心筋組織工学のための生物由来注射用材料の作製

Matrix gels produced from decellularized, pericardial and myocardial tissue retain native extracellular matrix components including diverse proteins, peptides, and glycosaminoglycans. These tissue matrixes can be used for tissue engineering. In this video matrix gels are fabricated by decellularizing pericardial tissue to yield acellular extracellular matrix.

The extracellular matrix is then processed to create a solubilized liquid version. Next, the solubilized matrix is tested in a small animal model to determine its feasibility as an injectable scaffold. Histological and immunohistochemical analysis demonstrate that in vivo, the injected material forms a porous fibrous scaffold that allows for vascular cell infiltration.

Hi, my name is Sonia Zeki. I'm a graduate student in the lab of Dr.Karen Chrisman at the University of California San Diego. Today we will fabricate Solubilized ECM from decellularized tissue and demonstrate in vivo testing for cardiac tissue engineering applications.

These techniques are important for the development of an injectable biomaterial therapy for the treatment of heart failure. Post myocardial infarction demonstrating the surgical procedure be Pam, shoot McGuffin, a veterinary neurosurgeon in our laboratory. So let's get started.

Begin this protocol by decellularizing the tissue to leave just the extracellular matrix or ECM first place porcine pericardium in a beaker of deionized water and wash it by stirring for 30 minutes at room temperature. After washing, place the tissue in one times PBS containing 1%SDS and 1%penicillin streptomycin stir continuously for 24 hours at room temperature. The next day, return the tissue to deionized water and stir continuously for five hours, shaken a 50 milliliter tube until all suds are gone.

Hematin and eoin staining should reveal the absence of nuclei in decellularized tissue. Place the remainder of the decellularized tissue in a 15 to 50 milliliter conical tube. Then place the tissue in the minus 80 degrees Celsius freezer.

Lyophilize the frozen samples by freeze drying under very low pressure and very low temperature until completely dry. Depending on the system used and the water content of the samples, this may take anywhere from 12 to 72 hours. Once the decellularized tissue is completely dry, transfer it to a Wiley Minim mill.

Set up with a 40 gauge mesh, grind the tissue into a fine powder. Once the majority of the sample has come through the filter, remove the collection jar. This sample is now referred to as extracellular matrix or ECM powder.

A second lyophilization step may be performed at this time, if not continuing immediately to the next step. This ensures the accuracy of the recorded weight to solubilize the ECM. Weigh out the desired amount of ECM powder into a sterile ation vial.

Add sterile filtered hydrogen chloride solution containing one milligram per milliliter. Pepcid to the scintillation vial with the ECM powder so that the ECM is at a concentration of 10 milligrams per milliliter. Add a stir bar and place the vial on the stir plate.

Stir continuously for 48 to 60 hours, periodically scraping down the sides of the vial with a sterile WHE spoon or spatula. To ensure digestion of all the ECM powder. As the ECM is digested, it will go into solution.

Once the ECM has been solubilized, place the sample on ice and adjust the pH to 7.4 using sodium hydroxide and hydrochloric acid. Once the pH has been adjusted, add 10 times PBS so that the final sort concentration of the solution is one times PBS. For example, if you have 900 microliters of ECM solution, after pH adjustments, 100 microliters of 10 times PBS should be added.

Determine the concentration of the solubilized ECM solution by tallying up the volumes added to neutralize and the volumes removed to test the pH. Then add one times PBS to reach the desired final concentration here. Six milligrams per milliliter.

On the day of surgery, prepare a 75 microliter injection of solubilized ECM in a 0.1 milliliter syringe tipped with a 30 gauge needle. Maintain anesthesia of an intubated female Harlan dolly rat with 2.5%ice of fluorine. Add eye, then verify anesthesia by performing a toe pinch.

Administer three milliliters of lactated ringer solution by subcutaneous injection in the lower abdomen for hydration. Next, place the animal in a supine position on the surgical table and gently tape down the limbs. Use clippers to remove the hair on the abdomen and vacuum up the free hair.

Make a series of 50 microliter injections of 2%lidocaine along a diagonal from the xiphoid process to the lower right hand side of the abdomen. Then scrub three times with Betadine starting in the middle and moving outward. Repeat the scrubbing with isop profile alcohol.

Then cover the animal using a surgical drape with a pre-made circular window. Secure with a surgical drape. Using a number 10 scalpel.

Make a three to four centimeter incision from the xiphoid process to the lower right hand side of the abdomen. Locate the xiphoid process and dissect vertically down through the muscle to the right, being careful to avoid the large vessel on the right. Next, use scissors to cut laterally through the muscle exposing the diaphragm.

Be careful to avoid the liver using a 36 inch length of three zero Vicryl suture and a pair of hemostats. Drive one needle through the xiphoid process and pull the suture halfway through. Tape the ends of the suture together and fix them to a point above and behind the animal, lifting the xiphoid up and exposing the diaphragm with another 36 inch length of three zero Vicryl suture.

Drive a needle through the muscle closest to the xiphoid process and again, pull through and fix the end to another point to the right of the surgical table, fully exposing the surgical cavity. Next, using a small pair of rat tooth micro forceps, grab the center of the diaphragm, pull it out, and make a very small incision with blunt scissors. Once this hole has been made, the lungs will retract.

Make a two to three centimeter incision vertically through the diaphragm to visualize the heart. Then insert a three inch swab to the left of the cavity and push the lungs outta the way. Secure the swab in place using a towel clamp.

Using another swab, move the right lung outta the way in order to locate the pericardial sack. Then using a pair of micro forceps and a pair of large serrated forceps, tear through the pericardial sack, exposing the apex of the heart. Grab the apex of the heart with the right tooth micro forceps and insert the needle to inject the solubilized matrix beveled edge to the left.

A third of the way up from the apex parallel to the epicardial surface. Be sure to avoid the great cardiac vein. Inject the solubilized matrix gel and wait a couple of seconds before removing the needle.

The tissue should whiten at the site of injection, swab away blood in the cavity, and remove the towel clamp and swab holding the right lung using curved micro hemostats, make an initial knot with a 30 inch length of five zero proline suture. Then use the rat tooth micro forceps to close the diaphragm with a continuous suture and a tapered needle before closing entirely. Insert PE one 60 suction tubing and close around using a 10 milliliter syringe to evacuate the chest cavity.

As the suture is tightened, the diaphragm should be concave. Turn the ice of fluorine down to 1%Take out both three zero Vicryl sutures holding the cavity open. Hydrate the muscle wiping excess away with a swab or sterile gauze.

Use a new length of three zero suture to close the muscle layer with intermittent sutures. Then turn the isof fluorine off. Clean the area and use staples to close the skin.

Then apply a triple antibiotic ointment to the incision site. Allow the rat to recover under 100%oxygen. When the animal starts to breathe around the ventilator, remove the trachea tube.

Once the animal is back on its feet, administer a subcutaneous injection of 0.05 milligrams per kilogram of buprenorphine hydrochloride analgesic, and return it to the home cage on a sterile towel. Cardiac tissue from rats injected with soluble matrix gels was sectioned and stained with hemat, toin, and eoin for gross tissue analysis at various time points. Following surgery at early time points, the injected region appears as a pink fibrous network that has spread interstitial at later time points.

Cell influx is observed after two weeks. Degradation of the injected matrix is seen. If the ECM was labeled with biotin before injecting, it can be more directly visualized by staining the biotin with HRP conjugated streptavidin to visualize vessel ingrowth and formation in the matrix injection region.

The slides here have been co stained with a fitzy. Conjugated is selectin that binds to endothelial cells shown in green, and an antis smooth muscle. Actin antibody shown in red stem cells may also be identified near the injection region.

Here they have been visualized with an immunohistochemistry stain for C kit. After watching this video, you should have a good understanding how to create solubilized ECM from decellularized tissue and tested in vivo for cardiac tissue engineering applications. Don't forget that while we use pericardial tissue, this general procedure to be modified, to be used with any tissue source.

Thank you for watching and good luck with your experiments.