The overall goal of this procedure is to investigate cell interactions between lung cancer cells and cancer associated fibroblasts in a three-dimensional co-culture method. This is accomplished through the primary culture of human lung cancer associated fibroblasts from collected lung tissue that is minced into one millimeter sized pieces. The second step is to prepare collagen gels embedded with fibroblasts by mixing a collagen solution using the trypsin eyes cells, and transferring to a six well plate to gelatinize.
Next lung adenocarcinoma cells are plated onto the surface of each gel to prepare a dimensional co-culture of cancer cells and fibroblasts, and the gel is separated from the edge of the well to generate a floating culture. The final step is to perform the air liquid interface culture and invasion assay through exposure of the contracted gels to air by placing them on a mesh situated in a six well plate. Ultimately, histological examinations are used to evaluate cancer cell invasion and morphological changes.
The main advantage of this procedure over existing methods like two dimensional monolayer culture, is that multicellular interactions can be investigated in three dimensional culture of condition. This procedure begins with the primary culture of fibroblasts from collected lung tissue as described and referenced in the text protocol. In this video, mouse lungs are used instead of human lung cancer tissue.
Place the lung tissue sample on a 10 centimeter tissue culture dish without culture medium. Cut the tissue into small sections of approximately one millimeter in size using sterile forceps and a scalpel During this process, avoid making the section dry, otherwise, the efficiency of cell outgrowth is impaired. Place small lung tissue sections apart from each other to maintain an empty area surrounding each piece.
Make scratches on the surface of the tissue culture dish using a scalpel blade. Alternatively, coverage of tissue pieces enhances the efficacy of cell outgrowth and cell propagation following several passages. First place silicone grease at two points, 1.5 centimeter apart in each well.
Next place minced tissue pieces individually in the center of each. Well then attach the cover slip to the surface of the plate using silicone grease. Gently add culture medium to the dish, or six well plate before the tissue sections dry out.
Efficient cell outgrowth is lost for the most part. If the samples dry out, do not pour the medium too rapidly to avoid detachment of the tissue sections for collagen gel formation. Wash fibroblasts with one x phosphate buffered saline or PBS and add tryin one to detach the outgrowing cells after trypsin ization for approximately five minutes.
At 37 degrees Celsius, detach the cells using nine milliliters of DMEM supplemented with 10%FBS collect the cells suspended in the medium in a 10 milliliter tube and centrifuge them at 200 to 300 times G to form cell pellets.Resus. Suspend the resulting pellets in 100%FBS at a density of half a million cells per milliliter. Cool the reagents, pipettes, and tubes for collagen gel preparation in a refrigerator prior to collagen gel preparation.
Next, prepare the collagen gel by mixing the fibroblast suspension in FBS Type one A collagen five X-D-M-E-M and reconstitution buffer vigorously pipette to ensure a homogeneous mixture avoiding bubble creation during the pipetting process. Then add three milliliters of the mixture to each well of a six well plate and allow to gelatinize in the incubator at 37 degrees Celsius without disturbance for 30 to 60 minutes for cancer cell culture on the collagen gel Resus. Suspend the cancer cells in the 3D co-culture medium at a density of one times 10 to the fifth cells per milliliter.
Poor two milliliters of the resuspended, A 5 49 lung adenocarcinoma cell solution onto the surface of each gel. Modify the cell number of cancer cells or fibroblasts in co-culture depending on experimental conditions to perform gel contraction. Incubate gels overnight at 37 degrees Celsius so the cancer cells can adhere to the collagen gel.
Then detach each and generate a floating culture in each well. Of the six well plate with an angled 21 gauge needle or small spatula to separate each gel from the edge of the well every two to three days. Refresh the wells with two milliliters of 3D co-culture medium.
Measure the size of each gel every day for five days. Finally, see the text protocol for collagen gel analysis. After culturing the collagen gels at 37 degrees Celsius for five days.
Expose the contracted gels to air by placing them on a mesh. In new six well plates make the mesh from commercially available cell strainers used for flow cytometry. Gently fill each well with 11 milliliters of 3D co-culture.Medium.
Position the gels onto the mesh using a sterile spoon and remove any remaining medium from the gels on this condition. Gels are submerged in the medium while the upper surface of the gel is exposed to air. Define this culture condition as air liquid interface.
This co-culture method mimicking the tumor microenvironment is a useful tool to investigate the interactions between cancer cells and fibroblasts embedded in collagen gels. A 5 49 cells were co-culture on a collagen gel embedded with the fibroblasts and the layer of a 5 49 cells was exposed to air by placing the gel onto a mesh in medium. Shown here is a representative picture of the gel placed on the mesh in a well of a six well plate histological analyses with hemat, toin and eoin.
Staining of cross-sections of a three dimensionally cultured gel revealed invasion of a 5 49 cells into the collagen gel embedded with lung cancer associated fibroblasts. Following this procedure, other methods like gene expression or knockdown can be performed in order to answer additional questions like specific gene function in tumor storm interaction.
