The breast cancer cell spheroids described herein will allow researchers to investigate molecular mechanisms of cancer cell endothelial cell interactions that are involved in proliferation and metastasis of cancer cells. The main advantage of this 3D model is that it provides a more realistic cellular arrangement that improves the reliability of inferences regarding the pathophysiology and treatment of breast cancer. Demonstrating the procedure will be Giovanna Azzarito, a PhD student from my laboratory.
Begin by treating or transfecting the plated cells for 24 hours, or as desired. For identifying cell distribution in spheroid, wash the cells with one milliliter of Hanks'Balanced Salt Solution or HBSS, containing calcium and magnesium and stay in HUVECs using 0.5 micrograms per milliliter blue dye. And MCF-7 cells using 0.5 micromolar green dye diluted in the respect of growth medium.
Place the plate into a 37 degree Celsius and five percent CO2 incubator for 30 to 40 minutes. Wash the cells with one milliliter of HBSS without calcium and magnesium. Then add one milliliter of enzymatic detachment re-agent, which is 0.25%trypsin for HUVEC and 0.5%trips in four MCF-7 to each brown dish using a P1000 pipette.
Elect each cell type separately in a five milliliter round bottom polystyrene tube, and add one milliliter of 10%Fetal Calf Serum or FCS medium using a P1000 pipette to stop the enzymatic reaction. Then centrifuge the cell suspensions at 250 times G for five minutes. Carefully aspirate the supernatant and suspend the cells in one milliliter of steroid free medium.
Use 100 microliters of each cell suspension diluted with 10 milliliters of Diluent 2 to determine the total cell number. Turn on the machine and flush by pressing the function and start buttons, with Diluent 2 solution in a cell counter vial. Use set up as four and press start to measure the blank with fresh Diluent 2 solution.
Change the scintillation vial with 100 microliters of cell suspension in 10 milliliters of Diluent 2 solution. Use set up as four and press start to measure the samples. Note the number of cells per milliliter on the digital display, Then prepare five milliliters of each cell suspension and mix them to get a final volume of 10 milliliters in a one to one ratio, using manual repeating pipette to pipette out 100 microliters of the cell suspension into each well of the 96-well U-bottom plate.
Place the plate into an incubator under standard tissue culture conditions and check for a spheroids formation after 48 hours. Take pictures of this spheroids at 40 X magnification using a phase contrast or fluorescence stereo microscope. For preparing the hanging drop culture mix the cell suspensions in a one-to-one ratio to get a final volume of two milliliters.
Use a P20 pipette to see the cell mixture in the form of 15 microliter drops on the inverted lead of a 10 centimeter Petri dish. Invert the lid with the drops and add five milliliters of EBM-2 base medium at the bottom of the dish to avoid evaporation of the drops. Collect approximately 50 spheroids in a 1.5 milliliter tube with a gut tip of a P200 microliter pipette.
Allow them to settle to the bottom of the tube by gravity and then discard the supernatant. Fix this spheroids with 500 microliters of 4%PFA for one hour at room temperature. Boil 2%Nobel Algor Solution in PBS using a hot plate with a magnetic stir for three to five minutes to dissolve the augurous powder completely and cooled down to around at 60 degrees Celsius.
Remove the PFA with a P1000 pipette, wash this spheroids with 500 microliters of PBS and allow them to sediment. Once settled discard the supernatant carefully pipette 600 microliters of Agora solution into the 1.5 milliliter tube with spheroids and placed the tube immediately in a centrifuge with a horizontal rotor at 177 times G for two minutes. Add a short string in the middle of the Agro Solution to easily remove the plug from the tube.
Solidify the Agros plug on ice, or at four degrees Celsius add 500 microliters of PBS into the 1.5 milliliter tube to avoid drying out the pellet. Acquire images of spheroid sections using a stereo microscope. Calculate the amount of solution necessary to add a 10 microliters per well of a mixture of calcium Aam and ethidium homodimers diluted using EBM-2 in a one to 50 ratio.
Add the staining mixture to the spheroids and place the plate in the incubator under the standard, the tissue culture conditions for 30 to 60 minutes. Acquire pictures at 40 X magnification using the fluorescence stereo microscope. In U-bottom plates the spheroids formation was observed 48 hours after seeding of MCF seven cells.
However, the spheroids formation was not observed in the case of endothelial cells or lymphatic cells. Seeding the MCF seven plus endothelial cells or MCF seven plus lymphatic cells at one-to-one ratio also resulted in spheroid formation. Time dependent sequential growth of MCF seven spheroid was recorded ranging from 24 to 120 hours.
The spheroids size increased from around 100 micrometers to around 200 micrometers over at five days. With an increased growth rate observed in the spheroids treated with the growth stimulator. The structure of the spheroids and the shape of their cells showed no abnormalities following transfection with control oligonucleotides in the presence of Lipofectamine.
Cells expressing their proliferative marker Ki-67 were distributed homogeneously in this spheroids. Cleave to caspase three staining of this spheroids showed apathetic cells after four days of culture. Studying of the cells in this spheroids with CD-31 and Ki-67 revealed that 55%of the cells are epithelial 45%are endothelial and 25%of cells are proliferating.
To assess the percentage of live and dead cells four day old spheroids were stained with calcium Aam and ethidium-homodimer. A freshly thought spheroid also stain for dead and live cells revealed that spheroids are very sensitive to freezing conditions. When following his protocol, make sure that healthy cells are used to mix aphrodite Another thing to remember would be to avoid damaging these aphrodite during DP cap to obtain a good sections also ensurer to palate the aphrodite before the across polymerizes.
This model could be further improved by including other cells, such as fibroblasts involved in tumor progression and using it to assess the efficacy of therapeutic agents targeting a cancer growth.
