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EMBRYONIC STEM CELLS PROTOCOL
9 s8 X! K# I" J! w# ?& B y. \0 gAllan Bradley's Lab, Baylor College of Medicine, Houston, Texas 胚胎干细胞基本操作
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EMBRYONIC STEM CELLS PROTOCOL
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# d* d7 P* P/ U4 | If you are embarking in growing ES cells, be prepared to refeed p9 H; A' q; k& p
them DAILY. All procedures should be carried out using sterile + R7 g5 a- L# @% U0 }. s
techniques. The growth and maintenance media for ES cells is M15 MEM
9 v" G3 B& i9 S+ [5 g( |(no pyruvate, high glucose) 15% FBS, 1X GPS, 1XBME. Handling ES cells: 1 W! q# F! S, V7 {
growth, maintenance, passing, freezing and thawing is conducted in a ( Y1 s- o1 j- C+ z/ A
manner to protect and maintain the quality of the cells and keep them in
: j$ n y( V% pa pluripotential state. Serum quality is critical for successful growth # \9 e. ?, L" y+ K6 k! k) n
of ES cells and especially true for blastocysts. The quality of the
; t$ f3 ~8 Z9 j7 m: Q8 X# b* Pfeeders is very instrumental. Remember also that in passing, freezing,
5 A1 o, D3 v! iand electroporating ES cells; it is best that the cells are still at 5 S0 _( j w4 g$ z# g
exponential growth (80% confluence) for optimal results.
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THAWING (QUICK THAW)! [7 y. V6 e( N1 g" p4 B& Q
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1. Remove cells from the freezer and quickly thaw in a 37oC waterbath.8 r/ L" u9 r Q$ h3 ?
$ x6 b5 L/ G- d1 R5 L. t8 B2. Transfer the cell suspension to a sterile 15 ml tube. Add 10 - + L: g f2 K% j m r
12 mls of M15 media to 1 ml of cell suspension.. N4 d, K5 e- f/ s% R
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3. Gently mix and pellet the cells by centrifuging @ 1000 rpm for 7 minutes.: Z* ], {+ ~" G! u
1 Z" @$ x* Y6 J7 w$ P; `4 m: r/ R4. Aspirate off supernatant and resuspend cells into 6 mls of M15,
) ^3 y! e# m' h5 Yand plate out cells in a 6-cm feeder plate.
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" G7 r! h# X( A, r0 h* b# e* E5. Refeed cells daily with fresh M15. Upon 80-85% confluence, cells - b% V/ e: U! x) Y9 O
need to be passage or freeze. (M15 media: DMEM, 15% FBS, 1X GPS, 1XBME)
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* n/ \% k) ]* x: `9 P' LPASSAGE OF ES CELLS( c* v9 L. }# @( k# M2 n
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ES cells typically should be passaged every 2-4 days (apart from colonies 0 \* Q; _ h' [+ w: l% D
under selection). If passaging is neglected the cells will differentiate
1 A. ?/ x3 L% ?! c [and you will select for variants that might have lost totipotency. Cells
: x# y3 t2 f5 J. Smust be fed when media begins to turn orange. Yellow media (acid pH) is
# P" @& C5 i/ _+ ?very bad for ES cells and should be avoided at all costs. If you are
# ?9 v" Z0 X+ `( A @planning to passage and believe that the cells might turn yellow ' O3 |8 v* ]; g+ M- d# [
overnight feed last thing in the evening and again the next morning
. z3 |' u, U' y& Y+ h/ \, S: Lbefore passaging. DO NOT PASSAGE CELLS WHEN MEDIA IS YELLOW.! D8 q: [6 h M/ _- ~) p3 ^
2 T* m# p$ E# j, w" {1. Check cells under the microscope for 80-85% confluence. c+ Y& @9 ?1 `
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2. Refeed cells 3 - 4 hours before passing them. (VERY IMPORTANT)
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3. Aspirate media off. Wash one time with PBS. Add 500 祃 of
, F$ i$ m" {+ p0 l- i7 ]4 htrypsin 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.
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5. Add media, M15 to inactivate the trypsin. About 2 mls to 1 x 7 U( V8 J- X3 z; G" ^
6-cm dish or 4 - 5 mls to 1 x 10-cm dish.
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6. With a transfer pipette, pipet up and down several times to , S& H R* k. P& ~
separate the cells and break any colonies.
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; q* E( k8 J* `$ O8 ]# A7. Determine the number of feeder plates you need, depending upon
' }3 {$ p7 z$ D8 Tthe passage you are doing. Add fresh media, M15 to the feeder plates (to ( `7 T8 L0 y; b. S9 J+ S) V: R
1 x 6-cm feeder dish: 6 mls of media; 1 x 10-cm feeder: 12 mls of 6 @6 i1 D% Y$ K4 @$ R5 f
media). Split ratios for ES cells can vary from 1:1 to 1:10. Do not * N( x( w" z# ~: r6 B
exceed 1:10. + v( a u, i/ S, [' Y
The area relationships for the various dishes are as follows:
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) z) s8 j5 o# K) NDish Media Trypsin Area (cm2) Diameter (actual)! ?% x7 u; q& ~
(6 mm) 96 well 200 祃/well 30-50 祃 0.3 0.6 cm
+ s9 z' v3 k% K! u; {# ` c: F(10 mm) 24 well 1.0 ml 200 祃 1.8 1.5 cm0 @) G) q2 H+ |0 ]) G; }7 A
(30 mm) 6-well plate 3-4 mls 400 祃 9.6 3.5 cm4 ^; D& B6 q* N: K% O7 C# Z
(6-cm) dish 6 mls 0.6 ml 21.2 5.2 cm
8 T1 z* k2 }6 d0 g9 S(10-cm) dish 12 mls 1.5 ml 60 8.7 cm
; ?; ^" K# H7 S/ L7 `- V e5 h(15-cm) dish 30 mls 2.5 ml 154 14 cm- a1 g1 Y5 I" Q7 D( p3 r0 T0 Z
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Some typical passaging ratios:
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1:6 = 1 x 60 mm to 2 x 90 mm ; _7 u% G5 H/ A6 X3 Y
1:6 = 1 x 30 mm to 1 x 90 mm
/ U' E8 O0 E& E$ A* _0 @" h1:4 = 1 x 30 mm to 2 x 60 mm
1 q' K" r6 {( h9 R1:5 = 1 x 24 well to 1 x 30 mm (6-well plate)
) Z, C, {. Z/ L3 q8 o0 [0 i+ Q1:6 = 1 x 96 well to 1 x 24 well
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! @5 ?! @/ ^" W, Y9 q$ v8. Aliquot the cell suspension into plates in the volume specified & f6 @) i" L( W Z5 x5 M6 Y- C4 c, E
for each plate. Remember to use Feeder plates. Always check the feeders
\1 n& \) q# ]/ ?before using them. They should be confluent, no gaps, not contaminated \! g! e% U: C$ N7 N
and not dividing. Use feeders that are older, (1-2 weeks old), the
) z/ o1 R7 W9 a( gadvantages are many: any contamination is assessed, also any dividing + q# I3 P% J( G7 C
run-away cells can be detected, and the passage will be earlier. Also,
& f7 c, V" b5 \5 |6 I' d2 Qolder feeders have settled nicely and flattened.
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- ]" f8 B# D/ ]9. Mix to have a uniform cell distribution. Return plates to the TC
5 y1 H: _' m6 P4 B! f: w37 oC incubator.
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FREEZING ES CELLS (SLOW FREEZE)
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* X6 W$ m+ w: m5 `" N1. Check cells under the microscope for 80-85% confluence.
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, {% U. A* Q0 G5 x" [( i7 h: P2. Refeed cells 3 - 4 hours before passing them.
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; I) A: C& t1 B" P# I6 f0 I3. Aspirate media off. Wash one time with PBS. Add 500 祃 of
5 G8 G" W M" j; d% utrypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.
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j7 {0 b: {( L) C. l% W2 S' x2 B4. Incubate @ 37oC for 15 minutes.
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5. Add media, M15 (M15 media: DMEM, 15% FBS, 1XGPS, 1XBME) to 4 z8 p0 r% @' A
inactivate the trypsin. About 2 mls to 1 x 6-cm dish or 4 - 5 mls to 1 x @% |' C! _% S: B
10-cm dish.
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j& f0 L0 |6 R( g6 g8 ~$ x% Z% ~6. With a transfer pipette, pipet up and down several times to 2 [: q' g& C" E
separate the cells and break any colonies. Collect cell suspension in a ; G0 h; @3 F# Q y: e8 ^
centrifuge tube and add more media to count.
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. ~. a( f# r! |3 H& C9 [7. Count a 200 祃 aliquot and calculate the total cell number. From 3 x# _: E) m( z9 |6 n
this, calculate the volume of media required to give a final density of
7 d4 Y) A8 o9 k, {" |3.0 x 107 cells/ml. This density is very important, do not deviate from
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/ _) L! F; W4 \ ]2 R8. Pellet cells by centrifuging @ 1000 rpm for 7 minutes.) b) Q0 R" C5 Q1 ]' G7 x/ p. S
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9. Aspirate off supernatant and resuspend the pellet in 1/2 the
' q- p4 s; g6 b5 q% u1 H6 Uvolume calculated in Step 7 above, with M15 media.7 s7 y4 g: k, z+ N& Y# h {1 o
( w7 c$ I# O' g- h4 A10. Add 1/2 the volume with 2X Freezing Media (60% DMEM, 20% FBS, 20%
. n; i& L* g. d* ?+ D3 E- ^# gDMSO, freshly prepared); the cell suspension is diluted as a result: 10%
& M* X" _2 h. X& hDMSO is the final conc. Add the freezing media dropwise, mixing well
2 a/ c5 q3 T& Fafter each addition.
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11. Aliquot the suspension into sterile freezing vials, pre-labeled
+ R" S2 C5 f* Fwith the cell type (AB2.2, AB1, etc.), clone number, passage number and 4 X" T. U) A/ N) h& V
date. A typical aliquot would have 0.3 ml - 0.4 ml of ES cells (@ a ; |( O0 |5 j: @$ ^4 t' [0 ~9 s
density = 3.0 x 107 cells/ml) this is about 9 x 106 cells - 12 x 106 ' L# m, M6 f% b+ t6 w9 L
cells total/vial.
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12. Place vials into a freezing container. It is critical that the
5 v, p$ M3 D$ b* |; t/ Zfreezing rate is not faster than 1/minute. Do not use any untested : q" a& E, B: ^: y
styrofoam container since freezing rates vary greatly and this will most
' C3 U4 z6 h# d) S$ T3 Y' u8 R/ Y( O" qlikely result in death of most of your cells. Freeze cells overnight at % o1 r. M: Q8 U, v: d0 }+ J
-70oC, (24 hours).
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13. Next day, transfer cells to the Liquid Nitrogen freezer (or
2 ^$ r; i0 l3 T$ A( q-135oC freezer). |
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