Name: preparation of intact bovine tail intervertebral discs for organ culture
Uploaded: 2012-02-02T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture at JoVE.com

इस परियोजना स्विस राष्ट्रीय विज्ञान फाउंडेशन (# SNF 310030-127586/1) द्वारा समर्थित किया गया.

1. Intervertebral डिस्क फसल काटने वाले <ol><li> पूरे लंबाई गोजातीय पूंछ एक स्थानीय बूचड़खाने से प्राप्त होता है, यदि संभव हो तो त्वचा के बिना त्वचा की उपस्थिति के बाद से संदूषण का मौका (चित्र 2) बढ़ जाती है. </li><li> बड़े काटन...

<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>

सफल अंग संस्कृति के लिए पहला कदम को यकीन है कि explant दूषित नहीं होना चाहिए है. पूंछ है इससे पहले कि आप प्रक्रिया के साथ शुरू चमड़ी किया जाना चाहिए. कोई भी जानवर एक बाँझ प्रयोगशाला में लाया बाल संदूषण के मामल...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियां (वैकल्पिक) </td></tr><tr><td colspan="4"> ताजा गोजातीय पूंछ से गोजातीय intervertebral डिस्क स्थानीय बूचड़खाना (आदर्श पोस्टमार्टम घंटे के भीतर और त्वचा के बिना) से, ऊतक. </td></tr><tr><td> Pulsavac प्लस एसी सिस्टम </td><td> Zimmer inc., स्विट्जरलैंड </td><td> 00-5150-486-01 </td><td> हिप - स्प्रे सिर के साथ और एसी बिजली की आपूर्ति के साथ सर्वश्रेष्ठ प्रदर्शन (8 ए.ए. बैटरी पैक के साथ एक लेकिन यह भी काम करता है कम सुविधाजनक है) </td></tr><tr><td> उच्च क्षमता फैन स्प्रे w / स्पलैश शील्ड, 12.7cm लंबाई </td><td> Zimmer inc., स्विट्जरलैंड </td><td> 00-5150-175-00 </td><td> वहाँ कई स्प्रे सिर उपलब्ध हैं, हम सफलतापूर्वक परीक्षण </td></tr><tr><td> स्केल्पल # 22 और # 10 ब्लेड </td><td> स्वान-रंडी, इंग्लैंड </td><td> # 10: 0201 # 22: 0208 </td><td></td></tr><tr><td> स्केल्पल ब्लेड धारक # 3 और # 4 </td><td> Hausmann, जर्मनी </td><td> # 3: 06.103.00 # 4: 06.104.00 </td><td></td></tr><tr><td> Lutz औद्योगिक ब्लेड </td><td> Lutz, जर्मनी </td><td> 1022.0884 </td><td></td></tr><tr><td> फास्फेट खारा (पीबीएस) buffered </td><td> Invitrogen, स्विटज़रलैंड </td><td> 10010-023 </td><td></td></tr><tr><td> Dulbecco संशोधित ईगल मध्यम (DMEM) </td><td> Gibco, स्विटज़रलैंड </td><td> 11960-044 </td><td></td></tr><tr><td> Lactated घंटी समाधान (ग्लूकोज के बिना) </td><td> Bichsel, स्विटज़रलैंड </td><td> 133 0002 </td><td></td></tr><tr><td> 6-अच्छी तरह से बहु - अच्छी तरह से थाली </td><td> TPP, स्विटज़रलैंड </td><td> 92006 </td><td></td></tr><tr><td> Betadine समाधान </td><td> मुंडीफार्मा, स्विटज़रलैंड </tघ&gt; <td> 10055025 </td><td></td></tr><tr><td> सर्जिकल त्वचा मार्कर </td><td> Porex सर्जिकल, स्विट्जरलैंड </td><td> 9560 </td><td></td></tr><tr><td> बड़े काटने बोर्ड </td><td></td><td></td><td> किसी भी ब्रांड का संभव है </td></tr></tbody></table>

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.

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

इस प्रोटोकॉल एक अंग संस्कृति के लिए अनुत्रिक गोजातीय intervertebral डिस्क के लिए कटाई के लिए तकनीक दिखाता है<em&gt; इन विट्रो में</em&gt; अंग संस्कृति.

बरकरार अंग संस्कृति के लिए गोजातीय टेल intervertebral डिस्क की तैयारी

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

Research

JoVE Journal

Bioengineering

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

और अंडाशय Oviducts के तीन आयामी अंग संस्कृति के लिए Alginate Hydrogels

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 ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

3 डी आतंच की तैयारी स्टेम सेल संस्कृति अनुप्रयोगों के लिए scaffolds

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 ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

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

3 डी सेल संस्कृति मॉडल का मास स्पेक्ट्रोमेट्री इमेजिंग के लिए तैयारी रणनीतियाँ नमूना

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

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

बहु अंग चिप - लंबी अवधि के बहु-ऊतक Coculture के लिए एक microfluidic प्लेटफार्म

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

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

Correlative माइक्रोस्कोपी का उपयोग बरकरार कोशिकाओं में epidermal वृद्धि कारक रिसेप्टर की स्तुईचिओमेटरी का अध्ययन

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

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

एक<em&gt; इन विट्रो</em&gt; अंग संस्कृति म्यूरीन intervertebral डिस्क के मॉडल

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

निर्माण और एक अंग का सत्यापन-ऑन-चिप प्रणाली एकीकृत इलेक्ट्रोड के साथ सीधे Transendothelial विद्युत प्रतिरोध को बढ़ाता है

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

खरोंच परीक्षण का उपयोग कर गोजातीय Cortical हड्डी की कमजोरी आकलन

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

खिंचाव, दोहरी चैनल, Microfluidic अंग चिप्स का स्केलेबल निर्माण

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