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EMBRYONIC STEM CELLS PROTOCOL! k: J; f# u/ Q; R: P& F
Allan Bradley's Lab, Baylor College of Medicine, Houston, Texas 胚胎干细胞基本操作 / K: a1 Z8 a B' V4 H4 ~! n7 w
6 E3 U# g+ Y9 T, AEMBRYONIC STEM CELLS PROTOCOL2 Y) l z/ ]# b3 S
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/ p/ n' K8 o$ H+ o* |( [% z; P# J If you are embarking in growing ES cells, be prepared to refeed # j2 `9 V) y$ ~) C! a1 L
them DAILY. All procedures should be carried out using sterile ( z* M3 u, n9 V/ G/ j
techniques. The growth and maintenance media for ES cells is M15 MEM
( u+ u( k9 r5 B8 B- E0 w# G* L' f(no pyruvate, high glucose) 15% FBS, 1X GPS, 1XBME. Handling ES cells: * F, |( Q B6 h- o, U0 X
growth, maintenance, passing, freezing and thawing is conducted in a
* {5 w( e; b. y3 Fmanner to protect and maintain the quality of the cells and keep them in
9 S4 c8 W" Q$ _a pluripotential state. Serum quality is critical for successful growth 1 ~5 e# k- q$ |% ^8 l9 D5 w
of ES cells and especially true for blastocysts. The quality of the
, [( P$ N0 u6 A/ K+ E0 u: vfeeders is very instrumental. Remember also that in passing, freezing, # ?* _( N! `2 A; t: q& _
and electroporating ES cells; it is best that the cells are still at
3 u+ Y# |$ b: b* H- f5 t# O4 Mexponential growth (80% confluence) for optimal results.
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# c1 g# X) \4 @, }. iTHAWING (QUICK THAW)* f( b% `. m4 F; o5 e
0 o+ y- _( k- P( a }* K# j1. Remove cells from the freezer and quickly thaw in a 37oC waterbath.
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2. Transfer the cell suspension to a sterile 15 ml tube. Add 10 - : }9 M- T0 r* e
12 mls of M15 media to 1 ml of cell suspension.3 I( l+ w/ q$ N9 h) b; [ i
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3. Gently mix and pellet the cells by centrifuging @ 1000 rpm for 7 minutes.$ l4 J z; `3 y3 ?
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4. Aspirate off supernatant and resuspend cells into 6 mls of M15,
% \& m1 y7 w6 {4 K8 c0 Q; land plate out cells in a 6-cm feeder plate.
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/ r+ K. j5 p& ^. W9 a4 I% W8 ^5 {5. Refeed cells daily with fresh M15. Upon 80-85% confluence, cells
8 i- Q" k9 s# X$ \# g; ^# }" B. Wneed to be passage or freeze. (M15 media: DMEM, 15% FBS, 1X GPS, 1XBME)* v" p6 p- }) e1 c3 ^, J# b
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PASSAGE OF ES CELLS
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. q& r9 ^3 j6 P hES cells typically should be passaged every 2-4 days (apart from colonies
; u5 _4 Z' _3 x7 s1 Aunder selection). If passaging is neglected the cells will differentiate
* _1 Q, {) l: w" Y+ `: Fand you will select for variants that might have lost totipotency. Cells
' F. \8 i$ h/ B2 I7 S* \1 hmust be fed when media begins to turn orange. Yellow media (acid pH) is 7 {7 }! [/ j# V( K2 \
very bad for ES cells and should be avoided at all costs. If you are
4 Z% c+ R1 s0 o; }$ |* C9 \% fplanning to passage and believe that the cells might turn yellow
: c+ G# w" p+ ~/ }# x2 T1 e: c1 ?overnight feed last thing in the evening and again the next morning 8 Q9 B/ w! x' ^2 F. V
before passaging. DO NOT PASSAGE CELLS WHEN MEDIA IS YELLOW.: S9 j( {) s; d. O' x
5 p1 h/ d ^/ b. M) T! G3 E* L+ E; y7 O1. Check cells under the microscope for 80-85% confluence.. D$ v9 U9 `5 o5 m" a
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2. Refeed cells 3 - 4 hours before passing them. (VERY IMPORTANT)
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9 q! l; O) `. V! r0 `8 p+ N- `5 t3. Aspirate media off. Wash one time with PBS. Add 500 祃 of
5 | {# d: J6 D' U% |trypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.
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4. Incubate @ 37 oC for 15 minutes." H/ F7 h! |3 ~
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5. Add media, M15 to inactivate the trypsin. About 2 mls to 1 x ; h; x8 @, S e- }: b [/ e9 V* {
6-cm dish or 4 - 5 mls to 1 x 10-cm dish.# ~3 L0 ]- J( [- X$ N0 ^$ w
0 V- Z/ p5 k0 K6 N6. With a transfer pipette, pipet up and down several times to 0 F, m5 j8 ^, D3 |- ^; t
separate the cells and break any colonies.7 q7 }) [+ R$ u2 u' F4 ]) H* b" v
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7. Determine the number of feeder plates you need, depending upon
7 A+ {) T% _% l5 `0 r8 x" h& othe passage you are doing. Add fresh media, M15 to the feeder plates (to
) x6 m" V/ x) k5 ]7 t# x1 x 6-cm feeder dish: 6 mls of media; 1 x 10-cm feeder: 12 mls of
* o/ l! B; O3 C; Nmedia). Split ratios for ES cells can vary from 1:1 to 1:10. Do not + r# G* c9 O( V6 }2 N
exceed 1:10.
, v5 g" b+ l# o The area relationships for the various dishes are as follows:5 }3 c3 f& H5 M
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Dish Media Trypsin Area (cm2) Diameter (actual)
6 K$ \6 r5 |0 M- v. P( E(6 mm) 96 well 200 祃/well 30-50 祃 0.3 0.6 cm
0 v) S" b6 d3 h: }3 K(10 mm) 24 well 1.0 ml 200 祃 1.8 1.5 cm5 u1 b$ b$ q5 P$ k. D( K" v8 Q
(30 mm) 6-well plate 3-4 mls 400 祃 9.6 3.5 cm* ] f% K/ O' a4 S
(6-cm) dish 6 mls 0.6 ml 21.2 5.2 cm
6 X7 a: F. M) S(10-cm) dish 12 mls 1.5 ml 60 8.7 cm' C' m+ `& D7 q+ L
(15-cm) dish 30 mls 2.5 ml 154 14 cm% G# K3 M5 E7 [( E5 _' l
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: v& O+ m: \* y' S7 w. USome typical passaging ratios:
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1:6 = 1 x 60 mm to 2 x 90 mm
% g. `) e7 w f6 }) _1:6 = 1 x 30 mm to 1 x 90 mm
9 U4 H% Q/ R J0 C1:4 = 1 x 30 mm to 2 x 60 mm
/ S/ Y+ A; K/ T9 z! u) k0 V3 F1:5 = 1 x 24 well to 1 x 30 mm (6-well plate)
3 B" w' E& ^4 t" ~ v1:6 = 1 x 96 well to 1 x 24 well
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/ m! e/ x4 A' x( F( G+ \( d8. Aliquot the cell suspension into plates in the volume specified 9 d+ t$ G* j2 }$ R$ E9 P5 D
for each plate. Remember to use Feeder plates. Always check the feeders
( p N7 T3 F4 b/ Tbefore using them. They should be confluent, no gaps, not contaminated + O; i) r" C# x
and not dividing. Use feeders that are older, (1-2 weeks old), the
8 o7 M, k6 D! j9 ^$ z0 o5 g' a& \advantages are many: any contamination is assessed, also any dividing
6 u* L* ~4 \6 L) C3 S, O5 n! Rrun-away cells can be detected, and the passage will be earlier. Also,
1 Y( G1 j% d: S O8 Molder feeders have settled nicely and flattened.7 N9 V; R& S7 d2 i
6 D+ `( g0 I* g9. Mix to have a uniform cell distribution. Return plates to the TC $ R( ~1 ]5 f7 b% X/ N4 \
37 oC incubator.! I2 F) c' O" u5 r) G
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FREEZING ES CELLS (SLOW FREEZE)
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1. Check cells under the microscope for 80-85% confluence.
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9 ]0 D) a" d, u2. Refeed cells 3 - 4 hours before passing them.* b3 h9 W6 ^* X5 a- n2 B! s
: E* h3 F; p2 b) Y- k. n3. Aspirate media off. Wash one time with PBS. Add 500 祃 of
+ l$ h+ V3 g, c5 i4 p9 `trypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.
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; x4 v x9 @4 x4. Incubate @ 37oC for 15 minutes.
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5. Add media, M15 (M15 media: DMEM, 15% FBS, 1XGPS, 1XBME) to
5 @+ @; ?5 }' G/ t) t4 Dinactivate the trypsin. About 2 mls to 1 x 6-cm dish or 4 - 5 mls to 1 x - ^# d+ u( d* z7 g3 ~0 M
10-cm dish.6 A3 @9 w0 C- x
9 C5 z5 e: h$ M9 {7 A6. With a transfer pipette, pipet up and down several times to , D1 q8 Z' t# O: O. t% E
separate the cells and break any colonies. Collect cell suspension in a
: r, b0 \2 n: Q+ icentrifuge tube and add more media to count.
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7. Count a 200 祃 aliquot and calculate the total cell number. From ! ]* `" z8 _/ g( i0 G! n9 u& ?
this, calculate the volume of media required to give a final density of
; d+ b0 _9 I8 A% x# U0 N1 P3.0 x 107 cells/ml. This density is very important, do not deviate from : X- v% N; K2 z7 n
it. , s+ I. h) m1 ^8 o! W7 K
, u. }) `% N) y0 _8. Pellet cells by centrifuging @ 1000 rpm for 7 minutes.6 M% V1 ~; g& P. q5 Y" z
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9. Aspirate off supernatant and resuspend the pellet in 1/2 the
. h$ k; A0 K# _6 Y Ivolume calculated in Step 7 above, with M15 media./ v8 `5 r5 C* u0 h8 S& C: S8 ~( W& I
. q2 l/ A1 g2 i8 z10. Add 1/2 the volume with 2X Freezing Media (60% DMEM, 20% FBS, 20% ; w& O4 S. @/ K2 }7 a
DMSO, freshly prepared); the cell suspension is diluted as a result: 10% - K) {- j7 e) N! I( g
DMSO is the final conc. Add the freezing media dropwise, mixing well - h2 @- i' n& B
after each addition., x# B% U8 `& h% ^; r' [
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11. Aliquot the suspension into sterile freezing vials, pre-labeled 1 D2 F' C+ n6 M9 U8 {8 W
with the cell type (AB2.2, AB1, etc.), clone number, passage number and + t) Y+ z) S) Y. C0 {8 |/ i: e
date. A typical aliquot would have 0.3 ml - 0.4 ml of ES cells (@ a
- v4 ]4 I* b' \ b4 }1 T* ldensity = 3.0 x 107 cells/ml) this is about 9 x 106 cells - 12 x 106 6 V% O" j$ T. D/ N" N- S
cells total/vial.% n \+ p( ?" J, H# h
& D5 _ c) j6 l3 L1 s K5 f3 w$ @$ L12. Place vials into a freezing container. It is critical that the 4 S9 m0 {0 @1 [
freezing rate is not faster than 1/minute. Do not use any untested
# w0 t1 w% R, x# rstyrofoam container since freezing rates vary greatly and this will most " X! Y/ m! L) e, E
likely result in death of most of your cells. Freeze cells overnight at
8 s/ M M9 |: L4 \; x+ ^, n, V-70oC, (24 hours).
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) y9 M& U; l5 c3 X% R) x! k% J13. Next day, transfer cells to the Liquid Nitrogen freezer (or
+ [% o. E0 y! P5 c h-135oC freezer). |
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