Questo progetto è stato sostenuto dal Fondo Nazionale Svizzero (FNS # 310030-127586/1).

1. Disco intervertebrale raccolta <ol><li> Tutta la lunghezza della coda bovina si ottiene da un mattatoio locale, se possibile senza la pelle in quanto la presenza della pelle aumenta la possibilità di contaminazione (Fig. 2). </li><li> Preparare a bordo di grandi dimensioni di taglio e la preparazione sterile stazione di lavoro e gli strumenti sopra un tagliere (Fig. 2). </li><li> Preparare sotto la garza sterile cappa a flusso laminare sterile imbevuto di cloruro di sodio 0,9% contenente sodio citrato 55mm e mettere in ciascun pozzetto della piastra 6-bene. </li><li> Preparare una bacinella e diluire 1:100 Betadine soluzione con acqua di rubinetto. </li><li> Immergere la coda tutta bovina in una bacinella contenente soluzione di Betadine 1% per 5 min. </li><li> In breve coda a secco con garza sterile e posizionarlo sul posto di lavoro sterile. </li><li> Rimuovere con cautela muscolare intorno alla coda, con lama di bisturi # 22. </li><li> Tritare via le parti della coda che non sono necessari, di solito entrambe le estremità della coda, che contengono relativamente grandi e molto piccoliIVD (Fig. 3). </li><li> Tagliare ulteriormente il muscolo ei tendini di tutto il IVD con lama di bisturi # 10. Attenzione a non tagliare l&#39;anello esterno del disco. </li><li> Marchio anteriore del DIV con un pennarello la pelle chirurgico (questo passaggio è facoltativo, aiuta a determinare il centro di rotazione assiale nel nostro bioreattore). </li><li> Individuare il punto di connessione e IVD vertebra muovendo la coda delicatamente. Sentite il confine tra IVD e ossa con il lato opaco della lama di un bisturi. Il sito di scissione dovrebbe essere 1-2 mm di distanza del DIV verso la vertebra. Luogo fatto su misura titolare industriale lama sul sito di scissione (Fig. 4). </li><li> Cleave IVD e vertebra martellando sulla parte superiore della misura portalama industriali (Fig. 3). </li><li> Ripetere sul lato opposto della IVD-vertebra connessione. </li><li> Avvolgere isolato IVD con garza sterile inumidita con sodio cloruro 0,9% contenente sodio citrato 55mm. </li><li> Continua l&#39;isolamento della IVD al numero del campione desiderato (di solito circa 6 possonoessere raccolta con diametro compreso tra 10-20 mm). </li><li> Collegare il sistema di lavaggio a getto (Zimmer, inc.) Con la soluzione sterile di Ringer lattato, 5L borse sono di dimensioni utili (in alternativa PBS sterile o soluzione salina allo 0,9%). </li><li> Tenere il IVD con una pinza e jet-lavaggio entrambi i lati della superficie di placca mediante Zimmer Pulsavac jet-lavaggio del sistema (Fig. 1C). La pistola a getto dovrebbe essere girato in superficie placca con un angolo tra i 30 - 60 ° per circa 30 anni su ogni lato. (Fig. 1 e 7) </li><li> Mettere il IVD indietro al 6 pozzetti e avvolgere con una garza umida, mentre il lavaggio a getto d&#39;un altro campione del disco. </li><li> IVD sono pronti per la cultura d&#39;organo ea valle applicazioni (ad esempio, la vitalità delle cellule, il carico meccanico, la cultura d&#39;organo) (Fig. 1D). </li></ol> 2. Rappresentante Risultati Misure di outcome per giudicare la preparazione di successo disco intervertebrale può essere un esperimento di diffusione in cui un colorante fluorescente piccolo peso molecolare (ad esempio Procion rosso)è pronta come una soluzione all&#39;1% in PBS 2. Il IVD è poi mantenuto con almeno 100 ml di soluzione colorante e il colorante viene poi consentito di diffondere passivamente nel IVD per 24 ore sotto gonfiore libero. Il IVD è poi flash-congelato in azoto liquido e riportato a temperatura ambiente e disidratati da una serie di trasferimenti di acetone (primo pre-raffreddata a -80 ° C, -20 ° C, poi a 4 ° C e, infine, a temperatura ambiente ). Il disco può essere incorporato in poli-metil-acrilato (PMMA) e tagliare con una lama affilata per la produzione di 100 micron di spessore sezioni. Queste sezioni possono poi essere visualizzati da qualsiasi microscopio a campo chiaro standard, ma preferibilmente con l&#39;utilizzo del microscopio a fluorescenza dal Procion rosso emette una fluorescenza rossa (fig. 5-6). Il trabeculi della superficie ossea appaiono molto pulita dopo la fase di getto lavaging (Fig. 7). Parametri per una cultura organo di successo sono prima di tutto l&#39;assenza di contaminazione, la vitalità cellulare mantenuto (fig. 8-9), l&#39;altezza del disco egenerale &quot;integrità disco&quot;, misurata con sezioni istologiche e safranina O / verde veloce macchia o Meyer Hematoxilin 3. Abbiamo sviluppato i protocolli e le macro pubblicati altrove 4,5 a macchia tessuto vivo del disco e la vitalità cellulare per la scansione in 3D utilizzando un microscopio confocale a scansione laser e il kit di colorazione Live / Dead (Molecular Probes, Invitrogen, Basilea, Svizzera). Abbiamo inoltre sviluppato una macro e routine per eseguire il conteggio delle cellule automatizzati per valutare la vitalità cellulare dei tessuti 3D o vettori 3D utilizzando la piattaforma NIH ImageJ 4-6. Protocolli alternativi sono stati proposti per determinare la vitalità cellulare delle cellule disco digestione iniziale tramite digestione sequenziale della matrice extracellulare con pronasi e collagenasi di tipo 2 e poi a macchiare la sospensione cellulare 7. Una precedente versione del protocollo coinvolto un passo di incubazione in PBS contenente dieci volte più concentrato concentrazione della penicillina / streptomicina per 10 minuti dopo la raccolta dei dischi precedenti organo culture. Con la fase di lavaggio a getto questo passo diventa obsoleto dal momento che questo passo sembra economici per scopi coltura. <img alt="Figura 1" src="/files/ftp_upload/3490/3490fig1.jpg" /> Figura 1. Panoramica delle fasi metodologiche per preparare bovina coccigeo dischi intervertebrali con piastre laterali intatte e un sottile strato (1-2mm) di osso. A) Rappresentazione schematica del disco intervertebrale (IVD) e dei suoi percorsi nutrizionali. B) Procedura per tagliare il IVD con una lama personalizzato dell&#39;assicurazione e un industriale lama affilata. C) passo lavaggio Jet per garantire la vitalità delle cellule e alta per permettere lo scambio di nutrienti mantenendo le placche terminali e 1-2 mm di osso su entrambi i lati. D) Infine l&#39;IVD può essere coltivato nella camera esemplare di un bioreattore o essere utilizzato per qualsiasi applicazione a valle. <img alt="Figura 2" src="/files/ftp_upload/3490/3490fig2.jpg" /> Figura 2 TOP:. Completi coda lunghezza bovine fresche (idealmente obtaINED entro 2-3 ore post-mortem per garantire la vitalità delle cellule alte. In basso: X-ray immagine di una coda bovina di età compresa tra circa 6 mesi che illustrano l&#39;esistenza di cartilagini di accrescimento (GP), centro secondario di ossificazione, tra le placche ossee (EP) e gli organismi vertebra (VB). <img alt="Figura 3" src="/files/ftp_upload/3490/3490fig3.jpg" /> Figura 3. A) Strumenti per sezionare la coda bovina in condizioni sterili. B) su misura porta lama per tagliare-out segmenti motional dalla coda bovina in azione durante il taglio del disco intervertebrale. C) Vista laterale della lama su misura porta con una lama standard industriali (Lutz, Germania). <img alt="Figura 4" src="/files/ftp_upload/3490/3490fig4.jpg" /> Figura 4. Disegno e immagine della coda bovina dopo aver rimosso il muscolo circostante e legamenti che illustrano i siti tenendoti unito a tagliare i segmenti di movimento. Parziale vertebra dovrebbe essere &quot;rotto&quot; wi° la misura portalama idealmente 1-2 mm di distanza dal placche cartilaginee per assicurarsi che la superficie totale delle ossa. <img alt="Figura 5" src="/files/ftp_upload/3490/3490fig5.jpg" /> Figura 5. Dimostrazione della pulizia efficace del jet-lavaggio passo prima (A) e dopo (B). Notare la pulizia delle superfici ossee dei segmenti del disco intervertebrale dopo la procedura di spruzzatura. <img alt="Figura 6" src="/files/ftp_upload/3490/3490fig6.jpg" /> Figura 6. Free-gonfiore esperimento di diffusione di preparati al momento dischi intervertebrali escisse coda bovina (IVD) ha lasciato per 24 ore in soluzione Procion 1% rosso, a dimostrazione dell&#39;effetto blocco da parte della cartilagine di accrescimento. Una radiografia digitale sagittale di un IVD ancora contenenti la cartilagine di accrescimento e un secondo strato di tessuto osseo immagine di microscopia a fluorescenza di taglio sagittale attraverso l&#39;espianto coccigeo bovina disco intervertebrale con placca cartilaginea ed ossea dopo 24h libera diffusione. GP: cartilagine di accrescimento, 2ndry CO: centro secondario di ossificazione. <img alt="Figura 7" src="/files/ftp_upload/3490/3490fig7.jpg" /> Figura 7. Free-gonfiore esperimento di preparati al momento dischi intervertebrali escisse coda bovina (IVD) ha lasciato per 24 ore in soluzione Procion 1% rosso, dimostrando la pulizia effetto del trattamento getto lavaggio. Sagittale sezioni spesse (~ 100 mm) di escisse IVD bovina con piastre laterali ~ 1,5 mmthick ossei sottoposti a trattamento a getto lavaggio (a sinistra) laterali di controllo senza trattamento (Destra) jet-lavaged trattamento. Ingresso mostra il modello di spruzzo, che è stato utilizzato dalla ferita Zimmer sistema debridement esperimenti di diffusione 24 ore gratuitamente bovina espianti disco intervertebrale in rosso Procion 1%. <img alt="Figura 8" src="/files/ftp_upload/3490/3490fig8.jpg" /> Figura 8. Immagini Live / Dead di proiezioni di confocale stack Imaging (250μm) prelevati da bovinsegmenti del disco intervertebrale e preparati con tale metodo di lavaggio jet-tenuto sotto free-gonfiore per 0 e 14 giorni rispettivamente per il nucleo polposo (NP), l&#39;interno anello fibroso (IA) e l&#39;anello esterno fibroso (OA). Cellule verde = cellule vive macchiate da calceina AM (acetil metilico), globuli rossi = nucleo morto cellule colorate con etidio omodimero-1. <img alt="Figura 9" src="/files/ftp_upload/3490/3490fig9.jpg" /> Figura 9. Vitalità delle cellule del disco intervertebrale utilizzando la microscopia confocale a scansione laser sul tessuto vivo e la scansione dello stack 3D. La vitalità delle cellule del nucleo polposo (NP) e Annulus fibrosi (AF) sul-21 giorno-cultura in free-gonfiore condizione. (Media ± SEM, N = 6) Le differenze statistiche sono state testate utilizzando non parametrico di Kruskal-Wallis firmato somma rank test tra i gruppi. State riscontrate differenze significative tra il giorno 21 e tutti i punti altro momento della NP. Mentre in AF differenze significative sono state trovate tra il giorno 7 agiorno 0. (* P &lt;0,05, ** p &lt;0,01).

<ol>
	<li>Lee, C. R., Iatridis, J. C., Poveda, L., Alini, M. <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 (Phila Pa 1976). 31, 515-522 (1976).</li><li>Chan, S. C. W., Gantenbein-Ritter, B., Leung, V. Y., Chan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreserved+intervertebral+disc+with+injected+bone+marrow-derived+stromal+cells:+a+feasibility+study+using+organ+culture.">Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture.</a> Spine. J. 10 (6), 486-496 (2010).</li><li>Chan, S. C., Ferguson, S. J., Wuertz, K., 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=Biological+Response+of+the+Intervertebral+Disc+to+Repetitive+Short+Term+Cyclic+Torsion.">Biological Response of the Intervertebral Disc to Repetitive Short Term Cyclic Torsion.</a> Spine (Phila Pa 1976). , (2011).</li><li>Gantenbein-Ritter, B., Sprecher, C. M., Chan, S., Illien-J&#252;nger, S., Grad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+protocols+for+live/dead+staining+in+three-dimensional+carriers.">Confocal imaging protocols for live/dead staining in three-dimensional carriers.</a> Methods Mol. Biol. 740, 127-140 (2011).</li><li>Gantenbein-Ritter, B., Potier, E., Zeiter, S., van der Werf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+three+techniques+to+determine+cell+viability+in+3D+tissues+or+scaffolds.">Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds.</a> Tissue Engineering Part C Methods. 14, 353-358 (2008).</li><li>Rasband, W. 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> ImageJ. , (1997).</li><li>Haschtmann, D., Stoyanov, J. V., Ettinger, L., Nolte, L. P., Ferguson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+intervertebral+disc/endplate+culture+model:+analysis+of+an+ex+vivo+in+vitro+whole-organ+rabbit+culture+system.">Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system.</a> Spine. 31, 2918-2925 (2006).</li><li>Gantenbein, B., Gr&#252;nhagen, T., Lee, C. R., van Donkelaar, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+organ+culturing+system+for+intervertebral+disc+explants+with+vertebral+endplates:+a+feasibility+study+with+ovine+caudal+discs.">An in vitro organ culturing system for intervertebral disc explants with vertebral endplates: a feasibility study with ovine caudal discs.</a> Spine. 31, 2665-2673 (2006).</li><li>Korecki, C. L., Kuo, C. K., Tuan, R. S., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervertebral+disc+cell+response+to+dynamic+compression+is+age+and+frequency+dependent.">Intervertebral disc cell response to dynamic compression is age and frequency dependent.</a> J. Orthop. Res. 27, 800-806 (2009).</li><li>Korecki, C. L., MacLean, J. J., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+effects+on+intervertebral+disc+mechanics+and+biology.">Dynamic compression effects on intervertebral disc mechanics and biology.</a> Spine (Phila Pa 1976). 33, 1403-1409 (1976).</li><li>Jim, B., Steffen, T., Moir, J., Roughley, P., Haglund, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+intact+intervertebral+disc+organ+culture+system+in+which+degeneration+can+be+induced+as+a+prelude+to+studying+repair+potential.">Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential.</a> European Spine Journal. , 1-11 (2011).</li></ol>

Non abbiamo nulla da rivelare.

Il primo passo per la cultura d&#39;organo di successo è quello di assicurarsi che l&#39;espianto non dovrebbe essere contaminato. La coda deve essere scuoiati prima di iniziare la procedura. Qualsiasi peli portato in un laboratorio sterile potrebbe essere problematico in termini di contaminazione. La coda bovina dovrebbe idealmente essere il più fresco possibile (questo influenza la vitalità iniziale delle cellule). Inoltre, la fase di lavaggio betadine si raccomanda di ridurre il rischio di ulteriori contaminazioni...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td colspan="4"> Fresco tessuti bovini disco intervertebrale da code bovina, da macello locale (idealmente entro le ore post-mortem e senza pelle). </td></tr><tr><td> Pulsavac Inoltre AC Sistema </td><td> Zimmer inc., Svizzera </td><td> 00-5150-486-01 </td><td> Migliori prestazioni con l&#39;hip-spruzzo testa e con alimentatore AC (quello con l&#39;AA 8 batterie fa anche il lavoro, ma è meno conveniente) </td></tr><tr><td> Ad alta capacità Fan Spray w / Splash Shield, lunghezza 12,7 centimetri </td><td> Zimmer inc., Svizzera </td><td> 00-5150-175-00 </td><td> Ci sono teste di spruzzo diversi a disposizione, abbiamo testato con successo questa </td></tr><tr><td> Lame di bisturi # 22 e # 10 </td><td> Swann-Mortsu, l&#39;Inghilterra </td><td> # 10: 0201 # 22: 0208 </td><td></td></tr><tr><td> Porta lama bisturi # 3 e # 4 </td><td> Hausmann, Germania </td><td> # 3: 06.103.00 # 4: 06.104.00 </td><td></td></tr><tr><td> Lutz industriale lama </td><td> Lutz, Germania </td><td> 1022.0884 </td><td></td></tr><tr><td> Tampone fosfato salino (PBS) </td><td> Invitrogen, Svizzera </td><td> 10010-023 </td><td></td></tr><tr><td> Dulbecco modificato (DMEM) </td><td> Gibco, Svizzera </td><td> 11960-044 </td><td></td></tr><tr><td> Soluzione di Ringer lattato (senza glucosio) </td><td> Bichsel, Svizzera </td><td> 133 0002 </td><td></td></tr><tr><td> 6-ben più pozzetti </td><td> TPP, Svizzera </td><td> 92006 </td><td></td></tr><tr><td> Betadine soluzione </td><td> Mundipharma, Svizzera </td&gt; <td> 10055025 </td><td></td></tr><tr><td> Pelle marcatore chirurgico </td><td> Porex chirurgica, Svizzera </td><td> 9560 </td><td></td></tr><tr><td> Tagliere grande </td><td></td><td></td><td> Ogni marchio è possibile </td></tr></tbody></table>

preparation of intact bovine tail intervertebral discs for organ culture

Il disco intervertebrale (IVD) è il comune della spina dorsale di collegamento vertebra per vertebra. Funziona per trasmettere carico della colonna vertebrale e dare flessibilità alla colonna vertebrale. Si compone di tre comparti: il nucleo polposo più interno (NP) che comprende dal fibroso (AF), e due placche cartilaginee che collega il NP e AF al corpo vertebrale su entrambi i lati. Dolore discogenico forse causata da malattia degenerativa del disco intervertebrale (DDD) e di ernia del disco è stato identificato come uno dei maggiori problemi della nostra società moderna. Per studiare i possibili meccanismi di degenerazione IVD, nei sistemi d&#39;organo coltura in vitro con le cellule disco dal vivo sono di grande appeal. La coltura in vitro di intatta IVD bovina coccigeo è avanzata ad un sistema modello di rilevante, che permette lo studio di meccano-biologico aspetti in un ambiente ben controllato fisiologiche e meccaniche. IVD coda bovina può essere relativamente facile ottenere numeri più alti in unod sono molto simili ai IVD umano lombare rispetto alla densità cellulare, popolazione di cellule e dimensioni. Tuttavia, le tecniche precedenti bovina caudale raccolta IVD mantenendo piastre laterali di placche ossee e cartilaginee fallito dopo 1-2 giorni di coltura in quanto i percorsi nutrizione erano ovviamente bloccato da sangue coagulato. IVD sono i maggiori organi avascolare, quindi, le sostanze nutritive alle cellule del NP dipendono esclusivamente sulla diffusione capillare attraverso le gemme dal corpo vertebrali adiacenti. Presenza di detriti delle ossa e sangue rappreso sulle superfici placca può ostacolare la diffusione di nutrienti verso il centro del disco e sulla vitalità cellulare compromesso. Il nostro gruppo ha stabilito un protocollo relativamente resoconto di &quot;crack&quot;, la IVD dalla coda con un basso rischio di contaminazione. Siamo in grado di permeabilize l&#39;appena tagliata superfici ossee placca utilizzando un sistema di lavaggio chirurgico jet, che rimuove i coaguli di sangue e detriti di taglio e in modo molto efficiente riapre il percorso di diffusione nutrizioneal centro del DIV. La presenza di placche di crescita su entrambi i lati l&#39;osso vertebrale deve essere evitata e deve essere rimosso prima della cultura. In questo video, abbiamo delineare le fasi cruciali durante la preparazione e dimostrare la chiave per una cultura organo di successo mantenendo alta la vitalità delle cellule per 14 giorni in libera cultura gonfiore. Il tempo della cultura potrebbe essere estesa quando opportuno ambiente meccanico può essere mantenuta utilizzando bioreattore meccanici di carico. La tecnica ha dimostrato qui può essere esteso ad altre specie animali quali suini, ovini e leporino isolamento caudale e lombare IVD.

Watch this Scientific Journal Video about Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture at JoVE.com

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

Questo protocollo illustra una tecnica di raccolta coccigeo dischi intervertebrali bovina per la cultura d&#39;organo per<em&gt; In vitro</em&gt; Organo cultura.

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  ...

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14.4K Views.  University of Bern. Questo protocollo illustra una tecnica di raccolta coccigeo dischi intervertebrali bovina per la cultura d'organo per In vitro Organo cultura.

Video: Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

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 ...

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Bioingegneria

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparazione del 3D fibrina ponteggi per le applicazioni delle cellule staminali Cultura

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Campione strategie di preparazione per la Spettrometria di Massa Imaging di 3D Models Cultura cellulare

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

Studiare la Stechiometria di Epidermal Growth Factor Receptor in cellule intatte con correlativa Microscopia

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

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 ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Fabbricazione e validazione di un sistema di organo-on-chip con elettrodi integrati direttamente quantificare transendoteliale resistenza elettrica

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales &#8211; nano-, micro-, meso-, and macroscopic &#8211; fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

This study assesses the fracture toughness of bovine cortical bone at the sub-meso levels using microscopic scratch tests. This is an original, objective, rigorous, and reproducible method proposed to probe fracture toughness below the macroscopic scale. Potential applications are studying changes in bone fragility due to diseases like osteoporosis.

Valutazione di fragilità dell'osso corticale bovino utilizzando Scratch test

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Fabbricazione scalabile di Stretchable, Dual Channel, organo di Microfluidic chip

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Piattaforma di coltura d'organo del segmento anteriore per il monitoraggio delle lesioni Open Globe e delle prestazioni terapeutiche

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

Ricostituzione della citoarchitettura e della funzione dei tessuti epiteliali umani su un chip d'organo open-top

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...