Single organs or tissues were cut out and placed whole in cold trypsin overnight, trypsin was removed, organs or tissues were briefly incubated, cells were dispersed in medium, and cultures were diluted and inoculated.
Operation method
Scheme 12.7 Chicken embryo organogeny
Principle
Single organs or tissues were cut out and placed whole in cold trypsin overnight, trypsin was removed, organs or tissues were briefly incubated, cells were dispersed in medium, and cultures were diluted and inoculated.
Materials and Instruments
Fertilized eggs DBSS Crude trypsin Move 1. Remove the embryos (fertilized eggs incubated for 10-13 days (>10 days requires a permit in the UK)) as described previously (see Scheme 12.2 ) and place in a sterile BSS. For more product details, please visit Aladdin Scientific website.
Culture medium Petri dishes
Forceps Iris knife Pipettes Test tubes Culture flasks Scalpels
2. Remove the head. 
3. remove the eye and carefully dissect to remove the lens, aqueous humor, and vitreous. 
4. Hold the retina with two sharp forceps and gently separate the pigment layer from the nerve layer and connective tissue (10-day embryos require a dissecting microscope.) Use 0.25% trypsin (0.25 crude trypsin (Difco 1 : 250 or equivalent) prepared in RPMI1640 or S-MEM and placed on ice. If pure trypsin is used, the concentration can be reduced, e.g., Sigma's "crystalline grade" or Worthington's "grade IV" can be formulated to a concentration of 0.05%-0.1%) and a brief soak in 1 mmol/L EDTA solution (to facilitate easy separation of the two tissues). (This will facilitate the separation of the two tissues). Place the separated tissues on one side. 
5. Pierce the roof of the skull with curved forceps, remove the brain with a spoon, and place the brain and retina together on one side of the dish. 
6. Disconnect from the middle of the torso (where the liver appears pink through the abdominal wall); if cut in the diaphragm it will pass between the heart and the liver, but sometimes the liver appears in the anterior rather than the posterior half. 
7. Remove the heart and lungs by gently probing into the anterior half of the cut (pick out the organs and confirm before cutting) and place the separated heart and lungs on one side of the dish. 
8. Probe into the back half of the section and remove the liver. Since the intestine is sandwiched between the two lobes of the liver, it can be removed as well, and the separated liver and intestine are placed on one side of the dish. 
9. Fold back the wall of the posterior half of the body cavity on the dorsal side of the inner wall and see the long lobulated kidneys arranged in parallel on both sides of the midline.
10. Gently slide the tip of the knife to the dorsal surface of the kidneys and peel the kidneys from the dorsal medial wall of the carapace (dissecting scopes are required for 10-day-old embryos), detach the kidneys, and place them on one side. 
11. Place the two scalpels on the midline of the posterior unend, with the tips forward, and advance them alternately to squeeze the spinal cord out like toothpaste. 
12. Turn the posterior trunk of the embryo over, tear the skin from the back and upper legs, and collect the skin and place it on one side. 
13. Cut the muscle from the femur of each leg and collect it together. 
14. Transfer all these collected tissues and any other tissues covered separately into test tubes containing 1 ml of 0.25% trypsin on ice to confirm that the tissue mass has sunk into the trypsin solution.
15. Allow to stand at 4°C for 6 to 18 hours.
16. Carefully aspirate the trypsin without stirring the tissue mass; shaking or slowly rotating the tube will aid digestion.
17. Incubate the tissue block in the remaining trypsin at 37°C for 15-20 min.
18. Inoculate two 25 cm2 flasks of each tissue type with 4 ml of medium per flask.
19. Add 2 ml of medium to the tubes containing the tissues and the remaining trypsin and gently pipette up and down to disperse the tissues.
20. Allow large pieces of tissue to settle.
21. Pipet the supernatant into the first culture flask, mix well, and transfer 1 ml of the diluted suspension into the second culture flask. This makes the cell densities in the two flasks different, which eliminates the need for cell counting, and the test determines the appropriate concentration of cells for use in future experiments.
22. Change the medium as needed (e.g., brain after 24 h; retinal pigment layer for about 5-7 days) and examine morphological characteristics and function. 