To begin, obtain PANC-1 cell culture and wash the cells with nine milliliters of Hank's balanced salt solution in three milliliter increments. Incubate the cells with two milliliters of trypsin for about 10 minutes. To neutralize the suspension, add three milliliters of DMEM supplemented with 10%FBS.
After washing the cells seven times with one milliliter media increments, collect each neutralized aliquot in a 15 milliliter tube. Similarly, obtain trypsinized human pancreatic stellate cells, or HPaSteC, using trypsin neutralization solution and stellate cell media. Mix the required amount of both the cell suspensions in 11 milliliters of DMEM with 10%FBS avoiding froth formation.
Gently dispense 100 microliters of cell suspension mix into the culture plate and incubated at 37 degrees Celsius with 5%carbon dioxide. On day three, add 50 microliters of DMEM with 10%FBS to each well along the side of the well. On day 10 or 11, aspirate the spent media without disturbing the solution near the halo and add 100 microliters of fresh media along the side of each well.
On day 14 or 17, using a one milliliter pipette, remove media from each well until the halo is reached. Align the plate against the background of the hood to locate the spheroids at the bottom. Gently pipette around 50 microliters of media near the spheroids to disturb it for pooling.
The spheroids obtained on day 14 were found to grow up to an average volume of 0.048 cubic millimeters.
