The overall goal of this procedure is to sandwich collagen embedded cancer cells within a fibrin gel to generate a 3D culture system for the monitoring of satellite tumor invasiveness and formation. Our 3D picture system can offer new avenues for investigating various cell and many key variants during cancer cell apoptosis or migration or to evaluate the efficacy of anti-cancer cell loss. An attractive feature of this 3D cell culture model is the ability to change the concentration of the extra cellular matrix or use another extra cellular Matrix for the assembling of the 3D system.
Demonstrating the procedure will be Marie-France Cote. Research assistant, my laboratory, and Audrey Turcotte, a grad student under my supervision. Begin by blending freeze dried rat tail tendon type one collagen for five two minute mixes at the high speed setting to uniformly disperse the collagen.
While the collagen is being incorporated, harvest the tumor cells of interest with the appropriate enzyme detaching solution. Followed by Trypan blue exclusion in numeration. Dilute the dissociated cells to a 50, 000 cells per plug concentration in DMEM.
When the collagen is ready, quickly add 1, 250, 000 cells to the collagen solution with pipetting to homogeneously disperse the cells. Immediately dispense 200 microliters of the single cell suspension into each well of a 96 well plate. Gently tapping the plate on the work area surface after each addition to remove any air bubbles and to distribute the solution evenly within the wells.
When all of the cells have been added, place the plate in a 37 degree Celsius incubator for at least two hours, adding 100 microliters of culture medium to each well after the first two hours if the cells are to be cultured overnight. To prepare the first fibrin gel layer, bring the freeze dried fibrinogen to room temperature before opening the vial to avoid hydrate crystal formation. Then transfer the fibrinogen to a glass beaker and add 37 degrees Celsius HBSS drop wise to the powder to solubilize the fibrinogen fragments.
Next, use a spatula to break down the larger protein pieces, agitating the beaker from time to time as appropriate to facilitate mixing. Pipette the suspension several times to dissolve the remaining protein, and filter sterilize the solution through a 22 micron filter. Then immediately overlay the surface of each well of a 24 well plate with 200 microliters of the fibrinogen, taking care to avoid air bubbles.
Once all of the wells have been thoroughly coated, tilt the plate at a 45 angle, then drop 1.5 microliters of thrombin solution into the center of one well and gently swirl the plate horizontally for one to two seconds. After treating five more wells with thrombin in the same manner, place the plate in a stable horizontal position under a laminar flow hood for five to 10 minutes, until the solutions gel. For immediate placement of the collagen plug, carefully tilt the plate to confirm that the fibrin gels are completely polymerized.
Then place the 96 well plate of collagen gel plugs next to the 24 well plate of fibrin gels and add one drop of HBSS into each collagen plug well. Next, use a micro spoon to transfer a single collagen plug into the center of each of the first fibrin gel layers in the 24 well plate. When all of the plugs have been transferred, add 300 microliters of fibrinogen solution to each well, followed by the addition of 2.25 microliters of thrombin as just demonstrated.
To coat the collagen plugs with a thin layer of growth factor reduced basement membrane before their placement, transfer each plug into a 1.5 milliliter centrifuge tube on ice containing 100 microliters of pure growth factor reduced basement membrane. After two minutes of soaking, transfer a single coated collagen plug into the center of each fibrin layer and incubate the plugs at 37 degrees Celsius for five minutes to solidify the growth factor reduced basement membrane. Then add the second fibrin layer as just demonstrated.
When the gels are fully polymerized, fill each well with 400 microliters of the appropriate culture medium and supplements. Next, add an Antifibrinolytic agent to the wells. And incubate the 3D cultures under the appropriate conditions for the tumor cell line being tested.
On the appropriate cell feeding days, tilt the plate at a 30 to 35 degree angle and incline a pipette against the wall of each well to aspirate the medium without disturbing the gels. Then add 400 microliters of fresh cell culture medium and Antifibrinolytic agent to each gel culture. This 3D cell culture assay allows a direct visualization of cancer cell migration from the collagen plug into the adjacent fibrin gel for the establishment of secondary tumors, allowing a comparison of the behavior or known metastatic cells to that of non or weakly metastatic cells.
For example, in this experiment, numerous satellite tumors dispersed randomly throughout the fibrin gel containing the metastatic cell line B16 F10 while no satellite tumors were seen for the weakly metastatic B16 F0 cell line. By contrast, these murine mammary gland carcinoma cells which are poorly metastatic but very invasive locally form a migration front that extends radially into the fibrin gel. After 14 days of culture, the mammary gland carcinoma cells pull away from the migratory front to form stellar shaped structures closely resembling mammary gland structures.
Chemotherapeutic agents can be tested on the embedded cancer cells as in this representative experiment where an increasing amount of DNA fragmentation were observed concomitant with the increased incidents of chemotherapeutic agent induced cancer cell apoptosis. When mastered, this technique can be completed in approximately six hours or spread across two days. Depending on the initial collagen plug incubation period.
While I'm attempting this procedure, it's important to prevent the formation of gaps at the interface between the central and the peripheral gels as they can reduce the ability of the cancer cells to invade the fibrin gel. Following this procedure, metallographic or Proteomic analysis can be performed in the satellite colonies, the collagen plugs, or the cancer cells to identify the differential expression of genes and proteins of interest. After its development and this technique paved the way to researchers in the field of cancer biology to explore the migration apoptosis metastatic suppressive gene expression can end angiogenesis to a variety of cancer cell lines.
After watching this video, you should have a good understanding of how to embed cancer cells in a collagen gel and sandwich them within a fibrin gel to monitor the invasiveness and formation of satellite tumors.
