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EMBRYONIC STEM CELLS PROTOCOL9 X; _+ G- A5 U4 {% U
Allan Bradley's Lab, Baylor College of Medicine, Houston, Texas 胚胎干细胞基本操作
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- C. R/ I6 i+ g7 |1 [EMBRYONIC STEM CELLS PROTOCOL
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( A7 w* b# H( X7 N: V If you are embarking in growing ES cells, be prepared to refeed
' y9 o. o5 w9 m! J) h" O5 z0 @them DAILY. All procedures should be carried out using sterile
6 i5 [& K5 l. B! U# c# J: v. e4 ]5 xtechniques. The growth and maintenance media for ES cells is M15 MEM ! @' @, I6 ~6 r) O; K9 Q
(no pyruvate, high glucose) 15% FBS, 1X GPS, 1XBME. Handling ES cells: * ~9 t; B1 C/ S% F
growth, maintenance, passing, freezing and thawing is conducted in a
: Z2 Q ` s: W1 L3 Mmanner to protect and maintain the quality of the cells and keep them in ) c" \6 r* P, m0 ]: P
a pluripotential state. Serum quality is critical for successful growth
9 G* ?( j% \% xof ES cells and especially true for blastocysts. The quality of the # H2 l" H8 x p/ o* K
feeders is very instrumental. Remember also that in passing, freezing, ' D0 x0 T1 j) v# |4 ?3 ]9 L
and electroporating ES cells; it is best that the cells are still at : e- v' _5 M+ X3 W$ x
exponential growth (80% confluence) for optimal results.' z% ?5 [& R3 [7 `; E. y
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3 q9 S% j& x; p- S, q7 i1 N: |THAWING (QUICK THAW)
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1. Remove cells from the freezer and quickly thaw in a 37oC waterbath.0 o- m6 G$ u! u8 N' q
9 n2 g$ C* F6 b1 [% s0 p2. Transfer the cell suspension to a sterile 15 ml tube. Add 10 - 6 y% ?5 f& T+ s
12 mls of M15 media to 1 ml of cell suspension.
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' C& V1 {( ~, m* S6 j' h) ^, e3. Gently mix and pellet the cells by centrifuging @ 1000 rpm for 7 minutes.
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' v: l2 m8 h t# k7 S x( o4. Aspirate off supernatant and resuspend cells into 6 mls of M15, 7 }; u# L& I [8 o7 Y
and 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
; M V0 D3 G$ K' ineed to be passage or freeze. (M15 media: DMEM, 15% FBS, 1X GPS, 1XBME)1 J; ]2 q" v+ G7 t
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PASSAGE OF ES CELLS5 p" m+ _! ~) U8 E8 z/ t
, ?2 w. A, H) P: j; g; n6 @ES cells typically should be passaged every 2-4 days (apart from colonies , p' [! l y( Y$ B& r; C% f
under selection). If passaging is neglected the cells will differentiate
" _ M7 D3 S5 ?, ^% s1 sand you will select for variants that might have lost totipotency. Cells 9 t: q' j! s9 B- b2 t5 q
must be fed when media begins to turn orange. Yellow media (acid pH) is . x# j+ |: T2 K9 _* l* v! }1 _, a
very bad for ES cells and should be avoided at all costs. If you are
5 {( Z- a' t/ I5 }& \) _planning to passage and believe that the cells might turn yellow : ^( Q0 _' s. V& s
overnight feed last thing in the evening and again the next morning
( a4 t# m7 k2 Hbefore passaging. DO NOT PASSAGE CELLS WHEN MEDIA IS YELLOW.
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: H. z1 K( i3 S- G7 I2 s1. Check cells under the microscope for 80-85% confluence.
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! q+ J/ o6 w! R# F& _: E1 n2. Refeed cells 3 - 4 hours before passing them. (VERY IMPORTANT)
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2 B2 a7 P8 V% q% P1 \9 x3. Aspirate media off. Wash one time with PBS. Add 500 祃 of
9 P. ^2 [+ \, N9 a$ W! D* n. V* Atrypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.
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" ^3 m& u4 ] b& o q2 K& I6 }% I4. Incubate @ 37 oC for 15 minutes.
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5. Add media, M15 to inactivate the trypsin. About 2 mls to 1 x
# a% `; c$ N- \' U5 @; V5 \8 \/ f6-cm dish or 4 - 5 mls to 1 x 10-cm dish., w! f! F+ [8 y& t
- J- u( \" u1 l: s' w6. With a transfer pipette, pipet up and down several times to
) x* ^! F( l. ]) b8 T+ ^separate the cells and break any colonies.
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7. Determine the number of feeder plates you need, depending upon
/ d0 L3 W6 f& S0 l4 P1 v1 Lthe passage you are doing. Add fresh media, M15 to the feeder plates (to + t _$ D( |1 q( ~1 A# F
1 x 6-cm feeder dish: 6 mls of media; 1 x 10-cm feeder: 12 mls of
6 r; T# m. Z+ Y8 G% dmedia). Split ratios for ES cells can vary from 1:1 to 1:10. Do not
. _4 o4 Y7 u9 B: v5 r2 s0 Qexceed 1:10. ! Q6 D G# O# @; K7 U/ s
The area relationships for the various dishes are as follows:0 ]4 _6 U1 H9 k8 ^' \8 I# c! N
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. [1 o* m3 o |. ?8 l+ G$ ZDish Media Trypsin Area (cm2) Diameter (actual)1 o+ _! D% B2 A I
(6 mm) 96 well 200 祃/well 30-50 祃 0.3 0.6 cm
) ?; j$ a5 w0 t0 {0 ?; Q! R(10 mm) 24 well 1.0 ml 200 祃 1.8 1.5 cm
5 h0 |7 ~* M* t6 h(30 mm) 6-well plate 3-4 mls 400 祃 9.6 3.5 cm3 y& |" V! C2 w. {# }- Z
(6-cm) dish 6 mls 0.6 ml 21.2 5.2 cm
/ e# r" M$ c* m4 R(10-cm) dish 12 mls 1.5 ml 60 8.7 cm. x- u% T% {& \
(15-cm) dish 30 mls 2.5 ml 154 14 cm
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Some typical passaging ratios:8 ^7 ^: s8 V* M- |& W5 j
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1:6 = 1 x 60 mm to 2 x 90 mm
+ {' C! j9 N' W5 @( [. N1:6 = 1 x 30 mm to 1 x 90 mm
5 l. Q# z* F' g* i1:4 = 1 x 30 mm to 2 x 60 mm $ j) y3 m3 [5 ]2 M9 f6 ^8 ?0 e
1:5 = 1 x 24 well to 1 x 30 mm (6-well plate)
9 u" @. @ a8 \9 |) }1:6 = 1 x 96 well to 1 x 24 well
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8. Aliquot the cell suspension into plates in the volume specified * l: l2 x6 a/ J: a$ S
for each plate. Remember to use Feeder plates. Always check the feeders
* c; e4 G' U* gbefore using them. They should be confluent, no gaps, not contaminated ! k6 W& f Q9 A% ?
and not dividing. Use feeders that are older, (1-2 weeks old), the
7 T/ u/ j9 U$ K& Dadvantages are many: any contamination is assessed, also any dividing 3 F* T& H0 B: {2 K4 d
run-away cells can be detected, and the passage will be earlier. Also, 1 M6 b% \# P5 r; N+ F
older feeders have settled nicely and flattened.
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9. Mix to have a uniform cell distribution. Return plates to the TC + x1 |+ N# b) {3 H+ T' i
37 oC incubator.
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FREEZING ES CELLS (SLOW FREEZE)0 Y7 t! M1 |2 e# Q) Q; J
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1. Check cells under the microscope for 80-85% confluence.
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2. 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 Y9 u8 P8 q, s s2 p) L
trypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.- y8 V- g! r6 Z
! y- v5 ^5 a" `3 x* [- X4. Incubate @ 37oC for 15 minutes.6 ~4 s/ _, ?. {; u; O
( z. p# m- G3 [# \# J% D' Y5. Add media, M15 (M15 media: DMEM, 15% FBS, 1XGPS, 1XBME) to
' C$ g5 X" z9 z9 ginactivate the trypsin. About 2 mls to 1 x 6-cm dish or 4 - 5 mls to 1 x ( w+ @" T, j2 ~2 v& Q# {
10-cm dish." H) c) c: C3 d0 F! y7 ~) y
5 j9 l: P' A) p6 z* b! k3 r6. With a transfer pipette, pipet up and down several times to ! @! C; V/ c) T# J
separate the cells and break any colonies. Collect cell suspension in a
5 h8 b) T2 c6 h1 [3 x/ ~centrifuge tube and add more media to count.
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6 H% W9 ~. \# o: d4 m' ^9 C7. Count a 200 祃 aliquot and calculate the total cell number. From
* I+ D+ f' q( T0 [4 G' S# z3 [5 }; ythis, calculate the volume of media required to give a final density of - W+ W# H, l# K7 s4 _: d/ ?2 |
3.0 x 107 cells/ml. This density is very important, do not deviate from
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3 O1 F, t5 d8 c6 Q, i" G8. Pellet cells by centrifuging @ 1000 rpm for 7 minutes.
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9. Aspirate off supernatant and resuspend the pellet in 1/2 the
3 U# ]- J) r& J8 ]" H6 lvolume calculated in Step 7 above, with M15 media.
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$ E; u" `8 N" H e$ C10. Add 1/2 the volume with 2X Freezing Media (60% DMEM, 20% FBS, 20% - W6 y8 q6 B9 Q- o( |( _
DMSO, freshly prepared); the cell suspension is diluted as a result: 10%
+ i" Y# V+ }* H" cDMSO is the final conc. Add the freezing media dropwise, mixing well
$ r! W9 [7 W1 M. Iafter each addition.
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11. Aliquot the suspension into sterile freezing vials, pre-labeled ' z: }6 R3 K2 r3 Y+ P6 Z
with the cell type (AB2.2, AB1, etc.), clone number, passage number and 4 g5 Z+ f( c1 l
date. A typical aliquot would have 0.3 ml - 0.4 ml of ES cells (@ a
" u" J5 [+ ]5 t. x. `* j8 Jdensity = 3.0 x 107 cells/ml) this is about 9 x 106 cells - 12 x 106
0 s0 G# n( D# K& Qcells total/vial.: R+ t+ j& z9 V' D# N! t
a$ y! Z: v/ y$ }0 {1 H12. Place vials into a freezing container. It is critical that the
2 [0 i: t- ] S9 qfreezing rate is not faster than 1/minute. Do not use any untested
; n2 i; w- ^* x( L2 ~7 W0 ?styrofoam container since freezing rates vary greatly and this will most
4 }& a( v# u0 m0 b% Jlikely result in death of most of your cells. Freeze cells overnight at * k% h& N" j) w
-70oC, (24 hours).
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5 K2 o: } }2 {1 Y13. Next day, transfer cells to the Liquid Nitrogen freezer (or
$ \ Y! l2 [- P) Z5 Y-135oC freezer). |
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发表于 2009-3-3 14:28
