Serum free ES MEDIA
Add all of the following to make ~1 liter w/o growth factors:
1L
Knockout DMEM/F12 (Invitrogen cat#: 12660-012)
5ml N2-SUPPLEMENT (Invitrogen cat#: 17502-048)
10ml B27 (Invitrogen cat#: 17504-044)
6.5 ml 7.5% BSA (in PBS) (Invitrogen cat#:15260-037)
10ml Pen/Strep 100x
10 mL Glutamine
-----------------------------------------------
Keep in dark. We find that this media is good at least 4 weeks at 4 C. For longer
storage, freeze at –20C. We generally aliquot in 50 ml tubes and add growth
factors as below for a working solution that is good for a maximum of 1 week.
Working Solution
For ES maintenance add MTG to 1.5x10-4 M final (Sigma M6145), LIF (1-2%
conditioned media or 1000 U/ml for recombinant) and human BMP4 10ng/ml
(R&D) and Glutamine. If you are storing you media in frozen aliquots, add these
reagents at the time of thawing.
*Alternatively- BMP4 can be replaced with two inhibitors PD0325901 (Cayman
Chemical Item#: 13034 (1uM)) and CHIR99021 (Cayman Chemical Item#:
13122 (3 uM)). This condition may work better for problem ES cell lines, which
display spontaneous differentiation in the standard media.
For 50 mL Aliquot…
Human BMP4 (@ 10 ug/mL)
LIF (2% final)
MTG (2 mL PBS, 26 ul MTG)
1:1000 dilution
1:50 dilution
1:1000 dilution
add 50 ul
add 1000 ul
add 50 ul
All cultures done in 6 well dishes that have been gelatinized.
Adapting Feeder/Serum Free
The following protocol can work for ES cells grown in serum containing media
with or without feeders. Your ES cells should have good morphology and be
fairly dense (>50% confluent) before the first serum free passage. Split newly
thawed ES lines at least 1-2 times using normal ES cell culture before attempting
to adapt SF. Trypsinize cells with 1 ml TrypLE Express (Invitrogen/Gibco
12605) 2-3 min. at 37C or until the cells start to detach. Pipet up and down
vigorously to get single cell suspension and dilute 1:10 in media base (DMEM or
IMDM with 0.05% BSA). Spin down and plate. Plate cells at differing
concentrations, 1:2-1:4, on gelatinized plates in SF media. Use the well
containing the cells with the best morphology for the next passage (usually 1:2 or
1:3).
Perform splits every day splitting 1:2 to 1:4 as above. If your ES cells were in
good shape, there should be very little cell death and they should adapt fairly
easily. If your cells were no so good or not dense enough, there may be cell death
for a few passages as they adapt.
By 3-5 splits your ES cells should be adapted. For general passaging, a 1:6 split
every 2 days is usually good.
Optional 2 step depletion:
Some ES cell lines have trouble being adapted and differentiate very easily. For
these lines the first SF passage is done on feeders. Feeders can be plated the night
before with serum containing media. Split ES cells as described above for the
normal protocol except plate on the feeders in SF media. Perform the next splits
as described above w/o feeders. This method takes an extra passage or two to
dilute out the feeder cells.
Tips for hard to adapt cell lines
I have found that media that has been made fresh works better for problem ES
lines.
The morphology of serum free ES cells is different than ES cells on feeders but
the cells should form colonies with smooth/shiny edges. You will be able to see
individual cells, unlike ES cells on feeders. They can also “mound up” like ES on
feeders but not quite so much.
Below is shown a mixed culture of undifferentiated and differentiating cells.
There is a strip of differentiating cells running down the center of the photo while
all of the other colonies display good morphology. Notice how the differentiating
cells are larger, flat, and do not have shiny edges. Good cultures should have
little to no differentiated cells.
Freezing
We freeze in DMEM or IMDM, 40% serum, 8% DMSO, 1.5-3 million cells per
tube (1 well of 6 well plate). We find that serum free ES are extremely sensitive
to freezing. Freezing needs to be performed carefully to prevent high levels of
cell death and differentiation. Therefore, it is very important to use ice cold
freeze media and cold cryo tubes, transfer to the freezer quickly and to freeze
slowly at –80 (Styrofoam racks work well). We have also found that they freeze
better if the cells were not too confluent, (<3 million/well in 6 well plate), and the
cells are fed fresh media 2-3 hours before harvesting. At thaw, dilute 1:10 in
media base (DMEM or IMDM), spin down and plate into one well of 6 well dish.
After thaw, a good freeze should be ready to split the next day. Poor freezes can
generally be rescued by by passaging 1 or 2 times.