3 |; z* x {# t# p, F1 A0 M7 u我有！怎样给你发过去，我不会。作者: 老姜 时间: 2013-1-31 17:55
Optimized Protocol for Deriva tion of Human Embry onic! l: K% H0 m$ H" F
Stem Cell Lines 0 p6 G L5 {) y; v7 l& T1 O/ vMaría Vicenta Cam arasa & Víctor Miguel Galvez&# t& @: f& p" M" M
Danie l Roy Brison & Dani el Bac hiller" n- Q( H" T: U7 c" ~4 a" G
#Springer Science+Business Media, LLC 2012 ! F3 s% P; L6 B! A' \ k$ ?% KAbstr act For t he pa st 12 ye ar s, th e b iolo gy an d a pplic atio ns2 _# u! |0 z% l* F9 \
of human embryonic stem cells (hESCs) have received great& d% ^+ n' r* z1 d& |
atte ntio n f rom t h e s cien tific comm unity. D e riva tive s of the first% S. f6 R2 p8 @, b% W
hESC line obtained by J. Thomson ’s group (Science 282 , i/ c: Y1 b' ^6 q4 m(5391):1145 –11 4 7 , 19 98 ) have been used in clinical trials in s4 R# {! x( I- W _ L. hpa ti en ts with sp ina l co rd inju ry, an d othe r h ESC lin es ha ve 5 T- P5 C/ j3 E. o- V8 Mno w b een u sed t o g en era te cell s f or use in tr eatin g blin dn ess8 K! L9 A2 L) ~" _+ a
( L a n ce t 3 7 9( 9 81 7) : 7 13 –72 0, 20 12 ). In addition to the classical9 x: ~- L, R. K9 t
pr ot oc ol b ase d on m ou se o r hu man f ee der l aye rs u si ng op en- R* Q2 w2 J: E( Q
cu lt ur e m eth ods ( In Vitro C ellu lar & Dev elop men tal B iolo gy -Animal 46(3 –4):386 –394 , 20 10 ; Stem Cells 23(9):1221–1227, : V3 _ w/ S4 e# a2005 ; Nature Biotechnology 24(2):185 –187, 2006 ;Human 2 J+ U/ p( v+ m/ Z9 V+ a- e: @Reproduction 21(2):503–511, 2006 ; Human Reproduction 20; {! r _2 j! W3 h w
(8):2201 –2206,2005 ; Fertility and Sterility 83(5):1517–1529, . j" a% ^( u- e- y, o! A2005 ), novel hESC lines have been derived xeno-free (without " X& A- e8 @" J: O! ~9 fusing animal derived reagents) (PLoS One 5 (4):1024–1026, + d' p+ X6 X" }6 g, m; I2010), feeder-free ( without supporting cell m onolayers) ( a$ f9 l2 B1 G% l0 r8 S(Lancet 365(9471):1601 –1603, 2005 ), in microdrops under . x3 {6 G% p W# L) g# aoil (In Vitro Cellular & Developmental Biology - Animal 46 " c& J$ H) R; j5 o1 m(3 –4):236 –41,2010 ) and in suspension with ROCK inhibitor3 f. p- U) [5 O; Z& c# S
(Nature Biotechnology 28(4):361 –4, 2010 ). Regardless of the1 ]3 ?8 p7 L# p8 b+ |
culture system, successful hESC derivation usually requires 2 z- C$ P( R; f0 l% ~optimization of embryo culture, the careful and timely isola-tion of its inner cell mass (ICM), and precise culture condi-tions up to the establishment of pluripotent cell growth during5 m) K3 `5 u& ?' C8 I# }1 A
hESC line derivation. Herein we address the crucial steps of) ?+ Q) L' n% a: k! j5 k5 e a
the hESC line derivation protocol, and provide tips to apply % @8 b E0 {9 wquality control to each step of the procedure.- ~9 p9 O! p5 |! n H! `7 ]0 ?
Keyword sHuman embr yonic stem cell s* p# ]4 `$ j8 ~5 p" t! z
. # S, ]* O; V3 c8 q6 i6 w& EDerivat ion E% X! U; o! ?5 o8 G- N! d. { B' r
. " ~+ P* n/ F8 |' p/ x- a8 sBlast ocyst# d6 E ^9 \" Y+ b& I2 c' [# X1 ^
. ; j& p* M. [! P2 lCultur e o ptimizatio n , Y8 Y1 ?* y% J; A# e' `) `( i1 FIntroduction* p% A* z( i5 Z! {& k9 @& F
To da te, hundreds of human embryonic stem cell (hESC) line s 6 {7 h8 Q5 `: W. \, s% X& _9 T( l, u$ ahave been de rive d f rom surplus embryos after as sisted repro-duction techniques. Reported derivat ion e fficiencies range from5 z) Z" V$ }6 l l
7 to 100 %. This wide span pr ob ably refl ects the di versit y of the& Q4 g* c5 I: X) M8 l
methodologies involved. M any var iables of the procedure s uc h 1 n* |" K! N: X& w# X4 Aa s em br yo g r ad e, timi ng of e mb ry o c ul tur e , zo n a pe ll uc id a (ZP)2 x6 j. Y$ }& T' L7 s$ h
removal and inne r cell mass (ICM) isol at ion a nd type of feeder 9 o* m6 ^" x7 p6 l" l9 l* K5 l' n' f* Mlayer, ac count f or the d ifferences among laboratories. Neverthe-less, d espite the v ariabilit y, al l hESC li nes derived s hare growth * \- N( R" r8 T5 L+ r3 x D6 l! b) e" hproperties and expression of com m o n st em ce ll m ar ke rs , i nd i -cating that diverse culture conditions select for unique charac-teristics. The International Stem Cell Initiative II has analysed + H! U! Z) S6 X. C5 x/ ^8 d! Mth e genetic stability of more than one hundred hESC lines , |8 i; S8 ~- j4 u: m+ ousing consensus protocols for karyotyping and genotyping. 6 L7 N! ?: X9 PThis echoes the importance of defining robust and common0 k- b9 Q8 [& u- A
criteria for the assessment of both existing and newly derived : a( \9 a8 m5 O5 s- ucell lines, and for the validation of advances in culture con-ditions. [13].: R, V4 _7 F' t
M. V. Camarasa (*) . u, t' N, \6 _% ?( g:. O; U! f) e! q2 J8 R! _3 z
V. M. Galvez9 M, Q* _1 E4 O h: e5 O. q1 `& ]
:6 a* z0 h X+ d: n4 ?: U: h
D. Bachiller ) G/ Y& X2 b) e$ OCaubet-Cimera Fundation, Centre for Advanced Respiratory9 o8 J9 P! i$ P
Medicine, Recinte Hospital Joan March, + G& @ D1 n" g2 k: Q' A( |" U. CCtra Sóller km 12, ( |4 T8 O- u" b8 p6 |9 y0 j07110 Bunyola, Illes Balears, Mallorca, Spain5 G, X2 f' u" M6 C3 O
e-mail: telomerasi@yahoo.es & u! X i9 ]- o. ?M. V. Camarasa9 W4 v0 U& g5 w. ~
e-mail: mavi.camarasa@caubet-cime ra.es: F; \9 ]% v8 j2 `
D. R. Brison" Z$ l$ y4 X" n
North West Embryonic Stem Cell Centre, Faculty of Life Sciences, 4 o1 d* I7 S5 lCore Technology Facility, University of Manchester, " _0 d$ @/ K) M1 j {. D9 ~* z" Y46 Grafton Street, 6 B& V3 s: P) q. e+ _. T. \( W* QManchester M13 9NT, UK# G# X9 F3 p. W& `3 |
D. Bachiller5 j8 j" s# s/ {! m: J
Consejo Superior de Invest igaciones Científicas (CSIC)," m4 E* w1 o5 h; l1 h' e$ G. K( w
Mallorca, Spain & @8 R' X7 s# m8 U: S' eStem Cell Rev and Rep # C- H! T5 x$ P! i" u# X/ HDOI 10.1007/s12015-012-9377-4 4 c. Y n" h, |4 B& {& P0 ?This protocol focuses on t he main first steps of the 2 X/ r5 J9 T% A2 bderiv ation of hESC lines, i.e. embryo culture and gradi ng,, `* p( e6 x+ }/ v" Z- ~, b# ~
and precise mani pulation of their ICMs and first outgr owths . h- H& @- y/ ^" l+ {: ^, Qof pluripot ent stem cell s. The Notes secti on includes alter-native procedu res as well as commenta ries and clarific ations- G; Y! ]% K Z$ }1 A7 V# z
on the techniques described in the Methods section. Assuming: ~/ T- `- m0 w1 u% v
experience in mammalian basic tissue culture technique, this / ^ K# \0 I0 I& y: W/ oprotocol should aid anyone wishing to derive and culture: t& n- {" p/ H2 _0 b
hESCs to set up a rapid and reliable method. This protocol! |) m& v. f$ ^: F( d
reflects continuous work optimizing the derivation, culture9 x; t( I8 k& Z! a
and characterization of hESC lines over more than 10 years, # n! `, z& ]5 z. Icomprising activities carried out in four different laboratories. 5 r, F! o# H: K- gAdva ntages of the present p rotocol over others previous-ly pub lished in clude: optimiz ation of f eede r cell yield,# {% p8 Q. F9 w- [
increase of I CM cell production and adjustment of the 7 F/ Z5 f$ n6 J+ M+ M2 ]# |deriv ation protoc ol accordi ng to embry o grade. 5 F. k7 [8 e% HFeeder cell yield is optimised by culturing tissue aggregates8 ~5 v' i( r3 @& u
until they grow as monolayers. Embryo culture is optimized H. n# j- M& Q! b
so that growth of the pluripotent cell population is favoured( {" c1 J% M& V) F* ?+ \
over that of the non-pluripotent trophectoderm (TE) cells. ( \& m V6 d8 }$ i F1 T* j O. pFinally, the fire pulling of glass Pasteur pipettes to form thin % K; Y: M( j2 Y1 q8 [: e2 Fopen and closed ends that match the size of the ICM or initial . n* }. c: v4 c8 U; b2 Coutgrowth to isolate, increase effectiveness of mechanical5 R I( Q3 M% J p2 y" d
splitting techniques. 4 \- f) h& C6 ]6 ]& QMateri als E- |; B9 w- O) E
Mate rials a nd Rea gents + }5 E. w3 M& a8 U/ O1. Bacteriological Petri dishes, 100 mm Ø, Fisher Scientific& ^$ l8 v9 n5 x/ v
#09-720-500.8 F, r& \& K; \2 M+ n' Z
2. Cen tre-wel l Organ Cultur e Di sh, 60 mm tissue culture 6 I* z# m* t' L8 d' ^! ytreat ed, BD #353037." F: h8 I0 W W/ o, D
3. Lon g glass Pasteu r pipet tes, Fisher # 1367820C .& `1 X. f$ C* D5 \5 ~; G. n( d" y
4. Mr Frost y® Nalge ne, Sigm a #C1562-1E A.8 k! S! }. l4 ~2 d/ h6 `
5. Se rological p ipette 1 ml, Cor ning #4485.# o% M4 C! B- r0 V; n# E
6. Se rological p ipette 5 ml, Cor ning #4487.. s5 {' w7 ~+ L! |% y
7. Se rological p ipette, 10 ml, Cor ning #4488. _% L+ k( E( g0 g6 x) D8. Se rological p ipette, 25 ml, Cor ning #4489.1 P$ C& T0 C9 K# r
9. Se rological p ipette, 50 ml, Cor ning #4490. 4 S5 A& {' ^# X2 ~# c5 n10. Tissu e culture dishes, 150 mm Ø, Cor ning #430599. + a$ e$ g8 r5 A11 . Tissue cult ure flas ks F1 00, 100 cm - O8 ~. x" Z8 J2 " e9 W: [# Q, F2 P* M; [Corning #3816 .+ u& \- q, B8 d
12. Tissu e culture plates, 4-well , BD #353654. " O- N+ _, O0 F4 n/ b4 o/ b( B13. Tissu e culture plates, 6-well , Cor ning #3516.5 Y; _" l4 K( r6 h0 ~' ]* A
14. Basic Fibrob last Gro wth Factor, #45103P- 100. 0 a6 A6 h: V4 _" a$ [* m- b1 Z* z15. Beta-mercap toet anol, Sigm a #M-752 2.' R0 A4 z9 q5 ?+ z. z' }' x8 @* W+ [
16. Collagen ase IV, Invit rogen #17104 -019.7 D3 Q5 r9 M" h- ]
17. DMEM medi um, Lonza #12-614 F.2 ?0 a8 `# o! ]( L" b
18. DMSO, Sigma # D2650.; B8 g* w9 X! {, S% ] p% `7 T$ I
19 . Fe tal bov in e serum (FB S) , Australia n origin Lo nza0 Q* Z% p9 Z4 y* H/ Q6 m ^
# D E1 4- 70 1 F. Tes t b at ch e s f o r fi b ro bl as t c e l l g r owt h. Be st& C# f- A4 Y! [( m7 l
origins a re US and US approved, followed by Au str a l i a n ) Z5 ^5 J; X' R6 {. j! C9 jand Australian/USDA approved. Poor quality serum is a: k4 n0 L4 u7 f; W( Y( |2 |
confirmed cause of feeder cell failure in hESC support./ ~4 H& ^/ U! j' w8 Z' R( {9 M
20. G1, Vitr olife #10128.7 s5 z) ]( {. a
21. G2, Vitr olife #10132. 7 @/ {" [4 G" J; G: P- \( H# d22. Gelatin, porci ne, Sigm a #G-1890. 9 _4 \; T' s( K" W23. Knockout (KO) DM EM, Invitrogen #10829- 018.1 f/ d G8 L1 X* i
24. Knockout serum replacement ( KO-SR), Invitrogen4 C& V7 f; q' M0 H0 C
#10828- 028./ e# d' v) }; {$ _3 \# k
25. L-Glutam ine, Lonz a #17-605 E.: D8 p( L' i0 ?, f0 Q/ m" K
26. MEM Non-essential aminoacids (NEAA), L onza # : y; g9 O) B) T, f6 H13-114E. 5 [! n4 \ B! O8 i! U6 _27. Mitomy cin C, Sigm a #M0503 . Hazard: this subst ance 8 P4 M/ k, K: G, A6 O% lis very toxic and procedures for safe hand li ng and- U' m! d, G2 e4 c- G
dispos al need to be in place. Do not brea the dust. C$ }* n+ U6 B9 \+ HAvoid contac t wi th eyes, on skin, on clot hing. Avoid! `, e# V4 ^4 J
prolonged or repeated exposur e.6 l# }0 B) {; o$ D- {
28. OVOIL, Vitrolife #10029. 3 {- |2 F" `+ H5 e8 U29. PBS with Ca, [ g1 F( ~7 p; p3 e( V# Z
+2 5 _" v: A! N( z/Mg5 t! w: s7 v: A8 e
+2 : v& `) l! R u, Lonza #17-513 F.* B8 h# K% P2 r; @
30. PBS, without Ca4 L4 s+ z4 W+ f- m+ W
+2 T3 Y5 n: @# s+ n2 i0 R- t
/Mg % \" F7 [: c" }, ]( w( j) _: j3 P+2/ B: A8 k' n' h1 ~+ q
, Lonza #BE17 -516F.- j8 x: u' K3 F
31. P re g na nt MF- 1, C F-1 o r MF-1 × CD- 1 fe ma le s, 1 2. 5 –13.5 ' ~4 Y4 I& V7 a0 h g+ ddp c, Charles River Laboratories. 2 W4 \; }, t7 Q: h. H32. Thawkit I, Vitrolife #10067.: d3 G3 @. T2 x. f
33. Trypsi n-EDTA, Cambre x # CC-5 012., ]1 f9 c# p4 [7 R
34. Tyrode ’s solut ion, acidic . Sigm a #T1788. : d! N3 h. I) @8 C7 h" Y2 w+ ISolut ions7 X' y& s0 K7 G- E) T6 v) Q+ _
1. Complete DMEM medium: DM EM plus 10 % v/v FBS,6 M% E$ f b8 d3 h! B& R% p' ^
1 % v/v L- Glutamine 200 mM, 1 % NEA A. 8 W: z7 Q$ r6 m/ z9 \% x2. Freezing medium: 90 % v/v FBS plus 10 % v/v DMSO.& |4 f" F% E D# M% F1 l2 F+ m: t
3. Gelatin solution: 0,1 % w/v gelatin in tissue culture grade! _- f! e! F- h' J; F3 A
water. To avoid contaminants and residues (detergent,7 H' x; V, A; `) o) ~
traces of other chemical, etc.), use a new 500 ml Pyrex- M0 b' j1 m! B4 j6 e" A& |
bottle. Autoclave the gelatin solution and store it at RT. # y) z3 h) B( p, l" d) j0 p4. HE S m ed ium: K O - DME M supp lemen ted w ith 20 % K O-S R, - v! O# r( d1 l- h. _. V- F9 S2 mM L -Glutamine, 1× N EAA, 50 mMβ-Mercaptoethanol) C8 @# B% h0 f6 j- D" m, {# ?5 X$ K
and 8 ng/ ml human basic fibroblast growth fact or. : K5 M. U( l7 n5 y1 r- ZMethods/ x; ?; f- i$ S0 L; p
ESCs are derived from preimplantation stage embryos. The ( Q8 x! y$ }: J0 {2 n; W% C. x% V, Umost common procedure involves culturing embryos to the * e: U7 Q. Y3 i6 t' Z8 D# |* B# [blastocyst stage (day five to seven after fertilization), isolating . g0 w9 K* T; ?/ V, Jtheir ICMs, and culturing the latter on mitotically inactivated' a% I3 J3 [1 r2 h" i: K; z
fibroblast cell layers. The resulting pluripotent cell popula-tions are selected by morphological and growth criteria. Novel9 K8 Z) w% E( |, m5 X
hESC lines are then established after continuous subculture of " G( G8 e/ i0 P* g& h. T, Q ethe initial outgrowth. A hESC population can be considered to : z: ^$ A' r2 G+ v' K* rbe an established line when it reaches passage 8 with approx-imately 3 × 10 2 ]- C6 Z( v# Z0 c/ j72 z! M& I, @3 I" T* h
cells, as defined by the UK National Clinical # p9 j2 d& n0 S9 r+ M2 Ghuman Embryonic Stem Cell Forum [ 14].. ?$ L$ R0 \$ Z, s
Stem Cell Rev and Rep * |3 k# Z. c6 V% H6 V/ pAfter eight to ten co nsecutive p assages, the novel line is+ U9 G7 c: P4 i: u
sufficiently expanded to check its viabi lity by freezi ng and # T9 `2 Z: c, t. a' n( Xthawing . " t5 n$ D# k) j0 _$ nTa b l e1 defines the term‘one v olume’ for each of the culture / J- w1 a. x* N/ o/ ~v es se l s d es cr ib ed t hr o ug ho u t t he manuscript. All incubations are) s2 F) R9 I5 t5 K0 t" d
performed in c ondition s o f 3 7° C, 95 % h um idity an d 5 % CO' \) v( q/ K5 u( V4 ~/ W
2 3 T( a5 F( l; w; o# |7 G [% f4 ^inair(SeeNote1).Figure 1 depi ct s t he expect ed time schedule * c7 s- Y9 N4 ~* ]" j; R4 `" |to complet e a hESC d erivation p ro toco l, provide d that t he feeder1 V4 c- d( H2 R, k: H. o
source has b ee n p revi ou sly v alidated. 9 Z3 l0 D2 n$ U: _! _* G/ X& u& TFeeder Layer Preparat ion& | F/ O7 E; a4 J# b
Primary cult ures of feeder cells have tended to be used for- r, T- i: B$ l: u
d er iva ti on o f n ov el h ES C li ne s, b u t r ou ti ne c u ltu re a ft er 5 ^! i9 @2 D& Q' U G; k: lderivation can be performed on f eeders produced from1 X" F k; s! [# s( j
estab lished cell lines, or prim ary feeder cells whi ch have ) F; b* y. V) X" w5 Jbeen immort alised. Altern atives to the use of prim ary mous e8 N3 d |' @" n( X4 x
embr yonic fibr oblasts (pME Fs) as feeder layer s for hESC" ?/ _% a1 l3 c3 V: }0 e
culture are discussed in Not e 2.' s, @$ q& X' G1 e% f5 z, V3 A. _
Reg ardless of the type of feeders used, every feeder layer : j% I$ G1 Y4 H9 ?4 v4 Mmust be con ditioned before stem cells are seeded on top of) E2 }! }! _. ^8 R4 T
it. To do so, feeder cult ure medium is disca rded; feeders are2 _' y8 q5 k5 F5 t% y$ {, }0 B9 J
then washed three times with one volume of PBS each time4 u! [3 y8 i6 j4 ^7 {; S1 [8 R
and once with half a volume of stem cell medium (HES).& K5 G# f) `% Z. S% \6 F( G3 _4 q) S9 ^" T
Plates are ready for use, from 30 ′ to several hours afterwards. 3 q/ `- Q" o! Y. E( m+ GMEF Isol ation) T) M4 d+ F9 h l- Z$ h
1. Sacrifice a pregnant female (12.5 –13.5 dpc) by cervical* l3 `, K# |2 Q; ~- ~
dislocation. See Note 3 for tips on strain choice.) a: r+ {' q8 k/ x5 F; L5 ^
2. Di ssect out the uterine horns and place them into a . k2 M( \& e# A. K; _& }7 `10 0 mm Ø Petri dish containing one volume of ster ile# P9 m% ~/ f3 e2 x& R) @
PBS 1×. Transf er the plate to a laminar flow hood to ; V1 b" o' Y; U1 q) A& O. J) A; Fco ntinue with the procedu re.5 \2 V3 a2 T7 f3 I
3. Cut open the whole length of the uteru s, so vite line+ c5 t! |9 d. w3 N7 E& q
sacs with embr yos and placentas are partially relea sed.: X! C" q+ [) v* v8 f
4. S e parate each embryo from its p lace nt a a nd out of the; _- w) v6 ?* r1 d& K9 Y
viteline sac and surrounding membranes, and transfer& ` z; i7 t F& K) N3 z
them all to a ne w Petri dish with on e volume o f fresh PBS.! f7 P/ N& W; c* P, [7 x9 s
5. Wash the embryos and transfer them ag ain to a new 5 L, \6 p/ i0 K/ L% _Pe tri dish with one vo lume of fresh PBS. : C8 _# {- P0 z9 P% `' r6. Decapitate the embryos and separate the visceral tissues ) Q5 E- }/ X5 y2 ~3 xfrom the bodies. Collect carcasses aside in the working- r ^7 ?3 K% G* }% e
plate. Aspirate and discard the PBS and the visceral+ x! x1 x7 N8 y& ~2 Q" p. E# H$ @% ^
tissues carefully with a 10 ml serological pipette. Leave1 P2 @4 K B# D& U# ~ G
the rest of the tissues in the plate.1 m6 J! m$ r1 U$ _3 W" N6 t
7. Mince body walls into small pieces until a homogeneous, R: b% {* L- e6 R# M2 O6 Z
suspension is formed. 8 e$ u1 I6 t# e" v8. Add 5 ml of trypsin-ED TA 1x to the plate and transfer) L' m% ~7 Y1 Q0 w- u4 ^! S
all contents to a conica l 50 ml tube. * [% K3 a3 q/ y( l; H; V4 F. V9. Incubat e diges tion at 37°C and 5 % CO % u3 @/ L4 P1 l: l2 . a: O. l0 ]% K) ]- S; L2 n+ Afor 30 min.+ N( j& e- l4 J( W- i
Hom ogeniz e the cell suspension by adding 45 ml of 3 s* V9 ~" I! C6 P4 N; sfresh cult ure medium and pipet ting up and down for) ^3 F1 _) G; @5 ^) v( [
ten times, and cen trifuge to recover the cells (700 g for) x6 o4 p1 Z& m! i7 K
2 min ).. s( a* l7 A0 Q/ e# C
10. Discard supern atant, ad d 50 ml of fresh culture medi a) F: a0 A9 Z I0 R& J3 q( O& ?* X
and resus pend cells. + c- w; [! U( u" C6 m: f5 T% G11 . Cen trifuge the cells at 700 g for 2 min and disca rd. @3 a: c! N* p `, ^
supern atant. ?$ ] a. O2 O5 o' @7 @" g$ J# f12. Resuspend t he cell s in 5 ml of complete D MEM3 V5 z# Q# f! M. J
medium.; @* ]2 z7 Y/ W4 T. O
13. Seed cell suspen sion at a rate of three embryos per 7 M: q# ?/ u8 k) f& y" z) n1 x LF100 flask. (General ly four F100 will be set up at the ) i2 h6 Y" t$ g" pbe gi nn ing o f the procedure per dissec ted p regn ant + x' ^/ T2 t* Xfemale). : n7 s" [+ n$ m14. Incubate at 37°C and 5 % CO * W4 K, i( {1 u4 L& s$ y Y2" U7 Z' |* }, S! F, K) g
unti l 90 % confl uence ( t) {6 V' f& W* C* N* sis reached (approx imately two to 4 days).) V2 U2 N% Y& z
15. Split the cells twice by tryps inisati on, seeding them3 h' |. y# H$ Y
onto n ew flasks/dish es be twee n 1:3 and 1:6 split ; t1 C( ^3 l# c4 b- oaccording to growth rate. $ _ n8 k& |5 J1 A6 d# wAfter t wo passages , t he ce lls s hould be growing as 4 B) l4 w1 k. Bmonolayers. Freeze 90 % of p assage 2 c el ls in vi als * ~# u/ ]+ N4 p. g' K( t+ K. W- Ccont aining four to ten millions units each. The frozen 4 Q) b1 O' R/ ^9 Dcells wi ll const it u te a s tock of act ive pMEFs for fut ure& J* U7 g' J* k% d4 B7 k6 W
use. ' F1 b1 K: K: P6 d' _To continue with the production of inactivated feeder, {9 s5 c5 ~( n& k* X
cells, expand the remaining 10 % of the original culture7 Z! i! F( m3 J' T9 h& k9 Q
up to pa ssa ge fou r. S ee d t he cel ls on to new f las ks/4 D d0 k& @3 F( W) W5 P% u7 I0 {" h
plates bet ween 1 :3 and 1:6 spli t according to grow th ' G% X. A0 ?5 U1 t; v+ q1 |rate each pa ssage. Then inac tiva te an d freeze passag e9 g0 r; t6 J; {# b# ]
four cells as explained below. See Note 4 for tips on % ~9 e3 m/ Y7 A0 i# b1 T4 cthese st eps. 9 Y* @; Z* c: vGene ration of Batches of Frozen Feeder Cells by Mit omycin ; g' w2 h( @6 q; c5 PC Inact ivation ; i4 U) U% p. @. o8 z1. Tr eat 80 % confluent, exponentially growing,5 M8 s3 u/ x2 v6 r$ x. W
pa ssage four pMEF populat ions (between 48 and d8 f8 k( B5 {8 O9 6 P150 plates), with 20 ml of 10 μg/ml Mitomycin C in% ], q/ W# f/ ]# Q* p+ e
complete DMEM medi um, f or 2– 3 h at 37°C a nd 3 U( h/ v' b9 P. v& a5% CO28 i" [5 ^, C" o' N- B" i
." ]7 g# S/ L9 l8 e' T
Table 1 Definition of the vessels and volumes used throughout culture : F) k) G& h& y. Vprocedures0 M; R; L' U- ?8 \+ ]8 m$ l
Dish/plate Area cm 8 {; m1 j$ g+ v2 R+ u2! z/ U5 z6 w' c5 q& f+ J
/well One Volume ml/well ( o1 K" w2 [* C& c4-well plate 1.4 0,5 7 p: I$ K& G6 i24 well plate 2.0 1 " ?' g) E4 L4 ^- D8 h, P# `One-well 60 mm plate 2. 9 1; x: {/ b: d2 b; e) o6 f2 @1 T1 \
6-well plate 9.5 3+ K+ R( q5 M# A1 m. D
60 mm Ø dish 21.3 58 y" `; \* e0 z: ^: @
100 mm Ø dish 58.1 20. i: m' w$ T& b* R
F100 flask 100.0 258 m1 d, q4 Y; ~. X
150 mm Ø dish 176.7 500 v* |* U! W9 t% |' m) G7 o, w8 N3 S
Stem Cell Rev and Rep 4 O5 o( y! v1 e2. Aft er inactivation, disca rd the supern atant and wash* b q# e0 z- u0 Y d4 ]1 i v& O
cell m onolayers 3 times w ith on e volume o f PBS + o$ B4 `5 s: `wi thout Ca) v; i/ q/ C1 m) Z- }, y g! c
+2 : _6 U0 q) e. q/Mg . N# K* c; S# Y+2/ a, f- X; ?& S! x, I+ z) |
. + I- u Z# Y7 w5 A3. Trypsi nise the cell s (2 ml per F100 flask or 3 ml per " w7 N. L: m& u. Z" r2 i1 ?! _P1 50 p late) for 2 – 7 min at room temperat ure (RT) . 8 ~ s S% E4 M c2 |4. Tap the sides of the flasks/dishes to dislodge the cells,2 `6 h& K8 q% x. C2 g
an d harves t them into 50 ml conica l centr ifuge tubes .* T: Z4 Z1 I( A" h4 y2 b
Po ol the contents of tw o to three P150s in one tube., r' r2 |, C/ R V9 n/ U6 d
5. Add 44 ml of compl ete medi um per tube.9 n: r7 q* M H; g
6. Cen trifuge tubes at 700 g for 2 min .( m+ O4 o* P0 n; P" m, o6 |
7. Br eak pellet manually by finger vortexing (see Note 5). ' t' f7 |' I. X& e q# g$ P9 qResuspend each pellet in 30 ml of complete DMEM and( }, d* q$ \ s. ?" B
count in a Neubauer chamber.& q/ q2 x2 @& c* k% w
8. Co unt v ia ble ce lls and calcu la te the numbe r of 4 d( a- Z2 m q3 R0 X! g/ L# rcryovi als needed, at a rate of 1– 4 million cells per vial.& b! Q' T" @/ X, J8 D
9. Cen trifuge cell suspen sions at 700 g durin g 2 min .6 x. a$ h2 a Z% r& O. S
10. Label cryovi als with batch numbe r, amoun t of cells 0 @4 p# @2 R/ Qand met hod of inact ivation, date and user ID. ; A, h5 R9 E8 G2 O3 X- m* o11 . Discard s upernatants from s tep 9 above and break; z4 N" M2 {8 Z {/ ?
pell ets carefully by finger vortexing. - k2 L5 S7 J& b12. A d d c ol d f reezing m edium to the pel let s a t 0,5 m l per v ial. # `8 N5 i# o- W; s* ?5 B6 pDisp ense cell s uspension i nto labelled cryovials and2 G6 B# ?' x+ l D! t
transfer them immedi ately to a precooled Mr Frost y® con-tainer, and then into an − 80°C freezer (see Note 6). , m% |1 ~4 n% S- b" WFeede r Layer Plating for IC M Culture" i3 U7 \! X; H# g$ u) h
1. Dispense one volume of gelatin 0.1 % ont o the ) Z* H6 R0 N* ~4 L$ m( z8 xrequi red number of dishe s/wells, and incuba te them, g% Z- i4 p- d. Y( D0 B
at 37 °C for 30 min. . X/ Q7 E" q% Z7 g, ^1 o- }; U( G2. Equ ilibrat e 10 ml of fresh compl ete DM EM medium,- k, l1 {: i, J& Q+ i
in a 15 ml conical tube, in the CO$ u9 G' Y1 I% {. e( b2 a, W5 Y5 w
2) g8 j/ X, Q% r3 T" K4 p8 s) B
incuba tor per each X0 g* o! l: w( u# m" P
1 – 2 vial (s) to be thawe d (see Note 7).; U/ j+ R5 F2 x1 N: t& r: m V
3. Once the coated dishe s and the medi a are equil ibrated,- F. G7 C. t% _. K8 f+ K& u( T
thaw the requi red number of cryovi als to plate 0.2– 0.30 {9 S* u1 A+ B' E) C3 A
mil lions of inactivat ed pMEFs per one-well 60 mm: P/ `0 n" G3 d
dish. Spray the frozen vials with 70 % ethano l. Roll ) ?; i6 f0 `. x8 m- athem in your hands, until only the last smal l piece of. d4 r* @! O8 E8 i' m: h
ice remains. Tr ansfer the c ontent of the vi als i nto$ H8 @9 X9 u+ d; q
the equilibrated compl ete D MEM. In order t o 7 I4 r( D: x1 e0 }1 Y+ X8 ]/ oavoid cell dam age i t is n ot advi sed t o thaw m or e( a$ `* A- O( I, c, s! T1 `- j
than four vials at a time.: _/ k% \( o) T6 m& { @+ u9 A4 D
4. Cen trifuge the tubes at 700 g for 2 min . Discard the * U! T& r; [' }$ N* m, v0 R' _s upern atant a nd gently break the pellet b y f in ger 5 F- Y8 z9 k3 W. Z. a2 xvo rtexing.! r# M Z3 k- c- u+ \
5. In order to elimin ate resi dual DM SO, repeat step 4.& O' Z0 W4 c2 U% \; W9 ]& u8 O3 ?3 s2 \
6. Dur ing the second centr ifuga tion, aspir ate the gelatin( B$ h& M& Y& Z4 D( K0 [( }1 J7 B. F
solut ion from the plate(s) and dispe nse half a vo lume " U% B" [/ O5 E Q: i; @of prew armed compl ete DM EM medium on each one. . D; U. [+ E, G3 S d7. Br eak pellets from step 5 above by finge r vortexing,2 }& J" W6 ?" w4 M7 J2 `, {
an d resus pend them in half a volum e of the wel l(s) to" M# M, [$ w, E+ X5 F" h8 \
plat e. : J1 A" m8 h3 x4 F: F9 e8. Di spense cells dropw ise onto the medium- containing " @6 s N+ c/ u! g- ywel l(s).: X9 S* x8 c, @" _0 k1 O
9. Place plate(s) at 5 % CO ; Y7 m! N: w. m" N8 N2 ; |$ z5 N" Q4 m9 O- x) Rat 37°C, and c arefully shake $ F. d# d& v) uthem horizontal ly in all direction s to distrib ute the cells 6 @% I) r( x) e$ K* |ev enly.! ?0 D4 K% r) A) ^
10. In c u ba te f ee d er s f r om 6 h to o v e rn ig h t to le t t he m ! H5 V/ }# P2 a2 a/ Q( ~/ j- fattach. Optimal feeder density corresponds to a conflu-ent monolayer; higher and lower densities will result * v, M, D) K: L: u K9 Xeither in reduced hESC growth and increased differenti-ation, respectively.1 X3 x' x4 l* T/ n' o q7 L
Se e Not e 8 for comments on feeder layer prepar ation.9 [$ a) R$ n5 q* A: l- X
Embryo Culture * L% Z3 g( D" a2 MSurplu s embryos from clin ical in vitro fertilizati on (IVF)4 @ P5 Z2 W& E- x Q# R
procedu res can be supplied, fresh or frozen , at any stage7 C0 D* u' {# D2 C: y) p- X5 b
from day one to day six post- fertilizati on. It is firmly estab-lished now that low-grad e blast ocysts produce h ESC lines " P# j1 v! w% R% T9 R- Lsucces sfully [ 15, 16]. The refor e, the derivatio n of stem cell4 c# M( w9 q. Q
lines is almost guaranteed for a ny Re search Pr ogramme ! A' f) Y% t- @9 C! N. s" j$ uestab lished in coll aboration with an IVF clin ical depart ment. $ x# X* s5 Y- V* \8 |/ v" n3 O' fIn this section of the proto col we will detai l the steps& K+ j, M( y! L/ w8 m
involv ed in embryo cult ure for hESC deriv ation. It is advis-able to use the same clinical grade embr yo culture reagent s( F$ a0 a8 Y. d ], v8 D$ a$ m
that are used in the clinical procedu res. Altern ativ ely, it is8 N* f* F% {+ G5 d% b+ D" e
sim ple to p repa re the m from thei r compo nents [ 17]. Se e: B* |5 \. n, n1 \1 @7 |7 |6 b
Note 9 for tips on embr yo cult ure.3 {, l& R6 h% A- C9 \+ {
We will detail Vitrolife® protocols, and advi se to fol low4 G3 f' L; C/ A% J) p. `/ B8 H
manufacturer instructions in ca se of different brand reagents./ y7 M# B) a% e' j6 j6 B6 {% F" \4 ~
The cul ture system us ed was a two-stage one, i .e. changing" | w7 R/ w1 ^. h/ F4 J
cul tur e m edia formulati on on day 3 or s ix-eight cell stage o f' p) t' Z* I+ X7 c" d. u3 S
development (from G1 to G2 series ), un til blastocyst formation .2 { l Q( P. z$ z% d5 |, }
Fig. 1 Time schedule for the % v& I" L3 d4 [& \+ r4 b4 W! icompletion of a full derivation( V$ Q; q, N+ i$ q
experiment , t0 W: |- O5 } q0 H, J& yStem Cell Rev and Rep5 o$ V/ l% J0 o$ l: m/ M
Embryos are cultured in m icrodrops under oil, to* a( V$ q- Y, L, m% [
prevent evaporation a nd to minimise pH changes. The& _0 ]( o5 ]$ ^& h( N' u
following protocol corresponds to t he culture of fresh or2 I6 z: F6 u1 \1 Q
thawed cl eavage-stage embr yos. In the ca se of receiving; X4 U, }3 U. z. v3 \
frozen embryos, it is crucial that the thaw ing i s per-formed in the reagents corresponding to their freezing. 5 w) [% c7 [$ Z: z1 WFollow manual instructions from the appropriate thaw-ing kit. After thaw ing, follow the protocol below for6 E. E) x/ B6 y
embryo culture.7 D& N7 N3 Z2 ]: O
1. Equilibrat e OVOIL® was hed wi th 1:10 volume of G1 or 9 o* M- N- E( h' q0 E4 y8 m0 [/ GG2 for 6 h to overni ght at 37°C and 5 % CO2 * m/ T# g$ Q& v6 w% N7 m+ }.1 k; |4 B0 M) m4 q" {2 F# [6 }0 H
2. Equilibrat e G1 or G2 medi a as required (see Tab le 2 ) for % R1 f" _* l; B- k, _( qa maxi mum of 3 h.' \5 @0 C7 N; @# g+ ~
3. To set up embr yo cult ure plates, pipet te 25μ l of the . r4 ~* Z$ |3 i' pequilibra ted G1 or G2 media per embr yo on embr yo/ z2 Q5 P6 {8 k
culture d ishes/plates . Cover the drop(s) with equil ibrat-ed OVO IL®, and add 25 μ l more of the medium to the 0 L- N: c/ i3 Q1 R7 mformed drop(s).3 U* U* f; T# n5 N
4. Assess embryo development daily (see Fig. 2 for a + F8 j; w4 f+ R, w pdi agram of g rowth pattern and c orresponding images / f; n& @- N$ O9 Yof embryo development), and transfer them to fresh$ p% y* u2 a3 m1 p
media d rops every secon d day (see Ta ble 2 for 2 O6 N x" _, e) m& U( ]' v* Tmedia c hange guideline). P re pare new drops as) z2 w7 H6 `, s0 `0 a8 I
describedinsteps 1to 3above.If embryoculture) p! k/ i& V$ C' J" T; [- }
star ted i n G1, remember that once embryos r each 8 m7 q" R5 b0 s h5 zda y 3 o r the six-eigh t-cell stage, they must be 2 Q, Y8 o0 c' W. P6 ]: o# l0 icultured in G2 medium." M& ]7 b2 A' p: H% n
5. When embr yos reach da y six of develo pment , or the" W+ ]6 Y( n! Z" T' t' L' s
ICM of the blast ocyst is visible, it is time to set up the 8 }5 r( Z' u6 m0 N6 y& efeeder plat es to seed the former./ v: @# z* y' G& a/ C _& \
ICM Isolatio n an d Se eding ) f6 C! w V- a) g3 SDuring denudation and ICM isolation i t is a dvised to2 x; h( [* Y7 {. c
process each em bryo indivi dually. Pulled glass Pasteur2 d: I4 {$ _& J& r
pipettes or ca pillaries a re recommended, or commercial-ly avai lable em b ryo h andli ng p ipettes used f or clini c al s; M N2 e! B! @7 F. E" yIVF p rocedur es. 5 ^# d- y9 A* s3 l4 ACon dition the feeders before the d enudation as follows: 1 V) y3 l& {+ q0 B+ ^/ b5 N, Gbetween 1 and 3 h before the seeding e liminate f eeder- x. t3 H$ ?# ~! t) C1 |
mediu m. Wash three times wi th 1 ml of PBS with Ca( l7 K* M3 {/ n& D
+2 5 @" N. a9 n5 K3 Y- F# R/ C& q/0 ^) e: F% r9 M# E
Mg 0 R7 B0 T$ {1 i+2( m" z9 Z: B# A5 u
and once with 0.25 ml of HES medium. Add 0.5 ml of ; v- F: {* b; K/ U) V; HHES medium and equilibra te for at least 20 min at 37°C and % s0 ~% O J/ |# i4 E5%CO* r; E8 `* I! c# p! N" p. v
2 * ~% E! u# N; X$ H0 p5 F. At this point the new feeders are ready to recei ve : Y! m3 C Q6 d. _- dthe IC M. % `+ X1 Q" F$ z' ?" W# RZona Pelluc ida Remova l " @2 M) s) E; p' P, t9 d- M1 N5 t1. Equ ilibrat e separa tely ac id tyrod e’s (AT) 1×, G2 me-dium and HES medium at 37°C and 5 % CO/ b! ?# p: E( ~, X
2* B8 E2 P; c7 k$ z# R
, for a, Z, ]" d: i, r
min imum of 20 min and a maximum of 6 h.: \ L" L( B* M; L3 r' h% R7 y2 W
2. Bef ore any mani pulation is perfor med, images of the8 a3 d! h; Y( ~5 L1 h- Q
blast ocysts at different planes (×40) should be taken' I& T7 @8 G% ^
for further asses smen t of its grade. Afterw ards return it* j3 y; }! t4 a6 ^
to the incuba tor. ; y5 x( j- c' d! [3. Pu ll 2 long ster ile glass Pasteur pipet tes wi th the aid of* L( u7 }1 D. Z6 J, U4 d \
a Mecker or Bunsen burner. Leave one of them opened z" x+ J* h# b' S2 P! U; gan d rounded , and close the other one leaving a small3 k* M9 [! m6 W" l$ S! A# l
rounded ball at the tip (Fig.4 ). Check the thickness of ; n* {# U4 S7 |" f! _& cthe tips under the s tereomicroscope and r epeat the3 y) L# A* ~6 N) }( i+ B* G7 h
procedure unt il a couple of pipettes whose sect ion' z6 Y1 p" }) p( J) l
mat ch the diam eter of the blastocyst are produce d.) N& Z# e) K$ N3 ]! f
4. Se t three drops of AT forming a row in a 60 mm tissue1 _- b2 ~" q7 Q' ?
cu lture dish, an d mark thei r position at the rim of the1 x4 P! k: p+ |* `
d i s h. A dd a n ot he r t h r e e dr op s o f G 2 m e d ium i n a & b3 l) h& J; {' |& Psecond row at the middle of the dish, and three drops0 z% ?* k& p- J, d
of HES media at the bott om, again aligned in a row : Z8 o$ V2 ?4 s$ H' p* D(S ee Fig. 3 for drop distrib ution). " R8 n6 _8 ?+ F4 _2 S9 J5. Se t anothe r three drops of approximat ely 100 μ lof 2 v% U( I' F4 j0 P+ T. U) gHES medi um in a new 60 mm dish. 4 A' i' }6 _' Y; L% F( r7 y( |6. PicktheembryoupinG2 mediumwitha 275 μ m " q+ c6 W) t7 G2 ]2 Qcapil lary mi cropipette, a nd t ransfer it to the left AT : z% y7 l9 e; y/ y. C: kdrop of the plate prepared in step 5 a bove. Stop- _2 @: Q+ j1 F9 k& j( m
dispensing G2 medium into the AT drop a s soon5 f {# f8 K# \! j
as the embryo has been releas ed. Empty the pipette 3 j" P- g2 ^% z- t0 V7 Jin the border of t he dish to discard r esidual G2( P" k- h4 [+ C6 ]$ o
medi um. * Y, u R" s+ @3 t/ @4 q( F6 k7. Wash the pipette twice in the second AT drop.% }" v& G5 M& }( F1 }. M. E5 C4 i! M$ }
8. Return to the fi rst drop, pi ck u p the embryo and 0 _$ f4 K' G$ p! wtrans fer it to the third d rop. Monitor unde r the micro-scope the dissolution of the ZP (0 – 2 min).+ J5 T4 r4 c1 D, M( D/ s9 b
9. W hen the troph ectoderm (TE) of the embr yo is ex-po sed by the disso lving ZP, transfer the embr yo se-qu entially to the three G2 drops. Use the first drop as a, O* K5 j G7 s. y3 d( P
pre-w ash, dispe nsing the minim um amoun t of AT into : k3 p- [- z# |3 m3 h3 j% q& Nthe G2, the second to load the pipet te with fresh G2 / j$ x3 r" R- @6 cmedi um, and the third to was h the blastocyst . ( x/ t/ Z8 `+ K7 Z3 o7 B10. Tr ansfer the zona-free (denuded) blastocyst to the % ]$ O% R- B6 P8 Mdrops of the HES medi a using the washing procedu re7 h5 k3 S; g/ w g2 f3 R# y9 i) v
as described above for G2. & r/ A- ^2 }! ]" D: I11 . Immedi ately trans fer the blas tocyst to the drops of ; x/ @. c1 O/ Ithe HES medi a prepared i n a sepa rate plate in s tep ( d' U& v* _0 L& [1 _2. Follow the s ame procedure of trans ferring to the0 g. B$ C) ]6 Q9 J' ]* y
left drop, washing t he pipette and loading i t in the ' ~3 s1 p3 s1 _6 Z* P5 b- Qsecond intermediate drop and transferring the5 ~& x; M; T3 g' ?
embryo t o the third drop. P l a ce the p lat e i n to the % N% P7 W6 e, K6 a! QCO7 s, c4 L6 A. V2 n" M! g
2 . q: O1 J3 L9 l; ?incubator. ) ^# A6 L6 v& R3 g7 ]% x h( d12. Usin g the ima ges take n in step 2 above, asses s the ( T6 v% q# _7 ~. e9 b. r! ~deriv ation grade of the denuded blastoc yst (see em-bryo grading s cheme in r eference [18]) and a ppl y * V! S) r) |1 m) tTable 3 for determin ing the best met hod of ICM iso-lation. Depen ding of the outcom e, g o to protocol A or / _% c5 K) ]* v1 v" F+ SB be l o w. S e e No te 10 fo r c om me nt s on t h es e& h0 h! e; T; [, p$ l
procedu res.+ W: F( L5 H. w* J
Stem Cell Rev and Rep 4 g- y% ]* _# G. w! t) ?; mICM Seedin g by Pipett ing (A) * |5 h# A5 N- I4 T% a1. Choose an open pulled long Pasteur pipette of the same3 A0 _2 `: v: ]! Z' ~ J9 t' }
diameter as the denuded blastocyst (see shape in Fig. 4a ).7 C+ K- k9 @6 C4 p3 `
2. Load half of the tip with fresh war m HES medi um from; y7 A+ v- }% d! M
the drops set up to equil ibrate before denudat ion (ste p 5 2 L) n% ~6 i5 G! a: Q8 Gon zona pell ucida remo val proto col).8 k% H2 r* w; ~' q
3. Aspirate the denuded embr yo and pipette it in and out 7 _; ]0 r1 s- @2 O% Jwhile checki ng for loose ning of the TE cells. f" [- A+ o4 M' h3 S3 i1 v7 o/ i( f5 Q4. Aspirate the i solated cell clumps and dispense them % o: @( g; u+ O7 E! O8 z0 I7 Vsepara tely on the feeder plate. $ ~. [( K- Y5 m. W) b5. Ta ke an image of the seeded fragments of the bl as-tocyst. Pay attention to l ocate the ICM and record6 ]- W3 y1 }3 G! S/ U1 Q
its posi ti on. . W; m: y0 M: aTable 2 Embryo culture% z0 P# L. G& j) Q" y3 f
protocol 8 |2 o0 T+ u& `7 |, h) i9 yDay of development Stage Medium Image recording Media change, U4 k ?8 d0 f- I- x1 U
1 Zygote (2pn) G1 − G1) D C/ K) A' ^8 q3 @
22– 4 cells G1 Yes − Y! ]( L* L9 G4 I/ Y) h+ u36– 8 cells G2 Yes G1 to G2 Y$ f( @" w# K0 G& j% y
4 Compacting morula G2 Yes − / i8 a- N+ A: t N/ W" o! s. l5 Expanding G2 − Fresh G2$ h2 r$ w4 y# e& S3 e: |
6 Blastocyst G2 Yes − 0 \ f2 T. s3 t7 R& P) u7 J7 Hatching G2 Yes − : O/ P1 Y0 x K1 O. D! v0 rFig. 2 Dagram of early human* t8 l2 a7 h! u
embryo development. a-a’ 2 w: L6 z4 h' m. E. ^/ otwo-cell stage embryo, 1 day $ G3 D) H, e( b* L% Oafter fertilization; b-b’ four-cell3 L% y. S! ?/ u
stage embryo, 2 days after - _: [4 t- Z/ j3 [fertilization; c-c’ eight-cell 0 N, p& g& ]. b9 { estage embryo, 3 days after f & k: N6 e2 ]/ b( b" _) b% rertilization; d e a rl y e xp an di ng $ P+ T( N2 `1 b2 |9 g" }, P7 tblastocyst;d’ morula containing4 y$ |3 S1 h! N+ T( P; m$ [
m o re th an 1 6 ce ll s, 4 d ay s a f t er ) E' a; J7 n" N u4 U( rfertilization; e early blastocyst, 5 b" a J/ b8 V5 days after fertilization; f& `5 f. K8 R& H
hatching blastocyst, around . a: h1 |( T Z% I7 days after fertilization. ZP:: y1 q& A( @. \3 [8 V' ~
Zona Pellucida. ICM: Inner1 y- u; ]! a w$ K4 c& v. n
Cell Mass. TE:& n& t0 r- S5 N6 x; g0 U/ t. Z
Trophectoderm + U; D6 e8 X9 x' i6 KStem Cell Rev and Rep 7 c! B0 k, \- _4 |: V6. Tr ansfer the plate to the incubator, avoiding abrupt( f/ e: q$ M4 h# g. f' z
movements t hat may separate the s eeded cells from3 c. Q/ H. f# Q/ a: O% w: G: R
the feeders.! b) q7 E) v4 @2 x4 Q( @2 I
7. Inspect the plat es daily. Cha nge the medium every other, ~. m1 h. w9 n# E2 d9 e
day. * t2 J, y0 E0 j5 X+ ~5 m% M0 H8. Prepare new feeder cell plates as soon as pluripo tent ! N" I7 q5 @; l; X. ggrowth is detected (see Not e 11 for deriva tion timelines $ W/ T& _; E, A' V- Y1 V1 |and Fig. 5 for ima ges on deriv ation progre ss).6 A9 c8 b7 E/ ?, ^
9. Split part of the emerg ing pluripotent colony when( o+ l3 h5 _+ w, Z1 _1 m
resi dual TE cells begin detach ing from the bottom of: M/ d( e+ u/ x/ T
the plate. Follo w Secti on 4 below to do so., P1 O& f7 v* l! |
ICM Seedin g by Mechan ical Cut ting (B) : x& K. X0 A4 b' q. |/ p q1. Aspira te the denuded blastocyst w ith a pulled open1 R' z8 U. r" r. \
pipette (Fig. 4b) and dispe nse drops of approxi mately , s, S5 s9 b" o2 t; @, C. o }20μ l on the dry area of the 60 mm dish containing fresh* o7 \0 V6 I: b. g$ p) j+ @3 Y
HES drops, until the blasto cyst is dispe nsed in one of7 b# X# A3 e( | q7 G3 {
them. 2 ?, f3 ~2 W5 I/ k2. Drag the denuded blastocy st across the dish, away from. \7 b$ K5 U% c, b
the HES drops. The volume of the embr yo-conta ining# q: k2 A# d# b
drop will diminish up to the point that the embryo% `& r y$ P; O8 C! m1 p4 M
flattens and sticks to the plastic. At this point you shoul d2 C3 G6 r) {) L! R
be able to locat e the IC M: it is much brigh ter and less" p! X+ _5 h5 R1 u+ n. I" R) Q
flat tened than the dim TE. Sa ve the pipett e without* V0 n: L0 m: K9 x) d5 [
emptying it for follow ing cutt ing steps . " t7 h5 s/ Z! ?) U; p* t/ b3. Pick a close d pulled (hold ing) pipette (Fig.4c ) with the ) i! N( K- j6 @/ u; X4 fleft hand (rig ht for left-hande d) and hold TE by pressing. _+ A2 f) u3 P
its border agains t the plate. 7 q$ ^3 R/ ]' D; W8 z! ]1 T/ {4. Pick the open (cutting) pipet te still contain ing medium: N- ?. O+ Q b. F& c) z
with the right hand (left for left-hande d). With its edge, " Z- z: g$ R7 E& ~& xcut out as much transparent TE as possible. Imm ediately 5 l. {% v( X. \# K( X, `/ Trelease the HES medi um to a void damag ing the ICM. : n9 B! X1 ]! L. c$ s/ y9 K5. Scrape loose the ICM contai ning piece, which will be/ E' i, J2 R+ h- J: B5 w
adhered t o t he plastic. M onito r the ICM w hile it/ x" o! t" l# V& b# E# P' T
Fig. 3 Shapes of long glass pipette tips for ICM and stem cell colony( |% H( k' H9 y6 K4 K( R
processing. a transferring pipette with open rounded end; b cutting! @1 t+ Z* A& _, ~# A' T1 U. B8 T9 o
pipette with open bevelled end; c holding pipette with a fine rounded% \# X9 O9 x( ? o
closed end; d curved hook with closed end for outgrowth scraping;e-g1 h$ ?$ l! B2 A& y) N: C# B; [ I
flat hooks for colony cutting and scraping. h detail of the tip of the2 R* @, D8 F' `, ?, w3 O
holding pipette depicted in C/ Q3 N" _/ R6 Z9 z! u
Table 3 Choice of method for ICM isolation according to blastocyst - i5 z8 W, s( H* g1 bgrade" z7 g. D" K+ `* ] O$ v
ICM0123 0 `( [% W0 ^6 STE ) m+ C' l. s' v* P0AA 1 A A A/B' l( O9 k9 ^1 [6 v' Z
2ABB 3 ABBB2 r& M. S" m& G9 Y
TE score - 0: no cell layer formed. 1: few big cells forming a loose 7 I: F8 M5 H, O2 m2 T3 Ylayer. 2: cells forming a medium coherent layer. 3: many cells in a tight8 Z/ L3 K/ E8 M& {3 k* S
cellular layer. ICM score - 0: no visible inner cell mass, inner to the TE # S0 _7 a: [/ zin any plane of the blastocyst. 1: ICM with less than 10 compacted/ e$ W4 k$ _. }' Q0 F7 D9 R) @+ u
cells, or loosened mass with up to 20 cells in the central focused plane, n5 v6 c; c: s, D$ |! L: z: F3 f
of the blastocyst. 2: ICM with more than 10 compacted cells, or more# Q$ V% u( v6 ?& B0 w
than 20 loosened cells in the central focused plane of the blastocyst. @7 F# m. J9 b* R
under the stereomicroscope. 3: ICM with more than 20 compacted 8 H( _2 |* b6 S9 P) tcells, or more than 25 loosened cells in the central focused plane of the 0 ^6 d1 @2 |4 q! {blastocyst. Method A refers to pipetting for ICM isolation. Method B % d2 J0 S9 p) j7 W+ n& A- Tconsists of mechanical cutting of the ICM ' f( ~# @1 g0 C. E9 Q, f& w* j. ~$ jFig. 4 Derivation plate. Embryos are dispensed in AT in drop 1, and 4 V* m+ w* J/ s7 G3 C: D1 I; ntr an sf e rr ed s er i al l y ac c or d in g to t he fl o w o f th e ar ro w s. AT: Ac id 4 z. R7 T7 j- {* r* YTyrodes. G2: embryo culture medium. HES: Human Embryonic Stem- f1 [9 B: t5 i$ K2 z
cell medium / I: A4 {7 Y o4 C w& w" cStem Cell Rev and Rep) i3 n; t) Z- V, p6 i- P8 D
detaches and float s. Qui ckly aspir ate medium from the 5 b$ e8 w2 ]5 P1 i- H3 `2 I- H0 eHES d rops i n t he p late and a dd t h em to the ICM -contai ning drop to compensate for t he evaporation 1 O: E! [' A. Q: D Eoccurred durin g cutting. 6 b T: r1 z/ `( z6. Aspirate the floating piece( s) and spread them onto the . a# b" [2 J$ [7 h1 p y/ kfeeder dish. Try to avoid pure TE fragments , recogni zed5 o: q% _1 ^/ i3 i. `
by thei r trans parency.! N) k' [' K0 s; z9 B2 f- F
7. Take an ima ge of the fragments seeded, record ing the |1 u9 s2 n7 E
position of the IC M piece on the dish. ' U4 }; _! b4 i `2 z6 H* e" x; O9 Z8. Inspect plates dail y, feedi ng them each second day until2 L1 {" ^; `: [8 P: H4 ^ \& I- x
pluripot ent cell grow th is obv ious.- G2 L# o( N6 E$ ^8 i7 H
9. Prepare new feeder cell plates as soon as pluripo tent* e1 C" @7 I+ |# B! b
growth is detected (see Not e 11 for deriva tion timelines, b# X" o$ F( j `+ Q7 G0 O
and Fig. 5 for images on deriv ation progre ss). Go to ; r9 h( T$ O# o0 BSection 4 below for the firs t spli t of the pluripot ent cells. f" Q5 i5 X# p' w" y! M! _Split of First Pluripoten t Out growth |' P4 k- C9 A! p6 O" Y+ GWhen the pluripot ent outgr owth(s) reach the stage de picted" }2 R h; O1 I, ]+ I
in Fig. 5c , they are ready to b e split. Mechan ical cutting can3 e, N6 A" K' D; b
be perfor med wi th a glass pipette of any of the shapes A, D, " | ~, ^4 A3 ?+ x3 Y. ZE or F depicted in Fig.4 . In a ny case, prepar e feeder plates$ M2 H0 a) L: @& V q7 q# h1 F$ R' p
the day before splitting. If t he pluripotent outgrowth is; v+ a% F2 m& T9 I; }/ H
isolated from the residual TE, a closed p ipette and a & N$ y! n' u6 P. Pscrapin g met hod could be more effective. If stem cells are % x/ b! I. K$ y7 u! q$ @) Xgrowing in clumps surro unded by TE, a n o pen pipette to cut 3 o* p" |0 }* B( Qsome of them will be preferred. It is wise to leave some i. `* o2 H( y" z5 I4 Z G) eoutgrowth(s) behind, u nt il split cells ar e s uccessfully8 T" j- N+ n) O9 A! T
growing in the new feeder dish. $ e) ?' p* x9 X( uNotes 2 h5 w% D9 `' r4 fIsolatio n and culture of mous e embr yonic fibroblas ts and4 Y+ B. ]. X+ p: R; B5 }
feeder preparatio n$ Q" q2 a7 i1 |6 E' c, M
Note 1. We describe here the deriv ation of hESC line s in / D3 M6 P4 n# L5 w: watmospher ic oxygen; howe ver, the use of ph ysio-logical concentrati ons of oxygen is curren tly gain-ing suppor t. Newly publi shed data report a higher% q# r* p& s% d* M" P+ d
efficiency in stem cell deriv ation and lack of chro-mosome X inact ivation in hESC line s estab lished 3 d" N) S* K5 i" j: \# F' gunder such condition s [19]. : }# Y; l' F+ L k& X) M% kNote 2. Pr imary MEFs a re the first cells to be us ed as - f7 o. D2 S( S, G! d vfeeder layers for hESC culture. Alternatively, , {- B8 v& i+ x. a) G' Xeither transfor med [ 20] or n aturally immortal-ised [ 21] M EFs, as well as human fibroblasts' N/ c h. C5 ~; @. ^: X
from several origins, includi ng foreskin [ 22]/ Z8 k' y3 {% ]5 b" v1 f& ^
and placenta primary cells [8 ]andanimmor-talised placental f ibroblast cell l ine [23]have& l1 _) W7 Q2 R: M- c+ n, F
be en u sed su cce ssfully as fee ders f or hESC9 E. H) v! j9 C
deri vation and cul ture. Primary lines f rom hu-man f oreskin f ibroblasts (HFF-1) have al so7 D! u% W: F! y
been used reproducibl y to cult ure h ESC li n es. 6 [# H' L) q! r* [" S |+ O- KIn the case o f HFF-1 cells, i rradi a tion i s the( P! V, d! s8 `
pref erred method of i nactivation. All m et hods ' I( `$ t: t& z) N' t& J( N- E3 ^described i n this paper are equally valid r e-gardless of the t ype of feeder s used. 2 h' Y y( e+ h; q; p$ YNote 3. Mice stra in has proven to be an importan t facto r. q0 X& X; n. h. p" I: s- F9 T
fo r th e q ua li ty o f th e fe ed e r ce lls d e riv ed fro m . l5 F$ K! S2 \: G% TFig. 5 Micrographs showing G# f/ b1 b+ X' X' A. V
evolution of the pluripotent and9 r- I7 ?# q- `& X
TE cells during a derivation ( \8 o8 I& v) e& [. p* o. Kexperiment. a ICM 1 day after ' \7 y9 Q5 {% g. a! t" iseeding. b ICM cell expansion 3 R) Z% D( e, W) L6 mafter 4 days in culture. Arrow+ Y% G- _/ o# R" @
points to the pluripotent4 h; w. Z/ @ Q5 h& j0 E. d3 Z1 w
outgrowth, which looks like a/ ?! r" Z* L, u" `. e5 P( d# f: {
clump at this stage; c colony5 @3 C4 |& _0 I. W1 i
outgrowth 2 days after splitting 9 m+ F" N# y5 tthe outgrowth in panel B: arrow 6 j" E9 r( l0 \. [! dpoints to the expanding J- P2 _; `5 R L9 s* E5 J
pluripotent cells out of the7 V7 C8 M) w3 H0 Q' A
initial hESC clump; d residual 8 f' I; H0 U' dTE cells 3 days after ICM ' I/ j0 k: k. b; U4 Useeding; e TE differentiated ; Z# i; L8 G1 U6 f! X% Mcells 4 days after seeding; f TE & O w2 v3 |( f' k4 `4 `2 Kcells detached from the plate6 T1 t- e) k4 \. r! C' g
9 days after seeding; g detail of# Y# R8 z% N* Z0 O @& Q: G- {
the emerging pluripotent colony # E7 t# h5 ]5 c( z* e2 X( uof picture C;h early hESC4 i K9 y) _8 k$ R2 k
colony at passage 4; i regular8 X2 v, Q5 M, G( {9 L
colony of an established line at $ g1 K% f' W, J+ Zpassage six $ F' E6 r+ [& ~1 B+ ]Stem Cell Rev and Rep ! T8 F8 g6 [6 ?; k3 R) A9 }1 [them . In our hands, embryos from the CF- 1 and6 Y- }: i) F2 b H% N
MF-1 line s, and from the cross between MF-1 and+ K+ n h$ T B$ u
CD-1, are much better than inbre d CD-1 embr yos./ Y3 p3 q1 q- g% y
Note 4. Isolated pME Fs can be frozen at passag e zero, and . p$ t9 X. T- ~expanded at a late r time to prepar e feeders . Nev-ertheless , the expansi on up to passag e tw o before0 r! b+ y! u% S- O% W% C
freezing has the advantage of increa sing the num-ber of cells obtained at passage four. It has to be( L4 [& H9 G# y, X; S, M& k
noted that in the isolati on procedu re describ ed in 0 u) c, ~, p8 L+ H# [the Meth o ds secti o n, an d d ifferentl y fro m oth er $ h" s/ j/ e$ V' F Xpublished protocols [ 24], tissue chunks are not/ ?5 y5 e- c* ~* {1 ]% O* l
discarded before seedi ng after first trypsiniz ation. 9 l: t3 L" H1 P0 uThis procedu re yiel ds signi ficantly higher numbe r 6 q9 ?) b5 e' _, }) G+ V# [of cells. It is advis ed to culture chunk s a nd clumps # U2 {& O# n! v! N0 T. W) pseparately from singl e cell suspen sions, and tryp-sinise them until they produce monol ayers. A cou-pl e of passages from one isolation/mouse will- a9 t2 d6 h2 t
normally be suffici ent to produce enough numbe r+ Y A9 N. y5 a! D( @
of vials for cult uring up to six new stem cell lines) k" @" L0 @& ^+ K" Y
during 1 year. @! @# W- t* T Z* f* ONote 5. Finger vorte xing is a simple way to mix a solution. h2 t; A+ @0 z
or disag gregate a pellet in a test tube. Hold the top $ P( v% k* K' O, mof the tube secure ly in one hand and draw the ring, 3 Q/ @ x Z' \) F7 W) mmiddle and index finge r of the other hand sequen-tially towards you, tappi ng the tube. This creat es a - c+ U0 Z! |1 p( L% G2 W) Uwh irlpo ol eff ect inside the t ube, w hic h can be# \: |) b* `2 e+ A% G
adjusted in inte nsity by speed. It is milder than4 v2 a7 K; W8 ]% h' T! \
mechanical vortexing and yields healthier cells . k3 |7 `' E! I) j) X ~after centr ifugation .- {9 ~+ W( G$ W$ ^
Note 6. Freez ing fibro blasts wi th a slow freezing met hod is - v0 i; @' G4 c% h) ? g& F) knot an issue , and they can be stor ed at − 80°C for $ U9 A# M! s9 r Y# a! \$ r1 N6 i2 S. mmonths without a decreas e in subseq uent plating% X, @$ e2 ?8 J4 w9 e1 ?0 u- y
efficiency. The proto col described herei n uses the ; B- V& s) J# H% o {1 \slow rate freezi ng container Mr Frost y, which has & }! I+ t" m0 F8 g+ v5 sbeen vali dated and used for nearly 20 y ears i n $ a. L/ N- ?' h$ @& H; Pmammal ian cell freezing [ 25]. It is a cheap and3 J+ U% p9 w6 l5 C0 D7 P6 ]
time savin g o ption when compa red to alte rnative K6 S+ e! Y. [+ R. S Ymethods such as contr olled-rate freezing and vitri-fication, which may be suitable for more delicate ; x q$ W! ~0 l8 Nmaterials like embr yos and hESCs ." [! b# j" H) D
Note 7. It is conveni ent to dilute freezi ng medium in com-plet e DM EM medi um at a rate of 1:10 to avoid + x4 | w. m, C2 p5 j% ftoxi city and different iation during cultu re. ' v6 {, ?1 p6 T& v& D/ PNote 8. MitomycinCis thepreferredmethodtoinac-t ivate M EFs, a nd detail ed st eps to p erform the 3 [( [, v4 s, G9 L5 ~& V' G- J) Rprocedur e are included in the text. Alternative-ly, γ - or X-ray irradiation can be used. In a ny + B: f. q# S9 t2 M+ qcase, t he hESC supporting capaci ty of each% w( ]3 O) A) N3 @
new batch of feeders has to be tested on 7 K) I9 I( ~ K; z7 d/ f# p. f4 Pknown hESC lines bef ore c onsider ing t he m, W( D2 [: e% m% a2 V+ @& y0 u% P
valida ted f or use. Once the new batch has been - U4 {+ W- S: N6 m% R6 B- A5 Lvalida ted f or hESC cultur e, it ca n be used in & {/ `- L2 Q# z) Z1 Gsubsequent derivation e xperim ent s. 3 b, }4 ?/ L6 h- eEmbryo and ICM Cul ture - R; D( _$ R) h& B3 A& TNote 9. The procedu res detai led here are based on the use) Q1 F u1 z2 p2 B7 d9 r
of high quali ty clin ical grade embr yos, but fre-quently, the quality of the embr yos donated for # g- d6 {- K' k0 i0 p/ ^research do es not correspon d to the standards: F7 i/ `0 o" X6 o. j) }+ Y) q! ~4 @
presen ted in Fig. 2 . If by day 6 of development , : n1 s! k& a3 o9 e# scompa cted cell s as in Fig. 2e – e are not form ed, # e! y/ K, z: u* u% zextending embryo c ultu re i n complete HES. Y% v, l |: K# F$ r+ o
medium for 1– 2 additional days will selec tively% N8 J7 i; l' c
favour the growth of the ICM ver sus the TE.) _# s7 V t) N
This appr oach is supported in the most recent + u" e$ x# P0 u4 e& O9 pli terat ure [ 15].6 [9 d" s' _! L0 O; C
Note 10. Reg ar ding ICM isolation, s om e authors use a n1 |7 q) d( v9 N' {0 O3 ]
extended acid t yrode’s treatment to weaken0 S8 \+ E5 G. e$ {7 V' K
the TE [26], thus facilitating the spreading( S" V2 g! E p6 M) [* m
of blastocyst onto the feeders. H owever, in I) B Y5 a! K+ aour hands, the efficiency of this method/ G3 k/ N8 ]7 r
d e p e nd s g r e at ly on th e T E g r ad i ng . A p pl y in g2 S* h& E% ]" @+ B8 _
the same AT t reatment to all embryos usually" s$ p( e5 _; l7 Z4 _1 |
results i n s evere damage to some of them.! U+ _5 k" @ r+ k% `; ]! I# i
Theref ore, fitting t he method for TE reduction 8 c9 M1 F0 K0 ?! I+ b v; ?/ ato the grade of each embryo (Table 2 )helps - B' L- ]5 j7 A" emaxi mizing the r ecovery of viable cells f rom 8 M9 b: T# a2 v8 {; V; [/ Rthe I CM. When TE is r el atively s mall com-pared t o the ICM, or when the ICM is not ; ?+ {& V: s( X4 g' n4 S6 Ucompact, a pipetting method (Method A) is# A- W; `; A: b, `! e
preferable. Alternatively, if the TE is robust ) Z, |) j" w2 {- q7 y+ o2 I, Yand t he ICM i s c om pact , mechanical cutting , m8 d& j3 e9 i: z8 r, b ^/ V+ ?(Met hod B) s hould be the method of choice. 8 w3 }+ p- v5 y( ]6 `Speed during embryo denudation and I CM % [" s& {( V3 ^( pisolat ion is criti cal, even if the m anipul ations$ h) E% _2 \* x( E
are performed on a heated stage. # S U5 d& I3 ^# z* U8 P) x# vNote 11. If healt hy, the first seeding of the cell s from the 6 U1 ]: f" Y) N! UICMs will att ach i n 6 h t o ove rn igh t . In o ur ' O0 S+ u0 { `9 q, `3 u1 `) d% Wexperi ence, those clum ps needin g more time to6 n5 @9 ~3 z) P7 E5 T
attach are not robust enough to yiel d deriv ations.7 Y/ `' L9 u) h; ]" Z+ [7 F+ Z9 p
The first outgr owth after the attachmen t of the : ?/ d/ `8 C2 r. M( H# rclump can be seen from days 3 to 16. At this - F/ K; _7 D" L) W5 epoint, the undifferentiated cells need the suppor t 4 q0 I9 b" X h8 r2 i. m ^! Oof fresh feeder layers to estab lish pluripot ent cell' M( `# Z4 h5 p& O1 x6 h
growth. TE cell s evolve invar iably to form syn-cytial cells that invade the feeder layer and die ! s0 z5 ?9 B' Sleavin g g aps i n it. It is critical to have fresh& f) w5 `7 t) h( l% N) X0 m
feeders ready by the time the TE cells and deriv-atives detach from the plat e. }9 { t/ c/ W& aAcknowledgments This protocol is the result of work funded by the & L! a1 p- q! H1 \North West Development Agency (NWDA) i n t he UK and the . ~$ u6 i' `& {( m9 t: F7 E! Q4 AMICINN-PLE2009-0091, IPT-20011-1402-900000 and FPI-CAIB ( T/ s3 K, [0 C+ Q1 e" GGrant FPI10 grants in Spain.( @4 e. Y3 ^1 x0 w6 w' H9 u
Con flic t of in terest Th e aut hors decl ar e no pot enti al con fl icts o f( ^$ Y, r. w' E0 R" U5 I4 U
interest.' W* d) T0 y7 e
Stem Cell Rev and Rep. V- ]! R, A7 p$ `2 M6 V
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17. Pool, T. B. ( 2004). D evelopmen t of culture media f or h uma n 3 R- j' G6 c7 X, h: O/ {assisted re produ ct iv e t echnolog y. Fertility and Sterility, 81 (2), 7 U' f- h" ? b9 {8 H287– 289. , R. `- v. p# N u' |6 p2 }18. Stephenson, E. L., Braude, P. R., & Mason, C. (2007). Interna-tional community consensus standard for reporting derivation of: y C! @! h9 u4 g1 H9 E0 r$ y
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feeder cell line for derivation and culture of hESc/hiPSc. Stem Cell * Q2 o/ l+ u/ d, E4 J2 C" z$ R8 w0 CResearch, 7 (2), 154– 162./ w2 ]0 m7 C. U1 ^- F2 X" Y
24. Schmidt, J. V. (2001). Embryonic Stem (ES) cell culture basics. + C: h- u6 v0 iCurrent Protocols in Toxicology, Chapter 15 , 15.1.1– 15.1.15.+ G3 j: d, |! H0 {( [' _
25. Venkatarama n, M. (1992). Effects of cryopreservation on immune& f0 z$ T% T. L' Y- P
responses: VI, an inexpensive method for freezing human periph-eral blood mononuclear cells. Journal of Clinical & Laboratory / q( b: d+ h( Y9 L( D( ^Immunology, 37 (3), 133– 143. & ?- H/ C+ B% \' w1 d26. Elle r s t r ö m , C . , S t r e h l , R . , M o y a , K . , A n d e r s s o n , K . , B e rg h ,$ ~7 B6 W2 V* ]$ z5 j- k
C., L un din , K. , H y lln er, J., & Semb , H. (2 006 ). D e rivat ion o f 3 L6 V$ r4 L, D# ba xeno-free h uman embryonic st em cell l ine. Stem C e lls , 24 , W1 V, _8 J* G, T(10), 2170 – 2 176 作者: 老姜 时间: 2013-1-31 17:57
还有非常精致的图片，没法发过去，不妨将你的Email发给我，我晚上给你发过去。有事88！作者: Jonathan 时间: 2013-1-31 18:05
回复 老姜 的帖子% L6 X, q: D3 Q6 Y& d5 @
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虽然不想抢你的风，但是我还是提供个pdf格式的吧5 W& C) p, n' K
[attach]49322[/attach]作者: 老姜 时间: 2013-2-1 01:15