Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

1. Isolation of Mouse Embryonic Fibroblasts (MEFs)

The following two steps are performed under non-aseptic conditions.

1. Sacrifice a pregnant mouse (CF1, Harlan, USA) at 13 or 14 d.p.c. (day post-coitum) by cervical dislocation.
2. Dissect out the uterine horns, briefly rinse in 70% (v/v) ethanol and place into a falcon tube containing PBS without Ca²⁺Mg²⁺ (Gibco, Invitrogen).

The following steps are carried out in a tissue culture hood under aseptic conditions and using sterile instruments.

3. Place uterine horns into a Petri dish and separate each embryo from its placenta and embryonic sac.
4. Dissect head and red organs, wash in PBS and place all embryos in a clean Petri dish. Finely mince the tissue using a sterile razor blade until it becomes possible to pipette.
5. Add 1 ml of 0.05% trypsin/EDTA (Gibco, Invitrogen), including 100 Kunitz units of DNase I (USB), per embryo.
6. Transfer the tissue into a 50 ml falcon tube and incubate for 15 min at 37 °C. After each 5 min of incubation, dissociate cells by pipetting up and down thoroughly.
7. Inactivate the trypsin by adding about 1 volume of freshly prepared MEF medium.
   MEF culture medium (components to make 500 ml of media, mix all components and filter):
   450 ml of DMEM, 50 ml of FBS (10% (v/v)), 5 ml of 200 mM L-glutamine (1/100 (v/v)), 5 ml of Penicillin-streptomycin (1/100 (v/v)).
8. Centrifuge the cells with low-speed (300 x g), 5 min, carefully remove the supernatant and resuspend cell pellet in warm MEF medium.
9. Plate approximately a number of cells which is equivalent to 3-4 embryos in each T150 (TPP) flask coated with 0.2% gelatine (Gelatine from bovine skin, Type B, Sigma) for 2 hr. The fibroblasts (P0, passage 0) are the only cells that have the ability to attach to the gelatine-coated flasks.
10. Ideally, cells are 80-90% confluent after 24 hr and at this stage a major part of P0 cells is frozen for future usage.
11. Expand the remaining T150 flask(s) of P0 cells till P3 or P4, then inactivate and use as feeders to replate hESCs or to produce conditioned medium (CM).

2. Inactivation and Plating MEFs (Feeder Cells Preparation)

All steps are carried out in a tissue culture hood under aseptic conditions.

1. Coat T150 flasks with 0.2% gelatine and incubate at RT for at least 2 hr.
2. Dilute mitomycin C in PBS (1 mg/ml) and filter.
3. Aspirate media from MEFs and wash with PBS without Ca²⁺Mg²⁺.
4. Place 20 ml of medium containing 10 μg/ml of mitomycin C on MEFs.
5. Incubate for 2 hr at 37 °C with mitomycin C containing medium then wash twice with PBS, trypsinize, centrifuge (for 5 min at 300 x g) and resuspend cells in warm medium.
6. Count cells and plate at a density of 56.000 cells/cm² in T150 flasks and use for CM production for the following 6 days.

3. Conditioned Medium (CM) Preparation

All steps are carried out in a tissue culture hood under aseptic conditions.

1. The day after plating inactivated MEFs at a density of 56.000 cells/cm² replace the MEF medium with hESC medium (UM, unconditioned medium) (0.5 ml/cm²) supplemented freshly with 4 ng/ml of FGF2.
2. Collect CM from feeder flasks after 24 h incubation and add fresh hESC medium containing 4 ng/ml of FGF2 to the feeders.
3. Repeat this procedure for the next 6 days. Each day store collected CM at -20 °C.
4. After 6 days mix all aliquots of medium and filter (Corning, 0.22 μm, PAS). Make 50 ml aliquots and store at -80 °C.
5. Supplement CM with additional 4 ng/ml of FGF2 before adding to hESCs grown on Matrigel.

4. Measurement of Activin A in Conditioned Media (ELISA)¹⁵

1. Bring all samples and reagents to room temperature.
2. Dilute capture antibody (HumanMouseRat Activin A MAb, R&D Systems) in PBS with 1% BSA, add to microplate (100 μl/well) and incubate overnight at RT.
3. After 24 hr wash the wells three times with PBST (PBS with 0.05% Tween 20) (300 μl/well) then block (1% BSA/PBS, 300 μl/well) for 1 hr at RT.
4. In this time prepare an Activin A (R&D Systems) standard curve, including 7 dilutions (concentration less than 30 ng/ml) and blank sample. The linear working range of the Activin A is between 0.25 and 32 ng/ml.
5. Add duplicates of standards and samples to the wells (100 μl/well) and incubate for 2 hr at RT.
6. Wash wells three times (300 μl of PBST).
7. Add the secondary (biotinylated) antibody (Human/Mouse/Rat Biotinylated Activin A MAb, R&D Systems) (0.25 μg/ml in 1% BSA/PBS) and incubate for 2 hr at RT.
8. Wash wells three times (300 μl of PBST), add Streptavidin-HRP (diluted in 1%BSA/PBS, R&D Systems) and incubate for 20 min at RT.
9. Wash wells three times (300 μl of PBST), add 100 μl of substrate solution (Quantikine, R&D Systems) and incubate for 30 min at RT in the dark.
10. Add 100 μl of stop solution (Quantikine, R&D Systems) to each well and mix gently.
11. Set Microplate reader (Molecular Devices Spectra Max 250, Global Medical Instrumentation, Inc., Minnesota) to 450 nm, with wavelength correction at 540 or 570 nm, to determine the optical density of each well.

5. Representative Results

The overall scheme of the isolation procedure is presented in Figure 1. The typical morphology of hESCs and hiPSCs cultured under different conditions is presented in Figure 2. The morphology of MEFs and inactivated feeder cells used to prepare CM is presented in Figure 3. In general, cells should be confluent 24 hours post-isolation and ready to be frozen or expanded. However, sometimes it might take 2-3 days before obtaining confluent cultures. The CM should be prepared from cells at passage 4 and not later. This is crucial because primary cells can only be expanded for 4-5 passages before the onset of senescence.

The cytokine, Activin A, is considered as the most critical factor secreted by feeder cells for the support of undifferentiated growth of pluripotent cells¹⁴. The measurement of the level of Activin A in CM (Figure 4) is a very convenient quantitative assay to monitor the quality of MEFs.

Figure 1. A schematic representation of the MEFs isolation procedure.

Figure 2. The typical morphology of undifferentiated hESCs cultured in the presence of (A) feeder cells, (B) conditioned medium and (C) defined medium. The typical morphology of hiPSCs cultured in the presence of (D, E) feeder cells.

Figure 3. The typical morphology of mouse embryonic fibroblasts (MEFs). (A) Passage 0 (P0) two days after plating/isolation, (B) inactivated feeder layer at a density of 56.000 cells/cm.²

Figure 4. Enzyme-linked immunosorbent assay (ELISA)-based measurements of the concentration of Activin A in conditioned medium (CM) prepared with mouse embryonic fibroblasts derived from the CF1 mouse strain. CM was collected for 6 days and then pooled. CM”1″ and CM”2″ refer to different batches of media and UM to unconditioned media. As the function of the conditioning process is Activin A secretion into the medium by MEFs, Activin A is almost undetectable in UM.