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

1. Intervertebral Disc Harvesting
<ol>
	<li>Whole length bovine tail is obtained from a local abattoir, if possible without skin since the presence of skin increases the chance of contamination (Fig 2).</li>
	<li>Prepare large cutting board and prepare sterile work station and instruments on top of a cutting board (Fig. 2).</li>
	<li>Prepare under the sterile laminar flow hood sterile gauze moistened with 0.9% sodium chloride containing 55mM sodium citrate and put into each well of the 6-well plate.</li>
	<li>Prepare a basin and dilute 1:100 Betadine solution with tap water.</li>
	<li>Immerse the whole bovine tail in a basin containing 1% Betadine solution for 5 min.</li>
	<li>Briefly dry tail with sterile gauze and place it on sterile work station.</li>
	<li>Carefully remove muscle around the tail with scalpel blade #22.</li>
	<li>Chop away the parts of the tail which are not needed, usually both ends of the tail, which contain relatively large and very small IVDs (Fig. 3).</li>
	<li>Trim further the muscle and tendons around the IVD using scalpel blade #10. Beware not to cut the outer annulus of the discs.</li>
	<li>Mark the anterior of the IVD with a surgical skin marker (this step is optional, it helps to determine the center for axial rotation in our bioreactor).</li>
	<li>Locate the IVD and vertebra connection point by moving the tail gently. Feel the border between IVD and bone with the dull side of a scalpel blade. The cleaving site should be 1-2 mm away of the IVD towards the vertebra. Place custom made industrial blade holder on the cleaving site (Fig. 4).</li>
	<li>Cleave IVD and vertebra by hammering on top of the custom-made industrial blade holder (Fig. 3).</li>
	<li>Repeat on the other side of the IVD-vertebra connection.</li>
	<li>Wrap isolated IVD with sterile gauze moistened with 0.9% sodium chloride containing 55mM sodium citrate.</li>
	<li>Continue the isolation of IVDs to the desired sample number (usually around 6 can be harvested with diameter between 10-20 mm).</li>
	<li>Connect the jet lavage system (Zimmer, inc.) with sterile Lactated Ringer's solution, 5L bags are a useful size (alternatively sterile PBS or 0.9% saline solution).</li>
	<li>Hold the IVD with forceps and jet-lavage both sides of the endplate surface using Zimmer Pulsavac jet-lavage system (Fig. 1C). The jet gun should be shot on the endplate surface at an angle between 30 - 60&deg; for about 30s on each side. (Figs. 1 and 7)</li>
	<li>Put the IVDs back to the 6-well plate and wrap with moistened gauze while jet washing another disc sample.</li>
	<li>IVDs are ready for organ culture and down-stream applications (e.g. cell viability, mechanical loading, organ culture) (Fig. 1D).</li>
</ol>
2. Representative Results
Outcome measurements to judge a successful intervertebral disc preparation can be a diffusion experiment where a small molecular weight fluorescent dye (e.g. procion red) is prepared as a 1% solution in PBS2. The IVD is then kept with at least 100 mL of dye solution and the dye is then allowed to diffuse passively into the IVD for 24h under free swelling. The IVD is then flash-frozen in liquid nitrogen and brought back to room temperature and dehydrated by a series of acetone transfers (first pre-cooled in -80 &deg;C, then -20 &deg;C, then 4 &deg;C and finally to room temperature). The disc can then be embedded in poly-methyl-acrylate (PMMA) and cut with a sharp blade to produce 100 &mu;m-thick sections. These sections can then be viewed by any standard brightfield microscope but preferentially by usage of fluorescence microscopy since procion red emits a red fluorescence (Figs. 5-6). The trabeculi of the bony surface appear very clean after the jet lavaging step (Fig. 7).
Parameters for a successful organ culture are first of all the absence of contamination, maintained cell viability (Figs. 8-9), disc height and overall "disc integrity", measured with histological sections and safranin O/fast green stain or Meyer's Hematoxilin 3. We developed protocols and macros published elsewhere4,5 to stain live disc tissue and to scan cell viability in 3D using a confocal laser scanning microscope and the live/dead staining kit (Molecular Probes, Invitrogen, Basel, Switzerland). We further developed a macro and routine to perform automated cell counting to estimate cell viability in 3D tissue or 3D carriers using the NIH ImageJ platform4-6. Alternate protocols have been proposed to determine cell viability of disc cells by initial digestion using sequential digestion of the extracellular matrix using pronase and collagenase type 2 and then to stain the cell suspension7. An earlier version of the protocol involved an incubation step in PBS containing ten times more concentrated penicillin/streptomycin concentration for 10min after the harvesting of the discs prior organ culture. With the jet lavage step this step becomes obsolete since this step seems beneficial for culturing purposes.
<img alt="Figure 1" src="/files/ftp_upload/3490/3490fig1.jpg" /> 
Figure 1. Overview of the methodological steps to prepare bovine coccygeal Intervertebral Discs with intact endplates and a thin layer (1-2mm) of bone. A) Schematic view of the intervertebral disc (IVD) and its nutritional pathways. B) Procedure to cut out the IVD with a customized blade-holder and an industrial sharp blade. C) Jet lavage step to ensure high cell viability and to enable nutrient exchange keeping the endplates and 1-2 mm of bone attached on both sides. D) Finally the IVD can be cultured in the specimen chamber of a bioreactor or be used for any down-stream application.
<img alt="Figure 2" src="/files/ftp_upload/3490/3490fig2.jpg" /> 
Figure 2. TOP: Full length fresh bovine tail (ideally obtained within 2-3 hours post-mortem to ensure high cell viability. Bottom: X-ray image of a bovine tail aged around 6 months illustrating the existence of growth plates (GP), secondary center of ossification, between the bony endplates (EP) and the vertebra bodies (VB).
<img alt="Figure 3" src="/files/ftp_upload/3490/3490fig3.jpg" /> 
Figure 3. A) Tools to dissect the bovine tail under sterile conditions. B) Customized blade-holder to cut-out motional segments from the bovine tail in action while cutting the intervertebral disc. C) Side-view of the customized blade-holder with a standard industrial blade (Lutz, Germany).
<img alt="Figure 4" src="/files/ftp_upload/3490/3490fig4.jpg" /> 
Figure 4. Drawing and picture of the bovine tail after removing the surrounding muscle and ligament illustrating the cleaving sites to cut out the motional segments. Partial vertebra should be "cracked" with the custom-made blade holder ideally 1-2 mm away from the cartilaginous endplates to ensure full bone surface.
<img alt="Figure 5" src="/files/ftp_upload/3490/3490fig5.jpg" /> 
Figure 5. Demonstration of the effective cleaning of the jet-lavage step before (A) and after (B). Notice the clean bony surfaces of the intervertebral disc segments after the spraying procedure.
<img alt="Figure 6" src="/files/ftp_upload/3490/3490fig6.jpg" /> 
Figure 6. Free-swelling diffusion experiment of freshly prepared excised bovine tail intervertebral discs (IVDs) left for 24 hours in 1% procion red solution, demonstrating blockage effect by the growth plate. A digital X-ray sagittal view of a IVD still containing the growth plate and a second layer of bone fluorescent microscopy image of sagittal cut through the coccygeal bovine intervertebral disc explant with cartilaginous and bony endplate after 24h free diffusion. GP: growth plate, 2ndry CO: secondary center of ossification.
<img alt="Figure 7" src="/files/ftp_upload/3490/3490fig7.jpg" /> 
Figure 7. Free-swelling experiment of freshly prepared excised bovine tail intervertebral discs (IVDs) left for 24 hours in 1% procion red solution, demonstrating cleaning effect of jet lavage treatment. Sagittal thick sections (~100 mm) of excised bovine IVDs with ~1.5-mmthick bony end plates that underwent jet lavage treatment (Left) control side without treatment (Right) jet-lavaged treatment. Inlet shows the spray pattern, which was used from the Zimmer wound debridement system 24h free diffusion experiments of bovine intervertebral disc explants in 1% procion red.
<img alt="Figure 8" src="/files/ftp_upload/3490/3490fig8.jpg" /> 
Figure 8. Live/Dead Images of projections of confocal Imaging stacks ( &#x0303;250&#x03BC;m) taken from bovine intervertebral disc segments prepared with this jet-lavage method kept under free-swelling for 0 and 14 days respectively for the nucleus pulposus (NP), the inner annulus fibrosus (IA) and the outer annulus fibrosus (OA). Green cells = live cells stained by calcein AM (acetyl methylester), red cells = dead cells nucleus stained by ethidium homodimer-1.
<img alt="Figure 9" src="/files/ftp_upload/3490/3490fig9.jpg" /> 
Figure 9. Cell Viability of intervertebral disc using confocal laser scanning microscopy on live tissue and 3D stack scanning. Cell viability of Nucleus Pulposus (NP) and Annulus Fibrosis (AF) over the 21-day-culture under free-swelling condition. (Mean &plusmn; SEM, N=6) Statistical differences were tested using non-parametric Kruskal-Wallis signed rank sum test among groups. Significant differences were found between day 21 and all other time points in NP. While in AF significant differences were found between day 7 to day 0. (* p &lt; 0.05, ** p &lt; 0.01).

<ol>
	<li>Lee, C. R., Iatridis, J. C., Poveda, L., Alini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+organ+culture+of+the+bovine+intervertebral+disc:+effects+of+vertebral+endplate+and+potential+for+mechanobiology+studies.">In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies.</a> Spine (Phila Pa 1976). 31, 515-522 (1976).</li><li>Chan, S. C. W., Gantenbein-Ritter, B., Leung, V. Y., Chan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreserved+intervertebral+disc+with+injected+bone+marrow-derived+stromal+cells:+a+feasibility+study+using+organ+culture.">Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture.</a> Spine. J. 10 (6), 486-496 (2010).</li><li>Chan, S. C., Ferguson, S. J., Wuertz, K., Gantenbein-Ritter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Response+of+the+Intervertebral+Disc+to+Repetitive+Short+Term+Cyclic+Torsion.">Biological Response of the Intervertebral Disc to Repetitive Short Term Cyclic Torsion.</a> Spine (Phila Pa 1976). , (2011).</li><li>Gantenbein-Ritter, B., Sprecher, C. M., Chan, S., Illien-J&#252;nger, S., Grad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+protocols+for+live/dead+staining+in+three-dimensional+carriers.">Confocal imaging protocols for live/dead staining in three-dimensional carriers.</a> Methods Mol. Biol. 740, 127-140 (2011).</li><li>Gantenbein-Ritter, B., Potier, E., Zeiter, S., van der Werf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+three+techniques+to+determine+cell+viability+in+3D+tissues+or+scaffolds.">Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds.</a> Tissue Engineering Part C Methods. 14, 353-358 (2008).</li><li>Rasband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li><li>Haschtmann, D., Stoyanov, J. V., Ettinger, L., Nolte, L. P., Ferguson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+intervertebral+disc/endplate+culture+model:+analysis+of+an+ex+vivo+in+vitro+whole-organ+rabbit+culture+system.">Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system.</a> Spine. 31, 2918-2925 (2006).</li><li>Gantenbein, B., Gr&#252;nhagen, T., Lee, C. R., van Donkelaar, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+organ+culturing+system+for+intervertebral+disc+explants+with+vertebral+endplates:+a+feasibility+study+with+ovine+caudal+discs.">An in vitro organ culturing system for intervertebral disc explants with vertebral endplates: a feasibility study with ovine caudal discs.</a> Spine. 31, 2665-2673 (2006).</li><li>Korecki, C. L., Kuo, C. K., Tuan, R. S., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervertebral+disc+cell+response+to+dynamic+compression+is+age+and+frequency+dependent.">Intervertebral disc cell response to dynamic compression is age and frequency dependent.</a> J. Orthop. Res. 27, 800-806 (2009).</li><li>Korecki, C. L., MacLean, J. J., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+effects+on+intervertebral+disc+mechanics+and+biology.">Dynamic compression effects on intervertebral disc mechanics and biology.</a> Spine (Phila Pa 1976). 33, 1403-1409 (1976).</li><li>Jim, B., Steffen, T., Moir, J., Roughley, P., Haglund, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+intact+intervertebral+disc+organ+culture+system+in+which+degeneration+can+be+induced+as+a+prelude+to+studying+repair+potential.">Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential.</a> European Spine Journal. , 1-11 (2011).</li></ol>

We have nothing to disclose.

The first step for successful organ culture is to make sure that the explant should not be contaminated. The tail should be skinned before you start with the procedure. Any animal hair brought into a sterile lab could be problematic in terms of contamination. The bovine tail should ideally be as fresh as possible (this affects initial cell viability). Furthermore, the betadine washing step is recommended to reduce the risk of contamination further. Instead of using a custom-made blade holder and a hammer to separate the IVD from the vertebra body, this could be performed by using a histological bandsaw (the diamond blade should be cooled with PBS or Ringer solution while cutting). Beware that the bandsaw, the sample holder and the cutting blade should be sterile. The technique demonstrated here is not limited to bovine tail IVD isolation but can be extended to other animal species such as porcine, ovine and leporine caudal or even lumbar IVD isolation. For IVD harvest in the region where spinal processes are present, it is important to remove all the processes first. 
The cell viability data given in figure 9 were collected under free-swelling for up to 21 days and can be used as a reference. This method maintains cell viability for 14 days under free-swelling. The drop in cell viability at 21 days could be due to the lack of mechanical loading which has been shown to be important in assisting nutrient diffusion across the endplates and to maintain disc cell viability3. It will be very likely that the cell viability will be even higher under hydrostatic compression and/or additional diurnal loading8-10. 
As an alternate protocol, the bovine discs can be prepared by removing all of the adjacent bone of the vertebrae by using a dental burr and just leaving the cartilaginous endplates11. IVD cell viability was maintained over 4 weeks by using this method. However, in this case torsion cannot be applied in a straightforward way to the motional segments since there is no stiff bone available to "grab" the disc. The concave endplates instead of a parallel surfaces on both sides also needs an adjustment of the specimen chamber in any bioreactor design to account for the loading profile differences11. Moreover, the evaluation of diffusion of molecules across bony endplates is also not possibly in these explants.
Significance of our work
<ul>
	<li>The usage of single-usage industrial blades to cut-out intact intervertebral disc segments for organ culture shortens the harvesting protocol considerably compared to protocols involving a cutting-step with a histological band saw. Also the trabecular structure of the bone is unaffected as it is the case from a grinding movement with a diamond saw blade.</li>
 <li>Jet-lavaging of the bony surface washes-out clotted-blood very efficiently and significantly improves cell viability of the nucleus pulposus (center of the disc) over time compared to untreated intervertebral discs.</li>
</ul>

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td colspan="4">Fresh bovine intervertebral disc tissue from bovine tails, from local slaughter house (ideally within hours post-mortem and without skin). </td>
 </tr>
 <tr>
 <td>Pulsavac Plus AC System</td>
 <td>Zimmer inc., Switzerland</td>
 <td>00-5150-486-01 </td>
 <td>Best performance with the hip-spray head and with AC power supply (the one with the 8 AA battery pack does also work but is less convenient)</td>
 </tr>
 <tr>
 <td>High Capacity Fan Spray w/Splash Shield, 12.7cm length </td>
 <td>Zimmer inc., Switzerland</td>
 <td>00-5150-175-00 </td>
 <td>There are several spray heads available, we tested this one successfully</td>
 </tr>
 <tr>
 <td>Scalpel blades #22 and #10</td>
 <td>Swann-Morton, England</td>
 <td>#10: 0201 
 #22: 0208</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Scalpel blade holder # 3 and #4</td>
 <td>Hausmann, Germany</td>
 <td>#3: 06.103.00 
 #4: 06.104.00</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lutz industrial blade</td>
 <td>Lutz, Germany</td>
 <td>1022.0884</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate buffered Saline (PBS)</td>
 <td>Invitrogen, Switzerland</td>
 <td>10010-023</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium (DMEM)</td>
 <td>Gibco, Switzerland</td>
 <td>11960-044</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lactated Ringer's solution (without glucose)</td>
 <td>Bichsel, Switzerland</td>
 <td>133 0002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>6-well multi-well plate</td>
 <td>TPP, Switzerland</td>
 <td>92006</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Betadine solution</td>
 <td>Mundipharma, Switzerland</td>
 <td>10055025</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Surgical skin marker</td>
 <td>Porex Surgical, Switzerland</td>
 <td>9560</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Large cutting board</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Any brand is possible</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture at JoVE.com

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

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  ...

preparation-intact-bovine-tail-intervertebral-discs-for-organ

Nanyang Technological University

Research

JoVE Journal

Bioengineering

14.4K Views.  University of Bern. This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

Video: Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

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

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

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 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

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.

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

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...