a protocol-Growing feeder-independent embryonic stem cells

guochaosh

Growing feeder-independent embryonic stem cells, U% B4 |& R) t& b) d" H% z& h
We use feeder-independent ES cell lines derived from the 129/Ola strain of mice5 z& z' U% q: k4 z
(Nichols et al., Development 110, p.1341, 1990). These cells are easy to maintain and
% m, a3 ?: Y! O% o. F" ~# e- q5 hsignificantly reduce the amount of tissue culture required. Parental cell lines (CGR8 and
3 }9 q) {* ^1 D- T& uE14Tg2A) were established from delayed blastocysts on gelatinized tissue culture dishes( I+ r, L2 L* L; Z) _
in ES cell medium containing leukocyte inhibitory factor (LIF) (Nichols et al., 1990).
7 ^6 ~/ `' Q' r( O1 `Sublines were isolated by plating cells at a single-cell density, picking and expanding
# `9 T. h2 y% R( F9 fsingle colonies, and testing several clones for germline competence. The majority of1 D1 Z& D0 [% J4 a
BayGenomics cell lines are derived from the E14Tg2A.4 subclone.& j, ~0 b" x( [5 X8 a. s( V6 Y
Tissue culture reagents5 @. V; t" o4 n! S
1. Dulbecco's phosphate-buffered saline (PBS) without calcium and magnesium
8 \8 A+ `* a0 P. @8 T6 D" M(Gibco-BRL).8 C, p1 `. F! a
2. 2 mercaptoethanol stock solution: Add 70 μl of beta-mercaptoethanol (Sigma) to
7 w/ o1 {6 X6 ]3 D8 J20 ml of distilled, deionized water (Gibco-BRL). Filter sterilize and store at 4°C
" m4 f8 y6 T# [5 F4 qfor up to 2 weeks.9 K5 Z0 h/ [' [- |# O
3. ES cell medium: 1× GMEM medium (Sigma, G5154) supplemented with 2 mM
6 {2 w/ F; m3 ]0 A2 p& `# A6 Eglutamine (Gibco-BRL), 1 mM sodium pyruvate (Gibco-BRL), 1× nonessential# i- U3 L3 h  E+ t  i0 L2 D- L- ]
amino acids, 10% (v/v) fetal bovine serum (characterized, Hyclone), a 1:1000
3 o4 v) |2 \3 D& A/ C# {dilution of beta-mercaptoethanol stock solution, and 500-1000 units per ml of
0 @! U# r; N$ Z( k7 T$ J$ zleukocyte inhibitory factor (Chemicon catalog # ESG1107).
+ a, C1 V( ?- ]4. Freezing medium: Add DMSO (Dimethyl Sulphoxide, tissue-culture grade,
  I: y8 O2 n3 r9 t; nSigma) to ES cell medium to a final concentration of 10% (v/v). Filter sterilize.2 Y# h( V, X$ y
Make fresh before use.8 T3 M9 W- t' }" p. f0 P
5. Trypsin solution: Add 100 mg of EDTA tetrasodium salt (Sigma) to 500 ml of- {. Z; a. g0 r  i7 ?
PBS. Filter sterlize and add 10 ml (for 1×) or 20 ml (for 2×) of 2.5% trypsin
+ o0 i3 z  s- }/ ?solution (Gibco-BRL) and 5 ml of chicken serum (Gibco-BRL). Store in 20 ml7 s/ _, H" _/ k
aliquots at -20°C. (Note: 2.5% trypsin solution should be aliquoted and stored at -0 A/ k! x, W1 w# m: p, _% Y
20°C to avoid multiple freeze-thawing cycles.)7 y9 e5 H+ g, c* l: o
6. 0.1% gelatin: Add 25 ml of 2% bovine gelatin solution (Sigma) to 500 ml of PBS.$ {( ~1 I2 ?7 u$ r8 ]- [% }
Store at 4°C.' p, n1 Y- B7 x" N+ j" e
7. Geneticin (Gibco-BRL): Dissolve powder in PBS to make a 125 mg/ml stock7 l$ |  w6 ~9 d" a5 z8 i* J* ]
solution (active concentration). Filter sterilize and store at -20°C. Add 0.56 ml to
1 D* V$ Q$ v5 R- [+ h- b  `each bottle (560 ml) of ES cell medium. (Note: The concentration of Geneticin
/ n( P# C4 u* c0 ~# a0 V: n8 j- p: Ishould be titrated to determine the minimum concentration that will kill! F% x$ G+ D  K; v1 k$ a( N
nontransfected ES cells in 5 days.)- h6 m* n) }7 ^" G! Z% u8 I. V( M
Thawing ES cells5 U: b6 O+ q8 b* d4 p
ES cells are frozen in medium containing 10% DMSO. Since DMSO can induce the
) ]7 a2 S. c1 @, e& Z9 c5 `9 ^differentiation of ES cells, we advise thawing the cells late in the day and changing the1 T1 I8 m/ H7 Y# V  j" G. w) N, @
medium the following morning to minimize the effects of residual DMSO.) f. P0 _- M/ X, |
1. Coat a 25 cm2 tissue culture flask with 0.1% gelatin and aspirate off immediately# R2 z, G6 _* M9 i- T
before use.; U3 n8 w- Z& M
2. Thaw ES cells (approximately 5×106 cells, equivalent to one confluent 6-well or" f+ ~. }, G( [$ V$ F. j5 H1 W
1/2 of a confluent 25 cm2 flask) in a 37°C water bath and dilute into 10 ml of  X; d- B' f, T# c, R* j0 K
prewarmed ES cell medium.( p! X# l% T8 S% ~, q! c
3. Pellet the cells by spinning for 3 minutes at 1200 rpm in a bench-top clinical
% _2 S, N& K9 ?) [0 Icentrifuge.
7 l$ i& Z8 Y8 ]$ o4. Aspirate off medium and gently resuspend cells in 10 ml of prewarmed medium.
7 K& f: p' b7 D+ O5. Transfer cell suspension to a 25 cm2 flask and grow at 37°C in a humidified 6%8 |# n! D, S# k3 \( _9 P
CO2 incubator.
% Z; j9 s7 `* }+ ^6. Change medium the following day to remove dead cells and residual DMSO.
6 }8 l9 u5 h0 v, sPassage and expansion of ES cell cultures
- P8 k% b. P- Y# A* E2 hES cells are routinely passaged every 2 days, and the medium is changed on alternate
: J! a$ Z3 W# o1 l0 @! j, Qdays. Thus, ES cells require daily attention. In our experience, feeder-independent ES
8 t3 S0 M# S& x5 |cells grow rapidly and quickly acidify the medium, turning it yellow. Allowing the cells$ {# g; W% U' C
to acidify the medium (by not changing the media every day or by passaging the cells at8 L& B+ B( s- l
too low a dilution) will cause the cells to undergo crisis, triggering excess differentiation
- L$ d! x. Z) R/ D& C) zand cell death, after which their totipotency cannot be guaranteed. Plating cells at too low+ Z, U( @7 [  J4 t& f. W5 ~: [
a density, insufficient dispersion of cells during passage, or uneven plating can cause) V& D9 [' Q" K# v6 N2 ~$ Y  U: C
similar problems, as the cells will form large clumps before reaching confluence and the
1 r1 w& N+ \' A$ Wcells within these clumps will differentiate or die. Germline transmission is a
6 j) Z: S3 ]$ b  c- i( v2 Zsignificantly reduced in cells that have been mistreated, even when they appear healthy at
2 n6 r; i. u! rthe time of injection.$ F/ t2 W4 k) s8 F2 ~8 o* }
1. For a confluent 25 cm2 flask of cells aspirate medium off and wash with 5-10 ml
) f3 m. s+ X& P5 p' ~3 H4 L* Hof prewarmed PBS, pipetting it away from the cells. Rock flask gently and
- x# U% ~& \+ s# b( V4 daspirate medium. Repeat.$ D3 ]. \3 S  g
2. Cover cells with 1 ml of 1× trypsin solution and return to 37°C incubator for 1-28 J* H8 _: K1 ^% W' n% r
minutes or until cells are uniformly dispersed into small clumps.
. k) C6 K. N7 a$ Z3. Add 9 ml of medium to inactivate the trypsin.9 h& o* [+ v* P! u4 {! j
4. Count cells and add 1 × 106 cells (usually 1/10 of a 25 cm2 flask) to a freshly
# l) N/ q6 p- vgelatinized flask.6 z: W8 W1 |- @' m' T5 M
5. To expand ES cells for electroporation (requiring a total of 1 × 108 cells), seed 3 ×
8 v, q7 r& M. W# T  f/ o106 cells (1/3 to 1/4 of a confluent 25 cm2 flask) into a gelatinized 75 cm2 flask  P7 J: e+ F' Y% a+ }- o
and add 30 ml of medium. Add 20 ml of medium on the following day. Once the! L6 |0 f: N! Q$ K! H
cells reach confluence, trypsinize the contents of the 75 cm2 flask and add 5 × 106" h. C$ w/ r  ?7 ~* b! k
cells (1/5 of a confluent 75 cm2 flask) to each of three 175 cm2 gelatinized flasks! j" a" N' ~! k% m- {: P% z4 r
containing 50 ml of medium. Add an additional 30 ml of medium the following
- }# @* ^: H* L. E1 n0 qday.
# ]' l+ k8 j6 C$ ^/ c+ ^Freezing ES cells& p: Z: G9 O2 ~3 P6 y7 |2 t
1. Trypsinize a confluent 25 cm2 flask of cells (approximately 1 × 107 cells) as) q' E5 r+ F* O$ j4 L  K/ E! s9 q4 H0 @3 ~
described above.
/ V" ^" H% p6 `4 e! o$ |( ?2. Collect trypsinized cells in 9 ml of medium and pellet for 3 minutes at 1200 rpm.
6 ^  c/ X- Y, b' X9 E3. Aspirate off medium and resuspend cell pellet in 1 ml of freshly prepared freezing
/ Q! o, ?0 R* X# B; G6 e; X. m6 `0 Qmedium. Aliquot 0.5 ml of cells into two cryotubes.
; s1 I4 r7 ]9 M" p& f1 w9 j4. Freeze the vials at -80°C overnight and transfer to liquid nitrogen for long-term
2 m, D1 L- Z0 jstorage.