a protocol-Growing feeder-independent embryonic stem cells

guochaosh

Growing feeder-independent embryonic stem cells
" e- y1 |$ y4 h9 WWe use feeder-independent ES cell lines derived from the 129/Ola strain of mice
( {+ u0 t0 O& L7 e% U! I8 P(Nichols et al., Development 110, p.1341, 1990). These cells are easy to maintain and
$ X+ S4 M7 C* I4 I9 s: Z; F5 y$ bsignificantly reduce the amount of tissue culture required. Parental cell lines (CGR8 and# J) s. c* t$ B3 ?
E14Tg2A) were established from delayed blastocysts on gelatinized tissue culture dishes. X* S& {/ u+ r% J- a, v0 i3 ^
in ES cell medium containing leukocyte inhibitory factor (LIF) (Nichols et al., 1990).
) D; T1 A# B; ~& m, DSublines were isolated by plating cells at a single-cell density, picking and expanding5 G3 d$ D& m1 A1 E
single colonies, and testing several clones for germline competence. The majority of8 k) A% w% G- I( `# a5 F9 n, l2 F
BayGenomics cell lines are derived from the E14Tg2A.4 subclone.
$ A: a/ j7 m# B0 YTissue culture reagents
5 ?( {5 ?& ^4 n. S; R1. Dulbecco's phosphate-buffered saline (PBS) without calcium and magnesium- E6 H1 z2 q2 R0 m6 X% s5 s
(Gibco-BRL).8 F$ U4 z( n; [* c! n; I
2. 2 mercaptoethanol stock solution: Add 70 μl of beta-mercaptoethanol (Sigma) to
  ?! r* B5 {: d! z( q0 ^! b& p20 ml of distilled, deionized water (Gibco-BRL). Filter sterilize and store at 4°C
( G$ G4 g; \4 I: bfor up to 2 weeks.) d, W- ~  ?9 A# ?) p
3. ES cell medium: 1× GMEM medium (Sigma, G5154) supplemented with 2 mM' }5 y! R) m6 S& @
glutamine (Gibco-BRL), 1 mM sodium pyruvate (Gibco-BRL), 1× nonessential
/ ?. t4 I' h# W2 i8 xamino acids, 10% (v/v) fetal bovine serum (characterized, Hyclone), a 1:1000
% ^: `6 }! I; x& ?7 \dilution of beta-mercaptoethanol stock solution, and 500-1000 units per ml of
2 |/ D2 w! F+ s  k( c/ _leukocyte inhibitory factor (Chemicon catalog # ESG1107).& ?8 E; R6 w1 Y$ ~2 }8 J4 f
4. Freezing medium: Add DMSO (Dimethyl Sulphoxide, tissue-culture grade,
$ q4 Y: l+ I. k" J$ x' K( N) c5 vSigma) to ES cell medium to a final concentration of 10% (v/v). Filter sterilize.
" ]! n" s' ]1 ~4 n' |Make fresh before use.0 O' n$ |8 D- ^7 A8 y" N4 Y
5. Trypsin solution: Add 100 mg of EDTA tetrasodium salt (Sigma) to 500 ml of- x) B( g- T3 l( ^. g2 M, c& J* [
PBS. Filter sterlize and add 10 ml (for 1×) or 20 ml (for 2×) of 2.5% trypsin8 F8 K3 b: l5 w% |
solution (Gibco-BRL) and 5 ml of chicken serum (Gibco-BRL). Store in 20 ml
5 N+ t. h2 Z/ @" o9 Ealiquots at -20°C. (Note: 2.5% trypsin solution should be aliquoted and stored at -
; D$ T& ]/ i. P9 `7 g  `) `20°C to avoid multiple freeze-thawing cycles.)
) G; A( P! f9 m! H) e6. 0.1% gelatin: Add 25 ml of 2% bovine gelatin solution (Sigma) to 500 ml of PBS.
7 _; [6 ^! L4 S, pStore at 4°C.4 S+ k( s, j7 W0 O
7. Geneticin (Gibco-BRL): Dissolve powder in PBS to make a 125 mg/ml stock( `+ a1 [& a6 m* A5 o2 p' B* f
solution (active concentration). Filter sterilize and store at -20°C. Add 0.56 ml to( X+ S7 ?$ T: A! \' g* R! c3 J
each bottle (560 ml) of ES cell medium. (Note: The concentration of Geneticin
; \' ~# W! p  `3 a1 ~$ R; y! }should be titrated to determine the minimum concentration that will kill
: t8 ~5 D6 n3 u5 f3 k% `0 Jnontransfected ES cells in 5 days.)
' _3 \$ P' j1 X% FThawing ES cells
; Q+ Y( T2 s4 r, U+ l& @( f+ OES cells are frozen in medium containing 10% DMSO. Since DMSO can induce the
. x8 F* f% a2 V9 r3 Sdifferentiation of ES cells, we advise thawing the cells late in the day and changing the
: T$ H% @6 I- vmedium the following morning to minimize the effects of residual DMSO.) [' L: ~4 o& f1 [) M/ [& s; F% a2 A
1. Coat a 25 cm2 tissue culture flask with 0.1% gelatin and aspirate off immediately
8 g3 ~4 I- Q- e6 B3 S# T. T, Sbefore use.
8 j, b1 Z; L1 Y* c: z/ @2. Thaw ES cells (approximately 5×106 cells, equivalent to one confluent 6-well or, f6 T/ Y6 G; r) m: O/ s0 ?; f
1/2 of a confluent 25 cm2 flask) in a 37°C water bath and dilute into 10 ml of4 a4 C6 c7 ?& w$ k6 Z
prewarmed ES cell medium.# i! F! V2 a/ {. ^# P, [, R2 L4 I
3. Pellet the cells by spinning for 3 minutes at 1200 rpm in a bench-top clinical" F( v  d5 ]& `2 |0 a
centrifuge.
/ d9 ]! j' W% A7 O4. Aspirate off medium and gently resuspend cells in 10 ml of prewarmed medium.0 j) e5 i7 V/ U' k0 E
5. Transfer cell suspension to a 25 cm2 flask and grow at 37°C in a humidified 6%
+ x2 \" {: {& j- }* FCO2 incubator.
2 `. v9 P2 ~& l+ k6. Change medium the following day to remove dead cells and residual DMSO.
: w) p/ m4 q/ K+ F% B) kPassage and expansion of ES cell cultures
/ q9 E$ u  r" f  Y, O  P9 m. N. a( @ES cells are routinely passaged every 2 days, and the medium is changed on alternate
$ q( v4 F. E% e9 Tdays. Thus, ES cells require daily attention. In our experience, feeder-independent ES
0 B  y  z+ D2 @+ ]cells grow rapidly and quickly acidify the medium, turning it yellow. Allowing the cells
$ \6 B' O- B4 {# K( O* W+ Gto acidify the medium (by not changing the media every day or by passaging the cells at. V3 m. E2 q( Q2 j8 w% d
too low a dilution) will cause the cells to undergo crisis, triggering excess differentiation
; X7 Q4 h2 ~$ k6 m+ \- W+ U/ Sand cell death, after which their totipotency cannot be guaranteed. Plating cells at too low
' g3 r7 h3 u) y: d$ Sa density, insufficient dispersion of cells during passage, or uneven plating can cause
* U0 ]/ `' ^1 G, T- Y( c, Ysimilar problems, as the cells will form large clumps before reaching confluence and the
) L2 o: D) K! I# Z; t+ D/ ?cells within these clumps will differentiate or die. Germline transmission is a
# Y4 U. K; ~* e7 `  Msignificantly reduced in cells that have been mistreated, even when they appear healthy at4 C# R0 P9 i* T* Z: m% f* [: I
the time of injection.1 E/ M2 C8 p( u1 r7 Y! A
1. For a confluent 25 cm2 flask of cells aspirate medium off and wash with 5-10 ml3 C0 {4 K; P, i! w
of prewarmed PBS, pipetting it away from the cells. Rock flask gently and
1 \" m, V3 E) X% t9 e# }9 \aspirate medium. Repeat., P4 }& H0 g& y$ b& A
2. Cover cells with 1 ml of 1× trypsin solution and return to 37°C incubator for 1-2
( s  d" b/ S! {  }' fminutes or until cells are uniformly dispersed into small clumps.
  a' s  x* C) G4 o3. Add 9 ml of medium to inactivate the trypsin.7 q; T3 X+ [" H. x
4. Count cells and add 1 × 106 cells (usually 1/10 of a 25 cm2 flask) to a freshly! R1 P  e, v6 i& y
gelatinized flask.
5 x3 N2 `5 y& y4 u. N" R5. To expand ES cells for electroporation (requiring a total of 1 × 108 cells), seed 3 ×
. A; x) L" |1 E1 Q, j106 cells (1/3 to 1/4 of a confluent 25 cm2 flask) into a gelatinized 75 cm2 flask
8 P# F$ q2 T# I- d9 w, R$ @and add 30 ml of medium. Add 20 ml of medium on the following day. Once the
# R& f( z" Q7 _/ pcells reach confluence, trypsinize the contents of the 75 cm2 flask and add 5 × 106! {4 i( i, a4 e" z0 r1 L: ~
cells (1/5 of a confluent 75 cm2 flask) to each of three 175 cm2 gelatinized flasks1 p: M3 r9 X) t9 ]& X+ Z: y
containing 50 ml of medium. Add an additional 30 ml of medium the following
5 ^9 j0 f3 f( [- s; K! w) m* \day.5 T, m9 U! r, k
Freezing ES cells4 W/ b6 j3 Y( P* Z
1. Trypsinize a confluent 25 cm2 flask of cells (approximately 1 × 107 cells) as& h  k2 ?  w5 z8 {
described above.3 E% T" V* M+ @! T
2. Collect trypsinized cells in 9 ml of medium and pellet for 3 minutes at 1200 rpm.; \4 h/ s  Z( D% k
3. Aspirate off medium and resuspend cell pellet in 1 ml of freshly prepared freezing4 @9 i! O- P8 d
medium. Aliquot 0.5 ml of cells into two cryotubes.
+ e' x) t/ x# X- b4. Freeze the vials at -80°C overnight and transfer to liquid nitrogen for long-term
, h- ~& I& `: G+ x; U* ustorage.