The overall goal of the following procedure is to generate a native matrix from fibroblasts for assessing tumor cell invasion. This is accomplished first by growing fibroblasts on plastic in a sorbic acid enriched media to generate the native matrix. In the second step, the native matrix is detached and released from the plastic growing surface, and then this matrix is seeded with tumor cells and lifted to the air liquid interface after a fixed period.
These 3D cultures are harvested for histological analysis of the tumor cell invasion. Ultimately, the effects of the native fibroblast matrix on tumor cell invasion can be evaluated by immuno histological analysis. So the advantage of this technique for generating 3D typic cultures is that we can assess native matrix produced by fibroblasts themselves in the absence of synthetic or foreign substrates, such as cross species type one collagen Begin by seeding 200, 000 fibroblasts per well in six well plates in fiberblast media, supplemented with freshly prepared IC acid.
Two phosphate refe the cells every two to three days with two to five milliliters of fresh media. A thick layer of cells embedded in extracellular matrix visible to the naked eye will form at the end of six weeks. Depending on the cells used, the visible matrix may form sooner or later and may begin to detach from the dish as seen here.
To release this layer from the tissue culture plate, gently scrape the circumference of the matrix with a one milliliter micro pipette tip, and then push the edges of the matrix towards the center of the well. The cells and native matrix should detach easily from the plate surface and now be floating in the media. Spread the native matrix out within the media to avoid it folding up into itself.
Let the native matrix float and remodel for five days, continuing to change the media every two to three days due to tensile strength and intrinsic remodeling. The matrix will contract drastically and become reduced to a smaller but thicker native matrix, at which time it will be ready to be utilized. For the invasion assay.
For the invasion assay. First use blunt forceps to transfer the native matrix gently to a nylon net. Once on the net, use a one milliliter micro pipette tip and blunt forceps to gently spread the matrix to lie as flat as possible.
Next, smear a small amount of Vaseline onto one end of one sterile clonal cylinder per matrix. Then place the cylinders onto the native matrices Vaseline side down to ensure a tight seal between the native matrix and the clonal rings. Now add 250, 000 cutaneous squamous cell carcinoma or SCC cells in carat site growth media to the cylinders.
After the cutaneous SCC cells have settled onto the native matrix, remove the cylinders, then lift the nylon net native matrix and cutaneous SEC cells to the air liquid interface, and onto a bent stainless steel wire mesh support. Finally, add Keratinocyte growth media, supplemented with as sorbic acid until the media level touches the bottom of the native matrix, changing the media every two to three days and harvesting at seven and 14 days post seeding of the cutaneous SCC cells. This technique opens up the possibility of examining and comparing the invasive behavior of tumor cells under different 3D stromal environments.
As seen in these images, invasion of the RDEB cutaneous SCC keratinocytes was significantly retarded in C seven over expressing native matrices in the right panels compared to the C seven deficient RDEB controls in the left panels. The development of this technique has enabled researchers to assess directly the influence of Native Matrix produced by fibroblasts on tumor cell processes such as invasion.
