器官培養のための無傷牛テール椎間板の準備

The overall goal of this procedure is to prepare intact intervertebral discs from bovine tail for culture and other downstream applications in our case to prepare intervertebral discs for culture in a bioreactor using complex mechanical loading. This is accomplished by first disinfecting the calf tail and then removing the muscles to obtain a clear view of the bony and disc structures. Next, a single intervertebral disc is cut keeping two to three millimeters of the partial vertebra attached on either side using the custom cutting tool.

The third step is to clean the bony surfaces of the intervertebral disc with the jet lavage system, with ensure diffusion of small solut such as glucose into the center of the intervertebral disc. The final step is to place the intervertebral disc into a stabilizing buffer or wrap it in sterile gauze to keep it moist for downstream applications. Ultimately, the success of the procedure is assay using a live dead stain to check for the presence of live cells 14 or 21 days after beginning intervertebral disc culture.

The following protocol is to show how invertible disc segments can be preserved for organ culture, which can help answering key questions in mechanobiology and tissue engineering. There's free reason why the bovine tail disc has become a kind of model system in tissue engineering. First, the cell density is very similar and very low like in a human introvert disc.

Second, the cell population there is the absence of not cordal cells, which is quite similar, also like to the human situation. Thirdly, also the dimension and the biochemical composition is very similar to the human situation. Generally, individual new to this method will struggle because it is hard to distinguish the bony endplate from the vertebra bone, and hence one will find it difficult to ensure the presence of cartilage growth plate.

We first had the idea of this method when we were looking for a fast and sterile technique to isolate the invertible disc with endplate for individual culture. Visual demonstration of these methods is important because it is difficult to locate a cutting site because the cutting site is located just a millimeter away from the boney plate. First, obtain a whole bovine tail from a local abattoir.

Ideally, the tail should be no more than four hours postmortem and should be procured with the skin removed. Since the presence of skin increases the possibility of contamination, ensure that all instruments to be used in the dissection process are sterile. Also create a sterile workstation by placing an autoclave cutting board on the work surface.

Then while working in a sterile lamina flow hood moisten a piece of sterile gauze with 0.9%sterile sodium chloride solution containing 55 millimolar sodium citrate. Place a piece of soaked gauze into each well of a six well plate. Next, fill a clean basin with one to 100 Betadine solution diluted with tap water and immerse the tail in the solution for five minutes.

After soaking, remove the tail. Briefly dry it with sterile gauze and place it on the sterile workstation First, use a number 20 or number 22 scalpel to remove muscle from around the tail. Then use a guillotine to chop away any unwanted parts of the tail, usually from the proximal and distal lens, which contain relatively large and very small intervertebral discs.

Then use number 10 scalpel to trim the muscles and tendons around the intervertebral discs. Be careful not to cut the outer annulus of the discs if necessary. For orientation purposes, mark the anterior of the intervertebral disc using a surgical skin marker.

Next, gently move the tail to locate the intervertebral disc and vertebral connection point. Use the dull side of a scalpel blade to fill the border between the intervertebral disc and bone. Then locate the cleaving site, which should be one to two millimeters away from the intervertebral disc towards the vertebra.

Place the custom made industrial blade holder on the cleaving site. This diagram very clearly shows the cleaving site, Cleve the intervertebral disc and vertebrae by hammering on top of the custom made industrial blade holder. Then move the blade holder to the other side of the intervertebral disc vertebrae connection and hammer through this side of the intervertebral disc vertebra connection to release the intervertebral disc.

Then rapidly isolated intervertebral disc with sterile gauze moistened with 0.9%sodium chloride containing 55 millimolar sodium citrate. Repeat the process to obtain the desired number of intervertebral discs. Usually around six discs of 10 to 20 millimeters in diameter can be obtained from one tail.

Once all of the invertible discs have been harvested, connect the jet lavage system to a three liter bag of sterile lactated ringer solution, sterile PBS or 0.9%sterile saline. Then hold an invertible disc with forceps and jet lavage both sides of the endplate surface for 30 seconds on each side while holding the jet lavage at a 30 to 60 degree angle in relation to the endplate surface, this image shows the effect of cleaning effect of the jet lavage. The top row of discs shows the invertible discs before cleaning, while the lower row shows the same discs after cleaning.

Once the first intervertebral disc is washed, wrap it in moist and gauze while washing the other invertible discs. After washing the invertible discs are ready for organ culture. In addition, a diffusion experiment may be performed to determine the success of the invertible disc preparation while working in a laina flow hood.

Prepare 40 milliliters of sterile DMEM culture media containing 5%fetal calf serum per disc. Transfer the media to a beaker and then submerge a single intervertebral disc in the media. Place the beaker on a shaking platform in a tissue culture incubator set to 37 degrees Celsius and 5%carbon dioxide.

The intervertebral discs may be cultured in this manner for around 14 days. Use of these custom made specimen chambers maintains an appropriate mechanical load, which allows for an extended culture time. First, prepare 40 milliliters of sterile DMEM culture media containing 5%fetal calf serum per disc as before.

Next, place the invertible disc into one of the custom-made culture chambers and add 40 milliliters of the freshly prepared media. Close the chamber under the flow hood. Place the custom made specimen chamber in a tissue culture incubator.

Add weight to the chamber according to the surface area of the discs. First, prepare 100 milliliters of a 1%solution of a fluorescent dye, such as proso red in PBS per intervertebral disc. Then immerse the disc in the solution and incubate overnight under the same conditions.

As for free swelling culture following the incubation, snap freeze the disc in liquid nitrogen and then immerse in minus 80 degrees Celsius acetone. Next, the intervertebral disc is brought slowly back to room temperature by 24 hour incubations in acetone at minus 20 degrees Celsius, four degrees Celsius, and finally, acetone at room temperature. After the disc is restored to room temperature, fully embed the disc in polymethyl methacrylate for one to two weeks according to standard procedures.

Once fully embedded, the disc is processed by cutting with a sharp blade and polishing with a grinder. Once processed, the diffusion of pro cy red throughout the disc can be evaluated by microscopy. These images show the results of a free swelling diffusion experiment.

Panel C shows the x-ray overview of an invertible disc with the two bony end plates prepared with our method. On the upper side, the growth plate was not removed imaged under fluorescent microscopy. It can be seen that the diffusion of the red fluorescent dye is blocked by the presence of the growth plate labeled gp, which thus acts as a barrier for staining the deeper layers of the cartilaginous end plate and the invertible disc.

The growth plate lies between the primary and secondary centers of ossification labeled co. If the growth plate has been successfully removed, the red fluorescent dye can diffuse unhindered to the nucleus pulposus labeled np. These images demonstrate the cleaning effect of jet lavage treatment.

The sagittal sections have a thickness of around 100 microns and the bony end plates are around 1.5 millimeters thick. Here, the intervertebral disc underwent jet lavage treatment and diffusion of the dye can be seen throughout the end plate. However, the dye does not diffuse well on the control side where jet lavage treatment has not been performed.

Here we see live dead confocal microscope images taken from bovine invertible disc segments prepared with this jet lavage method and kept under free swelling conditions for the nucleus pole processis labeled np, the inner annulus fibrosis labeled IA and Thea annulus fibrosis labeled oa. The green regions are live cells stained by calcium acetyl methyl lester, and the red areas are the nuclei of dead cells stained by aum. Homodimer one here, images from the same regions demonstrate that a high number of viable cells indicated by green fluorescence remain after 14 days under free swelling conditions.

This graph shows cell viability of the nucleus pulposus and annulus fibrosis regions of the intervertebral discs over the 21 day culture period mean results plus or minus the standard error of the mean for six samples. As shown. Statistical differences were tested using non-parametric Rosco Wallace signed ranked sum tests among groups.

Significant differences were found between day 21 and all other time points in the nucleus pulsus while in the annulus fibrosis. Significant differences were found between day seven to day zero Following this procedure that this cultural condition can be modified in order to answer additional questions like the entrance of mechanical loading, nutritional, or growth factors on the progression of this degeneration or regeneration, Our protocol accelerates the harvesting technique, how interpretable disc segments can be obtained for tissue engineering approaches measuring physiology or mechanobiology. In traditional methods, there is a histological, bandsaw used or bearing technique, which use about two hours longer than our protocol.