Proceed to perform the plating of primary human hepatocytes, or PHHs, after almost one hour of culturing the feeder cells. To do so, after 30 minutes of feeder cell culture, filter the pre-warmed hepatocyte thawing medium through a 0.2 micron polyethersulfone filter unit. Thaw the PHHs in a 37 degree Celsius water bath for one to two minutes.
Immediately after thawing, place the thawed cells on ice. Then, working in a biological safety cabinet, transfer the cells into the hepatocyte thawing medium. Using a 1, 000 microliter pipette, transfer one milliliter of the hepatocyte suspension in the thawing medium back into the vial, and wash the vial three to four times by gentle pipetting.
Combine the washing with the rest of the cell suspension in the tube, cap, and gently invert the thawed PHH suspension five times. Spin it at 100 G for eight minutes at room temperature. Carefully discard the supernatant by vacuum aspiration or pipetting without disturbing the cell pellet.
Then add three milliliters of complete plating medium to the wall of the conical tube containing the pellet. Rock the conical tube side to side to resuspend the cell pellet, and add five milliliters of complete plating medium. After counting the resuspended hepatocytes, adjust the cell suspension to the desired seeding density using complete plating medium.
Next, obtain the collagen-coated 24-well plate containing the previously plated feeder cells, and using a pipette or vacuum aspiration, remove the medium from the feeder cells. Immediately plate 500 microliters of the diluted hepatocyte suspension in each well containing the feeder cells. Shake the plate in a north-south, east-west motion by performing a back and forth motion in the north-south direction two times, followed by the same motion in the east-west direction.
Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for two to four hours. For the first 60 minutes of culture, shake the plate every 15 minutes as demonstrated previously. Following incubation, transfer the plate from the incubator to the biological safety cabinet.
After shaking the plate, use pipetting or vacuum aspiration to remove the complete plating medium. Immediately add 500 microliters of pre-warmed complete culture medium to each well. Images of PHHs from two donors cultured at various seeding densities with feeder cells were obtained on day seven and day 14 of the culture.
The various seeding densities showed visual differences of confluence, but maintained the typical hepatic cuboidal morphology.
