Primary cell culture-Preparation of primary chick embryo fibroblast (CEF) culture

How to prepare primary chick embryo fibroblast (CEF) culture?

• Primary cultures of chick embryo fibroblasts (CEF) are extensively used for the cultivation of viruses.
• Some viruses may not grow in continuous cell lines, because of which the primary culture is still used.

Materials required:

• Fertile chicken eggs, 10 days old
• 70% (v/v) ethanol 1 X phosphate-buffered saline (PBS) with calcium and magnesium
• Versene solution
• 0.25% trypsin
• 1X complete EMEM
• 1X PBS-D
• Strong light source (75-100W)
• Small sterile beaker
• Sharp scissors and forceps
• Sterile petri dishes
• 50-ml conical tubes 37 o C water bath
• 5-10 ml sterile pipettes
• 25 cm 2 or 75 cm 2 tissue culture flasks

Procedure of primary chick embryo fibroblast (CEF) culture

i. Remove embryo from egg

• Candle a 10-day old egg, by holding the egg in front of a bright light.
• Mark the position of the air sac with a pencil.
• Discard eggs with dead embryos or eggs without embryos.
• Use 70% ethanol to wash egg and place pointed-end-down in a small sterile beaker.
• Cut a large circular hole at the blunt end of the egg with a pair of sharp sterile scissors.
• Use sterile scissors or forceps to remove the shell above the air sac.
• Use sterile forceps to remove the membrane exposing the embryo.
• Use fresh sterile forceps to avoid cross-contamination.
• Gently and slowly remove the embryo so that it will free itself from the yolk and place it in a sterile petri dish.
• Care should be taken to not to touch the egg shell while removing the embryo.

ii. Prepare single-cell suspension from embryo:

• Use a fresh pair of sterile scissors to dissect the head, wings, feet, and body cavity contents from the embryo, and place into a 50ml conical tube.
• Remove contaminating blood/yolk by the addition and removal of 10ml of 1X PBS with calcium and magnesium.
• Macerate the embryo with dissecting scissors.
• Add 50ml of 1x PBS with calcium and magnesium to the tube and invert to wash the embryo. Allow the large pieces to settle to the bottom of the tube.
• Mix 25 ml of thawed 0.25% trypsin and 75 ml of versine solution and prewarm to 37 o C.
• Remove buffer from the macerated embryo using a pipette and add 20 ml of this prewarmed solution.
• Shake the tube gently for 15-20 min at 37 o C.
• Allow the large pieces to settle to the bottom of the tube.
• Use a pipette to transfer the supernatant, containing the cell suspension, to a tube containing 25ml of 1X complete EMEM. (Serum in the media will inactivate the trypsin).
• Centrifuge 10 min at 500 X g, room temperature.
• Remove the supernatant and resuspend the cell pellet in 20ml of 1 X complete EMEM. (The pellet will be loose, so care must be taken care when removing the supernatant.)
• Remove an aliquot of cell suspension and dilute 1:5 or 1:10 in PBS containing calcium and magnesium.
• Count cells using a hemacytometer.

iii. Culture of primary chick embryo fibroblast

• Prepare each 75-cm 2 flask required by adding 1X 10 6 cells and 30 ml of 1 X complete EMEM. Begin incubation.
• Wash a confluent CEF monolayer once with 15ml of 1 X PBS-D per 75-cm 2 flask.
• Mix 25ml of thawed 0.25% trypsin and 75 ml of versine solution and prewarm to 37 o C.
• Decant PBS-D and add 5 ml of the trypsin solution to the monolayer per 75 cm 2 flask.
• Incubate 5- to 10 min at room temperature until the cells begin to detach from the flask.
• Resuspend cells in the trypsin solution and disrupt cell clumps by pipetting up and down using a 5- or 10- ml pipette.
• Pipette cell solution into a conical tube of the appropriate size.
• Centrifuge 5 min at 500 X g, room temperature, to form a firm pellet.
• Decant supernatant and replace with the appropriate amount of 1 X complete EMEM medium. Resuspend cells by pipetting up and down.
• For preparation of a single 25 cm 2 flask:
• Pipette a 1 ml aliquot of the cell suspension into 10 ml of 1 ml of 1X complete EMEM into a new 25 cm 2 flask.
• For preparation of a large number of 25 cm 2 flasks:
• In a sterile bottle, pipet 10 ml of 1X complete EMEM for each flask to be prepared.
• Add 1 ml cell suspension to this medium per each flask to be prepared.
• Pipette 10 ml cell/medium mixture into each new 25 cm 2 flasks, swirling continuously.
• For preparation of a single 75 cm 2 flask:
• Resuspend cells from a single 25 cm 2 flask in 1 ml medium.
• Transfer to a 75 cm 2 flask containing 30 ml of 1X complete EMEM.
• Alternatively, subculture a 75 cm 2 flask into three 75 cm 2 flasks or nine 25 cm 2 flasks.
• Be careful while placing flasks in incubator with loosened caps and incubate 24 hr or until confluent.
• Freeze cells in cryotubes at ~10 6 cells/tube in 1 ml of freezing medium or subculture for experimental use.

Primary cell culture-Preparation of primary chick embryo fibroblast (CEF) culture

• chick embryo fibroblast culture
• Primary cell culture