a protocol-Growing feeder-independent embryonic stem cells

guochaosh

Growing feeder-independent embryonic stem cells" C$ Q0 E" i- x3 X; _
We use feeder-independent ES cell lines derived from the 129/Ola strain of mice) j- B2 B( g0 u9 X& e
(Nichols et al., Development 110, p.1341, 1990). These cells are easy to maintain and
8 |, z: ?7 X: j* ^6 ]; l7 `significantly reduce the amount of tissue culture required. Parental cell lines (CGR8 and! q  `, c* c1 l% P6 E; b1 p: Z
E14Tg2A) were established from delayed blastocysts on gelatinized tissue culture dishes
8 q9 Z, B6 b6 q( F4 v0 f4 Gin ES cell medium containing leukocyte inhibitory factor (LIF) (Nichols et al., 1990).
2 C  w6 G& H% n5 z; u" C* JSublines were isolated by plating cells at a single-cell density, picking and expanding4 c0 F: ]5 j' c* V( T9 `
single colonies, and testing several clones for germline competence. The majority of
  m+ C3 Y* \$ L1 B# Z+ m( b, zBayGenomics cell lines are derived from the E14Tg2A.4 subclone.. E3 R: I+ J' [# E' Y5 g, U7 Z1 {" Q- K
Tissue culture reagents
. p+ p  j  S& c# ?, M$ u# _/ \1. Dulbecco's phosphate-buffered saline (PBS) without calcium and magnesium% N- Z' f5 L, z" I
(Gibco-BRL).
* D, r7 j0 i# J* ?, @7 \, H# r2. 2 mercaptoethanol stock solution: Add 70 μl of beta-mercaptoethanol (Sigma) to# d  R5 u: o. \+ U
20 ml of distilled, deionized water (Gibco-BRL). Filter sterilize and store at 4°C- j$ E* X3 Q& B- h- Y! d
for up to 2 weeks.
, d% H* v, g/ I. m5 T: w9 m3. ES cell medium: 1× GMEM medium (Sigma, G5154) supplemented with 2 mM( R1 \2 G$ |9 r) _( G1 ~# G: F' r
glutamine (Gibco-BRL), 1 mM sodium pyruvate (Gibco-BRL), 1× nonessential5 h% I3 h0 X9 o) _6 Q$ x
amino acids, 10% (v/v) fetal bovine serum (characterized, Hyclone), a 1:1000
1 M9 `* B4 R6 h& {' bdilution of beta-mercaptoethanol stock solution, and 500-1000 units per ml of% q4 }( X+ J) v! I& P) X
leukocyte inhibitory factor (Chemicon catalog # ESG1107).( U) o% v% l: ~- ^
4. Freezing medium: Add DMSO (Dimethyl Sulphoxide, tissue-culture grade,+ m3 _% K" V: A1 y& y$ W
Sigma) to ES cell medium to a final concentration of 10% (v/v). Filter sterilize.
8 I; _! f( R0 l' w* e. yMake fresh before use.# A5 W4 F6 Z/ z9 c
5. Trypsin solution: Add 100 mg of EDTA tetrasodium salt (Sigma) to 500 ml of
. k  D: h5 w5 O; j4 j( iPBS. Filter sterlize and add 10 ml (for 1×) or 20 ml (for 2×) of 2.5% trypsin. N  p' a7 k- L. j0 o! {  ]
solution (Gibco-BRL) and 5 ml of chicken serum (Gibco-BRL). Store in 20 ml
; n" P1 h; D$ a0 Xaliquots at -20°C. (Note: 2.5% trypsin solution should be aliquoted and stored at -: ~4 S+ R. p+ S' L/ a8 r. [
20°C to avoid multiple freeze-thawing cycles.)9 l- z2 [7 k: w* s4 U
6. 0.1% gelatin: Add 25 ml of 2% bovine gelatin solution (Sigma) to 500 ml of PBS.9 G1 w" q3 p" Q# ?3 o0 V
Store at 4°C.9 K2 L+ K5 f- p% y1 o; R, M
7. Geneticin (Gibco-BRL): Dissolve powder in PBS to make a 125 mg/ml stock' D$ e$ w. t0 @* @+ s
solution (active concentration). Filter sterilize and store at -20°C. Add 0.56 ml to  ]9 G; @  D) b% u/ _7 [
each bottle (560 ml) of ES cell medium. (Note: The concentration of Geneticin; _1 ^! Y3 C1 z6 ^
should be titrated to determine the minimum concentration that will kill
4 O, @% b3 O6 u$ c  snontransfected ES cells in 5 days.). E2 W4 y% U9 b- W( p
Thawing ES cells
- ?) {# j9 K# O+ ~ES cells are frozen in medium containing 10% DMSO. Since DMSO can induce the
! m$ t# o" f- B% ?9 h- E  |% {differentiation of ES cells, we advise thawing the cells late in the day and changing the: y/ e, p! M9 ?- l
medium the following morning to minimize the effects of residual DMSO.8 \+ m/ J" D4 p  F1 B
1. Coat a 25 cm2 tissue culture flask with 0.1% gelatin and aspirate off immediately
7 C  s$ _( H" T2 K7 S" rbefore use.8 y$ i: X3 j( h" |* K
2. Thaw ES cells (approximately 5×106 cells, equivalent to one confluent 6-well or3 v# M: f+ O4 n# ^' m" i
1/2 of a confluent 25 cm2 flask) in a 37°C water bath and dilute into 10 ml of
6 p' F0 W5 [, {8 R  ?- Mprewarmed ES cell medium.6 t# d8 _( A) W  f9 i
3. Pellet the cells by spinning for 3 minutes at 1200 rpm in a bench-top clinical
' N+ R$ \: o9 ?" t5 \centrifuge.
2 {- B( e" x! _# E/ S* N4. Aspirate off medium and gently resuspend cells in 10 ml of prewarmed medium.( @* I* x4 _3 T' B2 j/ w
5. Transfer cell suspension to a 25 cm2 flask and grow at 37°C in a humidified 6%
  E, W1 f6 U; ~. e" d& z# s4 mCO2 incubator.
6 y* x2 p5 X1 U1 P" e; q6. Change medium the following day to remove dead cells and residual DMSO.
. _3 k, k& Q( F9 Q" q6 }, Z5 H( gPassage and expansion of ES cell cultures& s* a. T% }; U& ], k  K8 q- s
ES cells are routinely passaged every 2 days, and the medium is changed on alternate
" t; r( w* f& N) Q2 T. ^6 P& udays. Thus, ES cells require daily attention. In our experience, feeder-independent ES
# ?! I' G5 f+ {6 Icells grow rapidly and quickly acidify the medium, turning it yellow. Allowing the cells
6 z# D6 G0 h7 j: Jto acidify the medium (by not changing the media every day or by passaging the cells at& f% c! [$ ~8 c  v% @/ L
too low a dilution) will cause the cells to undergo crisis, triggering excess differentiation# R% ?- E& y% _/ x
and cell death, after which their totipotency cannot be guaranteed. Plating cells at too low
& x! A' ?5 z/ d) i( r9 R3 _# ca density, insufficient dispersion of cells during passage, or uneven plating can cause/ k9 ^) |+ w9 \3 L% `+ }
similar problems, as the cells will form large clumps before reaching confluence and the3 y/ N$ U  |. B6 [
cells within these clumps will differentiate or die. Germline transmission is a# c' D9 ^% ]: u! I- y4 Z
significantly reduced in cells that have been mistreated, even when they appear healthy at
; e) i$ y2 d9 Qthe time of injection.# Y8 O% b7 Y, S# V5 Q" `' @
1. For a confluent 25 cm2 flask of cells aspirate medium off and wash with 5-10 ml' V6 @7 H3 h8 L0 s9 w' J
of prewarmed PBS, pipetting it away from the cells. Rock flask gently and
# G0 n) v8 G4 f. U8 G$ S; Kaspirate medium. Repeat.
. S5 Y/ Z" g) |; i5 ~2. Cover cells with 1 ml of 1× trypsin solution and return to 37°C incubator for 1-27 G9 S; v( N; B1 W4 p: r7 n- ~5 g
minutes or until cells are uniformly dispersed into small clumps.' ~. x  D- c0 c7 Y6 l
3. Add 9 ml of medium to inactivate the trypsin.: L. J! d' [9 j
4. Count cells and add 1 × 106 cells (usually 1/10 of a 25 cm2 flask) to a freshly
7 n. a2 r7 L& b1 M+ wgelatinized flask.
3 ]  m1 t; n8 x0 F6 b( Q$ ?8 X5 v5. To expand ES cells for electroporation (requiring a total of 1 × 108 cells), seed 3 ×1 h$ `; c) e9 p+ P2 ~& C
106 cells (1/3 to 1/4 of a confluent 25 cm2 flask) into a gelatinized 75 cm2 flask/ k! j  m4 \2 A% ]6 Q: F7 q
and add 30 ml of medium. Add 20 ml of medium on the following day. Once the6 p& o, U' d5 B8 z1 y9 K
cells reach confluence, trypsinize the contents of the 75 cm2 flask and add 5 × 106: U3 Y4 ^$ k) @: Y! N9 Z
cells (1/5 of a confluent 75 cm2 flask) to each of three 175 cm2 gelatinized flasks
. k4 P. Y2 W# I1 f5 x" w! D2 qcontaining 50 ml of medium. Add an additional 30 ml of medium the following
, A; A3 X4 E- @+ F0 }. M5 Qday.) I* y4 z" K9 m* N) ~7 _, ]
Freezing ES cells- e: m* P7 E2 J- `1 }, x$ c/ l3 F
1. Trypsinize a confluent 25 cm2 flask of cells (approximately 1 × 107 cells) as& w( [5 E8 r/ G. h
described above.5 X. h: @& ]  |# `  \9 K
2. Collect trypsinized cells in 9 ml of medium and pellet for 3 minutes at 1200 rpm.9 `* ]# a( N1 N" ^' B
3. Aspirate off medium and resuspend cell pellet in 1 ml of freshly prepared freezing. y; l4 Z9 S1 Z% N; c; Y$ V$ J
medium. Aliquot 0.5 ml of cells into two cryotubes.- ~: w- u- y- L; O" |  N
4. Freeze the vials at -80°C overnight and transfer to liquid nitrogen for long-term
& i+ E; r0 B6 {storage.