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathomechanisms+of+discogenic+low+back+pain+in+humans+and+animal+models.">Pathomechanisms of discogenic low back pain in humans and animal models.</a> Spine J. 15 (6), 1347-1355 (2015).</li><li>Pelle, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+functional+studies+of+the+intervertebral+disc:+A+novel+murine+intervertebral+disc+model.">Genetic and functional studies of the intervertebral disc: A novel murine intervertebral disc model.</a> PLoS ONE. 9 (12), (2014).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+investigation+of+intervertebral+disc+degeneration+produced+by+needle-stab+injury+of+the+rat+caudal+spine.">Time course investigation of intervertebral disc degeneration produced by needle-stab injury of the rat caudal spine.</a> J Neurosurg: Spine. 15 (4), 404-413 (2011).</li><li>Liu, J. W., Abraham, A. C., Tang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+high-throughput+phenotyping+of+the+viscoelastic+behavior+of+whole+mouse+intervertebral+discs+using+a+novel+method+of+dynamic+mechanical+testing.">The high-throughput phenotyping of the viscoelastic behavior of whole mouse intervertebral discs using a novel method of dynamic mechanical testing.</a> J Biomech. 48 (10), 2189-2194 (2015).</li><li>Lin, K. H., Wu, Q., Leib, D. J., Tang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+the+contrast-enhanced+microCT+imaging+of+murine+intervertebral+discs.">A novel technique for the contrast-enhanced microCT imaging of murine intervertebral discs.</a> J Mech Behav Biomed Mater. 63, (2016).</li><li>Pasparakis, M. <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+tissue+homeostasis+by+NF-kappaB+signalling:+implications+for+inflammatory+diseases.">Regulation of tissue homeostasis by NF-kappaB signalling: implications for inflammatory diseases.</a> Nat Rev: Immunol. 9 (11), 778-788 (2009).</li><li>O&#8217;Connell, G. D., Vresilovic, E. J., Elliott, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+animals+used+in+disc+research+to+human+lumbar+disc+geometry.">Comparison of animals used in disc research to human lumbar disc geometry.</a> Spine. 32 (3), 328-333 (2007).</li><li>Lee, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+organ+culture+of+the+bovine+intervertebral+disc:+effects+of+vertebral+endplate+and+potential+for+mechanobiology+studies.">In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies.</a> Spine. 31, 515-522 (2006).</li><li>Chan, S. C., Gantenbein-Ritter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Intact+Bovine+Tail+Intervertebral+Discs+for+Organ+Culture.">Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture.</a> J. Vis. Exp. (60), e3490 (2012).</li><li>Holguin, N., Aguilar, R., Harland, R. A., Bomar, B. A., Silva, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+aging+mouse+partially+models+the+aging+human+spine:+lumbar+and+coccygeal+disc+height,+composition,+mechanical+properties,+and+Wnt+signaling+in+young+and+old+mice.">The aging mouse partially models the aging human spine: lumbar and coccygeal disc height, composition, mechanical properties, and Wnt signaling in young and old mice.</a> J Appl Physiol. 116 (12), (2014).</li></ol>

Gli autori di questo manoscritto dichiarano di non avere alcun interesse finanziario in competizione.

Questo protocollo descrive una cultura organo del murino FSU con enfasi sul monitoraggio dei cambiamenti biologici nel IVD. La corretta gestione di queste colture richiede attente tecniche sterili. In particolare, la dissezione passi 2.1-2.6 e la cultura passi 3.1-3.6 richiedono particolare cura per assicurare condizioni di sterilità sono mantenuti, e questi passaggi deve essere eseguita preferibilmente in una stanza procedura isolato con un flusso d&#39;aria HEPA per minimizzare contam...

Lombalgia (LBP) è il fattore principale per la disabilità globale e la perdita di produttività sul posto di lavoro, e gli americani spendono solo al di sopra di 50 miliardi di dollari sul trattamento LBP 1. Anche se prevalente, l&#39;eziologia della LBP rimane complessa e multifattoriale. Tuttavia, disco intervertebrale (IVD) degenerazione è tra i fattori di rischio più significativi per LBP 2. IVD è costituito da tre microstrutture: l&#39;annulus fibrosi esterno (AF), l&#39;interno del nucleo polposo (NP), e due placche cartilaginee che racchiudono l&#39;intera struttura prossimale e distale 3. Con l&#39;invecchiamento e la degenerazione, i componenti IVD cambiano strutturalmente, composizionale e meccanicamente 4. Queste modifiche comprendono la perdita di proteoglicani e idratazione del NP, diminuzione altezza del disco, ed è peggiorato competenza meccanica 5. Queste alterazioni sonospesso accompagnata da citochine che promuovono una risposta infiammatoria, così come neutrofili e radice dorsale ganglio intrusione nella cavità articolare culmina in una cascata di eventi che portano a sintomi LBP 6. Studiando i meccanismi di degenerazione IVD è una sfida per l&#39;uomo, perché spesso non è possibile per isolare la causa della degenerazione prima del verificarsi di lombalgia. Pertanto, un approccio riduttivo di semplificare il sistema sperimentale verso l&#39;organo IVD consente meccanicistico messa a punto delle variabili causali ed esaminando i loro effetti a valle 5. Il sistema è ridotto a solo la popolazione di cellule nativa e circostante matrice extracellulare, permettendo così l&#39;interpretazione diretta degli effetti degli stimoli esterni su IVD degenerazione. Inoltre, il basso costo e scalabilità di modelli murini, nonché il numero di animali geneticamente modificati 7, consentono tegli screening rapido mirato dei meccanismi degenerativi IVD e potenziali terapie. Qui, descriviamo un sistema di coltura organo murino in cui IVD cellulare e tissutale stabilità è mantenuta in 21 giorni, con particolare attenzione rivolta ai modelli omeostatici, meccaniche, strutturali e infiammatorie delle IVDs. Usando questo metodo, monitoriamo i cambiamenti funzionali IVDs&#39; in un modello di lesione indotta stab-8 di comprendere i meccanismi alla base degenerazione del disco. Inoltre, descriviamo diverse applicazioni di questo metodo cultura organo condurre ulteriori studi tra cui l&#39;imaging microCT mdc e tridimensionali ad alta risoluzione modellizzazione del IVD.

<table><tbody><tr><td>96 well plate</td><td>Midwest Scientific</td><td>TP92096</td><td>Used for biochemical assays</td></tr><tr><td>24 well plate</td><td>Midwest Scientific</td><td>TP92024</td><td>Used for organ culture</td></tr><tr><td>25 mL pipettes</td><td>Midwest Scientific</td><td>TP94024</td><td>Used for organ culture</td></tr><tr><td>10 mL pipettes</td><td>Midwest Scientific</td><td>TP94010</td><td>Used for organ culture</td></tr><tr><td>5 mL pipettes</td><td>Midwest Scientific</td><td>TP94005</td><td>Used for organ culture</td></tr><tr><td>500 mL bottle top filters, 22 &amp;micro;m</td><td>Midwest Scientific</td><td>TP99505</td><td>Used for filter media</td></tr><tr><td>10 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140098</td><td>Used for biochemical assays</td></tr><tr><td>200 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140900</td><td>Used for biochemical assays</td></tr><tr><td>1,000 &amp;micro;L pipette tips</td><td>Midwest Scientific</td><td>NP89140920</td><td>Used for biochemical assays</td></tr><tr><td>DMEM/F-12</td><td>Invitrogen</td><td>11330032</td><td>Used for culture media</td></tr><tr><td>Optiray 350</td><td>Guebert</td><td>19133341</td><td>Used for contrast enhanced microCT</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F2442</td><td>Used for culture media</td></tr><tr><td>Penicillin Streptomycin&amp;nbsp;</td><td>Sigma</td><td>P4333</td><td>Used for culture media</td></tr><tr><td>Tetrazolium Blue Chloride</td><td>Sigma</td><td>T4375</td><td>Used for biochemical assays</td></tr><tr><td>D-Luciferin</td><td>Sigma</td><td>L6152</td><td>Used for bioluminescence imaging</td></tr><tr><td>Chondroitin Sulfate</td><td>Sigma</td><td>C9819</td><td>Used for biochemical assays</td></tr><tr><td>10% Phosphomolybdic Acid Solution</td><td>Sigma</td><td>HT152</td><td>Used for contrast enhanced microCT</td></tr><tr><td>Safranin O</td><td>Sigma</td><td>S8884</td><td>diluted to .1% concentration (in water)</td></tr><tr><td>Fast Green FCF</td><td>Sigma</td><td>F7258</td><td>.001% concentration</td></tr><tr><td>Papain from papaya latex</td><td>Sigma&amp;nbsp;</td><td>P3125</td><td>Used for biochemical assays</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>Nucleic acid staining</td></tr><tr><td>Cyanoacrylate Glue</td><td>Loctite</td><td>234790</td><td>Adhesive&amp;nbsp;</td></tr><tr><td>1.5 mL Microcentrifuge Tubes</td><td>Fischer Scientific</td><td>S348903</td><td>Used for biochemical assays</td></tr><tr><td>&lt;strong&gt;Big Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BioDent</td><td>ActiveLife</td><td></td><td>For mechanical testing</td></tr><tr><td>Cytation 5</td><td>Biotek</td><td></td><td>Spectrophotometer</td></tr><tr><td>AxioCam503</td><td>Carl Zeiss AG</td><td></td><td>Microscope</td></tr><tr><td>VivaCT40</td><td>Scanco</td><td></td><td>MicroCT</td></tr><tr><td>Analytical balance</td><td>Denver Instrument Company</td><td>A-200DS</td><td>Analytical balance</td></tr><tr><td>Incubator HERAcell 150i</td><td>Thermo Scientific</td><td></td><td>Organ Culture</td></tr><tr><td>Dissection Scope</td><td>VistaVision</td><td></td><td>Used during dissection</td></tr><tr><td>Laser Micrometer</td><td>Keyence</td><td>LK-081</td><td>Measuring disc height</td></tr><tr><td>Microcentrifuge 5810 R</td><td>Eppendorf</td><td></td><td>Used for biochemical assays</td></tr><tr><td>Microtome</td><td>Leica&amp;nbsp;</td><td>RM2255</td><td>Used for histology</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Prism 7</td><td>GraphPad</td><td></td><td>For statistics</td></tr><tr><td>MATLAB R2014a</td><td>Mathworks</td><td></td><td>For modeling</td></tr><tr><td>Osiri-LXIV</td><td>Pixmeo</td><td></td><td>Open Source</td></tr><tr><td>MeshLab v1.3.3</td><td>Visual Computing Lab - ISTI - CNR</td><td></td><td>Open Source</td></tr><tr><td>PreView/FEBio 2.3</td><td>Utah MRL &amp;amp; Columbia MBL</td><td></td><td>Open Source</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Windows</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Dissection Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cohan-Vannas Spring Scissors&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>15000-02</td><td>Or any nice pair of spring scissors</td></tr><tr><td>Fine Scissors - Sharp&amp;nbsp; (small)</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>14060-09</td><td></td></tr><tr><td>Fine Scissors - Sharp&amp;nbsp; (larger)</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>14060-11</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>11252-40</td><td>At least 2; can also use #3&amp;nbsp;</td></tr><tr><td>Extra Fine Graefe Forceps, serrated</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>11150-10</td><td>At least 2</td></tr><tr><td>Micro-Adson Forceps, serrated</td><td>World Precision Instruments</td><td>503719-12</td><td></td></tr><tr><td>Micro-Adson Forceps, teeth</td><td>World Precision Instruments</td><td>501244</td><td></td></tr><tr><td>Scalpel Handle - #3</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10003-12</td><td></td></tr><tr><td>Scalpel Handle - #4</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10004-13</td><td></td></tr><tr><td>Scalpel Blades - #23</td><td>Fine Science Tools&amp;nbsp;&amp;nbsp;</td><td>10023-00</td><td></td></tr><tr><td>Insect Pins, size 000</td><td>Fine Science Tools</td><td>26000-25</td><td></td></tr><tr><td>27 G Needle</td><td>BD PrecisionGlide Needles</td><td>BD305109</td><td></td></tr></tbody></table>

an in vitro organ culture model of the murine intervertebral disc

Disco intervertebrale (IVD) la degenerazione è un contributo significativo per il mal di schiena. L&#39;IVD è una joint fibrocartilaginea che serve per trasmettere e smorzare i carichi nella colonna vertebrale. IVD consiste in un polposo ricco di proteoglicani nucleo (NP) e un ricco di collagene annulus fibrosi (AF) a sandwich da cartilaginee piastre terminali. Unitamente alle vertebre adiacenti, la struttura vertebre-IVD formano un&#39;unità funzionale spina (FSU). Questi microstrutture contengono tipi di cellule uniche e matrici extracellulari unici. cultura organo intero FSU conserva la matrice extracellulare nativa, fenotipi differenziazione cellulare, e le interazioni cellulari-matrice. Pertanto, le tecniche di coltura organo sono particolarmente utili per studiare i complessi meccanismi biologici della IVD. Qui, descriviamo un approccio high-throughput per la coltura di tutta la FSU del mouse lombare che fornisce una piattaforma ideale per lo studio dei meccanismi di malattia e le terapie per l&#39;IVD. Inoltre, descriviamo sapplicazioni everal che utilizzano questo metodo di coltura organo di condurre ulteriori studi tra cui l&#39;imaging microCT mdc e tridimensionali ad alta risoluzione elementi finiti modellizzazione della IVD.

Le figure 2-3 mostrano risultati rappresentativi della distribuzione dei proteoglicani, espressione NFKB, rigidità, viscosità, altezza del disco e peso umido per IVD coltivate mouse. Se coltivate correttamente, i parametri IVD del gruppo di controllo non dovrebbe essere significativamente diverso dal gruppo Fresh. Se la coltura è infetto o comunque compromessa, il gruppo di controllo sarà diverso dal gruppo fresco, specialmente nell&#39;espressione NFKB e distribuzione proteoglicani (risultati non m...

Watch this Scientific Journal Video about An In Vitro Organ Culture Model of the Murine Intervertebral Disc at JoVE.com

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

cultura organo intero del disco intervertebrale (IVD) conserva la matrice extracellulare nativa, fenotipi cellulari, e le interazioni cellulari-matrice. Qui si descrive un sistema di coltura IVD usando lombare mouse e IVD caudale nei loro unità spinali funzionali e diverse applicazioni che utilizzano questo sistema.

Un<em&gt; In Vitro</em&gt; Organ Culture Modello del disco intervertebrale Murine

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

an-in-vitro-organ-culture-model-of-the-murine-intervertebral-disc

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

9.5K Views.  Washington University in St. Louis.  cultura organo intero del disco intervertebrale (IVD) conserva la matrice extracellulare nativa, fenotipi cellulari, e le interazioni cellulari-matrice. Qui si descrive un sistema di coltura IVD usando lombare mouse e IVD caudale nei loro unità spinali funzionali e diverse applicazioni che utilizzano questo sistema. 

Video: An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Idrogeli di alginato per tridimensionale Cultura Organo di ovaie e tube

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Ricerca

Bioingegneria

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

Preparazione del intatto dischi intervertebrali coda bovina per la Cultura d&#39;organo

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

L&#39;ingegneria dei tessuti del colon in un modello murino

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

In vitro delle cellule Cultura modello per Tossico per inalazione Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Oculare Test (EIT) per identificazione del pericolo di sostanze chimiche irritanti oculari utilizzando Ricostruito umana Cornea come epiteliale (RHCE) Tessuto Modello

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Ovine Lumbar Intervertebral Disc Degeneration Model Utilizing a Lateral Retroperitoneal Drill Bit Injury

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration have been developed. The ideal animal model should closely mimic the human intervertebral disc with regard to morphology, biomechanical properties and the absence of notochordal cells. The sheep lumbar intervertebral disc model fulfils these criteria. We present an ovine model of intervertebral disc degeneration utilizing a drill bit injury through a lateral retroperitoneal approach. The lateral approach significantly reduces the incision and potential morbidity associated with the traditional anterior approach to the ovine spine. Utilization of a drill-bit method of injury affords the ability to produce a consistent and reproducible injury, of precise dimensions, that initiates a consistent degree of intervertebral disc degeneration. The focal nature of the annular and nucleus pulposus defect more closely mimics the clinical condition of focal intervertebral disc herniation. Sheep recover rapidly following this procedure and are typically mobile and eating within the hour. Intervertebral disc degeneration ensues and sheep undergo necropsy and subsequent analysis at periods from eight weeks. We believe that the drill bit injury model of intervertebral disc degeneration offers advantages over more conventional annular injury models.

Intervertebral disc degeneration is a significant contributor to back pain and a leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration exist. We demonstrate an ovine model of intervertebral disc degeneration, utilizing a drill bit, which achieves a consistent disc injury and reproducible level of disc degeneration.

Modello di degenerazione del disco intervertebrale lombare di ovini Utilizzando un lesione laterale retroperitoneale laterale

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous ...

Medicina

A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due to the lack of causative therapies, there is an urgent need for innovative experimental organ culture models to study the mechanisms involved in the progression of the disease, find therapeutic targets, and reduce the need for animal models. We here present a novel, three-dimensional organ culture model protocol mimicking the proinflammatory and catabolic microenvironment, which is present during IDD. 
Initially, bovine caudal IVDs were dissected, cleaned, and cultured in the tissue culture medium. Dynamic physiologic or pathologic loading was applied in a custom-made bioreactor for 2 hours per day. IVDs were assigned to a control group (high glucose medium, physiological loading, phosphate-buffered saline injection) and a pathological group (low glucose medium, pathological loading, tumor necrosis factor-alpha injection) for four days. Gene expression analysis from collected nucleus pulposus cells of the IVDs and enzyme-linked immunosorbent assay of the conditioned organ culture media was performed. 
Our data revealed a higher expression of inflammatory markers and reduced disc heights after loading in the pathological group compared to the control group. This protocol is reliable to simulate IVD inflammation and degeneration and can be further expanded to broaden its application scope.

This protocol presents a novel experimental model of proinflammatory, degenerative bovine organ culture to simulate early-stage intervertebral disc degeneration.

Un modello proinflammatorio di coltura degenerativa degli organi per simulare la malattia del disco intervertebrale in fase iniziale.

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due ...

Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To decode disc degeneration and to be able to develop regenerative approaches a thorough understanding of the cellular biology of the IVD is essential. One aspect of this biology that still remains unanswered is the question of how cells are spatially arranged in a physiological state and during degeneration. The biological properties of the IVD and its availability make this tissue difficult to analyze. The present study investigates spatial chondrocyte organization in the anulus fibrosus from early embryonic development to end-stage degeneration. An optical sectioning method (Apotome) is applied to perform high resolution staining analyses using bovine embryonic tissue as an animal model and human disc tissue obtained from patients undergoing spine surgery. From a very high chondrocyte density in the early embryonic bovine disc, the number of cells decreases during gestation, growth, and maturation. In human discs, an increase in cellular density accompanied the progression of tissue degeneration. As had already been demonstrated in articular cartilage, cluster formation represents a characteristic feature of advanced disc degeneration.

We present a method to investigate spatial chondrocyte organization in the anulus fibrosus of the intervertebral disc using an optical sectioning method.

Sezionamento ottico e visualizzazione del disco intervertebrale dallo sviluppo embrionale alla degenerazione

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To ...

Biologia

Generating a Pro-Inflammatory Organ Culture Model to Simulate an Early Intervertebral Disc Disease

Source: Saravi, B., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/62100">A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.</a> J. Vis. Exp. (2021).
This video demonstrates a technique for culturing bovine intervertebral discs in bioreactors to develop a three-dimensional organ culture model. Specific nutrient-deprived media and degenerative pressure conditions are applied to the discs, mimicking the pro-inflammatory and degenerative microenvironment observed during early-stage intervertebral disc disease. The disc heights are measured post-treatment to analyze the effects of nutrient deprivation and degenerative loading.

Generazione di un modello di coltura d'organo pro-infiammatorio per simulare una malattia precoce del disco intervertebrale

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Dissection of the bovine ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

Generating an Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease

Source: Saravi, B. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/62100">A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.</a>&#160;J. Vis. Exp. (2021).
This video demonstrates the development of a pro-inflammatory and degenerative organ culture model for early-stage intervertebral disc (IVD) disease using TNF-&#945;. It outlines the procedure for injecting TNF-&#945; into the intervertebral disc and explains the mechanisms through which TNF-&#945; induces inflammation and degradation of the IVD.

Source: Saravi, B. et al., A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.&#160;J. Vis. Exp. ...

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