We thank our co-authors from the original publications for their help and support. We thank Charlotte Emma Bamberger for helping to acquire the apotome images.

For the analysis of embryonic development and maturation, bovine discs were used. To evaluate degeneration of the IVD, human samples were analyzed.
Human IVD tissue was obtained from patients undergoing surgery for lumbar disc degeneration, disc prolapse, or spinal trauma in the Department of Orthopaedic Surgery, University Hospital of T&#252;bingen and the BG Trauma Centre T&#252;bingen. Full ethical committee approval was obtained before the commencement of the study (project number 244/2013BO2). Written informed consent was received from all patients before participation. The methods were carried out in accordance with the approved guidelines.
Bovine tissue was obtained from the Bavarian State Office for Health and Food Safety/Oberschlei&#223;heim and from a rendering plant in Warthausen (Germany). Local and veterinary authorities&#39; approval was received for tissue from dead animals.
1. Sample harvest
<ol>
	<li>Human IVD tissue: Place intraoperatively-obtained IVD samples immediately in Dulbecco&#39;s Modified Eagle Medium (DMEM) with 2% (v/v) of penicillin-streptomycin and 1.2% of (v/v) amphotericin B. Store at 4 &#176;C until further processing. Process the tissue within 48 h. Alternatively, store the tissues at -20 &#176;C for several weeks.</li>
	<li>Bovine IVD tissue: Ensure to harvest the tissue from the animals within 24 h after death. 
	Resect the bovine discs with the surrounding vertebrae from the dead animals en-bloc. Transport the frozen tissue on dry ice and store at -20 &#176;C until further processing. 
	&#8203;NOTE: If only fluorescence analyses are intended and no further biochemical quantification methods such as ELISA or PCR are planned, perform the tissue fixation as explained below. This allows to keep the tissue longer in storage before it needs to be processed. To prevent deterioration of the tissue matrix, perform fixation within 48 h after harvest unless the tissue is frozen directly.</li>
</ol>
2. Sample preparation
<ol>
	<li>Thaw the frozen tissue at room-temperature. Process the tissue as soon as no ice-crystals can be felt any more upon digital gentle compression of the tissue. 
	NOTE: Perform preparation of the tissue in DMEM in a Petri dish.</li>
	<li>Identify the origin of the human IVD tissue (anulus fibrosus, intermediate zone, nucleus pulposus, or cartilaginous endplate) based on macroscopic properties such as collagen density and orientation.</li>
	<li>Take the motion segment consisting of the bovine IVD disc with its two adjacent vertebrae and dissect the disc as a whole from the subchondral bone using a surgical blade (blade number 15).
	<ol>
		<li>Use two anatomic forceps to flip the disc to reach areas more centered. Perform the dissection. Ensure to resect the nucleus pulposus last as it is much thinner than the anulus, prone to tear, and it does not come off in a defined fashion easily.</li>
		<li>Identify the different areas of the cartilage.</li>
		<li>Cut out the area of interest from the whole disc using a surgical blade (blade number 20 or 22). Alternatively, use a cryotome blade. 
		NOTE: As bovine discs come en-bloc as a part of the spine, the discs can be prepared in-toto. This makes correct identification of the different types of cartilage much easier. When dissecting the disc in the fashion described above, the cartilaginous endplate remains on the vertebrae. If this area shall be investigated, it is best taken off the underlying bone with a chisel working in a slightly bent tangential direction.</li>
	</ol>
	</li>
	<li>In case that embryos are of a crown-rump length of smaller than 20 cm, ensure to process the embryos in toto to preserve the tissue architecture of the IVD. Do not perform any dissection of the vertebrae in these cases.</li>
	<li>Once the disc has been resected in-toto, identify the different areas of the cartilage.
	<ol>
		<li>Perform the decalcification in ethylenediaminetetraacetic acid (EDTA) (20% (w/v); pH = 7.4) at room temperature. Choose the volume depending on the sample size - the entire tissue must be well covered with EDTA.</li>
		<li>Change the decalcification solution daily which can go for up to 5 days depending on the size of the tissue.</li>
		<li>Verify that the decalcification is successful with a 20 Gauge needle that penetrates the vertebrae without notable resistance. 
		NOTE: The daily change of decalcification solution is important to prevent the chelate binder EDTA from saturation to maintain reaction effectivity. It also prevents bacterial colonization.</li>
	</ol>
	</li>
</ol>
3. Grading of sample age, integrity, and degeneration
<ol>
	<li>Classify the human disc tissue into one of the five following categories with the help of clinical information as well as X-ray and magnetic resonance imaging18 (Figure 3). 
	NOTE: Category I: To serve as near-healthy sample use anulus without any radiological sign of IVD degeneration derived from acute spinal trauma. 
	Category II: To illustrate a situation of acute inflammation with beginning degeneration use tissue from the intermediate zone from patients with clinical symptoms with a duration of less than 4 weeks. 
	Category III: To describe a situation where the inflammatory reaction had already had time to affect the tissue and cells take tissue from patients who were operated for a nuclear prolapse but with a symptom duration exceeding 4 weeks. 
	Category IV: For moderate disc degeneration select anulus obtained from surgery with interbody fusion for degenerative disc disease with a Pfirrmann score of 3 or 4 in magnetic resonance imaging19. 
	Category V: For end stage degeneration process anulus obtained from surgery with interbody fusion for degenerative disc disease with a Pfirrmann score of 5.</li>
	<li>Classify the bovine tissue based on the developmental stage/age of the animal into one of the eight categories, as displayed in Table 1.
	<ol>
		<li>Calculate the gestation age on crown-rump length of the embryos based on the formula suggested by Keller: 
		Gestation age in months = <img alt="Equation 1" src="/files/ftp_upload/62594/62594eq1v3.jpg" /> 
		&#8203;NOTE: Animals in the first 4 weeks of gestation present with a crown-rump length of 0.8-2.2 cm20.</li>
	</ol>
	</li>
</ol>
4. Tissue fixation
<ol>
	<li>Fixate the samples in 10x the volume of the sample of 4% (w/v) formaldehyde solution in phosphate buffered saline (PBS) over night at 4 &#176;C. 
	NOTE: The formaldehyde solution penetrates tissue at a rate of about 1 mm/hour from each direction. For very small or very big samples an adjustment of the exposure time could be necessary.</li>
	<li>Store the tissue in PBS at 4 &#176;C until further processing.</li>
</ol>
5. Histologic sectioning
<ol>
	<li>Embed the samples in water-soluble embedding medium on the cryotome knob.
	<ol>
		<li>Place the tissue onto the knob such that either an axial plane is generated or a plane that cuts the collagen type I lamellae perpendicular (e.g., a median sagittal sectioning plane). 
		NOTE: The tissue has to be fully covered by the embedding medium.</li>
	</ol>
	</li>
	<li>Section the embedded tissue at a thickness of 70 &#181;m in human samples and 40 &#181;m in bovine samples using a standard cryotome. 
	NOTE: The difference in section thickness is due to the difference in tissue integrity between the intact bovine disc and the highly degenerate human tissue.</li>
	<li>Collect the sections on a glass slide.
	<ol>
		<li>Encircle the tissue sections with a hydrophobic pen.</li>
		<li>Rinse the sections 3 times with phosphate-buffered saline (PBS) to remove the water-soluble embedding medium.</li>
	</ol>
	</li>
</ol>
6. Fluorescence staining
<ol>
	<li>Add 60 &#181;L of 1% (v/v) of DAPI (Exmax 358 nm, Emmax 461 nm) as well as 1% (v/v) of Actin Tracking staining (Exmax 540 nm, Emmax 565 nm) in PBS and incubate for 5 min at room temperature. 
	NOTE: The staining protocol described here is to visualize the nucleus using DAPI nuclear staining (blue) and the cytoplasm using Actin Tracking Stain (red). The IVD has a strong autofluorescence due to the collagen fibers in the green channel. The amount of fluid added to the sections in this protocol is intended for sections of a size of about 5 mm x 5 mm. For larger sections, this amount needs to be increased accordingly. Perform all the works in rooms without direct sunlight exposure and with dimmed lights to prevent bleaching the dye.</li>
	<li>Remove the staining fluid with a pipette and wash three times with 60 &#181;L of PBS each time.</li>
	<li>Add a suitable mounting medium and cover the sections with a cover slip. 
	&#8203;NOTE: Ensure that there are no air bubbles entrapped when adding the cover slip. This is best done by starting contact of the slip with the slide on one rim and then let the slip come down slowly.</li>
</ol>
7. Microscopic imaging and processing
<ol>
	<li>Place a slide with a stained section on the sample holder of the microscope. 
	NOTE: Due to the dense collagen type I network of the IVD, the scattered light makes the tissue difficult to visualize using conventional fluorescence microscopy. One way to address this problem is to perform optical sectioning using structured illumination. This also allows to render a three-dimensional projection of the entire specimen in both channels (blue and red). This is best done using the structured illumination setting and the Mosaic-mode with a 10x magnification objective lens to obtain an overview of the sample as well as 3D reconstructions of individual patterns.</li>
	<li>Start the structured illumination device.
	<ol>
		<li>Perform single field-of-view imaging with a suitable fluorescence microscope, fluorescence filters and adequate illumination. 
		NOTE: Adjust the exposure time for all the filters used in order to standardize the imaging acquisition. To get an accurate representation of the specimen at a higher resolution image the sections with a higher magnification (e.g., 20x objective).</li>
		<li>Postprocess the pictures by optimizing the intensity and brightness using an image optimization software compatible with the fluorescence microscope.</li>
	</ol>
	</li>
	<li>To visualize the section as a whole use the mosaic imaging technique
	<ol>
		<li>Open the acquisition settings (press on 6D- Acquisition) from the toolbar panel.</li>
		<li>Adjust the mosaic settings (in MosaiX Register) and define the number of columns and rows of field-of-view images that shall later be merged to one overview image.</li>
		<li>Press Setup and adjust the focus correction of individual tiles. 
		NOTE: It is almost impossible for a large tissue section to have the whole tissue surface in a single focus plane. Image tiles at different focal levels can be taken by &#39;MosaiX Acquisition&#39;.</li>
		<li>To start the acquisition of the image tiles, press Start.</li>
		<li>Stitch the imaged tiles by using the stitching function (Stitching button) with 20% overlap incorporated in the software.</li>
		<li>Postprocess the pictures by optimizing the intensity and brightness using an image optimization software compatible with the fluorescence microscope.</li>
	</ol>
	</li>
	<li>To analyze the spatial chondrocyte organization, use the 3D function incorporated in the software.
	<ol>
		<li>Adjust the z-stack settings. Define the scanning parameters: define the start and stop positions in the z-axis and the slice distance by activating Start/Stop button. 
		NOTE: The software automatically calculates the number of slices.</li>
		<li>To start acquisition of the image z-stacks, press Start.</li>
		<li>Postprocess the pictures by optimizing the intensity and brightness using an image optimization software compatible with the fluorescence microscope.</li>
	</ol>
	</li>
	<li>Export the pictures with a file format compatible with the image processing software. 
	&#8203;NOTE: Export the 3D reconstructions as individual images or/and as an interactive 3D model or in a video format.</li>
</ol>
8. Cellular pattern identification and density assessment
<ol>
	<li>Open the exported mosaic pictures of the entire tissue section in an appropriate image processing program.</li>
	<li>Define the areas subjected to cell density assessment by defining regions of interest of 500 &#181;m x 500 &#181;m in the pictures. 
	NOTE: All tiles that do not have an adequate image quality are excluded from analysis.</li>
	<li>Identify individual cellular patterns. 
	NOTE: Single cells are defined as individual cells - fully encapsulated within the adjacent matrix. Pairs are defined as two adjacent cells in close proximity (&#60;25 &#181;m) whereby the cells are interconnected through their matrixes (see Figure 2). String-formations are at least three chondrocytes aligned in line (from the middle of the nuclei &#60;25 &#181;m). These cells are encompassed by an intact matrix and matrix interconnections can be seen between each cell. Clusters represent multiple cells that are located in direct proximity to each other (&#60;25 &#181;m) and are encapsulated in a large lacuna devoid of matrix.</li>
	<li>Use a cell count plug-in for the quantitative analysis of the cellular patterns. 
	NOTE: The cytoplasm staining represents a verification method to identify the different spatial patterns.</li>
	<li>Calculate the cell density by dividing the counted cells by the size of the chosen region of interest.</li>
</ol>

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E., Roberts, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rumours+of+my+death+may+have+been+greatly+exaggerated':+a+brief+review+of+cell+death+in+human+intervertebral+disc+disease+and+implications+for+cell+transplantation+therapy.">Rumours of my death may have been greatly exaggerated': a brief review of cell death in human intervertebral disc disease and implications for cell transplantation therapy.</a> Biochemical Society Transactions. 35, 680-682 (2007).</li><li>Roberts, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disc+morphology+in+health+and+disease.">Disc morphology in health and disease.</a> Biochemical Society Transactions. 30, 864-869 (2002).</li><li>Lama, P., Kulkarni, J., Tamang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cell+clusters+in+intervertebral+disc+degeneration+and+its+relevance+behind+repair.">The role of cell clusters in intervertebral disc degeneration and its relevance behind repair.</a> Spine Research. 03, 15 (2017).</li><li>Sharp, C. A., Roberts, S., Evans, H., Brown, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disc+cell+clusters+in+pathological+human+intervertebral+discs+are+associated+with+increased+stress+protein+immunostaining.">Disc cell clusters in pathological human intervertebral discs are associated with increased stress protein immunostaining.</a> European Spine Journal: Official Publication of the European Spine Society, the European Spinal Deformity Society and the European Section of the Cervical Spine Research Society. 18 (11), 1587-1594 (2009).</li><li>Freemont, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+pathobiology+of+the+degenerate+intervertebral+disc+and+discogenic+back+pain.">The cellular pathobiology of the degenerate intervertebral disc and discogenic back pain.</a> Rheumatology. 48 (1), 5-10 (2009).</li><li>M&#252;llers, Y., 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=Quantitative+analysis+of+F-actin+alterations+in+adherent+human+mesenchymal+stem+cells:+Influence+of+slow-freezing+and+vitrification-based+cryopreservation.">Quantitative analysis of F-actin alterations in adherent human mesenchymal stem cells: Influence of slow-freezing and vitrification-based cryopreservation.</a> PLoS One. 14 (1), 0211382 (2019).</li><li>McCann, M. R., S&#233;guin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notochord+cells+in+intervertebral+disc+development+and+degeneration.">Notochord cells in intervertebral disc development and degeneration.</a> Journal of Developmental Biology. 4 (1), 3 (2016).</li></ol>

The authors have nothing to disclose.

Using fluorescence microscopy augmented by mosaic imaging and optical sectioning, we evaluated the spatial arrangement of chondrocytes in the anulus of the lumbar IVD throughout development, maturation, and degeneration. While degenerative tissue could be harvested from patients receiving spine surgery for disc degeneration, analysis of the embryonic period and maturation phase required the use of a model organism (bovine). High cellular densities were noted in the anulus during early embryonic development. In the furthe...

The intervertebral disc (IVD) is a cartilage-based structure that biochemically and with respect to cellular architecture, at first sight, resembles in many ways the articular cartilage1. Indeed, both IVD degeneration and osteoarthritis (OA) of articular cartilage are characterized by joint space narrowing due to cartilage wear, subchondral cyst and osteophyte formation, and subchondral sclerosis2,3. Despite these seeming similarities architecture and functional role of both tissues differ. While the matrix of articular cartilage is mainly formed of an arcade-forming collagen type II network, the IVD consists of three different types of tissue: the collagen type II-rich nucleus pulposus in the center takes up axial loads and transmits them to an encompassing ring of densely packed circular collagen type I fibers which is called anulus fibrosus. Their function is to absorb the translated axial pressures received by the proteoglycan- and water-rich nucleus with their tensile longitudinal fiber strength. At the top and bottom of each nucleus and anulus a hyaline cartilaginous endplate forms the junction to the adjacent vertebrae4 (Figure 1).
In articular cartilage, four distinct spatial chondrocyte patterns can be found: pairs, strings, double strings, small respectively big clusters5,6,7 (Figure 2). Changes in this pattern are associated with OA onset and progression8,9. Spatial chondrocyte organization is also indicative for a direct functional property of cartilage, namely its stiffness, underlining the functional relevance of this image-based grading approach10,11. These patterns can additionally be identified with already existing clinically available technology12. Due to the similarities between the IVD and articular cartilage, it can be hypothesized that characteristic chondrocyte patterns are also present in the IVD. Cluster formation is a phenomenon also observed in the degenerated IVD13,14.
When trying to analyze spatial cellular organization in the IVD, it is necessary to overcome several technical difficulties that are not present when investigating articular cartilage:
First, processing of the tissue itself is much more challenging than with the homogeneous hyaline cartilage which is largely composed of collagen type II. The IVD&#39;s main fiber component is collagen type I, which makes it much more difficult to generate thin histologic sections. While in the hyaline articular cartilage even thick sections can easily be analyzed due to the &#34;glass-like&#34; nature of the tissue, the collagen type I network of the IVD is optically highly impenetrable. For this reason, a strong background noise is a common problem in the histology of the IVD. A fast and cheap way to penetrate this optically dense tissue is the use of an optical sectioning device e.g., by means of an Apotome. In such an Apotome, a grid is inserted in the illumination pathway of a conventional fluorescence microscope. In front of the grid a plane-parallel glass plate is placed. This tilts back and forth thus projecting the grid in the image in three different positions. For each z-position, three raw images with the projected grid are created and superimposed. By means of special software, the out of focus light can be calculated out. The underlying principle is that, if the grid is visible, that information is in focus, if not it is considered to be out of focus. With this technique, well focused and high-resolution images can be acquired in a reasonable amount of time.
Secondly, the tissue is hard to come by from human donors. When doing total knee replacement, the entire surface of the joint can be obtained for further analysis during surgery. Although osteoarthritis of a diarthrodial joint is also a disease of the whole joint, there are nevertheless strong focal differences in the quality of the cartilage with usually some areas of the joint still being intact, for example due to reduced loading in that area. This situation is different in the IVD, where surgery is usually only performed when the disc is globally destroyed. When obtaining tissue from human donors from the operation room, the tissue is also highly fragmented and it is necessary to correctly allocate the tissue to one of the three cartilage types of the IVD before doing further analyses. To allow more detailed analyses of also larger tissue sections and to look into the embryonic development of the IVD the choice of an animal model organism is, therefore, necessary.
When choosing such a model organism it is important to have a system which is comparable with the human disc with respect to its anatomy and dimensions, its mechanical loading, the present cell population as well as its tissue composition. For the purpose of the presented technique here we suggest the use of bovine lumbar disc tissue: A critical property of the human disc resulting in its low regenerative potential is the loss of notochordal cells during maturation in the nucleus. However, in numerous model organisms notochordal cells can be detected their entire life long. Most of the few animals which lose their notochordal cells such as sheep, goats or chondrodystrophig dogs have an IVD that is much smaller than human discs. Only lumbar bovine discs present with a comparable sagittal disc diameter to those of human IVDs15.
A key factor leading to early disc degeneration is excessive mechanical loading. The intradiscal pressures of a standing cow in the lumbar spine are around 0.8 MPa with the spine aligned horizontally. Surprisingly these pressures are comparable to the lumbar intradiscal pressures reported for the erect human spine (0.5 MPa)15,16. Also the amount of water and proteoglycans in bovine discs is comparable to that of the IVD from young humans17. Therefore, although the actual movement pattern of the motion segments might differ in quadrupedal animals from the bipedal human, with respect to total loading and disc characteristics, the cow is much closer to human biology than other established animal models for the IVD such as sheep and dogs.
In this protocol we present a technique how to analyze changes in the IVD from the point of view of spatial chondrocyte organization from early embryonic development to end stage degeneration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Amphotericin B</td>
			<td>Merck KGaA,&#160; Germany</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion Microscope Slides SuperFrost Plus</td>
			<td>R. Langenbrinck, Germany</td>
			<td>03-0060</td>
			<td></td>
		</tr>
		<tr>
			<td>ApoTome</td>
			<td>Carl Zeiss MicroImaging GmbH, Germany</td>
			<td>462000115</td>
			<td></td>
		</tr>
		<tr>
			<td>AxioVision Rel. 4.8 with Modul MosaiX</td>
			<td>Carl Zeiss MicroImaging GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CellMask Actin Tracking Stain</td>
			<td>Thermo Fischer Scientific, US</td>
			<td>A57249</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems, US</td>
			<td>CM3050S</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fischer Scientific, US</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Gibco, Life Technologies, Germany</td>
			<td>41966052</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma-Aldrich, US</td>
			<td>60004</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Miscoscope - Axio Observer Z1 with Axio Cam MR3 and Colibri</td>
			<td>Carl Zeiss MicroImaging GmbH, Germany</td>
			<td>3834000604</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Merck KGaA,&#160; Germany</td>
			<td>104002</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J 1.53a, with Cell counter plugin</td>
			<td>National Insittute of Health (NIH), US</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Alexa Fluor 568 Phalloidin</td>
			<td>Thermo Fischer Scientific, US</td>
			<td>A12380</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopic Cover Glasses</td>
			<td>R. Langenbrinck, Germany</td>
			<td>01-1818/1</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP Pen Liquid Blocker</td>
			<td>Science Sevices&#160; GmbH, Germany</td>
			<td>N71310</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich, US</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich,US</td>
			<td>P5119</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>pf medical AG, Germany</td>
			<td>2023-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-tek O.C.T. Compound</td>
			<td>Sakura Finetek, Netherlands</td>
			<td>SA6255012</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using mosaic images, the architecture of the IVD with its dense collagen fiber network in the anulus and the softer nucleus can clearly be recognized (Figure 4). A continuous decrease in cellular density can be observed during embryonic development (Figure 5). While in the early stages of IVD development a cell density of 11,435 cells/mm&#178; in the bovine anulus fibrosus and 17,426 cells/mm&#178; in the bovine nucleus pulposus can be found, these numbers decre...

Watch this Scientific Journal Video about Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration at JoVE.com

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

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

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 ...

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2.0K Views.  University Hospital of Tübingen. We present a method to investigate spatial chondrocyte organization in the anulus fibrosus of the intervertebral disc using an optical sectioning method.

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

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...