Isolation of fibroblasts
Isolation of fibroblasts
The generalized protocol described below applies to mouse, rat, hamster, and chicken embryos. It is best to select embryos that are approximately half the time of full-term gestation; 10- to 13-day-old embryos contain the greatest number of undifferentiated mesenchymal cells from which fibroblasts can be derived. This experiment was obtained from the Cell Lab Guide (previous volume) by Peitang Huang.
Operation method
Isolation of fibroblasts
Principle
The general operating scheme described below applies to mouse, rat, hamster and chicken embryos. It is best to select embryos that are approximately half the time of full-term gestation; 10- to 13-day-old embryos contain the greatest number of undifferentiated mesenchymal cells from which fibroblasts can be derived. Figure 4.1 shows the removal and dissection of a mouse embryo, and Figure 4.2 shows the process of removing a chicken embryo. Move 1) Isolation of the embryo. Caveat PrecautionsCells cultured in this manner grow well for many passages, and after the first few passages, only fibroblast-like cells are viable and can be frozen as described in Section 2. For more product details, please visit Aladdin Scientific website.
For mammals (hamsters, mice and rats)
a. Rodents are executed using an approved protocol.
b. Immediately scrub the entire animal with 70% ethanol in a sterile ultra-clean table. If possible, place under UV light for 5 minutes.
c. Using sterile forceps and clippers, make a horizontal abdominal incision under the front leg and pull the skin toward the back leg with sterile forceps.
d. Using another sterile pair of scissors and forceps, make an incision through the abdomen, remove the uterus containing the embryo, and place it on a sterile Petri dish.
e. Remove the envelope around the embryo and place the embryo in a sterile flask containing sterile PBS.
f. Rinse the embryo to remove the PBS. Continue to rinse the embryo with PBS until the wash solution is clear.
For chicken embryos
a. Scrub eggshells with 70% ethanol.
b. Tap the rounded end of the egg with sterile scissors and gently remove the shell to expose the air sac.
c. Remove the white membrane covering the chorionic urothelium with a sterile regent.
d. Cut the envelope and remove the embryo by holding the neck of the embryo with curved forceps.
e. Discard the head and place the headless embryo in a wide-mouth beaker and rinse with PBS.
2) Transfer 6~8 embryos to a small sterile wide-mouth beaker and carefully strangle the embryos with new sterile scissors.
Rinse with PBS.
3) Transfer the grinded embryos to a 500 ml sterile beaker with a stirring rod under sterile conditions.
200-300 ml of sterile trypsin 0.25% (CIBCO/BRL) prepared in HBSS.
4) Stir gently for 15 minutes in a warm environment or in a 37°C incubator.
5) Allow the remaining tissue mass to settle slowly by gravity and transfer the liquid containing the unsuspended cells to a sterile, large volume centrifuge tube with bovine blood淸inactivated trypsin at a ratio of 1 ml bovine serum for every 10 ml supernatant.
6) Add fresh trypsin solution (see previous steps) to a 500 ml beaker containing residual undigested tissue mass and repeat steps 4 and 5.
7) Centrifuge the mixed cell suspension at 1200 r/min for 5 minutes and discard the supernatant.
8) Resuspend the precipitated cells with fresh sterile PBS and centrifuge again according to step 7.
9) Wash the cells repeatedly with PBS until clear.
The precipitate is resuspended with 10 ml of DMEM (CIBCO/BRL) containing 10% bovine serum and antibiotics (e.g. penicillin, streptomycin) to a final volume of 100 ml.
11) Allow any remaining large pieces of unbroken tissue or special particulate matter to settle.
Several layers of sterile gauze may be used to filter the cell suspension to remove any residual clumps.
12) To determine the concentration of cells present, add 0.2 ml of cell suspension to 1.8 ml of 1% acetic acid solution to lyse the red blood cells and count the cells using a blood cell counter (see "Cell Counting" in Section 2).
13) Inoculate 10 ml of Tissue Culture Solution with 1x10 7 to 4x107 cells per 10 mm dish and incubate in a C02 incubator at saturated humidity until the cells are well established.
14) When the cells are full, remove the culture medium and wash the cell layer twice with 10% warm Versene (0.53 mmol/L EDTA in PBS, GIBCO/BRL).
15) Discard the Versene and add the same volume of warmed 0.05% trypsin-EDTA (GIBCO/BRL).
16) Incubate at 37°C for 5 min.
17) Blow the cells gently with a sterile pipette and transfer to a sterile tube containing 1-2 ml of bovine serum.
18) Centrifuge at 1200/min for 5 min and discard supernatant.
19) Resuspend the precipitate in 5 ml of culture medium (see above), add another 15 ml of culture medium, and dispense into two 100 mm dishes.
20) Incubate the cells at 37°C in a C02 incubator with saturated humidity until the cells are full grown, and continue steps 14-20 to pass on the cells.