The overall goal of this procedure is to establish a tissue like three dimensional collagen matrix to study cell function. This is accomplished by first extracting collagen from rat tail tendons. Next, collagen fibers are reconstituted in the presence of fibroblasts to allow the fibroblasts to contract the collagen gel.
In the third step, tumor cells are seated on top of the matrix in the final step. The matrix is incubated on top of a metal grid to create an air liquid interface to allow invasion of the tumor cells into the matrix. Ultimately, immunohistochemical staining and fluorescence microscopy can be performed to analyze matrix synthesis cell migration, including tumor cell invasion and interactions between different cell types.
So this method is ideally suited to looking at invasion and metastasis, and also looking at tumor development. On a complex three-dimensional S, we can look at things like differentiation, cell survival and cell growth. But in particular, we can look at how tumor cells interact with stroma during key events of invasion.
One of the earliest parts of cancer metastasis itself To establish fibroblast cultures from skin implants. First place a few drops of MEM supplemented with penicillin, streptomycin, and fungi zone in the bottom of a Petri dish. Then place four millimeter punch biopsies acquired from a human forearm into the dish.
Next, finally, chop the biopsy into small pieces by rocking a number 24 scalpel blade against the bottom of the petri dish. Place the tissue slurry in a 25 centimeter squared tissue culture flask. Then pour primary fibroblast growth media over the tissue.
That is enough to cover the surface of the flask, but that is insufficient for the tissue to float. Incubate the flask for three days at 37 degrees Celsius in a humidified atmosphere of 5%CO2, and then add three milliliters of primary fiberblast growth media following another three days.Incubation. Change the media with fresh primary fiberblast growth media.
If the cells are nearly confluent, they may be split one to four into new flasks with fresh media. Begin by washing the rat tails in 70%Ethanol chill a bottle of 0.5 molar acetic acid at four degrees Celsius to remove the tendons from the rat tails. First, remove the skin from each individual rat tail.
Next, detach the tendon from the core of the proximal region of the tail. Finally, remove the tendon towards the distal region of the tail using tooth forceps. After the tendons have been removed, add 250 milliliters of 0.5 molar acetic acid to a glass conical flask.
Extract one gram of tendon per 250 milliliters of acid by stirring at four degrees Celsius for 48 hours after the tendon is extracted. Centrifuge the acid and tendon solution at 7, 500 times G for 30 minutes at four degrees Celsius, and then discard the pellet. Then add an equal volume of 10%weight per volume NACL to the supernatant and stir for an additional 30 to 60 minutes after the second stirring step.
Pellet the solution. This time at 10, 000 times, G, discard the supernatant and red dissolve the precipitate in 0.25 molar acetic acid with more stirring for 24 hours at four degrees Celsius. Next, dialyze the collagen solution against six to eight changes of six liters of approximately 17.5 millimolar acetic acid.
After the dialysis pellet the collagen at 30, 000 times G for 1.5 hours. Finally, transfer the supra natin into a sterile bottle and adjust the collagen concentration to about two milligrams per milliliter with the previously chilled 0.5 millimolar acetic acid, and then keep the collagen on ice. First, select three T 75 flasks of confluent primary fibroblasts.
Put a container of FCS at four degree Celsius to cool. Next, add 75 milliliters of the previously prepared rat tail collagen into a 100 milliliter glass pre cooled sterile bottle. Then add nine milliliters of 10 XMEM while stirring the mixture.
Add six milliliters of 0.22 normal sodium hydroxide slowing when nearing the last milliliter. To ensure the final color is between orange and pink. Trypsin eyes the fibroblasts and then pellet the cells at 400 times G for five minutes at room temperature.
During the spin assemble 36 35 millimeter plastic dishes. Resuspend the pellet in 10 milliliters of the pre cooled FCS, and immediately add the fibroblasts to the collagen. Mix and stir plate approximately 2.5 milliliters of the collagen fibroblast admixture per 35 millimeter dish as quickly as possible.
Avoiding bubbles and setting of the collagen. Place the dishes in the incubator for 10 minutes to allow the collagen to form a loose gel, and then add one milliliter of fiberblast growth media to each dish. Then detach the collagen fiberblast matrix from the sides of the dish using a pipette place the dishes back in the incubator.
On the next day, add another milliliter of Fiberblast growth media to the dishes. Then change the media every other day for about the next eight days. During this time, each collagen fibroblast matrix should contract from about 3.5 centimeters to about 1.5 centimeters in diameter before beginning this next step, sterilize all forceps and equipment with ethanol.
Then using blunt sterilized forceps, gently move a contracted matrix to a well of a 24 well plate, making sure the matrix does not fold. Next, select a flask of cells to be studied tripps knives and pellet the cells as before, and then resuspend the cells in media plate one milliliter of the cell suspension on top of the matrix, and place the contracted matrix in the incubator until the cells have grown to almost confluence on until top of the matrix. Approximately three to five days after autoclaving precut stainless steel grids for the creation of tripods prior to use place, a sterile grid in a six centimeter plastic dish add enough growth media to cover the grid.
Creating the air liquid interface is the most delicate part of this procedure. The matrix must be placed on the grid and then gently that medium must be aspirated off such that the matrix is still partly submerged from the bottom and is fed from the bottom. This creates a chemotactic gradient and allows the cells on top to start to invade into the matrix.
Cell behavior in this setup can then be assessed over 21 days or above. Place a matrix on the grid. Next gently aspirate the media until the bottom of the matrix is in contact with, but not covered by the media.
The air liquid interface should then look similar to that scene here. Next, transfer an infiltrated matrix onto a flat surface. Cut the matrix in half with a fresh scalpel, and then lift the matrix with the scalpel and transfer it to 4%PFA.
Fix the matrix overnight at room temperature in the following two figures, pancreatic ductal adenocarcinoma cells or P DAC cells were layered over an organotypic matrix. In the first figure, non-invasive P dac cells can be seen in a layer on top of the matrix, whereas in this second figure invasive P dac cells that have infiltrated the matrix are observed here, a living skin equivalent showing a stratified epidermis on a fibroblast contracted collagenous dermal component can be seen here. Invasive PD cells in green can be seen interacting with fibroblasts in red, which promote invasion of the PDX cells into the organotypic matrix.
The invasive PD cells shown here again in green are visualized within the collagen matrix, which is imaged using second harmonic generation, and shown in purple sites of matrix degradation are visible as dark holes within the purple matrix. In this figure C 8 1 61 melanoma cells invading a fibroblast contracted collagenous dermal equivalent are shown, As well as looking at the behavior of fundamental processes of fluorescent proteins during this assay. You can also use this asay along with immunohistochemical staining to look at cell proliferation markers and differences in cell survival, et cetera.
