These methods can provide mechanistic insights into immune cell-mediated tumor cell cytotoxicity and invasive behavior in a 3D setting. The main advantage of this technique is that this 3D spheroid co-culture model does not require the transfer of spheroids for multiple subsequent essays. Demonstrating this procedure will be Apsra Nasir, a PhD student from our department.
To generate the spheroids using aseptic technique in a cell culture hood add 500 microliters of molten agarose into an 81 microwell rubber mold taking care to avoid bubbles. When the agarose has solidified carefully flex the rubber mold to pop out the agarose casts into individual wells of a 12 well plate. To equilibrate the casts add 2.5 milliliters of cell culture medium supplemented with 10%fetal bovine serum into each well and place the plate into a cell culture incubator for one hour.
At the end of the incubation tilt the plate to remove the cell culture medium in the surrounding first, then in the seeding chamber and carefully seed 190 microliters of the prepared to tumor cell suspension dropwise into each cell seeding chamber. After a 15 minute incubation in the cell culture incubator add at 2.5 milliliters of medium to the outside of the cast and return the plate to the incubator for 48 hours. To set up a T-cell co-culture resuspend the appropriate number of T-cells in 190 microliters of inappropriate T-cell culture, medium per well until the 12 well-plate to allow removal of the cell culture medium surrounding the agarose cast first.
Then in the seeding chamber, without removing the spheroids use a light microscope to check whether any spheroids have been aspirated and holding the P200 loaded pipette about half a centimeter above each cast carefully seed the T-cells onto the casts in a dropwise fashion without dislodging the spheroids. When all of the casts have been seeded carefully returned the plate to the cell culture incubator for 15 minutes before adding fresh cell culture medium supplemented with fetal bovine serum to the outside of each cast for another 48 hour incubation. To embed the 3D co-cultures in type one collagen dilute stock collagen with serum free base medium to a final working concentration of three milligrams per milliliter and add 11 microliters of 10X PBS, add 1.2 microliters of one molar sodium hydroxide per 100 microliters of collagen.
After neutralizing the collagen solution on ice for one hour, remove the cell culture medium surrounding and within each cast as demonstrated. Carefully add the neutralized collagen one mixture to each cast in a dropwise fashion, and immediately returned the plate to the cell culture incubator for five minutes before inverting the plate for an additional one hour in the incubation. At the end of the incubation place the plate right side up in the biosafety hood and slowly add a fresh cell culture medium down the side of each well.
Then return the plate to the cell culture incubator for 48 hours. For immunofluorescent staining of the embedded 3D co-cultures fix each entire agarose cast including the spheroids in 5.4%formalin overnight at room temperature in a humidified chamber. The next day, remove the formalin surrounding the agarose cast and use sleek tip tweezers to grasp the corner of each collagen matrix to allow the casts to be peeled from the seeding chambers in a single fluid movement.
Place each collagen patch into a single well of an eight well chamber slide and add 250 microliters of 0.5%octoxynol all to each well. After one hour at room temperature replace the octoxynol with 250 microliters of blocking solution. After one hour at room temperature add a 250 microliters of the primary antibody cocktail of interest to each well and place a slide in a dark humidified chamber overnight at room temperature.
The next morning remove the primary antibody from each well and wash the wells three times with 300 microliters of IF buffer for five minutes at room temperature per wash. After the last wash, add a 250 microliters of inappropriate secondary antibody solution to each well for a one hour incubation at room temperature protected from light. At the end of the incubation remove the antibody and wash each sample three times with IF buffer as demonstrated.
After the last wash rinse the samples with 300 microliters of PBS per well and carefully remove the walls of the chamber slide. So that only the glass slide at the bottom remains. Add 200 microliters of mounting medium to the glass slide and carefully drop the cover slip onto the sample without creating bubbles then store the mounted samples in a dark dry place overnight before imaging by confocal fluorescence microscopy Here typical experimental setups for the assays and they yield of representative and analyzable samples at the end of the experiment for each protocol can be observed.
Embedding the 3D culture in type one collagen within the micro wells of an agarose cast allows monitoring and analysis of the invasion by image J.In this analysis of two primary murine, pancreatic cancer cell lines different spheroid shapes and invasive behaviors were observed. The first cell line one demonstrated a more compact spheroid formation and spiky invasion similar to that observed for single cell invasions. While the second cell line formed spheroids that were more loose and demonstrated a collective invasion pattern.
Co-culture can be performed with two different tumor clonal cell lines seeded at the same time or with tumor and T-cells upon tumor spheroid formation or a subsequent tumor T-cell interaction evaluations. For a detailed assessment of the invasive behavior immunofluorescent staining and con focal imaging can be performed in a high throughput manner. The agarose cast allows embedding of the whole 3D culture system in paraffin for serial sectioning and immunohistochemistry staining to quantify the spatial relationship between tumor and T-cells.
For example, T-cell infiltration can be identified and characterized by cell surface marker staining. This technique allows the analysis of patient samples as well as the use of stimulating and blocking drugs to probe tumor cell T-cell interactions.
