Boris
Greber
AG
Himmelbauer
lab use only
Embryonic stem
cell culture - basic procedures
This protocol does not aim at substituting
the reading of other general ES cell culture protocols in the literature such
as this excellent one:
M.P. Matise, W.
Auerbach and A.L. Joyner
Production of
targeted embryonic stem cell clones
in: A.L. Joyner (ed.)
- Gene Targeting, a practical approach (2nd edition, third chapter)
The Practical Approach
Series, Oxford University Press 2000 (book is available in our library)
Rather, this presents a brief summary
of the most important points of several protocols from different sources and
includes some of the author´s experiences with ES cells.
Important rules for ES cell culture
It is assumed that differentiation of
ES cells is to be prevented in order to keep the cells able to colonize the
germline.
- ES cells do not like to be alone,
chose appropriate size of dish at thawing, do not split by more than 1:10
- do not overgrow, split when
semiconfluent, otherwise cells will differentiate
- to prevent differentiation always
dissociate cultures into single cells after trypsinization
- do not grow cells for more than a
few weeks before starting an experiment
- important: always try to reduce
the time that cells are in culture
- water (endotoxins even in
destilled water) and serum quality are very important
- it is best to feed cells, i.e.
change medium, every day
- passage cells every 2-3 days to
prevent differentiation
Signs of
differentiation
In general,
differentiated ES cells do not always look the same depending on the reason for
them to differentiate (the main reasons are bad feeder layer quality and low
LIF concentration - see below).
* colonies are surrounded by flattened
cells that differ morphologically from the undifferentiated ES cells in the
centre
* large colonies with necrotic centres,
these appear as cells with defined boundaries
* colonies appear as individual cells
rather than as syncial mass. Often the cell nucleus is clearly visible in
differentiated ES cells
* flattened colonies that appear as
single-cell layers surrounded by a circle-like boundary
- keep a record of how often cells
are passaged after thawing.
- for passaging and freezing it is
best that the cells are at subconfluence.
- growing conditions should be
optimal regarding growth rate and plating efficiency - suboptimal conditions
can e.g. be due to a bad batch of FCS (see ingredients of medium below).
ES medium
ES cells are grown in dishes coated
with a feeder layer of mitotically inactivated mouse primary embryonic
fibroblasts (see accompanying protocol) at 37°C / 5%CO2
/ 95% humidity. In contrast to the feeder cells, ES cells form colonies rather
than a single-cell layer.
DMEM (high glucose, Gibco 41966-052, store
in fridge) minimal medium supplemented before use with
- 1/100 (v/v) L-glutamine (200mM: Gibco 25030-024, make up
sterile aliquots, store at -20°C), stable in solution for 10d only
- 1/100 (v/v) non-essential amino acids (Gibco 11140-035, store aliquots
in fridge)
- 1/500 (v/v) 2-mercaptoethanol (Gibco 31350-010, 0.1mM final
conc., store in fridge)
- 15% (v/v) FCS (e.g. Biochrom, heat-inactivated serum from
newborn calf, liquid, tested for ES culture, store in fridge)
- 1/100 (v/v) pen/strep (Gibco 15140-122, aliquots at -20°C)
- 1/10,000 (v/v) LIF ("ESGRO" from Chemicon, No.
ESG1107, 1000 U/ml final conc. or less (depending on properties of respective
cell line), make up 1/100 dilution in DMEM with 10% (v/v) or so serum to be
further diluted by 1/100 to achieve the working conc., very expensive, store in
fridge)
Remarks
- feeder layers and LIF are to
promote growth of ES cells and prevent differentiation.
- medium should be changed every day
or when it turns orange (timepoint depends on state of culture); discard cells
if medium has turned yellow (acid pH). However, color of medium also depends on
when it has been made up (freshly made medium turns orange more quickly)...
- store medium in fridge, warm up in
waterbath before use.
Thawing ES cells (quickly)
When thawing ES cells, always have
feeder plates prepared (mitomycin C treatment at least one day before use, see accompanying protocol).
- remove ES cells from
freezer/liquid nitrogen and quickly thaw in a 37°C waterbath.
- transfer cell suspension (frozen
stocks usually contain 106 or more cells/ml) to a sterile tube
containing several ml of warm ES medium.
- gently mix and pellet the cells by
centrifugation @ low speed for 5min.
- aspirate off supernatant (removal
of DMSO in freezing medium) and resuspend cells into 12ml (5ml) of warm ES
medium and plate out cells in a 10cm (5cm) feeder plate.
- refeed cells daily with fresh ES
medium. Upon subconfluence, cells need to be passaged or frozen or used for
doing experiments.
Passaging ES cells
Passage ES cells every 2-4 days (except having colonies under
selection), otherwise cells will differentiate.
- check cells under the microscope
for subconfluence.
- refeed cells 3h before passing
them (very important). Warm up reagents briefly before use.
- aspirate medium off, wash one time
with PBS (Gibco 14190-169, without bivalent cations). Add about 1ml (2ml) of
trypsin/EDTA (Gibco 25300-096) to a 5cm (10cm) plate and incubate at 37°C until
colonies float off.
- add several ml of ES medium to
inactivate the trypsin.
- with a transfer pipette, pipet up
and down several times to separate the cells and break any colonies.
- determine the number of feeder
plates you need, depending upon the passage you are doing. Carefully add fresh
ES medium to the feeder plates (5ml final
volume - fresh medium + cell susp. - to a 5cm feeder, 12 ml to a 10cm one, see
below). Splitting ratios for ES cells can vary from 1:1 to 1:10. Do not exceed
1:10.
The area relationships for the
various dish formats are as follows:
dish diameter area(cm2) rel.
area final vol. trypsin
96well 0.6cm 0.3 1/200 0,2ml/well ca.50µl
24well 1.5cm 1.8 1/ 30 1
ml/well ca.300µl
5cm 5.1cm 20 1/ 3 5ml ca.1ml
9cm (Nunc) 8.5cm
57 1 10ml ca.2ml
10cm (TPP) 10.0cm 79 1.4
12ml ca.3ml
Some typical passaging ratios:
1:6 = 1 x 5cm to 2 x 9cm
1:6 = 1 x 96well to 1 x 24well
- aliquot the cell suspension into plates in the volume specified for each plate. Always check the feeders before using them. They should be confluent, no gaps, not contaminated and not dividing. Use feeders that are older (about 1 week old), the advantages are many: any contamination is assessed, also any dividing run-away cells can be detected, and the passage will be earlier. Also, older feeders have settled nicely and flattened.
- mix to have a uniform cell
distribution. Return plates to the 37°C incubator.
Freezing ES cells (slowly)
* It is very important to reduce the
time the cells are in culture before freezing.
* Freeze at a density that allows
survival of the culture even if 90% of the cells die during the freezing and
thawing process. On the other hand, cell density must not be too high because
cells may differentiate in this case.
- check cells under the microscope
for subconfluence.
- refeed cells 3h before passing
them. Keep styrofoam box at -80°C and freezing vials as well as freezing medium
(10% DMSO (Sigma D 2650) in ES medium) on ice.
- aspirate medium off. Wash one time
with PBS. Add about 1ml (2ml) of trypsin/EDTA to a 6cm (10cm) plate and
incubate @ 37°C for a few min until cell clumps float off.
- add several ml of ES medium to
inactivate the trypsin.
- with a transfer pipette, pipet up
and down several times to separate the cells and break any colonies. You may
count an aliquot while collecting the cells by low speed centrifugation for
5min.
- for a 10ml plate, resuspend cells
in several ml (depending on desired cell titre: usually 106 or more
cells/ml) of pre-cooled freezing medium and immediately transfer to freezing
vials on ice (1ml per vial). Immediately transfer vials into a styrofoam
box pre-cooled at -80°C and then to the -80°C freezer. It is important to work
quickly.
- next day, transfer cells to a
liquid nitrogen freezer.