The overall goal of this procedure is to generate organotypic raft cultures from primary keratinocytes or established epithelial cell lines. This is accomplished by first making fibroblast collagen plugs. Next epithelial cells are layered on top of the plugs.
The plugs are then moved to the raft grids and maintained at an air liquid interface for 10 to 14 days. In the final step, the rafts are harvested. Ultimately, changes in epithelial differentiation can be evaluated by histological or immunohistochemical analysis That this method can provide insight into epithelial differentiation.
It can also be applied to other systems such as viruses that infect the cutaneous or mucosal epithelium as part of their lifecycle. Demonstrating the procedure will be Dan Anneker, a graduate student in my laboratory. First, pour chromic sulfuric acid into a glass beaker and immerse the metal raft grids in the acid for one hour.
Then continuously rinse the grids overnight with tap water. After the overnight rinse, rinse the grids for another three to five hours in double distilled water, let the raft grids dry. Then bend three sides of the raft, about 0.5 centimeters that equal distance from each other, autoclave the raft grids.
After preparing the collagen gel, trypsin is the J two fibroblasts, and then neutralize the tryin with medium. Transfer the fibroblasts into a 50 milliliter conical vial, and count the cells to determine the number of rafts that can be made. Then after spinning down the cells for four minutes at 244 times G at room temperature, aspirate the medium from the fiberblast pellet and resuspend the cells in an appropriate amount of reconstitution buffer DMEM and collagen for the number of rafts calculated.
Adding the collagen last to prevent the gel from solidifying too quickly. Once the collagen has been added, mix the solution quickly but gently to prevent the introduction of bubbles into the gel. If the gel mixture is too yellow, add a couple of drops of filter sterilized.
One normal sodium hydroxide. Mix gently until the gel turns a reddish orange color indicative of the correct pH. Now pipette three milliliters of the collagen fibroblast mixture slowly down the side of each well of a six well culture dish, avoiding generating bubbles, and then incubate the plate for 30 minutes at 37 degrees Celsius to allow the gel to solidify.
When the gel is solid, add three milliliters of E medium with EGF to the top of the solidified collagen gel, and return the plate to the incubator. After growing low passage keratinocytes to approximately 70%co fluency, trypsin is the keratinocytes, and then neutralize the trypsin with eed. With EGF, count the cells to determine the number of rafts that can be made.
After spinning down the cells resuspend the pellet in one milliliter of eed with EGF for each raft culture calculated. Now, remove the medium from the collagen gels by tilting the six well plate onto its side, and then add two milliliters of fresh eed with EGF to each gel. Next carefully, add one milliliter of resuspended keratinocytes to the two milliliters of eed already present in each of the wells.
Gently pipette the keratinocytes plus the two milliliters of media, and let fall dropwise onto the collagen plug. Place the plate in the 37 degrees Celsius incubator for two to four days to allow the cells to grow to co fluency. The cells are ready when the media changes to yellow one day after the media was replaced, or by day four, use sterile forceps to place a sterile metal raft grid bent sides down into a 10 centimeter dish.
Aspirate the medium from one collagen gel, and then slide a sterile spatula around the perimeter of the gel in an up and down motion to loosen the gel from the sides of the well. Moving the collagen plug from the six well plate to the grid is the most difficult part of this procedure. Take care to gently slide the spatula underneath the plug without splitting it and lay it down flat on the grid without creating any air bubbles between the plug and the grid.
Afterwards, you can use a spatula to smooth out any parts that have folded under during this procedure. To remove the gel, tilt the plate slightly, and place the spatula under the gel and lift. Lay the collagen gel on the metal grid without generating bubbles between the grid and the gel.
To make an air liquid interface add EED without EGF slowly to the bottom of the dish so that the raft grid is touching the media, but the collagen gel is not Finally incubate the rafts at 37 degrees Celsius. Maintaining the air liquid interface harvest the rafts after 14 days. As seen in this figure rafts made from HPV 31 immortalized primary human keratinocytes or H fk, 30 ones are notably thicker compared to rafts made from normal human for skin, keratinocytes or hks.
Thus, HPV proteins have the ability to maintain differentiating cells active in the cell cycle. Here, immunohistochemical analysis was performed on cross-sections of organotypic raft cultures generated from HK 30 ones as well as normal HKS using an antibody against cytokeratin 10 or K 10 in green. K 10 was not expressed in the basal layer indicated here by the arrows, and instead was found only in the supra basal layers, which consists of progressively differentiating cells.
In this figure. The expression of the HPV protein, E one, E four, a late protein that is restricted to the uppermost layers of the epithelium and represented here in green is shown as expected. No staining is observed in raft cultures from normal hks, as in the previous figure.
The arrows once again indicate the basal layer of the epithelium After its development. This technique paved the way for researchers in the field of cancer biology to study the pathogenesis of human papillomaviruses using a system that mimics epithelial differentiation in vivo. After watching this video, you should have a good understanding of how to make organotypic raft cultures from primary keratinocytes or established epithelial cell lines.
