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* C7 T3 v: ~5 z
We use feeder-independent ES cell lines derived from the 129/Ola strain of mice1 r: p" Y( S; m4 U0 ~1 q
(Nichols et al., Development 110, p.1341, 1990). These cells are easy to maintain and% y. `' t% K% E# a3 O9 P) [
significantly reduce the amount of tissue culture required. Parental cell lines (CGR8 and9 h" b- g6 h+ Z5 ^
E14Tg2A) were established from delayed blastocysts on gelatinized tissue culture dishes - q* B" T- R/ Jin ES cell medium containing leukocyte inhibitory factor (LIF) (Nichols et al., 1990). $ A; |, C! [% D' |Sublines were isolated by plating cells at a single-cell density, picking and expanding; c: G# ^3 T4 f+ V: O) C3 z
single colonies, and testing several clones for germline competence. The majority of 9 R7 I! F" C$ N# `( M& u: c, YBayGenomics cell lines are derived from the E14Tg2A.4 subclone. 6 Q5 z: z9 C% L dTissue culture reagents. s( U5 g5 E' i/ z9 I
1. Dulbecco's phosphate-buffered saline (PBS) without calcium and magnesium 0 v5 R" C; H [, m! n+ ?4 b7 e) Q(Gibco-BRL). - ]5 K' ^9 j6 q- K; S( c2. 2 mercaptoethanol stock solution: Add 70 μl of beta-mercaptoethanol (Sigma) to, R; D- U% i6 |. J
20 ml of distilled, deionized water (Gibco-BRL). Filter sterilize and store at 4°C2 J" w1 L) J& H/ q: p/ j3 d
for up to 2 weeks.& P' F) q+ N+ d: D. q2 v' E8 V
3. ES cell medium: 1× GMEM medium (Sigma, G5154) supplemented with 2 mM, n. p4 o- _: f2 m9 r. D
glutamine (Gibco-BRL), 1 mM sodium pyruvate (Gibco-BRL), 1× nonessential, B* A0 J( R, o6 ?
amino acids, 10% (v/v) fetal bovine serum (characterized, Hyclone), a 1:1000 ! p( a- c. y1 @4 e2 gdilution of beta-mercaptoethanol stock solution, and 500-1000 units per ml of3 O( K7 P" f1 \+ Z; c* k
leukocyte inhibitory factor (Chemicon catalog # ESG1107). ( F( i' D$ G3 B" A$ v4. Freezing medium: Add DMSO (Dimethyl Sulphoxide, tissue-culture grade,4 M' f# q& |, Q' ^5 M* l- z8 h8 V
Sigma) to ES cell medium to a final concentration of 10% (v/v). Filter sterilize.7 w; f6 a" J- M) _- B
Make fresh before use.2 S, N5 R8 g/ G0 P0 D! `( @- d7 l
5. Trypsin solution: Add 100 mg of EDTA tetrasodium salt (Sigma) to 500 ml of- ~ v* x p- ~$ T" x% _/ j
PBS. Filter sterlize and add 10 ml (for 1×) or 20 ml (for 2×) of 2.5% trypsin . ]* [' Y) @3 Q2 V) wsolution (Gibco-BRL) and 5 ml of chicken serum (Gibco-BRL). Store in 20 ml - q% x5 S3 u; b% T: S& ?aliquots at -20°C. (Note: 2.5% trypsin solution should be aliquoted and stored at -( v# G: W8 \% O6 Q) t5 n
20°C to avoid multiple freeze-thawing cycles.)$ Y# z4 d2 l) u) r2 C' r
6. 0.1% gelatin: Add 25 ml of 2% bovine gelatin solution (Sigma) to 500 ml of PBS. : S& b1 ^; _% E p# e7 U4 XStore at 4°C. - M3 V' e" I' ~, _7. Geneticin (Gibco-BRL): Dissolve powder in PBS to make a 125 mg/ml stock, m. \3 E: h# |- R% e. E
solution (active concentration). Filter sterilize and store at -20°C. Add 0.56 ml to9 l. |& Y T o, v6 @. p, K, F
each bottle (560 ml) of ES cell medium. (Note: The concentration of Geneticin# Z6 M' R" j* r( F7 I9 H$ n8 N( Q
should be titrated to determine the minimum concentration that will kill% z. {1 L. v& {! e% u0 ^
nontransfected ES cells in 5 days.) 9 d9 T4 K, _2 mThawing ES cells , _, c) T5 l% Y1 i6 v/ DES cells are frozen in medium containing 10% DMSO. Since DMSO can induce the( x7 [4 M+ J7 N
differentiation of ES cells, we advise thawing the cells late in the day and changing the- C) C! I' b& p' }; i( g. V
medium the following morning to minimize the effects of residual DMSO. ; L3 r6 w! N* ^8 O6 F( l; S1. Coat a 25 cm2 tissue culture flask with 0.1% gelatin and aspirate off immediately/ e! p/ c! Q( c/ |1 v5 L
before use.7 _ L' d2 J; e5 z$ T% J! r
2. Thaw ES cells (approximately 5×106 cells, equivalent to one confluent 6-well or 1 z& X. Q* _. g& K8 N; p1/2 of a confluent 25 cm2 flask) in a 37°C water bath and dilute into 10 ml of $ y% i: Y# L4 w# w, x& i, ?0 }) Bprewarmed ES cell medium. ! P! u7 c6 ^) M0 Q% ^3. Pellet the cells by spinning for 3 minutes at 1200 rpm in a bench-top clinical* y8 y& y. n' f* T
centrifuge.! v b' i# ?" T2 _/ }
4. Aspirate off medium and gently resuspend cells in 10 ml of prewarmed medium.' x9 A& y" [' {. b3 V$ ^
5. Transfer cell suspension to a 25 cm2 flask and grow at 37°C in a humidified 6% d& d! I) G$ h2 r" V
CO2 incubator." u8 } u! g) ?. g7 ?
6. Change medium the following day to remove dead cells and residual DMSO. + N2 {. ?' v- ~2 m$ e+ F& y0 iPassage and expansion of ES cell cultures2 S( j* }9 v. a" v
ES cells are routinely passaged every 2 days, and the medium is changed on alternate* k0 i" I3 W. B6 D
days. Thus, ES cells require daily attention. In our experience, feeder-independent ES Y" K/ [5 P% ]; N6 r
cells grow rapidly and quickly acidify the medium, turning it yellow. Allowing the cells 7 h$ K$ q& P1 mto acidify the medium (by not changing the media every day or by passaging the cells at - V. U' I% l% W! h( g# ztoo low a dilution) will cause the cells to undergo crisis, triggering excess differentiation # K+ a( l" }6 D) N6 Z6 s$ f8 x$ zand cell death, after which their totipotency cannot be guaranteed. Plating cells at too low e5 E8 ]2 P6 f8 `3 r
a density, insufficient dispersion of cells during passage, or uneven plating can cause 1 v& Y. L) E' K, |) |; A$ {! j3 o dsimilar problems, as the cells will form large clumps before reaching confluence and the. j6 R8 `, U6 P" e& j6 D
cells within these clumps will differentiate or die. Germline transmission is a 9 G) B) V0 {: d( z1 xsignificantly reduced in cells that have been mistreated, even when they appear healthy at. ` u2 e/ | a; K. Z
the time of injection. % h% t7 R8 i2 w* t* f2 U1. For a confluent 25 cm2 flask of cells aspirate medium off and wash with 5-10 ml . g8 n S8 w1 v: tof prewarmed PBS, pipetting it away from the cells. Rock flask gently and * a# B, w( a T* O3 y4 t% `aspirate medium. Repeat.5 \' [: `' l3 ?2 L( ~
2. Cover cells with 1 ml of 1× trypsin solution and return to 37°C incubator for 1-2 ! V) K( p' z3 Y) K Gminutes or until cells are uniformly dispersed into small clumps. + b$ k# I( L/ R' d1 X6 q) v1 j3. Add 9 ml of medium to inactivate the trypsin., N7 X6 x7 Z& H* G* V0 F" w
4. Count cells and add 1 × 106 cells (usually 1/10 of a 25 cm2 flask) to a freshly: Q' E, X' B0 I: Q: F, `
gelatinized flask. : U1 h% d, O8 {$ a: T9 _* I/ c5. To expand ES cells for electroporation (requiring a total of 1 × 108 cells), seed 3 × 9 C M1 a0 a5 C) ^) S4 y106 cells (1/3 to 1/4 of a confluent 25 cm2 flask) into a gelatinized 75 cm2 flask 8 |( l( A# H8 d) o: V. L' h( J. tand add 30 ml of medium. Add 20 ml of medium on the following day. Once the2 g! l. [. B! m E3 M; Z& G/ D
cells reach confluence, trypsinize the contents of the 75 cm2 flask and add 5 × 106 & P; n( H8 c/ Hcells (1/5 of a confluent 75 cm2 flask) to each of three 175 cm2 gelatinized flasks7 u' d6 T' g/ g* B
containing 50 ml of medium. Add an additional 30 ml of medium the following * k2 T+ y# K7 K$ Q, T% ^) aday.9 m# [* ~8 D1 R4 m
Freezing ES cells/ _! V( h5 X# D6 [0 r
1. Trypsinize a confluent 25 cm2 flask of cells (approximately 1 × 107 cells) as+ z/ B N. O5 C u, `( A( @+ i
described above.; u( `7 |6 c& D4 d9 m
2. Collect trypsinized cells in 9 ml of medium and pellet for 3 minutes at 1200 rpm. + l3 }% i" S( S% h2 E1 D+ X3. Aspirate off medium and resuspend cell pellet in 1 ml of freshly prepared freezing : a" Y8 @! F1 j) A/ y- P+ |8 B1 Umedium. Aliquot 0.5 ml of cells into two cryotubes.. Y8 K( a/ W) W* I
4. Freeze the vials at -80°C overnight and transfer to liquid nitrogen for long-term: ^1 ], Z6 L1 Y1 l8 ]+ C
storage.