-
ES
细胞培养
-
实验方
法
ES cell
culture media and solutions
ES
细胞培养需要较高的实验条件，培养血清要用纯度较高的（
ES
级别）的胎牛血清，为防
止细胞分化，需要在培养皿底部
铺种滋养层细胞（
Feeder cells
）并在培养基中加
入白细
胞抑制因子（
LIF
）
。培养细胞的平皿和吸管均为一次性聚乙烯材料。
(A)
D
MEM with high glucose
(B)
0
.1
mM
non-essential
amino
acids
(100
×
stock,aliquoted
,stored
at
4
℃
)
(C)
1
mM sodium pyruvate
(100
×
stock,aliquoted ,stored
at 4
℃
)
(D)
1
0
-4
M
β
?
mercaptoethanol
(100
×
stock,aliquoted ,stored
at-20
℃
)
(E)
2
mM
L
?
glutamine
(100
×
stock,aliquoted ,stored
at-20
℃
)
(F)
1
5%FBS
要用纯度较高的（
ES
级别）的胎牛血清
(G)
P
enicillin and streptomycin
(final concentration 50
μ
g/ml each)
(H)
1
000 U/ml LIF
白细胞抑制因子，抑制
ES
细胞分化
Preparation of EMFI feeder layers
Regents
Frozen vials of
primary embryo fibroblasts
Tissue
culture dishes
PBS without Ca2+ and Mg
2+
0.05% tripsin in saline /EDTA
DMEM +10%FBS
Mitomycin C
(stock 1 mg/ml in PBS stored in dark at
4
℃
and used within
two
weeks
,mitomycin
C
is
toxic
;wear
gloves
and
use
caution
when
handling )
Methods
1.
Thaw a frozen vial EMFI cells quickly
at 37
℃
.
2.
Add cells to
10 ml DMEM +10%FBS and centrifuge
(270
g,
5 min)
3.
Decant
supernatant , resuspend the cell pellet gently in
10 ml DMEM
+10%FBS, and
split
onto five
150
mm plates each
containing a total of
25
ml
DMEM
+10%FBS
.Mix
well.
注意摇动混匀，不要单纯只按一个方向摇动，
以使细胞较均匀地分布于平皿中，
。
4.
Incubate cells
at 37
℃
,5% CO
2
.
5.
When the cells form a confluent
monolayer (approx. three days )each
plate should either be:
(a)Thrypsinized
,split
onto
five
additional
150
mm
dishes
,and
grown
until they form a confluent monolayer
,or
(b)Directly treated with mitomycin
C
（丝裂霉素）
to inhibit cell
growth and division .
因为
ES
细胞与滋养细胞共培养时，未经丝裂霉
素处理
的滋养细胞增殖很快，
会与
ES
细胞竞
争养分，
此步丝裂霉素处理
可使滋养细胞失去增殖，但仍保持存
活。
6.
Remove the medium from the confluent
plates and 10 ml DMEM + 10%FBS
containing 100
μ
l
mitomycin C (1mg/ml stock).Swirl plates to ensure
an even distribution of medium.
7.
Incubate cells
at 37
℃
,5% CO
2
for 2-2.5h.
8.
Wash the monolayer of cells twice with
10 ml PBS per dish .
9.
Add 5 ml trypsin /EDTA to each plate .
10.
incubate
37
℃
,5% CO
2
until the cells come off the plate .
11.
Add 10 ml
DMEM +10%FBS to each plate and break any cell
aggregates
by
gently
pipetting.
12.
Centrifuge
cells
(270
g,5min)and
resuspend
the
pellet
in
DMEM+10%FBS.
5
13.
Count the cells and dilute to a
concentration of 2
×
10
cells/ml.
14.
Plate the
cells immediately onto tissue culture dishes
containing
DMEM+10%FBS for the
appropriate cell densities and volumes of medium
for different plate sizes.
15.
Allow
feeders
to
attach
at
least
2h
,but
preferably
overnight
,before
adding ES cells.
16.
Change the
medium to ES cell medium immediately before adding
ES
cells .Mitomycin C treated EMFI
feeders can be used for up to seven
days with medium changes every three to
four days.
Preparatiooon of a stock of
mitomycin C treated EMFI cells
Reagents
正常的滋养细胞贴壁生长于培养皿底面，呈
梭形，应当均匀分布，并将皿底完
全覆盖。
1.
thaw a frozen
via of EMFI cells quickly at
37
℃
.
2.
Add cells to
10 ml DMEM +10% FBS and centrifuge (270 g,5min).
3.
Decant
supernatant
,
resuspend
the
cell
pellet
gently
in
10
ml
DMEM
+10%FBS,
and split onto five 150 mm plates each containing
a total
of 25 ml DMEM +10%FBS .Mix
well.
4.
incubate cells at
37
℃
,5% CO
2 .
5.
When
the
cells
form
a
confluent
monolayer
(approx.
three
days
)each
plate
should
trypsinized
,split
onto
five
additional
150
mm
dishes ,and grown until they form a
confluent monolayer
6.
Remove the
medium
from the
confluent
plates and 10
ml DMEM +
10%FBS
containing
100
μ
l
mitomycin
C
(1mg/ml
stock).Swirl
plates
to
ensure
an even distribution of medium.
7.
Incubate cells
at 37
℃
,5% CO
2
for 2-2.5h.
8.
Wash the monolayer of cells twice with
10 ml PBS per dish .
9.
Add 5 ml trypsin /EDTA to each plate .
te 37
℃
,5%
CO
2
until the cells come off
the plate .(5-10min).
10 ml DMEM
+10%FBS to each plate and break any cell
aggregates
by gently pipetting.
fuge cells (270 g,5min)and resuspend
the pellet in freezing
medium
.Freezing
all
the
cells
from
each
plate
in
one
freezing
vial
in
1
ml
of
1
×
freezing
medium
and
store
at
-70
℃
for
one
day
.Transfer
the vials to liquid nitrogen.
make
feeder
plates
thaw
a
frozen
vial
of
mitomycin
C
treated
EMFI
cells quickly at
37
℃
cells to 10
ml DMEM +10%FBS and cenrifuge (270 g,5min).
supernatant , and resuspend the cell
pellet gently in 30 ml
DMEM +10%FBS.
cells
directly
into
tissue
culture
plates.
Depending
of
the
size
of
the
plates
required
put
10
ml
/100mm
plate,
5ml
/60
mm
plate,5ml/60
mm plate, or
1.5ml/35mm plate.
feeders
to
attach
preferably
overnight
,before
adding
ES
cells
or at least 2h if
using gelatinized plates .
the medium
to ES cell medium immediately before adding ES
cells .
Growth of ES cells on feeders plates
Equipment and reagents
100mm dishes containing feeder layers
ES cell medium briefly pre-warmed to
37
℃
0.
05% trypsin in saline /EDTA
PBS without Ca2+ and Mg2+
Gelatin ,0.1% solution in water
,autoclaved
Method
1.
Quickly
thaw
one
vial
of
frozen
ES
by
warming
in
your
hand
or
at
37
℃
,and
transfer
cells
to
a
12
ml
tube
containing
10
ml
of
ES
cell
medium
before
all the ice has
disappeared .
2.
Centrifuge at 270 g for 5min .
3.
Aspirate
supernatant
and
resuspend
pellet
in
10
ml
ES
cell
medium
and
plate on a 100 mm dish
with a feeder layer.
4.
Change
the
medium
the
next
day
by
swirling
the
medium
in
dish
to
collect
debris
,then
aspirate
.Add
fresh
medium
gently
to
the
side
of
the
plate
so that the feeder
layer is not disturbed.
5.
On
the
second
day
(cell
should
be
just
subconfluent)
wash
cells
twice
with
PBS and add 2 ml trypsin/EDTA.
6.
Incubate for
approx .5 min at 37
℃
until
cells begin to come off the
plate .