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EMBRYONIC STEM CELLS PROTOCOL
5 r3 Z" ?0 R. G. L: L! c8 x1 D6 bAllan Bradley's Lab, Baylor College of Medicine, Houston, Texas 胚胎干细胞基本操作 % s5 ?1 l, w: V7 z* u/ l* \+ _
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EMBRYONIC STEM CELLS PROTOCOL. z, a' W+ b2 Z8 J' J4 t7 b" M
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$ J1 K% @0 J/ \' Q2 y3 S If you are embarking in growing ES cells, be prepared to refeed
, T! ~0 ]4 Q0 s) a Fthem DAILY. All procedures should be carried out using sterile
1 z4 ` r' r+ }8 [8 N7 I9 ttechniques. The growth and maintenance media for ES cells is M15 MEM
8 F. U1 P: i; d% F: G(no pyruvate, high glucose) 15% FBS, 1X GPS, 1XBME. Handling ES cells: ! g4 G4 N8 J: O0 W
growth, maintenance, passing, freezing and thawing is conducted in a / _% M5 U3 G9 M; q, f3 x7 k; ^' |
manner to protect and maintain the quality of the cells and keep them in
6 c0 M. A7 ~6 X6 {4 h# Xa pluripotential state. Serum quality is critical for successful growth " t e+ l+ p! z# Q, [% c. s$ S
of ES cells and especially true for blastocysts. The quality of the " X$ K1 D. [) F8 L4 B( r0 r
feeders is very instrumental. Remember also that in passing, freezing, 7 |8 Q7 ]# u! I \$ r6 l
and electroporating ES cells; it is best that the cells are still at ! o6 J& _& L- X0 n3 ]) B$ H/ Q
exponential growth (80% confluence) for optimal results.
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THAWING (QUICK THAW)
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1. Remove cells from the freezer and quickly thaw in a 37oC waterbath.4 }/ E8 C! K( z/ q3 u0 U0 L
Y! X/ Q+ s u5 P# O0 g8 n2 l9 M, |2. Transfer the cell suspension to a sterile 15 ml tube. Add 10 - 9 P6 O1 M e P, e2 v6 ]9 T9 F
12 mls of M15 media to 1 ml of cell suspension.
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- Y7 M0 U, S4 _8 |3. Gently mix and pellet the cells by centrifuging @ 1000 rpm for 7 minutes.
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4. Aspirate off supernatant and resuspend cells into 6 mls of M15,
* \0 P- b9 k0 T- nand plate out cells in a 6-cm feeder plate.
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5. Refeed cells daily with fresh M15. Upon 80-85% confluence, cells
3 J4 [8 [! ~1 K5 v, {1 i8 S- J2 [need to be passage or freeze. (M15 media: DMEM, 15% FBS, 1X GPS, 1XBME)- O( K9 J) K& f2 O1 P5 U
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3 l& r6 u# X" N8 l! O* S4 [ zPASSAGE OF ES CELLS% y9 K" ~) p6 X) _7 _' s, H
) B4 W* x- n; A7 I3 W( kES cells typically should be passaged every 2-4 days (apart from colonies
+ T9 z8 ^5 ~% s7 N! e# R! dunder selection). If passaging is neglected the cells will differentiate
9 K' j) G8 K' d: V6 N, gand you will select for variants that might have lost totipotency. Cells . m% [# Y1 L) R; I- c0 ?9 P5 a+ \
must be fed when media begins to turn orange. Yellow media (acid pH) is ! }3 b2 _' J ?0 j0 {: N
very bad for ES cells and should be avoided at all costs. If you are
+ Y, `0 S+ `$ R! lplanning to passage and believe that the cells might turn yellow . v: O. r- I0 b0 B# ^3 R: S+ a
overnight feed last thing in the evening and again the next morning - s' L2 R- a8 ]( p0 r7 _. t
before passaging. DO NOT PASSAGE CELLS WHEN MEDIA IS YELLOW.
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1. Check cells under the microscope for 80-85% confluence., J, C+ J# v# S' v
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2. Refeed cells 3 - 4 hours before passing them. (VERY IMPORTANT)9 D1 v' _. D/ v2 p. o4 w# P
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5 I$ e$ O$ p6 V6 D0 w& L7 y1 g; g3. Aspirate media off. Wash one time with PBS. Add 500 祃 of 6 d6 f& T1 V; s9 F
trypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.
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" |! a& s$ j! \0 L7 N4. Incubate @ 37 oC for 15 minutes.
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5. Add media, M15 to inactivate the trypsin. About 2 mls to 1 x
8 v' ?4 B6 k. I. X; B$ e6-cm dish or 4 - 5 mls to 1 x 10-cm dish.# s0 X6 i2 K! S1 N* ?
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6. With a transfer pipette, pipet up and down several times to , m; P2 F5 Z8 B w0 \' t
separate the cells and break any colonies., c0 M# b l3 B+ |7 J
7 o" a# a+ O. X$ \/ l0 c) z' L7. Determine the number of feeder plates you need, depending upon
8 T j& c% O" I% `6 {: h/ |the passage you are doing. Add fresh media, M15 to the feeder plates (to
% H3 g- e3 h: s2 Z1 x 6-cm feeder dish: 6 mls of media; 1 x 10-cm feeder: 12 mls of
2 C' ^ X8 v- X" @; imedia). Split ratios for ES cells can vary from 1:1 to 1:10. Do not / O3 k. w/ L" p& k2 T
exceed 1:10.
$ x3 M6 T1 Q7 p9 L3 Y) f- e The area relationships for the various dishes are as follows:, j8 a2 W# _6 C% C/ f
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% Z0 H$ J# ]$ O+ X# ?Dish Media Trypsin Area (cm2) Diameter (actual), W: ?- A8 c* c+ ~/ @2 N5 D& v
(6 mm) 96 well 200 祃/well 30-50 祃 0.3 0.6 cm
8 k+ x3 x* e7 r8 ?6 U+ F; [(10 mm) 24 well 1.0 ml 200 祃 1.8 1.5 cm. z7 B, q( G1 L, o
(30 mm) 6-well plate 3-4 mls 400 祃 9.6 3.5 cm
* h. \7 v7 k+ A; _/ I0 e8 M(6-cm) dish 6 mls 0.6 ml 21.2 5.2 cm
# B( \* |' z+ X(10-cm) dish 12 mls 1.5 ml 60 8.7 cm& [6 Z7 p5 `: R7 i) @" I7 b" N# V
(15-cm) dish 30 mls 2.5 ml 154 14 cm
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# H- {2 z4 Z$ _1 U& G% W0 J: kSome typical passaging ratios:
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- }( i. a; [) U( L1:6 = 1 x 60 mm to 2 x 90 mm
1 \1 Y2 X+ ?: B! N5 W1:6 = 1 x 30 mm to 1 x 90 mm
6 P0 `5 K+ H* X' W2 ~1:4 = 1 x 30 mm to 2 x 60 mm
5 W2 y2 R, U& H# N1:5 = 1 x 24 well to 1 x 30 mm (6-well plate)
& j- }* b% {* D' ~% L' Q1:6 = 1 x 96 well to 1 x 24 well
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, |! |4 `6 ^3 ?) q8. Aliquot the cell suspension into plates in the volume specified
( c/ r9 |7 Z: M/ ofor each plate. Remember to use Feeder plates. Always check the feeders * N* O1 {2 }0 m4 L
before using them. They should be confluent, no gaps, not contaminated $ @; R0 E, P) V* T; f( e: L
and not dividing. Use feeders that are older, (1-2 weeks old), the ; P: m, J4 d9 `* [6 I
advantages are many: any contamination is assessed, also any dividing - \% F# n5 X3 j
run-away cells can be detected, and the passage will be earlier. Also,
O0 _7 t+ `7 ^" h# z# Z8 }4 }7 Lolder feeders have settled nicely and flattened.
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; \0 E9 J. x, T, h% I4 {4 F( h9. Mix to have a uniform cell distribution. Return plates to the TC
4 _" M- j0 K. m37 oC incubator.
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Z( X8 [" o2 @) k6 @. K4 HFREEZING ES CELLS (SLOW FREEZE)
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! V5 Y# H; P) o1. Check cells under the microscope for 80-85% confluence.
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$ M2 w- I2 d; f: Y( ` V$ M( F2. Refeed cells 3 - 4 hours before passing them.
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3. Aspirate media off. Wash one time with PBS. Add 500 祃 of
' E; t3 s' M6 Z3 M' a1 Htrypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.. w5 V" ]3 T7 q9 X: F
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4. Incubate @ 37oC for 15 minutes.
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5. Add media, M15 (M15 media: DMEM, 15% FBS, 1XGPS, 1XBME) to
$ w* Z* f! e8 |9 p) F7 ] }9 s# vinactivate the trypsin. About 2 mls to 1 x 6-cm dish or 4 - 5 mls to 1 x
9 \8 @+ M* U7 q10-cm dish.- }6 W' m# X) _. f* Q8 P
/ q# O" m( T4 P( N6 b6. With a transfer pipette, pipet up and down several times to - Q7 Y- L/ N7 T& y. G& B' I
separate the cells and break any colonies. Collect cell suspension in a
1 u) ^. [" _+ C" z; T Q4 wcentrifuge tube and add more media to count.: F6 \6 R- j/ G
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7. Count a 200 祃 aliquot and calculate the total cell number. From
! W( s# x& I' q- E, j: Bthis, calculate the volume of media required to give a final density of
2 X2 Q' @- h! z3.0 x 107 cells/ml. This density is very important, do not deviate from : ^3 |/ q5 b% m& w8 U+ [
it.
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0 `2 v. b) @ |% O8. Pellet cells by centrifuging @ 1000 rpm for 7 minutes.' v \) J6 p+ [$ s( r8 M
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9. Aspirate off supernatant and resuspend the pellet in 1/2 the ( Z7 s9 ^/ r$ Z0 C, }) p
volume calculated in Step 7 above, with M15 media.
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3 K7 j7 n; N6 S( s; l- o10. Add 1/2 the volume with 2X Freezing Media (60% DMEM, 20% FBS, 20%
% J# M8 U# ]- H4 `: e. n* JDMSO, freshly prepared); the cell suspension is diluted as a result: 10% * } z {# B8 W) O
DMSO is the final conc. Add the freezing media dropwise, mixing well & o/ {& N' D5 w6 a( o U$ J# u
after each addition.
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11. Aliquot the suspension into sterile freezing vials, pre-labeled 5 r0 b+ a' y6 o% H
with the cell type (AB2.2, AB1, etc.), clone number, passage number and 8 f& {1 t: i! _1 n
date. A typical aliquot would have 0.3 ml - 0.4 ml of ES cells (@ a
/ m' F; K. ~$ l$ f- l8 Qdensity = 3.0 x 107 cells/ml) this is about 9 x 106 cells - 12 x 106 : ^! b. K- H: g0 K
cells total/vial.3 _7 S5 J' C$ G- M: I
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12. Place vials into a freezing container. It is critical that the ) \/ N( x/ `2 L: Q5 k+ Y, Y' p) i/ ~
freezing rate is not faster than 1/minute. Do not use any untested / d" |' O* O5 D" [
styrofoam container since freezing rates vary greatly and this will most
2 V. y% Z4 D% ]% p1 ^likely result in death of most of your cells. Freeze cells overnight at
. a# T# W& L! D- z4 J-70oC, (24 hours).
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13. Next day, transfer cells to the Liquid Nitrogen freezer (or
6 ~2 s; b( M4 R: {% K-135oC freezer). |
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