Name: Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture (Video) | JoVE Journal
Uploaded: 2012-02-02T13:00:00.000+00:00
Duration: 13 min 37 s
Description: Learn to prepare intact bovine tail intervertebral discs for organ culture using a custom cutting tool and jet lavage system. This method enables bioreactor studies of mechanical loading effects on spinal tissue.

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This project was supported by the Swiss National Science Foundation (SNF # 310030-127586/1).

1. Intervertebral Disc Harvesting
<ol>
	<li>Whole length bovine tail is obtained from a local abattoir, if possible without skin since the presence of skin increases the chance of contamination (Fig 2).</li>
	<li>Prepare large cutting board and prepare sterile work station and instruments on top of a cutting board (Fig. 2).</li>
	<li>Prepare under the sterile laminar flow hood sterile gauze moistened with 0.9% sodium chloride containing 55mM sodium citrate and put into each well of the 6-well plate.</li>
	<li>Prepare a basin and dilute 1:100 Betadine solution with tap water.</li>
	<li>Immerse the whole bovine tail in a basin containing 1% Betadine solution for 5 min.</li>
	<li>Briefly dry tail with sterile gauze and place it on sterile work station.</li>
	<li>Carefully remove muscle around the tail with scalpel blade #22.</li>
	<li>Chop away the parts of the tail which are not needed, usually both ends of the tail, which contain relatively large and very small IVDs (Fig. 3).</li>
	<li>Trim further the muscle and tendons around the IVD using scalpel blade #10. Beware not to cut the outer annulus of the discs.</li>
	<li>Mark the anterior of the IVD with a surgical skin marker (this step is optional, it helps to determine the center for axial rotation in our bioreactor).</li>
	<li>Locate the IVD and vertebra connection point by moving the tail gently. Feel the border between IVD and bone with the dull side of a scalpel blade. The cleaving site should be 1-2 mm away of the IVD towards the vertebra. Place custom made industrial blade holder on the cleaving site (Fig. 4).</li>
	<li>Cleave IVD and vertebra by hammering on top of the custom-made industrial blade holder (Fig. 3).</li>
	<li>Repeat on the other side of the IVD-vertebra connection.</li>
	<li>Wrap isolated IVD with sterile gauze moistened with 0.9% sodium chloride containing 55mM sodium citrate.</li>
	<li>Continue the isolation of IVDs to the desired sample number (usually around 6 can be harvested with diameter between 10-20 mm).</li>
	<li>Connect the jet lavage system (Zimmer, inc.) with sterile Lactated Ringer's solution, 5L bags are a useful size (alternatively sterile PBS or 0.9% saline solution).</li>
	<li>Hold the IVD with forceps and jet-lavage both sides of the endplate surface using Zimmer Pulsavac jet-lavage system (Fig. 1C). The jet gun should be shot on the endplate surface at an angle between 30 - 60° for about 30s on each side. (Figs. 1 and 7)</li>
	<li>Put the IVDs back to the 6-well plate and wrap with moistened gauze while jet washing another disc sample.</li>
	<li>IVDs are ready for organ culture and down-stream applications (e.g. cell viability, mechanical loading, organ culture) (Fig. 1D).</li>
</ol>
2. Representative Results
Outcome measurements to judge a successful intervertebral disc preparation can be a diffusion experiment where a small molecular weight fluorescent dye (e.g. procion red) is prepared as a 1% solution in PBS2. The IVD is then kept with at least 100 mL of dye solution and the dye is then allowed to diffuse passively into the IVD for 24h under free swelling. The IVD is then flash-frozen in liquid nitrogen and brought back to room temperature and dehydrated by a series of acetone transfers (first pre-cooled in -80 °C, then -20 °C, then 4 °C and finally to room temperature). The disc can then be embedded in poly-methyl-acrylate (PMMA) and cut with a sharp blade to produce 100 μm-thick sections. These sections can then be viewed by any standard brightfield microscope but preferentially by usage of fluorescence microscopy since procion red emits a red fluorescence (Figs. 5-6). The trabeculi of the bony surface appear very clean after the jet lavaging step (Fig. 7).
Parameters for a successful organ culture are first of all the absence of contamination, maintained cell viability (Figs. 8-9), disc height and overall "disc integrity", measured with histological sections and safranin O/fast green stain or Meyer's Hematoxilin 3. We developed protocols and macros published elsewhere4,5 to stain live disc tissue and to scan cell viability in 3D using a confocal laser scanning microscope and the live/dead staining kit (Molecular Probes, Invitrogen, Basel, Switzerland). We further developed a macro and routine to perform automated cell counting to estimate cell viability in 3D tissue or 3D carriers using the NIH ImageJ platform4-6. Alternate protocols have been proposed to determine cell viability of disc cells by initial digestion using sequential digestion of the extracellular matrix using pronase and collagenase type 2 and then to stain the cell suspension7. An earlier version of the protocol involved an incubation step in PBS containing ten times more concentrated penicillin/streptomycin concentration for 10min after the harvesting of the discs prior organ culture. With the jet lavage step this step becomes obsolete since this step seems beneficial for culturing purposes.
<img alt="Vertebral column structure diagram, nutrient flow, disc crack, organ culture setup, research methods." src="/files/ftp_upload/3490/3490fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1. Overview of the methodological steps to prepare bovine coccygeal Intervertebral Discs with intact endplates and a thin layer (1-2mm) of bone. A) Schematic view of the intervertebral disc (IVD) and its nutritional pathways. B) Procedure to cut out the IVD with a customized blade-holder and an industrial sharp blade. C) Jet lavage step to ensure high cell viability and to enable nutrient exchange keeping the endplates and 1-2 mm of bone attached on both sides. D) Finally the IVD can be cultured in the specimen chamber of a bioreactor or be used for any down-stream application.
<img alt="Bone growth analysis; anatomical image and X-ray of tail vertebrae highlighting growth plates." src="/files/ftp_upload/3490/3490fig2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2. TOP: Full length fresh bovine tail (ideally obtained within 2-3 hours post-mortem to ensure high cell viability. Bottom: X-ray image of a bovine tail aged around 6 months illustrating the existence of growth plates (GP), secondary center of ossification, between the bony endplates (EP) and the vertebra bodies (VB).
<img alt="Bone fixation process; equipment in setup (A), clamp on bone (B), force testing device (C)." src="/files/ftp_upload/3490/3490fig3.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3. A) Tools to dissect the bovine tail under sterile conditions. B) Customized blade-holder to cut-out motional segments from the bovine tail in action while cutting the intervertebral disc. C) Side-view of the customized blade-holder with a standard industrial blade (Lutz, Germany).
<img alt="Vertebral structure diagram and sample showing vertebra, IVD, growth plate, and cleaving site." src="/files/ftp_upload/3490/3490fig4.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4. Drawing and picture of the bovine tail after removing the surrounding muscle and ligament illustrating the cleaving sites to cut out the motional segments. Partial vertebra should be "cracked" with the custom-made blade holder ideally 1-2 mm away from the cartilaginous endplates to ensure full bone surface.
<img alt="Skeletal tissue samples comparison; measurement, analysis; experiment with ruler scale." src="/files/ftp_upload/3490/3490fig5.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5. Demonstration of the effective cleaning of the jet-lavage step before (A) and after (B). Notice the clean bony surfaces of the intervertebral disc segments after the spraying procedure.
<img alt="Histological analysis diagram showing bone growth, featuring primary and secondary ossification." src="/files/ftp_upload/3490/3490fig6.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
Figure 6. Free-swelling diffusion experiment of freshly prepared excised bovine tail intervertebral discs (IVDs) left for 24 hours in 1% procion red solution, demonstrating blockage effect by the growth plate. A digital X-ray sagittal view of a IVD still containing the growth plate and a second layer of bone fluorescent microscopy image of sagittal cut through the coccygeal bovine intervertebral disc explant with cartilaginous and bony endplate after 24h free diffusion. GP: growth plate, 2ndry CO: secondary center of ossification.
<img alt="Histological analysis, vertebral disc. Microscopy image shows nucleus pulposus, annulus fibrosus detail." src="/files/ftp_upload/3490/3490fig7.jpg" data-altupdatedat="1788304762000" data-alt="Figure 7"> 
Figure 7. Free-swelling experiment of freshly prepared excised bovine tail intervertebral discs (IVDs) left for 24 hours in 1% procion red solution, demonstrating cleaning effect of jet lavage treatment. Sagittal thick sections (~100 mm) of excised bovine IVDs with ~1.5-mmthick bony end plates that underwent jet lavage treatment (Left) control side without treatment (Right) jet-lavaged treatment. Inlet shows the spray pattern, which was used from the Zimmer wound debridement system 24h free diffusion experiments of bovine intervertebral disc explants in 1% procion red.
<img alt="Cell viability assay results, fluorescent microscopy images, time-lapse comparison, days 0 to 14." src="/files/ftp_upload/3490/3490fig8.jpg" data-altupdatedat="1788304762000" data-alt="Figure 8"> 
Figure 8. Live/Dead Images of projections of confocal Imaging stacks ( ̃250μm) taken from bovine intervertebral disc segments prepared with this jet-lavage method kept under free-swelling for 0 and 14 days respectively for the nucleus pulposus (NP), the inner annulus fibrosus (IA) and the outer annulus fibrosus (OA). Green cells = live cells stained by calcein AM (acetyl methylester), red cells = dead cells nucleus stained by ethidium homodimer-1.
<img alt="Cell viability graph, intervertebral disc over time; NP and AF comparison; viability percentages." src="/files/ftp_upload/3490/3490fig9.jpg" data-altupdatedat="1788304762000" data-alt="Figure 9"> 
Figure 9. Cell Viability of intervertebral disc using confocal laser scanning microscopy on live tissue and 3D stack scanning. Cell viability of Nucleus Pulposus (NP) and Annulus Fibrosis (AF) over the 21-day-culture under free-swelling condition. (Mean ± SEM, N=6) Statistical differences were tested using non-parametric Kruskal-Wallis signed rank sum test among groups. Significant differences were found between day 21 and all other time points in NP. While in AF significant differences were found between day 7 to day 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/pubmed?term=((In+vitro+organ+culture+of+the+bovine+intervertebral+disc%3A+effects+of+vertebral+endplate+and+potential+for+mechanobiology+studies%5BTitle%5D)+AND+%22Spine+%28Phila+Pa++1976%29%22%5BJournal%5D)">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/pubmed?term=((Cryopreserved+intervertebral+disc+with+injected+bone+marrow-derived+stromal+cells%3A+a+feasibility+study+using+organ+culture%5BTitle%5D)+AND+%22Spine.+J%22%5BJournal%5D)">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/pubmed?term=((Biological+Response+of+the+Intervertebral+Disc+to+Repetitive+Short+Term+Cyclic+Torsion%5BTitle%5D)+AND+%22Spine+%28Phila+Pa+1976%29%22%5BJournal%5D)">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-Jünger, S., Grad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Confocal+imaging+protocols+for+live%2Fdead+staining+in+three-dimensional+carriers%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">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/pubmed?term=((Accuracy+of+three+techniques+to+determine+cell+viability+in+3D+tissues+or+scaffolds%5BTitle%5D)+AND+%22Tissue+Engineering+Part+C+Methods%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRasband%2C+WS+%22ImageJ%22">ImageJ</a>. , National Institutes of Health. Bethesda, Maryland, USA. (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/pubmed?term=((Establishment+of+a+novel+intervertebral+disc%2Fendplate+culture+model%3A+analysis+of+an+ex+vivo+in+vitro+whole-organ+rabbit+culture+system%5BTitle%5D)+AND+%22Spine%22%5BJournal%5D)">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., Grünhagen, T., Lee, C. R., van Donkelaar, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+in+vitro+organ+culturing+system+for+intervertebral+disc+explants+with+vertebral+endplates%3A+a+feasibility+study+with+ovine+caudal+discs%5BTitle%5D)+AND+%22Spine%22%5BJournal%5D)">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/pubmed?term=((Intervertebral+disc+cell+response+to+dynamic+compression+is+age+and+frequency+dependent%5BTitle%5D)+AND+%22J.+Orthop.+Res%22%5BJournal%5D)">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/pubmed?term=((Dynamic+compression+effects+on+intervertebral+disc+mechanics+and+biology%5BTitle%5D)+AND+%22Spine+%28Phila+Pa+1976%29%22%5BJournal%5D)">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/pubmed?term=((Development+of+an+intact+intervertebral+disc+organ+culture+system+in+which+degeneration+can+be+induced+as+a+prelude+to+studying+repair+potential%5BTitle%5D)+AND+%22European+Spine+Journal%22%5BJournal%5D)">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>

We have nothing to disclose.

The first step for successful organ culture is to make sure that the explant should not be contaminated. The tail should be skinned before you start with the procedure. Any animal hair brought into a sterile lab could be problematic in terms of contamination. The bovine tail should ideally be as fresh as possible (this affects initial cell viability). Furthermore, the betadine washing step is recommended to reduce the risk of contamination further. Instead of using a custom-made blade holder and a hammer to separate the ...

<table><tbody><tr><td>Fresh bovine intervertebral disc tissue from bovine tails, from local slaughter house (ideally within hours post-mortem and without skin). </td><td></td><td></td><td></td></tr><tr><td>Pulsavac Plus AC System</td><td>Zimmer inc., Switzerland</td><td>00-5150-486-01</td><td>Best performance with the hip-spray head and with AC power supply (the one with the 8 AA battery pack d&amp;#8211;s also work but is less convenient)</td></tr><tr><td>High Capacity Fan Spray w/Splash Shield, 12.7cm length </td><td>Zimmer inc., Switzerland</td><td>00-5150-175-00</td><td>There are several spray heads available, we tested this one successfully</td></tr><tr><td>Scalpel blades #22 and #10</td><td>Swann-Morton</td><td>#10: 0201 #22: 0208</td><td></td></tr><tr><td>Scalpel blade holder # 3 and #4</td><td>Hausmann, Germany</td><td>#3: 06.103.00 #4: 06.104.00</td><td></td></tr><tr><td>Lutz industrial blade</td><td>Lutz, Germany</td><td>1022.0884</td><td></td></tr><tr><td>Phosphate buffered Saline (PBS)</td><td>Invitrogen</td><td>10010-023</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>GIBCO, by Life Technologies</td><td>11960-044</td><td></td></tr><tr><td>Lactated Ringer&amp;rsquo;s solution (without glucose)</td><td>Bichsel, Switzerland</td><td>133 0002</td><td></td></tr><tr><td>6-well multi-well plate</td><td>Techno Plastic Products</td><td>92006</td><td></td></tr><tr><td>Betadine solution</td><td>Mundipharma, Switzerland</td><td>10055025</td><td></td></tr><tr><td>Surgical skin marker</td><td>Porex Surgical, Switzerland</td><td>9560</td><td></td></tr><tr><td>Large cutting board</td><td></td><td></td><td>Any brand is possible</td></tr></tbody></table>

preparation of intact bovine tail intervertebral discs for organ culture

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.

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

The overall goal of this procedure is to prepare intact intervertebral discs from bovine tail for culture and other downstream applications in our case to prepare intervertebral discs for culture in a bioreactor using complex mechanical loading. This is accomplished by first disinfecting the calf tail and then removing the muscles to obtain a clear view of the bony and disc structures. Next, a single intervertebral disc is cut keeping two to three millimeters of the partial vertebra attached on either side using the custom cutting tool.The third step is to clean the bony surfaces of the intervertebral disc with the jet lavage system, with ensure diffusion of small solut such as glucose into the center of the intervertebral disc. The final step is to place the intervertebral disc into a stabilizing buffer or wrap it in sterile gauze to keep it moist for downstream applications. Ultimately, the success of the procedure is assay using a live dead stain to check for the presence of live cells 14 or 21 days after beginning intervertebral disc culture.The following protocol is to show how invertible disc segments can be preserved for organ culture, which can help answering key questions in mechanobiology and tissue engineering. There's free reason why the bovine tail disc has become a kind of model system in tissue engineering. First, the cell density is very similar and very low like in a human introvert disc.Second, the cell population there is the absence of not cordal cells, which is quite similar, also like to the human situation. Thirdly, also the dimension and the biochemical composition is very similar to the human situation. Generally, individual new to this method will struggle because it is hard to distinguish the bony endplate from the vertebra bone, and hence one will find it difficult to ensure the presence of cartilage growth plate.We first had the idea of this method when we were looking for a fast and sterile technique to isolate the invertible disc with endplate for individual culture. Visual demonstration of these methods is important because it is difficult to locate a cutting site because the cutting site is located just a millimeter away from the boney plate. First, obtain a whole bovine tail from a local abattoir.Ideally, the tail should be no more than four hours postmortem and should be procured with the skin removed. Since the presence of skin increases the possibility of contamination, ensure that all instruments to be used in the dissection process are sterile. Also create a sterile workstation by placing an autoclave cutting board on the work surface.Then while working in a sterile lamina flow hood moisten a piece of sterile gauze with 0.9%sterile sodium chloride solution containing 55 millimolar sodium citrate. Place a piece of soaked gauze into each well of a six well plate. Next, fill a clean basin with one to 100 Betadine solution diluted with tap water and immerse the tail in the solution for five minutes.After soaking, remove the tail. Briefly dry it with sterile gauze and place it on the sterile workstation First, use a number 20 or number 22 scalpel to remove muscle from around the tail. Then use a guillotine to chop away any unwanted parts of the tail, usually from the proximal and distal lens, which contain relatively large and very small intervertebral discs.Then use number 10 scalpel to trim the muscles and tendons around the intervertebral discs. Be careful not to cut the outer annulus of the discs if necessary. For orientation purposes, mark the anterior of the intervertebral disc using a surgical skin marker.Next, gently move the tail to locate the intervertebral disc and vertebral connection point. Use the dull side of a scalpel blade to fill the border between the intervertebral disc and bone. Then locate the cleaving site, which should be one to two millimeters away from the intervertebral disc towards the vertebra.Place the custom made industrial blade holder on the cleaving site. This diagram very clearly shows the cleaving site, Cleve the intervertebral disc and vertebrae by hammering on top of the custom made industrial blade holder. Then move the blade holder to the other side of the intervertebral disc vertebrae connection and hammer through this side of the intervertebral disc vertebra connection to release the intervertebral disc.Then rapidly isolated intervertebral disc with sterile gauze moistened with 0.9%sodium chloride containing 55 millimolar sodium citrate. Repeat the process to obtain the desired number of intervertebral discs. Usually around six discs of 10 to 20 millimeters in diameter can be obtained from one tail.Once all of the invertible discs have been harvested, connect the jet lavage system to a three liter bag of sterile lactated ringer solution, sterile PBS or 0.9%sterile saline. Then hold an invertible disc with forceps and jet lavage both sides of the endplate surface for 30 seconds on each side while holding the jet lavage at a 30 to 60 degree angle in relation to the endplate surface, this image shows the effect of cleaning effect of the jet lavage. The top row of discs shows the invertible discs before cleaning, while the lower row shows the same discs after cleaning.Once the first intervertebral disc is washed, wrap it in moist and gauze while washing the other invertible discs. After washing the invertible discs are ready for organ culture. In addition, a diffusion experiment may be performed to determine the success of the invertible disc preparation while working in a laina flow hood.Prepare 40 milliliters of sterile DMEM culture media containing 5%fetal calf serum per disc. Transfer the media to a beaker and then submerge a single intervertebral disc in the media. Place the beaker on a shaking platform in a tissue culture incubator set to 37 degrees Celsius and 5%carbon dioxide.The intervertebral discs may be cultured in this manner for around 14 days. Use of these custom made specimen chambers maintains an appropriate mechanical load, which allows for an extended culture time. First, prepare 40 milliliters of sterile DMEM culture media containing 5%fetal calf serum per disc as before.Next, place the invertible disc into one of the custom-made culture chambers and add 40 milliliters of the freshly prepared media. Close the chamber under the flow hood. Place the custom made specimen chamber in a tissue culture incubator.Add weight to the chamber according to the surface area of the discs. First, prepare 100 milliliters of a 1%solution of a fluorescent dye, such as proso red in PBS per intervertebral disc. Then immerse the disc in the solution and incubate overnight under the same conditions.As for free swelling culture following the incubation, snap freeze the disc in liquid nitrogen and then immerse in minus 80 degrees Celsius acetone. Next, the intervertebral disc is brought slowly back to room temperature by 24 hour incubations in acetone at minus 20 degrees Celsius, four degrees Celsius, and finally, acetone at room temperature. After the disc is restored to room temperature, fully embed the disc in polymethyl methacrylate for one to two weeks according to standard procedures.Once fully embedded, the disc is processed by cutting with a sharp blade and polishing with a grinder. Once processed, the diffusion of pro cy red throughout the disc can be evaluated by microscopy. These images show the results of a free swelling diffusion experiment.Panel C shows the x-ray overview of an invertible disc with the two bony end plates prepared with our method. On the upper side, the growth plate was not removed imaged under fluorescent microscopy. It can be seen that the diffusion of the red fluorescent dye is blocked by the presence of the growth plate labeled gp, which thus acts as a barrier for staining the deeper layers of the cartilaginous end plate and the invertible disc.The growth plate lies between the primary and secondary centers of ossification labeled co. If the growth plate has been successfully removed, the red fluorescent dye can diffuse unhindered to the nucleus pulposus labeled np. These images demonstrate the cleaning effect of jet lavage treatment.The sagittal sections have a thickness of around 100 microns and the bony end plates are around 1.5 millimeters thick. Here, the intervertebral disc underwent jet lavage treatment and diffusion of the dye can be seen throughout the end plate. However, the dye does not diffuse well on the control side where jet lavage treatment has not been performed.Here we see live dead confocal microscope images taken from bovine invertible disc segments prepared with this jet lavage method and kept under free swelling conditions for the nucleus pole processis labeled np, the inner annulus fibrosis labeled IA and Thea annulus fibrosis labeled oa. The green regions are live cells stained by calcium acetyl methyl lester, and the red areas are the nuclei of dead cells stained by aum. Homodimer one here, images from the same regions demonstrate that a high number of viable cells indicated by green fluorescence remain after 14 days under free swelling conditions.This graph shows cell viability of the nucleus pulposus and annulus fibrosis regions of the intervertebral discs over the 21 day culture period mean results plus or minus the standard error of the mean for six samples. As shown. Statistical differences were tested using non-parametric Rosco Wallace signed ranked sum tests among groups.Significant differences were found between day 21 and all other time points in the nucleus pulsus while in the annulus fibrosis. Significant differences were found between day seven to day zero Following this procedure that this cultural condition can be modified in order to answer additional questions like the entrance of mechanical loading, nutritional, or growth factors on the progression of this degeneration or regeneration, Our protocol accelerates the harvesting technique, how interpretable disc segments can be obtained for tissue engineering approaches measuring physiology or mechanobiology. In traditional methods, there is a histological, bandsaw used or bearing technique, which use about two hours longer than our protocol.

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JoVE Journal

Bioengineering

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

This video demonstrates the establishment of primary human placental villous and decidual organ cultures in ex vivo conditions. These models are well-suited for studying pathogenesis at the human maternal-fetal interface.

Establishing Villous and Decidual Organ Cultures as Ex Vivo Models of Human Maternal-Fetal Interface

This video demonstrates a method to establish an infection model in placental tissues to study infection propagation. An ex vivo placental organ culture is infected with Listeria monocytogenes, a facultative intracellular parasite. The bacteria invade extravillous trophoblasts (EVTs) to enter the placental tissue and then propagate to other cells via cell-to-cell spread.

A Technique to Establish a Bacterial Infection Model in an Ex Vivo Organ Culture

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.

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

This video demonstrates the development of a pro-inflammatory and degenerative organ culture model for early-stage intervertebral disc (IVD) disease using TNF-α. It outlines the procedure for injecting TNF-α into the intervertebral disc and explains the mechanisms through which TNF-α induces inflammation and degradation of the IVD.

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

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.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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

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

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

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

This study presents a novel surgical technique for implanting a biomimetic artificial intervertebral disc (BioAID). The method ensures precise placement and stability, aiming to restore cervical spine function using a goat model. This technique shows promise for clinical application in both human and canine patients suffering from cervical spine myelopathy.

Surgical Technique for the Implantation of a Biomimetic Artificial Intervertebral Disc in a Goat Animal Model

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

Preparation of Complaint Matrices for Quantifying Cellular Contraction

Learn to prepare intact bovine tail intervertebral discs for organ culture using a custom cutting tool and jet lavage system. This method enables bioreactor studies of mechanical loading effects on spinal tissue.

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

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