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 ...

<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  ...

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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

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 ...

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 ...

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 ...

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales &#8211; nano-, micro-, meso-, and macroscopic &#8211; fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

This study assesses the fracture toughness of bovine cortical bone at the sub-meso levels using microscopic scratch tests. This is an original, objective, rigorous, and reproducible method proposed to probe fracture toughness below the macroscopic scale. Potential applications are studying changes in bone fragility due to diseases like osteoporosis.

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...