This article details the preparation of the extracellular matrix (ECM) from mouse tongue tissue. The process involves several steps including washing, freeze-thaw cycles, and treatment with various solutions to achieve decellularization.
The extracellular matrix or ECM - the non-cellular component of the tissue environment - consists of a complex network of structural and regulatory proteins and polysaccharides secreted by cells. The ECM provides structural and biochemical support to cells and regulates their growth.
To prepare the tongue extracellular matrix, begin by taking a freshly harvested mouse tongue. Wash it with ethanol to remove its lipid layer.
Next, subject it to alternating freeze and thaw cycles. Freezing promotes intracellular ice crystal formation, while thawing melts the ice crystals, resulting in the disruption of the cellular membrane.
Now, suspend the tongue in an antibiotic solution to inhibit bacterial growth. Subsequently, treat the organ with a hypertonic saline solution. In the presence of a high solute concentration, water flows out from the cells, causing them to shrink. This step aids in cellular death and detachment from the extracellular matrix.
Incubate the tongue in a non-ionic surfactant. The surfactant completely solubilizes the cellular and nuclear membranes, facilitating the release of intracellular contents into the solution.
Lastly, suspend the tongue in a solution containing calcium and magnesium ions, followed by nucleases treatment. The calcium and magnesium ions enhance the activity of nucleases to degrade any free nucleic acids in the solution.
Store the decellularized tongue tissue in a suitable buffer until further processing.
After isolating the tongues from euthanized mice according to the text protocol, immerse the tissue in 75% ethanol for 3 minutes. Then, transfer each tongue into a 1.5-milliliter microcentrifuge tube with 1 milliliter of 10 millimolar sterile PBS. To carry out cell lysis, freeze the tongues at minus 80 degrees Celsius for 1 hour. Then, thaw the tissue at room temperature for 45 minutes. Repeat the freeze-thaw two more times.
Next, in a 3.5-centimeter or 6-centimeter culture dish containing 75% ethanol, use a surgical needle to load each tongue onto a piece of surgical suture and, with a small piece of sterile tin foil, wrap the end of the suture. In a 3.5-centimeter or 6-centimeter culture dish, use 3 milliliters of PBS to rinse each tongue for 30 seconds. Then, in a wide-mouth bottle, prepare 90 micrograms per milliliter of ampicillin in 250 milliliters of sterile ultrapure water and add a stir bar.
Lower up to 5 tongues into the bottle until they are approximately 2 centimeters from the bottom and tighten the cap with part of the suture remaining outside the bottle. Place the bottle on a magnetic stirrer for 12 hours. Then, after the incubation, prepare 250 milliliters of 90 micrograms per milliliter of ampicillin in sterile 1 molar sodium chloride. Transfer the tongues and stir bar into the bottle and screw on the cap. Place the bottle on the magnetic stirrer for 24 hours.
To carry out cell lysis, prepare 90 micrograms per milliliter of ampicillin in 250 milliliters of sterile 2% Triton X-100 in PBS. Transfer the tongues and the stir bar to the bottle and tighten the cap. Then, stir the bottle for 48 hours. After preparing calcium chloride and magnesium chloride with ampicillin, move the tissue and stir bar into the bottle and stir the bottle for 24 hours.
Prepare 1 milliliter of 300 micromolar DNase in HBSS in one microcentrifuge tube for each tongue sample. Transfer the tongues into the tubes with the same portion of suture hanging outside. Then, incubate the tissue at 37 degrees Celsius for 24 hours. Transfer the samples back to a wide-mouth bottle of sterile PBS with ampicillin and place it on the stirrer for 24 hours. Following the incubation, store the prepared tongue extracellular matrix, or TEM, in sterile PBS at 4 degrees Celsius until use.
繁體中文
