Strategic Alliance for Bioscience Research and Education 

Mammalian and Plant Cell Culture and DNA Transfection

Generate Chick Embryo Primary Cells Experimental Design

Materials
5 – 8 Day Old Chick Embryo (2-eggs)* 70 % Ethanol Sterile Scissors, Forceps and Probes
Sterile Petri Plates Phosphate Buffered Saline (PBS) Cold Sterilized Trypsin **
Minimum Essential Medium (MEM) Fetal Calf Serum (FCS) *** Sterile Capped Falcon Tubes
T25 Culture Flasks 1 & 10 ml sterile pipettes 10% Serum with MEM
37oC incubator
* Obtained from Carolina Biological Supply House or other source – maintain in incubator prior to use.
** Trypsin can be purchased from a number of commercial sources including Sigma Aldrich or ATCC or prepared as 0.25%
*** Fetal Calf or Fetal Bovine Serum will perform well

Materials Preparation
Prepare sterile work area using 70% ethanol spray. Sterilize scissors and other tools in a beaker of 70% ethanol

Procedure (two eggs per station, work in pairs and use one egg at a time)
• Use a bright flashlight in a darkened room to candle a 5 to 8 day old egg to ensure that it is alive. This is
easily accomplished by holding the egg in front of a bright light source; the embryo can be seen as a
shadow. Circle the embryo with a pencil.

• Place the egg blunt end up in an egg carton, and wash the top of the egg with a mild detergent,
followed by swabbing with ethanol.

• Carefully puncture the top of the egg with the point of a pair of sterile scissors or teasing probe, and cut
away a circle of shell, thus exposing the underlying membrane (the chorioallantois). Do not let the
probe puncture deeper than 1 mm.

• With a second pair of sterile scissors, carefully cut away and remove the shell and chorioallantoic
membrane, exposing the embryo.

• Identify and carefully remove the embryo by the neck, using a sterile metal hook or a bent glass rod,
and place the embryo in a 100mm petri dish containing phosphate buffered saline (PBS). Wash several
times with PBS by transferring the embryo to fresh petri plates. After removal of all yolk and/or blood,
move the embryo to a clean dish with PBS.

Embryo One
• Remove the head, limbs and vicera of the first embryo. Be sure to remove the entire limb by pulling at
the proximal end.
• Mince the tissue from remaining tissue and place into a 50 ml sterile falcon tube or a glass culture tube.
Place 5 ml of trypsin (also called versene) onto the embryo. Cap the tube and allow the mixture to
incubate at 37oC for 20 min. Shake the tube every five minutes. The cells will dissociate from the tissues.
• After 20 min has passed, titurate the cell mixture with a 10 ml pipette to further dissociate cells.
• Transfer 10 ml of MEM containing 10% FCS (serum contains a protein, trypsin inhibitor which will stop the
proteolysis), cap the tube. Then shake the tube vigorously to isolate cells from the embryo.
• Allow the larger, undigested tissue pieces to settle and using a pipette, transfer most of the supernatant
into an equal volume of Minimal Essential Medium (MEM) + 10% Fetal Calf Serum (FCS).
• Centrifuge the cells in MEM at 1000 rpm for 10 minutes in a standard clinical centrifuge. Remove the
supernatant and resuspend the pellet in 25 ml of fresh MEM + 10% FCS.
• Remove 0.1 ml of the culture and determine cell concentration.
• Seed two 25 cm plastic culture flasks containing 5 ml of MEM with 10% FCS to a final concentration of
105 cells/ml.
• Label and place your cultures in the tissue culture incubator at 37° C and examine daily for cell density
and morphology.

Embryo Two
• Place the second embryo on it’s back and carefully cut open the body cavity of the embryo
 Strategic Alliance for Bioscience Research and Education 
Mammalian and Plant Cell Culture and DNA Transfection
Generate Chick Embryo Primary Cells Experimental Design

• Chose one tissue per group and remove one of the following tissues: lung, liver, heart, muscle (from a
limb near the backbone), or the intestines.
• Carefully cut the tissue into small 0.5 mm cubes and transfer into a culture tube containing 1 ml of
trypsin, shake the tube and incubate at 37oC for 20 min with futher agitation every 5 min.
• Following the incubation, transfer 4 ml of MEM containing 10% FCS to the tissue and trypsin mixture and
shake the tube vigorously. Allow the mixture to settle for 5 to 10 min.
• Transfer the supernate (allow a few smaller pieces of tissue to transfer) into a T25 flask.
• Label and place your cultures in the tissue culture incubator at 37° C and examine daily for cell density
and morphology.

For both cultures: Note any changes in the color of the media. Tissue Culture media has a pH indicator (Phenol
Red) added in order to check on the growth of cells. The media initially is a cherry red (with slight blue haze)
and turns orange and then yellow as the cells grow, thereby reducing the media. Should this color change
occur within 24 hours, the culture is most likely contaminated and should be disposed of.
• Examine the cultures using an inverted phase contrast microscope. This will allow observation of the cells
without opening or disturbing the growth.
• Make cell density determinations at 10 X magnification.
• Plot the cell density on a log scale vs. time of culture.
• Diagram the shape of the cells at each phase.
Notes
The cultures will develop differently than the suspension cultures. The viable cells will grow out of the trypsinized
pieces of tissue and will remain in contact with the bottom of the culture flask. They will continue to divide and
migrate until the entire bottom of the flask is covered with a single layer of cells (contact inhibition and the
formation of a monolayer).