This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.
The overall goal of this procedure is to process cardiac extracellular matrix to a hydrogel, which enables studying the effect of the matrix in various cell based assays. This method can help to answer key questions in the cardiac field, such as how extracellular matrix affects cell behavior. The main advantage of this technique is that it facilitates the use of the extracellular matrix to do large scale experiments in vitro or in vivo.
The applications of this technique can be extended towards a therapy for myocardial infarction because the matrix can provide essential survival cues. To begin the experiment, use a sterile scalpel with a number ten blade, to remove fat tissue from a previously obtained left ventricular myocardium. Cut the myocardium in cubes approximately 1 by 1 by 1 cm, using a scalpel.
Next, place the cubes into a sterile 50 mL tube. Store the tubes containing the cubes at 80 degrees celsius. Take the tube containing the prepared cubes out of the freezer and transport it on ice to the cryostat.
Set the object and chamber temperature of the cryostat to 15 degrees celsius. Chill empty 50 mL tubes and stamps in the chamber. Add a layer of cryosection medium onto the cold stamp, and let it freeze until it is white and solid.
Add a second layer of cryosection medium, and place a myocardium tube into the second layer. Make sure the cryosection medium and myocardium are completely frozen. Then, insert the stamp with frozen myocardium into the holder and tighten all the screws.
Trim the surface of the tissue until an even sectioning surface is obtained. Section the frozen myocardium with automatic sectioning to guarantee uniform cut sections. Approximately 30 to 40 sections can be sliced from one cube.
Place each slice, after sectioning, loosely in a pre-cooled 50 mL tube. Close the filled tubes and place them on ice. Transfer the frozen sections of all 50 mL tubes, into one sterile beaker.
Add 50 mL of lysis solution per 50 mL tube containing myocardium slices. Next, shake the myocardium slices in a sterile beaker. Strain the liquid with the tissue slices through a coarse strainer.
Then, transfer the tissue slices with a blunt tweezer to a new sterile beaker. Add 50 mL of 0.5%SDS in PBS per initial 50 mL tube of myocardium slices. Shake the beaker continuously for six hours at 100-150 rpm at room temperature.
After shaking, strain the liquid with the tissues slices through a coarse strainer. Using blunt tweezer, transfer the tissues slices to a new sterile beaker and wash the slices three times with 50 mL of PBS. Next, wash the slices overnight in PBS, with 1%penicillin streptomycin and 1%nystatin.
The next day, remove the washing solution, and add 25 mL of FBS preheated at 37 degrees celsius with 1%penicillin streptomycin and 1%nystatin, to the decellularized slices per initial 50 mL tube of myocardium slices. Incubate the slices for three hours at 37 degrees celsius to remove any remaining DNA from the matrix. Then, transfer the slices to a new sterile beaker and wash three times with 50 mL of PBS.
Place the ECM slices loosely in a new sterile six well plate. Freeze the ECM at 80 degrees celsius, and lyophilize it for two days in a lyophilize. For the pulverization of the ECM slice, use milling tubes containing 1.4 mm ceramic beads.
Fill the tubes loosely with lyophilized ECM, using sterile, blunt tweezers. Snap freeze the tubes in liquid nitrogen. Insert the frozen tubes into the milling machine and pulverize the ECM.
Set the duration to 30 seconds, adjust the maximum speed, and start the device. After pulverization, take out the tubes, and snap freeze them again in liquid nitrogen. Wash the microparticles from the tubes, by adding 1 mL of ddH20 per tube, and shake for complete solubilization.
Then, filter the heterogeneous solution through 200 micrometer mesh, into a 50 mL tube. Freeze the tube in liquid nitrogen. Replace the standard lid of tube, with a 0.22 micrometer filter to prevent the powder from flowing out of the tube.
Insert the tube in the lyophilizer, and lyophilize again to remove the water from the wash step. Dissolve the previously prepared lyophilized human ECM powder in pepsin solution, to a final concentration of 10 mg per mL. Transfer the solution to 2 mL tubes with a volume of 1 mL per tube.
Pipette the solution to mix thoroughly. Next, place the tubes in the shaker and shake at 1200 rpm for 48 hours at 27 degrees celsius. Take the tubes out of the shaker and place them on ice or a pre-cooled tube holder.
Mix 1/9 of the volume of the ECM solution of cold 10X PBS, with 1/10 of a volume of ECM solution of cold 0.1 molar sodium hydroxide, in a pre-cooled tube and place it on ice to neutralize the digested ECM solution and inactivate the pepsin. Finally, add the ECM solution to the neutralization mixture and mix thoroughly through pipetting or vortexing. Quantitative assays for individual ECM components revealed a more complete DNA removal and better preservation of total collagen, elastin, and glycosominoglycans as compared with decellularization by SDS, alone.
RT-PCR was used to quantify the expression of structural cardiomyocite proteins, early and late cardiomyogenic transcription factors, and endothelial cells surface marker genes in Murine ESC, undergoing spontaneous differentiation. Contact with cECM drives pluripotent stem cells, preferably toward a cardiomyocite like phenotype. Phase contrast light microscopy demonstrates that enzymatic digestion of the cECM for 48 hours, results in a more homogenized ECM solution.
Live/dead staining of human cardiac fibroblasts on cECM hydrogel, shows increased viability. Both cECM microparticles and cECM hydrogel enhance the metabolic activity of contractile HL-1 cells, in ischemic conditions, as compared to cells in standard culture. Once mastered, this technique can be done within one week from tissue to hydrogel, if it's performed properly.
After it's development, this technique paved the way for researchers in the field of cardiac regeneration, to explore the effects of cardiac extracellular matrix, in in vitro and in vivo, model systems.
