The derivation of TS cell lines from 3.5dpc mouse blastocysts is similar to
the derivation of embryonic stem (ES) cell lines. However, the success rate
is considerably higher and less expertise is required to recognize pluripotent
TS cell colonies.
Set up matings (natural or super-ovulated) between the mice
of interest.
Prepare 4-well plates of EMFIs (5x10
4 cells; 500 ul of 1x 10
5
cells/ml per well in TS medium the day before flushing. This is the same density
used to culture TS cells. [TS cell lines have also been derived from blastocysts
in the absence of EMFI cells, but with 70%EMFI-CM].
Replace the TS medium with TS + F4H medium (500 ul per well) on the morning
of flushing (day 1).
Flush and collect 3.5dpc blastocysts. Using sterile conditions, place one
blastocyst per well in the 4-well plates containing TS + F4H medium and culture
at 37
oC, 5% CO
2.
The blastocysts should hatch and attach to the wells in 24 to 36 hours (day
2).
On day 3, a small outgrowth is formed from each embryo. Feed each culture
with 500 ul fresh medium.
Day 4 is the day that the outgrowth is usually disaggregated. However, this
will depend on its size; it should be smaller than the size of the outgrowth
disaggregated for ES cell line derivation. The ideal size for TS cell derivation
is illustrated on p. 87, Figure 4b of Robertson reference above, or on p.267
Figure 26 in Hogan et al reference. Larger outgrowths will also work, but with
less efficiency.
Once suitable outgrowths have been chosen, they may be disaggregated by different
means. The microdrop technique described in Robertson and Hogan references
may be used. However, we perform the disaggregation directly in the wells
tey were cultured in. Remove the medium and wash the cells with PBS(500 ul).
Aspirate the PBS, add 0.1% trypsin/EDTA (100 ul), and incubate for 5 minutes
at 37
oC, 5% CO
2. Using a P2 pipetteman or a drawn Pasteur
pipette disaggregate the clump by pipetting up and down vigorously until the
outgrowth is reduced to a small clump of cells. Immediately stop the trypsinization
by adding 70% cond medium + 1.5x F4H (400 ul) and returning to the incubator.
Change the medium 8 hours after disaggregation (500ul 70% cond medium+ 1.5x
F4H).
On day 6, feed each culture (500ul 70% cond medium+ 1.5x F4H), and continue
to re-feed every 2 days.
Between days 6 and 10 (highly variable) TS cell colonies will begin to appear.
They look like flat, epithelial sheets with a distinctive colony boundary
(Tanaka et al). They are likely identical to the colonies described as "epitheloid
cells" on p.91, Figure 8d of Robertson etal, or the "epithelial-like cells"
on page 269-270 figure 3D of Hogan et al.
Continue to feed the cultures until TS cell colonies get sufficiently large
to cover about 50% of the well. Some differentiation will be observed at the
edges of the colonies. This is normal; they are most often giant cells and
other unidentified cell-types that may be between a stem cell and giant cell
phenotype.
Passage the half-confluent well of TS cells to a 6-well plate or 35mm dish
of pre-plated EMFIs (1x10
5 cells/ml- usually between days 15 and
20. Aspirate the medium and wash TS cells with PBS (500ul). Aspirate the PBS,
add trypsin/EDTA (100 ul), and incubate for 5 minutes at 37
o C/5%
CO
2. Stop trypsinization be adding TS + 1.5x F4H (400ul) and pipetting
up and down to get a near single-cell suspension. Transfer the cells to a
6-well plate or 35mm dish of TS+1.5x F4H medium (2.5ml) on EMFIs. This first
passage is crucial; this is the most likely time for the culture to differentiate.
Change the medium 8 hours after passage (3ml TS+1.5x F4H).
Feed the cells every two days (3 ml TS+1.5x F4H). Follow the "
Culturing
TS cell lines" guidelines provided. After one or two more passages on EMFIs,
TS cells may be cultured without them in the presence of 70% cond med+F4H.