This method can help answer key questions in oral cancer research field, such as how to build an appropriate T-assist model in-vitro. The main advantage of this technique is that reconstructed TSCC express tumor characteristics that bear a strong resemblance to clinical TSCC histopathology. When first had the idea for this method when we realized that 2-D cultured TSCC cells did not properly exhibit the native phenotype of TSCC.

Furthermore, we realized the importance of extracellular matrix for cancer survival evasion and differentiation. Visual demonstration of this method is critical as the preparation of tongue extracellular matrix and 3-D culture using a mini bioreactor are difficult to learn through words only, and are best as seen for clarification. After isolating the tongues from euthanized mice according to the text protocol, immerse the tissue in 75%ethanol for three minutes.

Then transfer each tongue into a 1.5 milliliter microcentrifuge tube, with one milliliter of 10 milliMolar sterile PBS. To carry out cell lysis, freeze the tongues at minus 80 degrees Celsius for one hour. Then thaw the tissue at room temperature for 45 minutes.

Repeat the freeze/thaw two more times. Next, in a 3 1/2 centimeter or six 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 1/2 centimeter or six centimeter culture dish, use three 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, ultra-pure water and add a stir bar. Lower up to the five tongues into the bottle, until they are approximately two 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 one 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%TritonX-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 one 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 tissues at 37 degrees Celsius for 24 hours. Transfer the samples back to a wide mouth bottle of steril 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 four degrees Celsius until use. To reconstitute a static model for TSCC seed one times 10 to the sixth single Cal-27 cells into a 3 1/2 centimeter culture dish. Add three milliliters of DMEM F12 medium containing 10%FBS into 90 micrograms per milliliter each of ampicillin and kanamycin.

Incubate the cells at 37 degrees Celsius for two to three days to 60%confluency. Then load the TEM onto the cell monolayer in the culture dish. Incubate the dish at 37 degrees Celsius and 5%carbon dioxide for 28 days.

Refresh the medium every day. Prepare a self-made stirred mini bioreactor by removing the plunger from a 10 milliliter syringe. Make a hole one centimeter in diameter near the lower terminal of the rod, and load a stir bar through the hole.

Make another hole a half centimeter in diameter at the center of the bottle cap of a plastic wide mouth bottle, and put the piston rod through the cap. Then cut half of a 50 milliliter centrifuge tube and weld it on the outer side of the bottle cap. Attach fish hooks to the rod by wrapping the rod with fishing lines tied to the fishhooks.

To prepare a dynamic cell culture, seed one times 10 to the sixth single Cal-27 cells in the mini bioreactor. Add 150 milliliters of DF12 medium containing 10%FBS, and 90 micrograms per milliliter each of ampicillin and kanamycin. Using the fishhooks attached to the rod, load the TEM onto the mini bioreactor, then tighten the bottle cap.

Place the mini bioreactor on a magnetic stirrer in a carbon dioxide incubator at 37 degrees Celsius. Active the mini bioreactor at 200 RPM for seven to 14 days. H and E staining confirmed that the protocol demonstrated in this video produced TEM with perfect decellularization compared to native tongue tissues with complete disappearance of nuclear staining, and only rare damage to the tissue integrity, while eliminating the cellular components.

In this figure, H and E staining reveals that 3-D reconstitution of TSCC using TEM and a self-made mini bioreactor produced typical TSCC pathological characteristics. The cells in stirred culture conditions demonstrated single-cell migration or collective migration in different lesion areas. In static culture conditions, Cal-27 cells also formed invasive structures in the TEM, however it took longer.

Though U2OS cells could live in the culture medium, they were not found in the TEM, indicating that the TEM is biocompatible with certain types of cancer cells, and that different tumor cells need different microenvironments to flourish. While attempting this procedure it's important to remember to operate under sterile conditions. After it's development this technique paved the way for the researchers in field of oral cancer to explore characteristics of TSCC cells in extracellular matrix samples.

After watching this video, you should have a good understanding of how to prepare the cellularized tongue extracellular matrix and fabricate an in-vitro model of TSCC.