Culturing fibroblasts in spheroid recapitulate tumor tissue much better than the commonly used 2D system on stiff plastic. It allows to study fibroblasts differentiation in a tumor-like environment. In this method, the rigidity of cell culture plastic work does not artifactually influence fibroblast differentiation.
Instead, the influence of cell matrix interaction on tumor induced differentiation of fibroblast can be analyzed in vitro. To begin this procedure, obtain the cell culture media, which is MEM supplemented with 1%heat inactivated FBS and 1%penicillin streptomycin. Also, obtain a six milligram per milliliter methylcellulose solution.
Mix one part of the methylcellulose with three parts of the cell culture media to prepare the spheroid formation solution. Resuspend freshly thawed immortalized normal fibroblasts or other cell types as listed in the text protocol in the spheroid formation solution. If cytokines or integrin inhibitors are included in the study, add these compounds to the cell suspension in order to imbed them into the spheroids during their formation.
Where appropriate, add inhibitor compounds together with 10 nanograms per milliliter of TGF beta one to trigger differentiation in immortalized normal fibroblasts. Distribute 100 microliters of the spheroid formation solution into each well of a round bottom 96 multi-well plate. Pipette the solution in each well up and down several times to mix.
Then, incubate the plate in the cell culture incubator at 37 degrees celsius with 5%carbon dioxide for 24 hours to allow one spheroid to be formed in each well. First, cut off the end of a pipette tip to enlarge the orifice. After the incubation is complete, use this cut pipette to collect the spheroids into one 1.5 milliliter reaction tube per experimental condition or per protein to be analyzed.
Centrifuge the spheroids at 1000 times G for about 30 to 60 seconds. Carefully remove the methylcellulose containing supernatant by pipetting making sure to not disturb the pelleted spheroids. Special care is to be taken when transferring the spheroids to the reaction tubes.
Make sure you don't have shears drift forces by cutting the tip. It's also important that you collect all the spheroids. During the washing step, make sure you don't lose the spheroids by aspiration.
To wash the spheroids add 50 microliters of one X PBS. Centrifuge at 1000 times G for 30 to 60 seconds. Then carefully remove the PBS by pipetting.
Depending on the protein to be stained, fix the spheroids either with 50 microliters of 4%paraformaldehyde and PBS for 20 minutes at room temperature. Wash the spheroids with PBS as previously described. Permeabilize the cells with 0.1%triton X and PBS for four minutes at room temperature.
Then, wash the spheroids in PBS three times as previously described. Add PBS containing 5%FBS and 2%BSA to the spheroids and incubate at room temperature for one hour to block non-specific protein interaction sites. Now, incubate the spheroids with 30 microliters of primary antibodies and PBS with 2.5%FBS and 1%BSA at four degrees celsius overnight.
The next day, wash the spheroids in PBS three times as described previously. After this, incubate the spheroids with 30 microliters of secondary antibodies and PBS with 2.5%FBS and 1%BSA at room temperature for 90 minutes. Wash the spheroids in PBS three times as previously described.
Next, incubate the cells with 30 microliters of Dappy staining solution for four minutes at room temperature. Wash the spheroids in PBS one time as previously described. Using a glass Pasteur pipette, place the spheroids on a glass slide and a drop of PBS.
Use a laser scanning microscope to image the spheroids by fluorescence microscopy at a magnification of 10 times. Take different optical cross section of the spheroid at different optical plains of the Z stack. A representative image of the immunofluorescent staining of the homo spheroids of normal pancreatic fibroblasts compared to the homo spheroids of immortalized cancer associated fibroblasts shows an increased signal for the alpha three subunit in the differentiated cells.
The fluorescence signal of the cells in the spheroid are then quantified with the Z stack images of the 3D spheroid and the transcriptional levels of the integrin alpha three subunit gene are determined by qPCR. All of these results demonstrate that integrin alpha three is upregulated in immortalized cancer associated fibroblasts as compared to the normal counterpart. This proves that integrin alpha three beta one can be considered a marker for pancreatic fibroblasts differentiation.
This method is ideally suited to capitulate self tissue generally found in organs and cancer masses where the extra cell matrix stiffness determines cell fate. Spheroids culture is a versatile model that can be combined with other analytical assays after spheroid dissociation such as quantity real time PCR and full cytometry. Paraformaldehyde used for fixation of the spheroids is a hazardous chemical agent.
Gloves and eye wear should be worn for protection.
