generating an organ culture model to simulate early-stage intervertebral disc disease

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

After the first dynamic loading cycle on day one, directly place the IVDs in a Petri dish in a vertical position and stabilize them with a tweezer. Inject recombinant TNF alpha with a 30-gauge insulin needle, slowly at a speed of approximately 70 microliters per minute into the IVDs of the pathological group. After injecting, pull the syringe halfway back and pull the plunger to create a vacuum that prevents the injected solution from leaking. Then, remove the syringe completely from the IVD.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Intervertebral disc (IVD) culture and loading
<ol>
	<li>Transfer the intervertebral discs (IVDs) 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% Penicillin/Streptomycin + 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&#160;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>
2. Intradiscal tumor necrosis factor-alpha (TNF-&#945;) injection
<ol>
	<li>Directly after the first dynamic loading cycle on day 1, place the IVDs in a Petri dish in a vertical position and stabilize the IVDs with a tweezer.</li>
	<li>Inject recombinant TNF-&#945; (100 ng in 70 &#181;L of phosphate-buffered saline (PBS) per IVD) with a 30-gauge insulin needle into the nucleus pulposus tissue of the pathological group. Inject slowly at a speed of approximately 70 &#181;L in 1 min.</li>
	<li>After injection, pull the syringe halfway back within the IVD and pull the syringe plunger to create a vacuum that prevents the injected solution from leaking back before removing the needle and syringe completely from the IVD. 
	&#8203;NOTE: Perform a pilot experiment by injecting PBS containing trypan blue dye to evaluate the distribution of the injected solution after loading and overnight culture.</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>TNF-alpha, recombinant human protein</td>
			<td>R&#38;D systems, Minnesota, USA</td>
			<td>210-TA-005</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine IL-8 Do.it-Yourself ELISA</td>
			<td>Kingfisher Biotech, St. Paul, USA</td>
			<td>DIY1028B-003</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>
	</tbody>
</table>

Source: Saravi, B. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62100">A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.</a>&#160;J. Vis. Exp. (2021).
This video demonstrates the development of a pro-inflammatory and degenerative organ culture model for early-stage intervertebral disc (IVD) disease using TNF-&#945;. It outlines the procedure for injecting TNF-&#945; into the intervertebral disc and explains the mechanisms through which TNF-&#945; induces inflammation and degradation of the IVD.

Source: Saravi, B. et al., A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.&#160;J. Vis. Exp. ...

Take an intervertebral disc (IVD), a cushion-like structure located between the vertebrae, from a bovine tail.
Position it vertically and inject recombinant TNF-&#945;, a pro-inflammatory cytokine, into the nucleus pulposus (NP), the gel-like core.
Withdraw the syringe halfway and pull the plunger to create a vacuum, preventing TNF-&#945; backflow. Then, remove the syringe.
Place the IVD in a bioreactor chamber containing media, and apply an intense pressure load daily.
TNF-&#945; binds to its receptors on NP cells, triggering a signaling cascade.
This activates initiator and executioner caspases, which degrade cellular proteins and induce apoptosis.
TNF-&#945; also activates the transcription factor NF-&#954;B, which induces inflammatory gene expression, leading to the release of enzymes that degrade the IVD&#39;s extracellular matrix.
Additionally, the intense pressure load induces mechanical stress, further degrading the IVD.
This reduces the IVD&#39;s height in comparison to untreated controls, establishing a degenerative and proinflammatory model of early-stage IVD disease.

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

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.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

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

科研

JoVE Journal

生物工程

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.

Ovine Lumbar椎间盘退变模型利用横向腹膜后位钻伤

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

医学

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

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

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