Chicken embryo fibroblast preparation experiment
Chicken embryo fibroblast preparation experiment
The attenuated replication-defective poxvirus Ankara (MVA), an alternative to standard poxvirus strains, can be used as a vector for the expression of exogenous proteins.MVA was derived from the poxvirus Ankara in chicken embryo fibroblasts by multiple passages (>570 passages). Although MVA can efficiently express recombinant proteins, it cannot be packaged into infectious virus particles in human and most mammalian cells, so chicken embryo fibroblasts specifically designed for MVA culture need to be prepared. This experiment focuses on the preparation of chicken embryo fibroblasts. Content from Compact Molecular Biology Laboratory Guide (5th Edition)
Operation method
basic program
Principle
Cultured chicken embryo fibroblasts were prepared from chicken embryos.
Materials and Instruments
9 days old chicken embryo Move 1. Place 10 9-day-old chicken embryos with the airy part facing upwards and spray with 70% ethanol. 2. Using sterile dissecting scissors, open the tops of the eggs and cut off the shells from the membranes, leaving the membranes intact. Remove the membrane with sterile forceps. Repeat the same procedure for the other eggs. 3. Remove the embryos and place them in sterile 100 cm2 petri dishes. 4. 4. Cut off the head and feet of each embryo and place the remaining body in a sterile 100 cm2 dish with 10 ml of additive-free MEM medium. Squeeze through a 10 ml syringe (5 chick embryos/syringe), mince the embryos and inject them into a sterile tryptic digestor. 5. Add 100 ml of trypsin/EDTA at 37°C and incubate for 5 min at 37°C with continuous stirring in a humidified 5% CO2 incubator. 6. Pour the liquid from the sterile tryptic digest flask into a 500 ml beaker with a two-layer gauze spout and transfer the filtered liquid to a 250 ml centrifuge flask. 7. Add 100 ml of fresh trypsin/EDTA to the sterile tryptic digest vial containing the remaining tissue and incubate at 37°C. 8. Pour the digest into another sterile tryptic digest vial. 8. Pour the digested liquid into another 500 ml beaker with a two-layer gauze spout and add the filtered liquid to the previous 250 ml centrifuge flask. 9. Centrifuge for 10 min at 1200 g at 4°C in a Sorvall RC-3B centrifuge. 10. Discard the supernatant and add to the centrifuge. 10. Discard the supernatant, add 10 ml of MEM-10 complete medium, resuspend the precipitate by repeated aspiration 10-15 times, and adjust the volume to 100 ml. 11. Transfer the suspension to a 250 ml centrifuge bottle and centrifuge at 1200 g for 10 min at 4°C. 12. 12. Resuspend the precipitate with 5 ml of MEM-10 complete medium and adjust the volume to 30 ml. 13. Add the cell suspension at 1 ml per bottle to 30 150 cm2 tissue culture flasks containing 30 ml of MEM-10 complete medium. 14. Incubate at 37°C for a few days until the cells are full grown, then transfer the flasks to a humidified 5% CO2 incubator and store at 31°C. CEF cell cultures can be stored at 31°C for 2-3 weeks. Primary CEF cells can also be used directly for viral growth or trypsinized CEF cells can be frozen in liquid nitrogen for later use. For more product details, please visit Aladdin Scientific website.
70% ethanol, trypsin/EDTA, no added MEM medium, complete MEM-10 medium.
Sterile dissecting scissors Sterile forceps 100 cm2 Sterile Petri dish 10 ml syringe Sterile trypsin digestion flask with magnetic stir bar CO2 incubator 500 ml beaker Gauze Sorvall centrifuge 250 ml centrifuge tube 150 cm2 Tissue culture flasks