To begin, wash the fresh human atrium sample in a 100 millimeter glass plate containing sterile HBSS. Gently shake the samples in the plate to remove any residual blood. Transfer the sample to a 100 millimeter plate wetted with a small volume of HBSS.
With crossed scalpels, chop the sample into two-millimeter-sized cubes. Next, transfer four small myocardial fragments to a 35 millimeter plate. Place a sterile cover glass over the fragments and apply gentle pressure.
Pipette 1.5 milliliters of DMEM into the plate. Then incubate the culture plates at 37 degrees Celsius, under 5%carbon dioxide supplementation. When the cultures have reached 85%confluency, remove the plate from the incubator and use sterile forceps to lift the cover glass.
Then place it upside down on a new 35 millimeter plate. Then add sterile PBS to rinse it. Now remove the myocardium fragments.
Then pipette DMEM out of the plate. Use sterile PBS to rinse the plate containing the outgrown cells. After discarding the rinse, add one milliliter of 0.25%trypsin EDTA to each plate, and incubate the plates at 37 degrees Celsius, with 5%carbon dioxide, for five minutes.
After incubation, remove the plates from the incubator and use a microscope to confirm the cell detachment. Next, add two milliliters of trypsin stop solution, or TSS, into each plate to block the trypsinization. Transfer the cell suspension into a sterile 15 milliliter tube.
Centrifuge it at 400 G for five minutes at four degrees Celsius. Aspirate the supernatant using a five milliliter serological pipette. Then add three milliliters of DMEM to the pellet and resuspend it.
Place the cell suspension on a 60 millimeter plate. Incubate the cells at 37 degrees Celsius, under 5%carbon dioxide supplementation. Next, pipette three milliliters of sterile 0.2%gelatin solution to each 60 millimeter plate.
After incubation, aspirate the solution and add three milliliters of sterile PBS until further use. Take the plate containing cardiac fibroblasts out of the incubator. Remove DMEM from the plate and rinse with sterile PBS.
After discarding the rinse, add one milliliter of 0.25%trypsin EDTA to each plate. Use a microscope to confirm the cell detachment. Next, add two milliliters of TSS into each plate To block the trypsinization.
Transfer the cell suspension into a sterile 15 milliliter tube. Centrifuge it at 400 G for five minutes at four degrees Celsius. Aspirate the supernatant using a five milliliter serological pipette.
Then add three milliliters of DMEM to the pellet and resuspend it. Remove the PBS from the 60 millimeter gelatin-coated plate and place the cell suspension into the plate. Culture the fibroblasts in a confluence state for up to 21 days.
After 21 days of culture, aspirate the culture medium. Then rinse the plates with three milliliters of PBS. After aspirating the PBS and adding one milliliter of decellularization solution, observe the plate under an inverted contrast microscope.
After two minutes, gently pipette four milliliters of PBS to dilute the decellularization solution in the plates. Aspirate the diluted solution. Then gently rinse the plates with PBS.
Finally, add one milliliter of PBS to the plates. Store the extracellular matrix-coated plates at four degrees Celsius until further use. The outgrowth of fibroblasts from the small fragments of native myocardium placed in culture was observed within three to five days.
Decellularization resulted in extracellular matrix coating on the plate surface. The composition and architecture of the in vitro-produced cardiac extracellular matrix was preserved due to decellularization.
