Cell are normally passaged every 2-3 days, this is important to avoid differentiation.

Signs of differentiation are:-

i) colonies are surrounded by flattened differentiated cells.

ii) large colonies with necrotic centres, these appear as cells with defined boundaries.

iii) colonies appear as individual cells rather than as a syncial mass.

iv) colonies are more \"rounded\" than \"flat\", they also have a clearly defined boundary, worse than this, they have formed free floating embryoid bodies.

Cells are passaged as followed:-

1. All reagents are warmed to 37

Medium

PBS

PBS/EGTA

TRYPSIN/EDTA

2. Remove medium

3. Wash with PBS (5ml/25cm2 flask, 10 ml/80cm2 flask), aspirate

4. Wash with PBS/EGTA, aspirate

5. Place flask on 37 warming tray for approx. 1 min or until individual cells can be seen in colonies.

6. Add 0.5 ml (25 cm2), 1 ml (80 cm2) TRYPSIN/EDTA and rock flask backwards and forwards until colonies float off, this should take ~ 1 min.

7. Using a 1 ml pipette, pipette up and down, (avoid making bubbles as these kill the cells), for approx. 1 min. Check that all colonies have been dispersed and that a single cell suspension has been achieved. Don\'t leave cells in TYRPSIN/EDTA for longer than 3 mins as it is quite toxic.

8. Neutralise trypsin by adding an equal volume of medium, mix by gentle pipetting.

9. Aspirate media from feeder flask, as this is ifferent media to ES cell media. Seed feeder* flasks with an aliquot of cell suspension, a 1:10 and 1:20 split is appropriate for a well growing culture with medium - large colonies that are not touching each other but are reasonably close together.

*Feeder flasks contain Mitomycin C treated (see protocol) Primary Mouse Embryo Fibroblasts (PMEFs) at a concentration of 0.3 x 106/25 cm2 flask, 1 x 106/80 cm2 flask, or Mitomycin C treated STO cells at a concentration of 1.25 x 106/25 cm2 flask, 4 x 106/80 cm2 flask. STO cells are smaller than PMEF\'s. (see charts).

If cells are to be grown in the presence of LIF only ie. no feeder layer, flasks or plates must be treated with 0.1% gelatin in PBS at 37 for at least 1 hr - 2 hrs. This is removed before the medium is added.