Mouse Embryoid Body Formation!

[Sumit Rai]

I have been trying to make embryoid body using the hanging drop method for
quite some time. But recently each hanging drop (30ul) has 2-3 EBs rather
than one big EB. I culture my ESCs using the following media:
DMEM-High Glucose
15% ESC qualified FBS
25mM HEPES
1X Glutamax
2mM Sodium Pyruvate
1X Pen/Strep
400uM Monothiglycerol (MTG)
1000 Units LIF/ml (Expressed by COS-7 cells)

When the plate is 70-80% confluent I split the cells with 0.25%
Trypsin/EDTA and seed 30ul hanging drops with 1200 cells/drop that are
cultured for 4 days. I make sure I have a single cell suspension by
pipetting them about 50-times through a pasteur pipette (Cell Viability is
about 95% as checked by Cell countess, Invitrogen).

For the past one year I was getting good EBs but recently I have started
getting this problem. I also checked for mycoplasma contamination but there
is none.

I recently tried tryipsinizing the mESCs for 5 min and then seeded cells
for EB fromation and to my surprise it worked. But again when I followed
the same procedure, I saw formation of 3-4 EBs in one hanging drop.

I was thinking about increasing the concentration of FBS from 15% to 20% so
that it increases the viscosity of the solution and the cells can come
close to each other forming a single large EB.

Any help will be greatly appreciated.

Sumti Rai,
Graduate Student,
UGA

[Sébastien Vigneau]

Hi Sumit,

If the goal is to differentiate cells (which EBs are typically used for),
you should not include LIF in the media. ES cells will not differentiate
and EBs will not compact in the presence of LIF, which may lead to "EBs"
fragmentation. Additionally, it is not common to use HEPES to buffer
DMEM-based media when cells are cultivated in a 5% CO2 incubator (HEPES is
generally used to buffer in normal atmosphere). The number of cells per
drop also seems a little large to me, so you may want to try with smaller
number of cells.

Alternatively, if you did remove LIF from the media and you still don't get
one EB per drop, with cell number and drop volume that used to lead to EBs,
maybe this is caused by a change in the geometry of the drop, for instance
due to a change in the plastic of the lid the drops are hanging from. Or
maybe the incubator is vibrating...

I hope this help.

Best,

Sébastien

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[Sumit Rai]

Hi Sebastien,

Thanks for the reply. You are completely right that during the formation of
EBs, LIF has to be removed allowing the stem cells to differentiate.To make
EBs I use LIF deficient media. Also, with respect to using HEPES, I am
trying to perform the experiment "as is" mentioned in a paper. It used to
work perfectly fine initially (for about 1.5 years) but now this problem
has risen. I guess I will try 1000 cells /drop. I have also tried seeding
the cells in 20ul drop so as to get the cells close to each other but
unofortunately this also doe not help.

Sumit