To begin, thaw the feeder cells in a 37 degree Celsius water bath for one to two minutes. Immediately after thawing, place the feeder cells on ice. Using aseptic techniques within a biosafety cabinet, add the freshly thawed cells to 10 milliliters of feeder cell thawing medium.
Then with a 1000 microliter pipette, take one milliliter of the resultant cell suspension in the thawing medium. Wash the empty cell vial with it and combine it with the rest of the cell suspension. Centrifuge the cells at room temperature and 400 G for four minutes.
Carefully discard the supernatant by aspirating or pipetting without disrupting the cell pellet. After resuspending the pellet in one milliliter of complete plating medium, count the feeder cells, then dilute the cell suspension using the complete plating medium. Next, plate 500 microliters of the diluted cell suspension per well in a collagen coated 24 well plate.
Agitate the plate in a north, south, east, west motion by employing a back and forth shaking in the north south direction two times, followed by the same motion in the east west direction. Finally incubate the plate before seeding the primary human hepatocytes in it. Examine the feeder cell attachment before thawing the hepatocytes for seeding.
