Derivation of mouse embryonic fibroblasts MEFs

Cull a pregnant ICR mouse (12.5-13.5 days post coitum) in accordance with local animal welfare guidelines. Use two methods of culling (e.g. CO2 or avertin exposure followed by cervical dislocation) to verify the animal is dead prior to dissection.

1. Place the mouse facing upward on the bench and soak its fur with 70% ethanol. Soaking the fur minimizes the risk of contamination with mycoplasma and other organisms and also prevents fur getting in the way of the dissection.

2. Dissect the abdomen, unravelling and pushing the intestinal tract to one side to reveal the uterine horns. Extract the uterine horns by snipping above the genitalia and just below the ovaries as shown in Figure 2(A).

3. Transfer the uterine horns to a fresh dish of 1x PBS supplemented with 1x Pen/Strep (PBS+P/S). Cut the uterine wall between individual embryos and using a forceps, tease the embryos into the PBS+P/S by tearing the mesometrium with two sets of sharp tipped forceps. Day 12.5-13.5 embryos should resemble those shown in Figure 2(B).

4. Transfer the embryos to a fresh 10 cm Petri dish with 10 mL PBS+P/S. Hold the head of the embryo with one pair of blunt forceps, and carefully remove all the red viscera using sharp forceps with a pecking motion.

5. Cut off the limbs and tail of the embryo using the forceps. Finally, remove the head of the embryo and transfer the remaining trunk to a new Petri dish with PBS+P/S.

6. Mince the eviscerated embryo sections very well with two razor blades and transfer the pieces to several milliliters of prewarmed 1x trypsin EDTA (2 mL/embryo). Triturate the cells continuously until the trypsin solution becomes cloudy with liberated cells. Be careful not to over-trypsinize the cells as this will lead to lysis, clumping and loss of cells. About 5 minutes continuous vortexing of the embryo pieces should be sufficient.

7. Neutralize the protease with MEF medium (1 mL/embryo) (serum in the medium contains trypsin inhibitor and so stops the reaction).

8. Let the cell suspension sit for 5 minutes. Adipose tissue will generally float to the top of the supernatant, and cell clumps will fall down to form a pellet. Remove

Figure 2 Murine embryo extraction for feeder preparation. (A) The uterus from a 12.5-13.5 dpc pregnant mouse is removed as follows: After a longitudinal section is made from above the diaphragm to the genitalia, the intestinal tract is unwound and pushed to one side, revealing the uterine horns behind. The uterine horns are held up with forceps and a sharp scissors is used to snip the mesometrium so they can be separated from the abdominal cavity. Subsequently, the embryos are removed from the uterine horns. (B) Typical embryos from day 12.5 to 13.5: the embryos should be similar in size and development to those shown.

Figure 2 Murine embryo extraction for feeder preparation. (A) The uterus from a 12.5-13.5 dpc pregnant mouse is removed as follows: After a longitudinal section is made from above the diaphragm to the genitalia, the intestinal tract is unwound and pushed to one side, revealing the uterine horns behind. The uterine horns are held up with forceps and a sharp scissors is used to snip the mesometrium so they can be separated from the abdominal cavity. Subsequently, the embryos are removed from the uterine horns. (B) Typical embryos from day 12.5 to 13.5: the embryos should be similar in size and development to those shown.

the fat with a pipette, and carefully transfer the cell suspension to a fresh 15 mL centrifugation tube without disturbing the pellet.

9. Pellet the cells by centrifugation (300 g/~ 1,000 rpm for 5 min).

10. Resuspend the cells in MEF medium (1 mL/embryo). Measure cell number by counting an aliquot of the cells using a haemocytometer and seed the flask or dish to be used for generating feeder layers with 1 x 105 MEFs/cm2.

11. Add additional MEF medium to the dishes/plates so the medium is at least 0.5 cm in depth.

12. Leave the cells to sit down overnight in the incubator (37°C, 5% CO2) before expanding these cultures and mitotically inactivating them.

Note: Remember MEFs and other primary cell cultures are a common source of microbial contamination in the cell culture facility. To reduce the risk of contamination, always soak euthanized mice in 70% ethanol prior to embryo extraction, dip uterine horns in 70% ethanol before embryo removal, and culture the MEFs in medium supplemented with 1 x Pen/Strep for 2 days after the MEFs are derived.

Note: MEFs should be specifically screened for mycoplasma (See Chapters 1 and 2 for more on mycoplasma) before being used as feeders for human ES cell culture. We recommend an enzymatic assay [Mycoalert mycoplasma detection kit made by Cambrex www.Cambrex.com] as a quick and straightforward way to screen cultures for mycoplasma. To increase the sensitivity of this assay, MEFs should have been cultured without antibiotics for at least 48 hr prior to the mycoplasma screening.

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