Chicken Embryo Fibroblast culture

CHICKEN EMBRYO FIBROBLAST CULTURE

Aim

Materials

  • 70% alcohol, swabs, small beaker 20-50 ml, forceps, straight and required curved, 9-cm Petri dishes, 11-day embryonated eggs, Balanced Salt solution (BSS), 250 ml conical flask, Magnetic centrifuge tubes, Haemocytometer, Growth medium with serum, culture flasks.

Procedure

  • Swab the embryonated eggs with 70% alcohol and place with blunt end uppermost in a small beaker.
  • Break the top of the shell and peal off to the edge of the air sac with sterile forceps.
  • Using a sterile forceps, remove the embryo from the shell and place it on a sterile Petri dish containing 20ml BSS.
  • Transfer to embryo to fresh sterile BSS and rinse
  • Transfer to another dish, dissect off unwanted tissues and chop finely with scalpels to about 3mm diameter.
  • Transfer by pipette to a 15-or 50 ml sterile centrifuge tube and allow the pieces to settle.
  • Wash by resuspending the pieces in BSS, allowing the pieces to settle and removing the supernatant fluid, two or three times and then transfer the pieces into a 250-ml conical flask and add 100 ml of trypsin.
  • Stir at about 200 rpm for 30 min at 370c by using a magnetic stirrer.
  • Allow pieces to settle collect supernatant, centrifuge at 1000 rpm for 5 min, resuspend pellet in 10 ml medium with serum and store cells on ice.
  • Add fresh trypsin into the conical flask and continue to stir and incubate for a further 30 min. Repeat steps 7 to 10 until no further disaggregation is apparent.
  • Collect and pool chilled cell suspensions and count by hemocytometer.
  • Dilute to 106 per ml on growth medium and seed as many flask as are required with app.2 X 105 cells per cm2.
  • Change the medium at regular intervals 2 to 4 days as dictated by reduction in pH.
  • Observe the flask under inverted microscope for the development of the cells.

Questions

  • What is the purpose of using alcohol in tissue culture techniques?
  • What is the role of using trypsin in cell culture?
  • Write about common media used in cell culture technique?
  • Write about common cell lines their origin and type?
Last modified: Wednesday, 20 June 2012, 6:23 AM