generating a pro-inflammatory organ culture model to simulate an early intervertebral disc disease

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

Start with rinsing the entire bovine tail thoroughly with tap water to remove dirt and hair on the surface. Then, immerse the tail in 1% betadine solution for 10 minutes to disinfect the surface. Use a scalpel number 20 to remove the soft tissue from the caudal spine to facilitate the identification of the intervertebral discs or IVDs.
Remove the spinous and transverse processes of the vertebrae with a bone removal plier. Cut transversely with bone pliers through the middle of each vertebral body to obtain individual motion segments, then put the collected motion segments in a Petri dish with a gauze wetted in Ringer&#39;s solution.
Locate the IVD and vertebrae by moving the motion segments gently. Then, identify the location of the growth plate by touching and finding the convex side of the bony end plate. Cool the blade of the bandsaw with Ringer&#39;s solution, and use it to make two parallel cuts in the growth plate of the IVDs, one on each side.
Transfer the IVDs to a clean Petri dish with clean gauze wetted with Ringer&#39;s solution. Scrape off the vertebral body and growth plate using the scalpel blade, leaving the end plate intact. Position the two surfaces flat and parallel for the loading procedure, and transfer scraped IVDs to a fresh Petri dish with gauze wetted with Ringer&#39;s solution.
Measure the disc height and diameter with a caliper. Then, clean the blood clots in the vertebrae bone with Ringer&#39;s solution using a jet lavage system. Disinfect IVDs in PBS and 10% penicillin-streptomycin, with shaking for 15 minutes. Rinse off the high-concentration antibiotics with PBS and 1% penicillin streptomycin.
Transfer discs to IVD chambers containing 5 milliliters of IVD culture medium, and place them in the bioreactor system with an incubator at 37 degrees Celsius with 85% humidity and 5% carbon dioxide. Culture the discs for four days within a bioreactor system by maintaining different loading conditions according to the experimental groups.
In the physiological control group, culture the IVDs with high glucose medium using a loading protocol of 0.02 to 0.2 megapascals and 0.2 Hertz for two hours per day. In the pathological group, culture the IVDs with low glucose medium using a loading protocol of 0.32 to 0.5 megapascals and 5 Hertz for two hours per day.
Between the loading procedures, place the IVDs in six-well plates with 7 milliliters of IVD culture medium for free swelling recovery. Measure the disc height daily with a caliper after the free swelling period and after dynamic loading for the experimental duration.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Dissection of the bovine intervertebral disc
<ol>
	<li>Rinse the whole tail thoroughly with tap water to remove dirt and hair on the surface. 
	NOTE: With intact, distal ends, a maximum of 9 intervertebral discs (IVDs) (coccygeal 1-9) per tail can be used for the experiments depending on the desired size of the IVDs. Considering the desired diameter between 15-20 mm, we used 12 bovine tails with 5 IVDs per tail for the experiments.</li>
	<li>Immerse the whole tail in a box containing 1% betadine solution for 10 min. Briefly dry the tail with sterile gauze and place it on a sterile drape. 
	NOTE: During dissection of the disc, humidify the tails with Ringer&#39;s solution wetted gauze to prevent dehydration. Store the tails (or left-over segments) wrapped in wet gauze until the whole dissection procedure is completed.</li>
	<li>Use a scalpel (No. 20) to remove the soft tissue as completely as possible from the caudal spine to facilitate the identification of the IVDs. Remove the spinous and transverse processes of the vertebrae with bone removal pliers. 
	NOTE: Select IVDs with the desired diameter. IVDs with a diameter range of 15-20 mm were used in the current study.</li>
	<li>Cut transversely with bone pliers through the middle of each vertebral body to obtain individual motion segments. Put motion segments in a Petri dish with gauze wetted with Ringer&#39;s solution.</li>
	<li>Locate the IVD and vertebra by palpation and by moving the motion segments gently. Make two parallel cuts with the band saw in the growth plate of the IVDs, one on each side of the IVD. Identify the location of the growth plate by touching and finding the convex site of the bony endplate part (hard) adjacent to the disc (soft) with a safety distance of approximately 0.5-1 mm from the IVD towards the vertebra. Ensure that the blade of the band saw is cooled with Ringer&#39;s solution while cutting the vertebrae.</li>
	<li>Transfer IVDs in a clean Petri dish with clean gauze wetted with Ringer&#39;s solution. 
	NOTE: The gauze should be moistened and not too wet to prevent swelling of the IVDs,</li>
	<li>Use the scalpel blade to scrape off the vertebral body (red/pink bone), growth plate (white cartilage), leave the endplate intact (yellow-pink). Make the two surfaces flat and parallel for the loading procedure. Transfer scraped IVDs to a fresh Petri dish with gauze wetted with Ringer&#39;s solution. 
	NOTE: Wear a chainmail glove to protect the hand while holding the IVD and scraping.</li>
	<li>Measure the disc height and diameter with a caliper. Clean the blood clots in the vertebrae bone with Ringer&#39;s solution using a jet lavage system.</li>
	<li>Transfer the IVDs to 50 mL plastic tubes, one IVD per tube. Add 25 mL of Phosphate-Buffered Saline (PBS) + 10% Penicillin/Streptomycin (P/S) per IVD and leave it shaking for 15 min on an orbital shaker at room temperature.</li>
	<li>Aspirate the supernatant and add 10 mL of PBS + 1% P/S per IVD for 2 min to rinse the IVDs.</li>
</ol>
2. IVD culture and loading
<ol>
	<li>Transfer discs to IVD chambers and add IVD culture medium (Dulbecco&#39;s Modified Eagle Medium (DMEM, 4.5 g/L high glucose DMEM for the physiological group and 2 g/L low glucose DMEM for the pathological group) + 1% P/S + 2% fetal calf serum + 1% ITS (contains 5 &#181;g/mL insulin, 6 &#181;g/mL transferrin, and 5 ng/mL selenious acid) + 50 &#181;g/mL ascorbate-2-phosphate + 1% non-essential amino acid + 50 &#181;g/mL antimicrobial agent for primary cells) and place in an incubator at 37 &#176;C, 85% humidity and 5% CO2.</li>
	<li>Culture the discs for 4 days within a bioreactor system according to experimental groups. In the pathologic group, maintain degenerative loading conditions at 0.32-0.5 MPa, 5 Hz for 2 h/day. In the physiological control group, use a loading protocol of 0.02-0.2 MPa, 0.2 Hz for 2 h/day. 
	NOTE: Position the IVDs in chambers containing 5 mL of IVD medium during the loading procedures. The volume depends on the size of the bioreactor&#39;s loading chambers. Between the loading procedures, place the IVDs in six-well plates with 7 mL of IVD culture medium for free-swelling recovery.</li>
	<li>For analyzing the changes in disc height during the experimental period, measure the disc height with a caliper after IVD dissection (baseline) and then daily after the free swelling period and after dynamic loading for the experimental duration.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ascorbate-2-phosphate</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Band saw</td>
			<td>Exakt Apparatebau, Norderstedt, Germany</td>
			<td>model 30/833</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Munndipharma, Frankfurt, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning ITS Premix</td>
			<td>Corning Inc., New York, USA</td>
			<td>354350</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Gibco by life technologies, Carlsbad, USA</td>
			<td>10741574</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM low glucose</td>
			<td>Gibco by life technologies, Carlsbad, USA</td>
			<td>11564446</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol for molecular biology</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>09-0851</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco by life technologies, Carlsbad, USA</td>
			<td>A4766801</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acid solution</td>
			<td>Gibco by life technologies, Carlsbad, USA</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin(P/S)</td>
			<td>gibco by life technologies, Carlsbad, USA</td>
			<td>11548876</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution, tablet</td>
			<td>Sigma-Aldrich, St. Louis, USA</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen, Sandiego, USA</td>
			<td>ant-pm-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulsavac Jet Lavage System</td>
			<td>Zimmer, IN,USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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

Take a bovine tail.
Remove the overlying tissue and vertebral processes, then cut transversely to obtain segments.
Identify the intervertebral disc or IVD, located between the growth plates and vertebral end plates.
Cut on either side of the IVD and place it in a dish containing gauze soaked in buffer to maintain tissue viability.
Scrape off any attached vertebral tissue and measure the IVD height.
Wash with buffer to remove blood remnants and disinfect the IVD in a buffer containing antibiotics.
Place the IVDs in bioreactor chambers containing media.
The media in the physiological group has high glucose, while in the pathological group, a low-glucose medium induces nutrient deprivation.
Place the chambers in the bioreactor.
For the physiological IVD, apply a gentle load daily. For the pathological IVD, apply an intense load at a higher rate daily.
Between the daily loading tests, allow the IVDs to recover in media.
After all loading procedures, measure the IVD heights.
A reduced height in the pathological IVDs indicates a degenerative and pro-inflammatory condition.&#160;

11 ビュー. 初期の椎間板疾患をシミュレートするための炎症誘発性臓器培養モデルの生成

ビデオ: 初期の椎間板疾患をシミュレートするための炎症誘発性臓器培養モデルの生成 - 実験

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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

アン<em&gt;インビトロ</emマウス椎間板の&gt;器官培養モデル

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

JoVE Journal

生物工学

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

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

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

側方腹腔ドリルビット傷害を利用した羊の腰椎椎間板変性モデル

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

医学

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

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

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

胚発生から変性までの椎間板の光学断面化と可視化

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

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

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