To begin the hanging drop method, pipette 20 microliters of a pre-prepared skin cell suspension onto the lid of a Petri dish. Now, cover the lid with the bottom half of the Petri dish and gently flip it to create the hanging drops. Add sterile water to the Petri dish to prevent evaporation of the full-grown medium from droplets.
Incubate the droplets for 48 to 72 hours at 37 degrees Celsius. Next, fill the wells of a new plate with the full growth medium. Using sterile cut pipette tips, transfer the cell spheres to the wells of the multi-well plate.
Incubate the transferred spheres for one day in the multi-well plate at 37 degrees Celsius before experimentation. For the limiting cell adhesion method, first add 100 microliters of 1%surfactant solution in PBS into each well of a U-bottom plate. Next, incubate the plate with the solution at 37 degrees Celsius for 24 hours.
Now, prepare the cell suspension in the desired cell density. Pipette out the surfactant solution from the wells, then seed 50 microliters of the cell suspension into each well. Finally, incubate the plate at 37 degrees Celsius for 24 hours to reach cell aggregates.
Spheres consisting of 10, 000 cells were easier to manipulate, as they were visible in the wells. The bigger spheres showed decreased stability. Different cell types formed spheres of different colors.
The perfectly rounded spheres were prone to losing their shape during the transfer in the hanging drop method. Accurate handling was required to reduce the deformation resulting in intact cell aggregates. Inaccuracy and inexperience resulted in sphere damage.
