tissue culture flask with only enough media to keep tissue moist not suspended this flask is then flipped over or partially tipped and 2 ml of media 1 is added to the bottom to keep the tissue fragments humidified with the flask inverted the fragments are stressed by gravity but tend to remain in contact with the flask surface due to the moisture from the media the flask is then placed in a 5 co 2 38 5 degree celsius incubator for 48 hours during this time some of the tissue fragments will adhere to the flask surface after 48 hours the humidifying media is removed the flask is flipped upright and 2 ml of media 2 bicarb dmem f12 10 fcs is gently added to bathe and immerse the tissue fragments cell growth from the tissue fragments is monitored daily and media is replaced every 2 3 days when cell growth is confluent around the tissue fragments the media is removed and the culture is washed with pbs penstrep the cells are then harvested via trypsin edta incubation when most of the cells have detached from the flask surface the digestion is stopped with the addition of 5 10 mls media 2 the suspension is then aspirated to break up cell aggregates and then centrifuged the resulting pellet is re suspended in media 2 and.