4 Notes
1. This technique can be applied to different cell types and multicellular models. Depending on the chosen cell type, optimization of the spheroid assembly step may be required. In fact,
differences in cell–cell adhesions can result in loose aggregates
or nonhomogeneous spheroids. Several strategies may help
to promote cell clustering including use of ultralow attachment
culture plates, higher densities of methylcellulose or using
other additives to modulate viscosity and compactness [13].
2. Even when completely dissolved, methylcellulose solution is
extremely viscous and difficult to filter. We suggest filtering
small volumes at a time and changing filters often to prevent
clogging. Nevertheless, it is likely that filtering might result
into a slightly lower percentage of methylcellulose in the final
stock. Alternatively, the solution can be autoclaved.
3. This protocol involves at least two different cell types. This
implies having to choose the optimal culture medium for
both cell types. If both cell types require specific media, the
two can be mixed in equal proportion. It is then recommendable to test the medium compatibility for all cell types prior
coculturing the cells.
4. Cell labeling with CellTracker: 2–3 days before the experiment,
HCC1806 cells and CAFs can be passed to a 25 cm
2 flask so
they will reach 70% confluency the day of spheroid preparation
step. Cell labeling can be performed directly in the flask,
removing culture medium and adding the CellTracker staining
solution.
5. Collagen concentration, polymerization temperature, and pH
strongly affect the mechanical and structural properties of the
obtained hydrogel. Therefore, it is important to maintain stable conditions of temperature across experiments to ensure
reproducibility. 5ÂCol functions as a buffer increasing the pH
of the ECM mixture until it becomes salmon-colored, indicating that a neutral pH has been reached. Consequently, it is of
critical importance to adjust the volume of the 5ÂCol solution
accordingly to stock concentrations of collagen I and Matrigel,
to maintain comparable gel porosity after polymerization while
using different stock solutions [14].
ä
Fig. 3 (continued) 50 μm. (b) Representative maximum fluorescence intensity z-projected images of three
time points (time ¼ 24, 32 and 39 h) extracted from time-lapse imaging of spheroid invasion acquired 24 h
after embedding in ECM (taken with 10Â objective). Images were taken every 30 min during 15 h. Images
showed in b(i) and b(ii) correspond to the dashed box depicted in (b). Asterisks show the position of CAFs over
time within the invasive strands. Scale bars: 50 μm
3D Invasion Assay with CAFs and Cancer Cells
253
1. This technique can be applied to different cell types and multicellular models. Depending on the chosen cell type, optimization of the spheroid assembly step may be required. In fact,
differences in cell–cell adhesions can result in loose aggregates
or nonhomogeneous spheroids. Several strategies may help
to promote cell clustering including use of ultralow attachment
culture plates, higher densities of methylcellulose or using
other additives to modulate viscosity and compactness [13].
2. Even when completely dissolved, methylcellulose solution is
extremely viscous and difficult to filter. We suggest filtering
small volumes at a time and changing filters often to prevent
clogging. Nevertheless, it is likely that filtering might result
into a slightly lower percentage of methylcellulose in the final
stock. Alternatively, the solution can be autoclaved.
3. This protocol involves at least two different cell types. This
implies having to choose the optimal culture medium for
both cell types. If both cell types require specific media, the
two can be mixed in equal proportion. It is then recommendable to test the medium compatibility for all cell types prior
coculturing the cells.
4. Cell labeling with CellTracker: 2–3 days before the experiment,
HCC1806 cells and CAFs can be passed to a 25 cm
2 flask so
they will reach 70% confluency the day of spheroid preparation
step. Cell labeling can be performed directly in the flask,
removing culture medium and adding the CellTracker staining
solution.
5. Collagen concentration, polymerization temperature, and pH
strongly affect the mechanical and structural properties of the
obtained hydrogel. Therefore, it is important to maintain stable conditions of temperature across experiments to ensure
reproducibility. 5ÂCol functions as a buffer increasing the pH
of the ECM mixture until it becomes salmon-colored, indicating that a neutral pH has been reached. Consequently, it is of
critical importance to adjust the volume of the 5ÂCol solution
accordingly to stock concentrations of collagen I and Matrigel,
to maintain comparable gel porosity after polymerization while
using different stock solutions [14].
ä
Fig. 3 (continued) 50 μm. (b) Representative maximum fluorescence intensity z-projected images of three
time points (time ¼ 24, 32 and 39 h) extracted from time-lapse imaging of spheroid invasion acquired 24 h
after embedding in ECM (taken with 10Â objective). Images were taken every 30 min during 15 h. Images
showed in b(i) and b(ii) correspond to the dashed box depicted in (b). Asterisks show the position of CAFs over
time within the invasive strands. Scale bars: 50 μm
3D Invasion Assay with CAFs and Cancer Cells
253