Primary and Passage Culture Protocol of Mouse Embryonic Fibroblasts

Mouse embryonic fibroblasts are suitable feeder cells for mouse or human embryonic stem cells and induced pluripotent stem cells. They can produce factors that inhibit the autonomous differentiation of embryonic stem cells and promote the proliferation of embryonic stem cells, which can effectively promote the proliferation of cells and maintain their undifferentiated characteristics and pluripotency.
At present, most laboratories use mouse fibroblasts as a feeder layer to isolate and culture embryonic stem cells. The feeder layer not only provides a stable external environment for the growth and propagation of embryonic stem cells but also maintains the characteristics of infinite proliferation and differentiation of embryonic stem cells.

Animals. Pregnant mice are prepared for 14 to 16 days of gestation.
Sterile anatomical instruments. 2 ophthalmic scissors, 2 ophthalmic forceps, 3 pairs of glass plates, 1 small pincer, and 1 tissue scissors.
MEF growth medium. High glucose type DMEM containing 10% fetal bovine serum.
35 mm, 60 mm and I00mm tissue culture dishes.

Female rats at 12.5 to 14.5 days of gestation are sacrificed with necks broken, and the uterus is exposed under sterile conditions.
Lift the proximal cervix with tweezers to separate the mesangium and cut the uterine horn.
The whole uterus is removed and placed on a plate with PBS. Wash with PBS three times, and discard the residual blood on the surface.
The uterus is cut open along the mesangial side of the uterus, and the embryo with the fetal membrane is taken out and placed in another plate containing PBS for full washing, and the surface red blood cells are discarded.

Tear the fetal membrane with small tweezers, remove the fetal rat, discard the fetal membrane, and wash with PBS three times.
The head, viscera, and limbs of the embryo are removed, and the trunk is placed in another plate containing PBS. Wash with PBS at least three times, and the red blood cells are fully discarded.
The residual embryo is broken up with ophthalmic scissors, and the paste tissue is transferred to a 20 mL flat-scale test tube. And 5 mL of trypsin solution and 40 μL of DNase crude product are added to avoid the release of DNA that would cause the solution to be too sticky.

Incubate at 37°C water bath for 30 minutes, or place overnight at 4℃, shake, and then pour the upper cell suspension into a 10 mL centrifuge tube containing 10 mL of MEF growth medium and mix.
Cells are collected after centrifugation at 1000 r/min for 6 minutes. The cells are then suspended in 15 mL of growth medium and counted with a blood cell counter plate.
Eight cells of 5×10⁷-1×10⁸ are obtained from ten 15-day-old fetal rats. Every 1×10⁶ cell is suspended in 10 mL of MEF growth medium and inoculated into a 100 mm culture dish. Cells are immediately attached to the wall after inoculation, and fresh MEF growth medium is replaced 24 hours later.
When the cell density reaches about 90%, the passage could be performed in 2-3 days.

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Cells can be passaged when they have grown for 3-7 days and overlap and cover the entire bottom of the culture flask.
Discard the culture supernatant.
Rinse twice with PBS (without calcium and magnesium).
Add 0.25% trypsin for digestion.
Under the microscope, when a small number of cells float and cracks appear in the cell layer attached to the wall, rinse the bottom of the bottle with a pipette and blow 5-6 times.
Immediately add MEF culture medium to terminate the digestion.
Centrifuge at 1000 rpm for 5 minutes. Discard supernatant.
Culture medium is added and this is blown to make a single cell suspension which is divided into each culture flask.
A small amount of tissue blocks in the primary cells has not been fully digested. Single-cell suspension should be made as far as possible during each subsequent passage process, and the tissue blocks will gradually disappear.
Primary embryonic fibroblasts mix with some miscellaneous cells. When it's generally transmitted to the third generation, the miscellaneous cells gradually reduce. Mouse embryonic fibroblasts are fusiform. However, when connected in pieces, the cells crosslinked with each other, and the shape is not typical.

The primary embryonic mouse fibroblasts isolated by the above method are not pure. The main components are fibroblast-like cells, but there are neuro-like cells, cardiomyocytes, and some cells of unknown type.
After passage, the number of miscellaneous cells decreases, and the cell composition tends to be single with the increase of passage algebra, but the proliferation ability of cells decreases, and the proliferation of cells stops after 3-5 generations. The feeder cells used in the experiment should be passed within 3 generations.
In addition, the gestational age of the embryonic mice used for isolation also influences the quality and purity of the isolated cells.
MEFs should be passed at a suitable density, neither too dense nor too thin. MEFs with a suitable density can be passed at a ratio of 1 to 3.
In general, primary cells isolated from tissues can only be a passage through 2-3 times. This is one reason why the method is limited.

For research use only. Not for any other purpose.