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标题: a protocol-Growing feeder-independent embryonic stem cells [打印本页]

作者: guochaosh 时间: 2010-12-3 18:42 标题: a protocol-Growing feeder-independent embryonic stem cells

Growing feeder-independent embryonic stem cells' c$ j. x0 l6 g7 Q6 O
We use feeder-independent ES cell lines derived from the 129/Ola strain of mice
(Nichols et al., Development 110, p.1341, 1990). These cells are easy to maintain and
significantly reduce the amount of tissue culture required. Parental cell lines (CGR8 and
E14Tg2A) were established from delayed blastocysts on gelatinized tissue culture dishes
in ES cell medium containing leukocyte inhibitory factor (LIF) (Nichols et al., 1990).
Sublines were isolated by plating cells at a single-cell density, picking and expanding
single colonies, and testing several clones for germline competence. The majority of
BayGenomics cell lines are derived from the E14Tg2A.4 subclone.
Tissue culture reagents" W( `+ Q; S  X% A' V, o
1. Dulbecco's phosphate-buffered saline (PBS) without calcium and magnesium, U  g# {2 `- a5 ~, W: t/ n
(Gibco-BRL).! k8 c; j0 c8 A; \2 C8 i8 }. J% `
2. 2 mercaptoethanol stock solution: Add 70 μl of beta-mercaptoethanol (Sigma) to; P. `: ?0 V( k) Y
20 ml of distilled, deionized water (Gibco-BRL). Filter sterilize and store at 4°C
for up to 2 weeks.8 g' [2 P; |$ A9 ]
3. ES cell medium: 1× GMEM medium (Sigma, G5154) supplemented with 2 mM
glutamine (Gibco-BRL), 1 mM sodium pyruvate (Gibco-BRL), 1× nonessential
amino acids, 10% (v/v) fetal bovine serum (characterized, Hyclone), a 1:1000! g2 B: i+ w: n' C- l
dilution of beta-mercaptoethanol stock solution, and 500-1000 units per ml of
leukocyte inhibitory factor (Chemicon catalog # ESG1107).
4. Freezing medium: Add DMSO (Dimethyl Sulphoxide, tissue-culture grade,4 y" Y6 @5 e. v7 k7 ]) {  d. e0 y
Sigma) to ES cell medium to a final concentration of 10% (v/v). Filter sterilize.9 [% f" C/ p! r9 S1 ]! O
Make fresh before use.' P4 h4 S2 F: Y2 r* h3 e
5. Trypsin solution: Add 100 mg of EDTA tetrasodium salt (Sigma) to 500 ml of# i, u, j) I% O5 b, n- G& O6 r! z
PBS. Filter sterlize and add 10 ml (for 1×) or 20 ml (for 2×) of 2.5% trypsin* C  U7 _, H. C* Y/ s4 F3 C5 {$ G
solution (Gibco-BRL) and 5 ml of chicken serum (Gibco-BRL). Store in 20 ml
aliquots at -20°C. (Note: 2.5% trypsin solution should be aliquoted and stored at -, B0 ?3 H8 X9 i
20°C to avoid multiple freeze-thawing cycles.)
6. 0.1% gelatin: Add 25 ml of 2% bovine gelatin solution (Sigma) to 500 ml of PBS.
Store at 4°C.4 ?; X) j6 ]) W0 f3 c# T: \. D3 L! C7 x, k
7. Geneticin (Gibco-BRL): Dissolve powder in PBS to make a 125 mg/ml stock. K& _" [/ J  R( d) ]4 P9 q
solution (active concentration). Filter sterilize and store at -20°C. Add 0.56 ml to% t' D" _4 `) ~( ~" U
each bottle (560 ml) of ES cell medium. (Note: The concentration of Geneticin3 ?! k9 B: u8 J
should be titrated to determine the minimum concentration that will kill
nontransfected ES cells in 5 days.)# `. }" l5 }3 \6 k  f
Thawing ES cells
ES cells are frozen in medium containing 10% DMSO. Since DMSO can induce the* R+ o- x3 x. b5 Q. ^
differentiation of ES cells, we advise thawing the cells late in the day and changing the8 d" P! z. G0 ]/ w
medium the following morning to minimize the effects of residual DMSO.
1. Coat a 25 cm2 tissue culture flask with 0.1% gelatin and aspirate off immediately
before use.: S. f" O4 I" G2 v3 ]: P" ~
2. Thaw ES cells (approximately 5×106 cells, equivalent to one confluent 6-well or/ e. |5 {3 U6 |9 N& j0 d
1/2 of a confluent 25 cm2 flask) in a 37°C water bath and dilute into 10 ml of* X3 r  L8 y4 ]$ u, C: K6 ^
prewarmed ES cell medium.
3. Pellet the cells by spinning for 3 minutes at 1200 rpm in a bench-top clinical7 S# ~! L) O7 g) [3 S  p- L
centrifuge.
4. Aspirate off medium and gently resuspend cells in 10 ml of prewarmed medium.
5. Transfer cell suspension to a 25 cm2 flask and grow at 37°C in a humidified 6%2 [, K( k  T' H9 ~- @* v# o+ t
CO2 incubator.
6. Change medium the following day to remove dead cells and residual DMSO.
Passage and expansion of ES cell cultures* V8 g  E/ t6 b+ s: u% [
ES cells are routinely passaged every 2 days, and the medium is changed on alternate. H# s7 B; _+ ~/ V
days. Thus, ES cells require daily attention. In our experience, feeder-independent ES& B4 U- D$ w# Q. |9 Q7 h5 _
cells grow rapidly and quickly acidify the medium, turning it yellow. Allowing the cells
to acidify the medium (by not changing the media every day or by passaging the cells at. S. n1 R- ]2 y& e: E
too low a dilution) will cause the cells to undergo crisis, triggering excess differentiation2 T* l5 @) d% t# _) C1 I( h
and cell death, after which their totipotency cannot be guaranteed. Plating cells at too low
a density, insufficient dispersion of cells during passage, or uneven plating can cause
similar problems, as the cells will form large clumps before reaching confluence and the& o4 j5 |3 d$ z  `3 M! A% K
cells within these clumps will differentiate or die. Germline transmission is a, D; t( Q* P2 [# S- K5 H$ F
significantly reduced in cells that have been mistreated, even when they appear healthy at. c8 ]  C8 k0 }, r
the time of injection.8 r) s: h0 Y* d7 o$ o& L: ~; g' s: [
1. For a confluent 25 cm2 flask of cells aspirate medium off and wash with 5-10 ml
of prewarmed PBS, pipetting it away from the cells. Rock flask gently and/ M* T, \( k9 g, d' H, n
aspirate medium. Repeat.5 y: H4 d) e; ^( Z6 h7 n8 l
2. Cover cells with 1 ml of 1× trypsin solution and return to 37°C incubator for 1-2' o* w; L8 w/ u- m4 W
minutes or until cells are uniformly dispersed into small clumps.
3. Add 9 ml of medium to inactivate the trypsin.
4. Count cells and add 1 × 106 cells (usually 1/10 of a 25 cm2 flask) to a freshly
gelatinized flask.9 W7 \4 r+ p$ A  _! l: `3 Z
5. To expand ES cells for electroporation (requiring a total of 1 × 108 cells), seed 3 ×7 B+ t( K" u* _, L) _( {* t- M; i
106 cells (1/3 to 1/4 of a confluent 25 cm2 flask) into a gelatinized 75 cm2 flask
and add 30 ml of medium. Add 20 ml of medium on the following day. Once the
cells reach confluence, trypsinize the contents of the 75 cm2 flask and add 5 × 1061 J% c9 m& R/ M& ^9 q" o
cells (1/5 of a confluent 75 cm2 flask) to each of three 175 cm2 gelatinized flasks
containing 50 ml of medium. Add an additional 30 ml of medium the following
day.
Freezing ES cells
1. Trypsinize a confluent 25 cm2 flask of cells (approximately 1 × 107 cells) as
described above.
2. Collect trypsinized cells in 9 ml of medium and pellet for 3 minutes at 1200 rpm.
3. Aspirate off medium and resuspend cell pellet in 1 ml of freshly prepared freezing( a) i1 N2 g+ _
medium. Aliquot 0.5 ml of cells into two cryotubes.- V6 s: N& Z, L! w6 _  E
4. Freeze the vials at -80°C overnight and transfer to liquid nitrogen for long-term/ z* A! i# x) N6 O! N
storage.

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