This work was supported by the Washington University Musculoskeletal Research Center (NIH P30 AR057235), Molecular Imaging Center (NIH P50 CA094056), Mechanobiology Training Grant (NIH 5T32EB018266), NIH R21AR069804, and NIH K01AR069116. The authors would like to thank Patrick Wong for his contributions in data collection.

All animal experiments were performed in compliance with the Washington University in St. Louis Animal Studies Committee.
1. Animals
<ol>
	<li>Obtain two strains of mice: 10-week old BALB/c (n = 6, BALB-M, BALB/cAnNTac) and 10-week old nuclear factor kappa-B-luciferase reporter animals (NF-&#954;&#946;-luc) bred on a BALB/c background (n = 6, BALB/c-Tg(Rela-luc)31Xen).</li>
	<li>Prior to dissection, euthanize animals with CO2 overdose at a flow rate of 2.5-3 L/min for 5 min followed by an additional 2 min of dwelling time.</li>
	<li>Disinfect the outside region of the animal by bathing it in a 70% ethanol bath for 2 min before dissection.</li>
</ol>
2. Dissection
<ol>
	<li>Make a longitudinal vertical cut on the dorsal surface of the mouse using small dissection scissors to expose the body cavity.</li>
	<li>Make two longitudinal vertical cuts on either side of the animal&#39;s spine starting from the first lumbar vertebrae (L1) to the caudal vertebrae (C8).</li>
	<li>Using a scalpel, fine forceps, and fine dissection scissors, carefully remove the spine from L1-C8 from the animal&#39;s body cavity.</li>
	<li>Carefully remove excess soft tissues surrounding the spinal column, while ensuring to not scrape or injure the IVD on the ventral side.</li>
	<li>Further dissect the spinal column into vertebrae-disc-vertebrae functional spinal units (FSUs) at the L1/L2, L3/L4, L5/L6, C3/C4, C5/C6, and C7/C8 discs.</li>
	<li>Rinse the FSUs in Hank&#39;s balanced salt solution supplemented with 1% penicillin-streptomycin for 2 min.</li>
	<li>Randomly assign the FSUs into three groups: uncultured and untreated FSUs (Fresh), cultured but untreated FSUs (Control), and cultured and stab treated FSUs (Stab).</li>
	<li>Snap freeze the Fresh FSU samples with liquid nitrogen immediately after dissection and store in a -20 &#176;C freezer.</li>
</ol>
3. Organ Culture Conditions
<ol>
	<li>Prepare 500 mL of sterile culture media of 1:1 Dulbecco&#39;s modified Eagle&#39;s medium: Nutrient Mixture F-12 (DMEM:F12) supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin.</li>
	<li>Using 10 mL serological pipettes, pipette 2 mL of culture media into each well of a 24-well culture plate.</li>
	<li>Following dissection and rinse, place each FSU into an individual well in the media-filled 24-well plate.</li>
	<li>Incubate the samples in a sterile incubator that maintains 37 &#176;C, 5% CO2, 20% O2 and &#62; 90% humidity.</li>
	<li>After an initial culture period of 24 h, mechanically injure the Stab group samples via needle puncture of the annulus fibrosus using a sterile 27-gauge needle as shown in Figure 1A.</li>
	<li>Change media every 48 h by preparing a new media-filled 24-well culture plate, and transfer samples from the old plate to the new using sterile tweezers. Aspirate the old media and dispose of the old culture plate in a biohazard waste bin.</li>
	<li>On the final day of culture, incubate all samples in media containing 0.75 mg/mL nitro blue tetrazolium chloride for 24 h.</li>
	<li>Afterwards, freeze all the FSUs with liquid nitrogen and store in a -20 &#176;C freezer until ready for mechanical testing or histology (Figure 1B).</li>
</ol>
4. Longitudinal Measurements NF-&#954;B
<ol>
	<li>Image the FSUs from NF-&#954;&#946;-luciferase animals at 1, 5, 13, and 19 days to assess NF-&#954;B expression.</li>
	<li>Add 10 &#181;L of 1 mg/mL luciferin solution to each well and incubate in a sterile incubator at 37 &#176;C for 10 min.</li>
	<li>Image the samples for bioluminescence using the imaging system with a 1 min exposure time and bin setting of 2.</li>
	<li>Concurrently, use the machine to take a photograph and overlay it with the bioluminescence image to determine the anatomical location of luminescence in the IVD.</li>
</ol>
5. Mechanical Assessment
<ol>
	<li>Determine the mechanical behavior of the IVDs by using displacement-controlled dynamic compression9.</li>
	<li>Using a dissection microscope, scalpel, and tweezers, remove the bony vertebral bodies of the FSU from each sample while keeping the cartilaginous endplates intact and attached to the IVD.</li>
	<li>Attach the isolated IVDs to a 1 cm x 1 cm x 0.2 cm aluminum plate using cyanoacrylate glue.</li>
	<li>Measure the disc height and width by taking an average of three measurements on the longitudinal axis of each disc using a laser micrometer. Calculate the disc height ratio by dividing the average disc height by the maximum disc width.</li>
	<li>Use the measured disc height to calculate the input strain values used in mechanical testing. Note that the average disc height was approximately 690 &#181;m &#177; 39 &#181;m.</li>
	<li>Place the samples in a phosphate buffered saline bath under the compression machine and preload the disc to 0.02 N.</li>
	<li>Cyclically compress the disc using a sinusoidal waveform at 1 Hz for 20 cycles at the 1% strain level and 5% strain level for 3 trials each and record the load and displacement values of the IVD. Allow 10 min of resting time between trials for the disc to relax and to prevent injury.</li>
	<li>Calculate the average stiffness from the loading phase of the last cycle, and calculate the loss tangent from the phase angle between load and displacement data.</li>
</ol>
6. Proteoglycan and Collagen Quantification
<ol>
	<li>Following mechanical testing, measure the disc wet weight by placing the isolated IVD in a pre-tared centrifuge tube and measuring the weight using an analytical balance.</li>
	<li>Digest the isolated IVDs in 250 &#181;L papain digestion solution (0.1 M sodium acetate, 0.01 M EDTA, 0.005 M cysteine HCl, pH 6.4) overnight at 65 &#176;C using a block heater.</li>
	<li>Centrifuge samples for 10 min at 2,000 x g and collect the supernatant fluid.</li>
	<li>Measure the proteoglycan content of the IVD by using the dimethylmethylene blue (DMMB) assay with chondroitin sulfate standards.</li>
	<li>Prepare DMMB solution (21 mg DMMB, 5 mL absolute ethanol, 2 g sodium formate in 1 L ddH2O).</li>
	<li>Pipette 30 &#181;L of standards and samples in triplicate in a 96-well plate, along with 250 &#181;L of DMMB solution in each well.</li>
	<li>Read the absorbance of the plate at 525 nm using a spectrophotometer.</li>
	<li>Measure the collagen content using a hydroxyproline assay kit according to manufacturer instructions.</li>
</ol>
7. Histology
<ol>
	<li>Fix a subset of samples in 4% paraformaldehyde for 24 h, de-calcify in 5% formic acid for 48 h, dehydrate with ethanol, and embed in paraffin.</li>
	<li>Using a microtome, section the samples sagittally at 10 &#181;m thickness and apply sections to glass slides. Stain the sections using Safranin-O/Fast Green and DAPI.</li>
	<li>Using a light microscope, image slides at 10X magnification.</li>
</ol>
8. Contrast-enhanced microComputed Tomography (microCT)
<ol>
	<li>Scan a subset of samples at the 0, 2, 5, and 7-day time points using longitudinal Ioversol contrast-enhancement10, and terminal phosphomolybdic acid (PMA) contrast-enhancement at the 7 day time point.</li>
	<li>4 h prior to microCT scan time, add 300 &#181;L of 350 mg/mL iodine Ioversol solution to the culture media for a final concentration of approximately 50 mg/mL iodine-containing Ioversol solution and incubate in a sterile incubator at 37 &#176;C for 4 h.</li>
	<li>Following incubation, wrap the sample in sterile gauze and place in a 1 mL microcentrifuge tube.</li>
	<li>Place samples in the microCT system and scan at 45 keVp, 177 &#181;A, 10.5 &#181;m voxel size, and 300 ms integration.</li>
	<li>Export data from the microCT as DICOM files and visualize using software (e.g., OsiriX).</li>
	<li>At the 7-day time point, fix samples using a 4% paraformaldehyde solution for 24 h, followed by 5% PMA solution for 3 days, and scan using the same settings as those used in step 8.4.</li>
</ol>
9. Three Dimensional Finite Element Modeling
<ol>
	<li>Segment the DICOM files of the IVDs using software with manual thresholding (e.g., OsiriX).</li>
	<li>Convert the NP and AF volumes of the IVD into voxel-based finite element meshes using Meshlab.</li>
	<li>Combine the microstructures of the NP and AF to form a complete IVD and apply experimentally determined boundary conditions in the finite element software.</li>
</ol>

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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+functional+studies+of+the+intervertebral+disc:+A+novel+murine+intervertebral+disc+model.">Genetic and functional studies of the intervertebral disc: A novel murine intervertebral disc model.</a> PLoS ONE. 9 (12), (2014).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+investigation+of+intervertebral+disc+degeneration+produced+by+needle-stab+injury+of+the+rat+caudal+spine.">Time course investigation of intervertebral disc degeneration produced by needle-stab injury of the rat caudal spine.</a> J Neurosurg: Spine. 15 (4), 404-413 (2011).</li><li>Liu, J. W., Abraham, A. C., Tang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+high-throughput+phenotyping+of+the+viscoelastic+behavior+of+whole+mouse+intervertebral+discs+using+a+novel+method+of+dynamic+mechanical+testing.">The high-throughput phenotyping of the viscoelastic behavior of whole mouse intervertebral discs using a novel method of dynamic mechanical testing.</a> J Biomech. 48 (10), 2189-2194 (2015).</li><li>Lin, K. H., Wu, Q., Leib, D. J., Tang, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+the+contrast-enhanced+microCT+imaging+of+murine+intervertebral+discs.">A novel technique for the contrast-enhanced microCT imaging of murine intervertebral discs.</a> J Mech Behav Biomed Mater. 63, (2016).</li><li>Pasparakis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tissue+homeostasis+by+NF-kappaB+signalling:+implications+for+inflammatory+diseases.">Regulation of tissue homeostasis by NF-kappaB signalling: implications for inflammatory diseases.</a> Nat Rev: Immunol. 9 (11), 778-788 (2009).</li><li>O&#8217;Connell, G. D., Vresilovic, E. J., Elliott, D. 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The authors of this manuscript declare that they have no competing financial interests.

This protocol outlines an organ culture of the murine FSU with emphasis on monitoring the biological changes in the IVD. The successful maintenance of these cultures requires careful sterile techniques. In particular, the dissection steps 2.1-2.6 and the culture steps 3.1-3.6 require special care to ensure sterile conditions are maintained, and these steps should be performed preferably in an isolated procedure room with a HEPA airflow to minimize contaminants. Because the dissection proc...

Low back pain (LBP) is the leading factor for global disability and lost productivity in the workplace, and Americans alone spend in excess of 50 billion dollars on LBP treatment1. Although prevalent, the etiology of LBP remains complex and multifactorial. However, intervertebral disc (IVD) degeneration is among the most significant risk factors for LBP2.
The IVD is made of three microstructures: the exterior annulus fibrosis (AF), the interior nucleus pulposus (NP), and two cartilaginous endplates that sandwich the whole structure proximally and distally3. With aging and degeneration, the IVD components change structurally, compositionally, and mechanically4. These changes include the loss of proteoglycans and hydration in the NP, decreased disc height, and deteriorated mechanical competence5. These alterations are often accompanied by cytokines that promote an inflammatory response, as well as neutrophil and dorsal root ganglion intrusion into the joint space culminating in a cascade of events that lead to LBP symptoms6.
Studying the mechanisms of IVD degeneration is challenging in humans because it is often not possible to isolate the cause of the degeneration before the occurrence of low back pain. Thus, a reductionist approach of simplifying the experimental system down to the IVD organ allows mechanistic fine-tuning of causal variables and examining their downstream effects5. The system is reduced to only the native cell population and surrounding extracellular matrix, thus enabling the direct interpretation of the effects of external stimuli on IVD degeneration. Moreover, the lower cost and scalability of murine models, as well as the large number of genetically modified animals7, allow for the rapid targeted screening of IVD degenerative mechanisms and potential therapies. Here, we describe a murine organ culture system in which IVD cellular and tissue stability is maintained over 21 days, with specific focus given to the IVDs&#39; homeostatic, mechanical, structural, and inflammatory patterns. Using this method, we monitor the IVDs&#39; functional changes in a stab-induced injury model8 to understand the mechanisms behind disc degeneration. Furthermore, we describe several applications of this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution modeling of the IVD.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well plate</td>
			<td>Midwest Scientific</td>
			<td>TP92096</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Midwest Scientific</td>
			<td>TP92024</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>25 ml pipettes</td>
			<td>Midwest Scientific</td>
			<td>TP94024</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>10 ml pipettes</td>
			<td>Midwest Scientific</td>
			<td>TP94010</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>5 ml pipettes</td>
			<td>Midwest Scientific</td>
			<td>TP94005</td>
			<td>Used for organ culture</td>
		</tr>
		<tr>
			<td>500 ml bottle top filters, 22um</td>
			<td>Midwest Scientific</td>
			<td>TP99505</td>
			<td>Used for filter media</td>
		</tr>
		<tr>
			<td>10 ul pipette tips</td>
			<td>Midwest Scientific</td>
			<td>NP89140098</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>200 ul pipette tips</td>
			<td>Midwest Scientific</td>
			<td>NP89140900</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>1000 ul pipette tips</td>
			<td>Midwest Scientific</td>
			<td>NP89140920</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>DMEM /F-12</td>
			<td>Invitrogen</td>
			<td>11330032</td>
			<td>Used for culture media</td>
		</tr>
		<tr>
			<td>Optiray 350</td>
			<td>Guebert</td>
			<td>19133341</td>
			<td>Used for contrast enhanced microCT</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F2442</td>
			<td>Used for culture media</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin&#160;</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td>Used for culture media</td>
		</tr>
		<tr>
			<td>Tetrazolium Blue Chloride</td>
			<td>Sigma</td>
			<td>T4375</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>Sigma</td>
			<td>L6152</td>
			<td>Used for bioluminescence imaging</td>
		</tr>
		<tr>
			<td>Chondroitin Sulfate</td>
			<td>Sigma</td>
			<td>C9819</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>10% Phosphomolybdic Acid Solution</td>
			<td>Sigma</td>
			<td>HT152</td>
			<td>Used for contrast enhanced microCT</td>
		</tr>
		<tr>
			<td>Safranin O</td>
			<td>Sigma</td>
			<td>S8884</td>
			<td>diluted to .1% concentration (in water)</td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma</td>
			<td>F7258</td>
			<td>.001% concentration</td>
		</tr>
		<tr>
			<td>Papain from papaya latex</td>
			<td>Sigma&#160;</td>
			<td>P3125</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>Nucleic acid staining</td>
		</tr>
		<tr>
			<td>Cyanoacrylate Glue</td>
			<td>Loctite</td>
			<td>234790</td>
			<td>Adhesive&#160;</td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge Tubes&#160;</td>
			<td>Fischer Scientific</td>
			<td>S348903</td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>Big Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioDent</td>
			<td>ActiveLife</td>
			<td></td>
			<td>For mechanical testing</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>Biotek</td>
			<td></td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>AxioCam503</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>VivaCT40</td>
			<td>Scanco</td>
			<td></td>
			<td>MicroCT</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Denver Instrument Company</td>
			<td>A-200DS</td>
			<td>Analytical balance</td>
		</tr>
		<tr>
			<td>Incubator HERAcell 150i</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Organ Culture</td>
		</tr>
		<tr>
			<td>Dissection Scope</td>
			<td>VistaVision</td>
			<td></td>
			<td>Used during dissection</td>
		</tr>
		<tr>
			<td>Laser Micrometer</td>
			<td>Keyence</td>
			<td>LK-081</td>
			<td>Measuring disc height</td>
		</tr>
		<tr>
			<td>Microcentrifuge 5810 R</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Used for biochemical assays</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica&#160;</td>
			<td>RM2255</td>
			<td>Used for histology</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 7</td>
			<td>GraphPad</td>
			<td></td>
			<td>For statistics</td>
		</tr>
		<tr>
			<td>MATLAB R2014a</td>
			<td>Mathworks</td>
			<td></td>
			<td>For modeling</td>
		</tr>
		<tr>
			<td>Osiri-LXIV</td>
			<td>Pixmeo</td>
			<td></td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>MeshLab v1.3.3</td>
			<td>Visual Computing Lab - ISTI - CNR</td>
			<td></td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>PreView/FEBio 2.3</td>
			<td>Utah MRL &#38; Columbia MBL</td>
			<td></td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Windows</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cohan-Vannas Spring Scissors&#160;</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>15000-02</td>
			<td>Or any nice pair of spring scissors</td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp&#160; (small)</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp&#160; (larger)</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>14060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>11252-40</td>
			<td>At least 2; can also use #3&#160;</td>
		</tr>
		<tr>
			<td>Extra Fine Graefe Forceps, serrated</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>11150-10</td>
			<td>At least 2</td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps, serrated</td>
			<td>World Precision Instruments</td>
			<td>503719-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps, teeth</td>
			<td>World Precision Instruments</td>
			<td>501244</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle - #3</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle - #4</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>10004-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blades - #23</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>10023-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Insect Pins , size 000</td>
			<td>Fine Science Tools&#160;&#160;</td>
			<td>26000-25</td>
			<td></td>
		</tr>
		<tr>
			<td>27G Needle</td>
			<td>BD PrecisionGlide Needles</td>
			<td>BD305109</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro organ culture model of the murine intervertebral disc

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.

Figures 2-3 show representative results of proteoglycan distribution, NF-&#954;B expression, stiffness, viscosity, disc height, and wet weight for cultured mouse IVDs. If cultured properly, the IVD parameters of the Control group should not be significantly different from the Fresh group. If the culture is infected or otherwise compromised, the Control group will be different from the Fresh group, especially in NF-&#954;B expression and proteoglycan distribution (results not shown). Figures 4-5</...

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

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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

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

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

Research

JoVE Journal

Bioengineering

9.4K Views.  Washington University in St. Louis. 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.

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

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

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

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

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 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  ...

Tissue Engineering of the Intestine in a Murine Model

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

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

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

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

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

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

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

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

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

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

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

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

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

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...