|
- 积分
- 1025
- 威望
- 1025
- 包包
- 2313
|
Optimized Protocol for Deriva tion of Human Embry onic; m4 o2 Z: T6 \; G/ J, y3 Z6 k0 g
Stem Cell Lines
7 g7 _, N1 | {* d: S; S. pMaría Vicenta Cam arasa & Víctor Miguel Galvez&
* E( q6 ~7 l+ P+ L: mDanie l Roy Brison & Dani el Bac hiller- l' }9 ]" \* X7 j ~
#Springer Science+Business Media, LLC 2012
1 l& d/ K3 R: L7 b2 d/ M% `# A! EAbstr act For t he pa st 12 ye ar s, th e b iolo gy an d a pplic atio ns/ ^- Q' a6 J3 P) p8 D% L
of human embryonic stem cells (hESCs) have received great; Y" i8 W) \( W) [9 c# _/ D+ @
atte ntio n f rom t h e s cien tific comm unity. D e riva tive s of the first& g& d5 I4 x" ^+ T
hESC line obtained by J. Thomson ’s group (Science 282( l8 k p" J" V& x) R& `% \
(5391):1145 –11 4 7 , 19 98 ) have been used in clinical trials in
# O! a8 e7 I3 L& Y; npa ti en ts with sp ina l co rd inju ry, an d othe r h ESC lin es ha ve" P' c8 I c& d9 V
no w b een u sed t o g en era te cell s f or use in tr eatin g blin dn ess4 p5 L5 F/ [6 C% i
( L a n ce t 3 7 9( 9 81 7) : 7 13 –72 0, 20 12 ). In addition to the classical
0 _8 k1 H/ D% l* _ e7 Q! epr 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
8 A7 j) t7 e* x: a* J$ |8 ~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,- n& ~5 o$ w) U" a
2005 ; Nature Biotechnology 24(2):185 –187, 2006 ;Human
3 P* z ]; V; f3 JReproduction 21(2):503–511, 2006 ; Human Reproduction 20
% ]# |+ k2 {% q7 y A$ x(8):2201 –2206,2005 ; Fertility and Sterility 83(5):1517–1529,. u* q+ C. H5 j! R3 @
2005 ), novel hESC lines have been derived xeno-free (without! f$ W8 ^" ]+ Q: ]
using animal derived reagents) (PLoS One 5 (4):1024–1026,
( X6 s: e+ `2 t! Y( H& P0 q2010), feeder-free ( without supporting cell m onolayers)
8 m1 E; p- U3 [& {; D. ^' j$ t; D4 [(Lancet 365(9471):1601 –1603, 2005 ), in microdrops under
3 [" x3 V5 L0 d0 j; G0 g: koil (In Vitro Cellular & Developmental Biology - Animal 46
, h# i# g3 A& ~; q. Y& R(3 –4):236 –41,2010 ) and in suspension with ROCK inhibitor, g3 \% o2 K7 Y1 l1 k' e
(Nature Biotechnology 28(4):361 –4, 2010 ). Regardless of the
# I! e; F3 v% f- g; x1 wculture system, successful hESC derivation usually requires/ B# b( Q3 U' Z$ T0 X% E9 J
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 during C0 ~$ f) {- H2 [
hESC line derivation. Herein we address the crucial steps of8 `8 J; o3 O* r2 o5 s' K
the hESC line derivation protocol, and provide tips to apply
5 r+ L. O( j t& _# }% vquality control to each step of the procedure.9 F( ]4 l$ H1 r; {3 v/ i
Keyword sHuman embr yonic stem cell s. a7 S) Z' `$ A9 j
.
' ?. W& J3 I% @. F! lDerivat ion! w! i4 k; w2 I# e2 i$ }
.$ Q q. u/ }, @2 g/ u5 h& K8 @
Blast ocyst
( a3 e" H' U6 A/ i+ [. z) h.
7 j. O J8 |1 |# {Cultur e o ptimizatio n
0 y, [$ u5 ~0 NIntroduction
+ u b- w" ^" OTo da te, hundreds of human embryonic stem cell (hESC) line s3 O/ Q9 i A6 j+ I' s4 Y; |
have been de rive d f rom surplus embryos after as sisted repro-duction techniques. Reported derivat ion e fficiencies range from
! A) F! ^/ c: N7 to 100 %. This wide span pr ob ably refl ects the di versit y of the
' E( c& S+ c+ vmethodologies involved. M any var iables of the procedure s uc h, z5 i7 n% i" q( b: Q/ c
a 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)
! n! K9 R y% m0 Tremoval and inne r cell mass (ICM) isol at ion a nd type of feeder+ M/ r1 X' `: E
layer, 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
# z" H3 u) v" w3 g2 O- d3 Bproperties 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
/ z4 {+ p7 T e5 tth e genetic stability of more than one hundred hESC lines
! a0 H; S" G$ v9 ?" z2 w, iusing consensus protocols for karyotyping and genotyping.' c2 k2 |' H+ f) e8 F% K
This echoes the importance of defining robust and common! ^3 J/ t% j j7 W
criteria for the assessment of both existing and newly derived
/ ?/ r) J1 i& R# z% {cell lines, and for the validation of advances in culture con-ditions. [13].- a7 E8 T2 O, C4 J; H8 B" D) u$ m
M. V. Camarasa (*): [0 q: T' G! V. H" I, p
:
5 M e5 V6 l; Z. S6 E4 LV. M. Galvez
6 k$ R: |. A( T- A% x4 j* O& ~. J, C:
% y# L: Q y& v& s; T- w, ~4 BD. Bachiller
( Z+ Z$ \ u4 x: Z* h T, ]' ]Caubet-Cimera Fundation, Centre for Advanced Respiratory: w+ z, g* b- e9 p
Medicine, Recinte Hospital Joan March,3 z1 N3 Y0 o9 Z5 B
Ctra Sóller km 12,( m9 h$ i: j" s- ?
07110 Bunyola, Illes Balears, Mallorca, Spain# Z6 N7 ~$ [! R8 q3 }' v
e-mail: telomerasi@yahoo.es
) h& [+ j' [, A, ?% W1 u+ V, `/ qM. V. Camarasa1 k/ Y& r+ K7 `, \& Y
e-mail: mavi.camarasa@caubet-cime ra.es1 `! t M1 m: N" H
D. R. Brison0 B9 w; _1 h! f: E7 p
North West Embryonic Stem Cell Centre, Faculty of Life Sciences,
5 U$ b9 p( e" L6 x3 C& KCore Technology Facility, University of Manchester,. n. o4 \9 R. t
46 Grafton Street, C- v8 Q+ F2 u& y( l9 u. A! l
Manchester M13 9NT, UK
( Z7 B8 c5 Y$ eD. Bachiller- c7 @: N- Y1 M9 z9 b; W; `$ D
Consejo Superior de Invest igaciones Científicas (CSIC),
/ p/ J7 Q" F/ V( A# o8 Y1 sMallorca, Spain
& |4 l2 c& I9 s7 l! V+ D; q& MStem Cell Rev and Rep
* |" t m4 A: k. N ~+ N; ADOI 10.1007/s12015-012-9377-4) S& n: P4 _- X1 U r
This protocol focuses on t he main first steps of the
) {- a! J+ X: A) n" Lderiv ation of hESC lines, i.e. embryo culture and gradi ng,
; n2 ?/ e7 }( ~; H) r- e+ Qand precise mani pulation of their ICMs and first outgr owths7 c9 ^2 ?& {% H6 l* `
of pluripot ent stem cell s. The Notes secti on includes alter-native procedu res as well as commenta ries and clarific ations5 P. D' W2 q6 h/ H5 f( P
on the techniques described in the Methods section. Assuming
2 J: D/ l( |: d4 Mexperience in mammalian basic tissue culture technique, this$ X8 W, [( ^& M% m
protocol should aid anyone wishing to derive and culture! P2 a( U2 |& }* G. C3 Q
hESCs to set up a rapid and reliable method. This protocol& r6 D5 a2 m9 F! V3 a' M+ q! q
reflects continuous work optimizing the derivation, culture3 s- Q! O+ |* Z& g$ n7 n1 h
and characterization of hESC lines over more than 10 years,- Y* F) B, O7 m7 e, L
comprising activities carried out in four different laboratories./ S: J" S( _) U* l h
Adva ntages of the present p rotocol over others previous-ly pub lished in clude: optimiz ation of f eede r cell yield,
7 ~ [, o7 Q9 W! ?6 B( S1 Cincrease of I CM cell production and adjustment of the
\4 g# {& y& F2 kderiv ation protoc ol accordi ng to embry o grade., \6 W: `% E5 R3 ?( y% U% ~5 P
Feeder cell yield is optimised by culturing tissue aggregates
5 V& i9 [* K: A* f* v, Auntil they grow as monolayers. Embryo culture is optimized M4 O+ T! x+ w, W
so that growth of the pluripotent cell population is favoured' H0 ^. v0 G: F3 o
over that of the non-pluripotent trophectoderm (TE) cells.$ ]6 b$ L; z' t' W) S" e& j# u- |
Finally, the fire pulling of glass Pasteur pipettes to form thin
" P1 S8 }7 p* Kopen and closed ends that match the size of the ICM or initial
% z0 x8 ^, a1 o- ]) ?outgrowth to isolate, increase effectiveness of mechanical
4 g$ l- ~; s6 [8 e' b" u% Q2 Fsplitting techniques.) G8 B! a7 \ x
Materi als
: Z( `( c+ r4 ~' dMate rials a nd Rea gents9 {. N* L5 Q3 I# A9 K/ b
1. Bacteriological Petri dishes, 100 mm Ø, Fisher Scientific
. b6 w4 I4 h) s3 O4 [#09-720-500.; A0 O) @/ b9 v- }7 N
2. Cen tre-wel l Organ Cultur e Di sh, 60 mm tissue culture/ U5 p. q3 p D) g" u2 l+ W" x& j
treat ed, BD #353037.
; N6 h& a8 z1 A, l; L, }$ t0 ~- F3 D3. Lon g glass Pasteu r pipet tes, Fisher # 1367820C .
9 n; `6 c5 t2 E) ]$ E4. Mr Frost y® Nalge ne, Sigm a #C1562-1E A.
5 `$ ]5 Z5 p9 \$ m* v4 P5. Se rological p ipette 1 ml, Cor ning #4485.
, m) O6 A5 k% K9 R3 `; I2 q* }6. Se rological p ipette 5 ml, Cor ning #4487.
6 X- t ~9 x' o9 k( e5 P8 p7. Se rological p ipette, 10 ml, Cor ning #4488.
4 q7 B2 G1 x- e% `8. Se rological p ipette, 25 ml, Cor ning #4489.' s- Q8 e2 C( Q) ?# q, l
9. Se rological p ipette, 50 ml, Cor ning #4490.
( ^/ O& Q, C) Y) [. z10. Tissu e culture dishes, 150 mm Ø, Cor ning #430599.# P9 ~! H3 {/ i$ y- ~0 L* e9 Y
11 . Tissue cult ure flas ks F1 00, 100 cm4 A8 }" t7 Y- M" t) i8 A. d, w* e
2
3 l8 A- M" u# u, y# u) s' W4 Y0 CCorning #3816 .' g& C2 t5 ]( o( e3 l$ b- G
12. Tissu e culture plates, 4-well , BD #353654.% p' F4 l4 U9 K) V
13. Tissu e culture plates, 6-well , Cor ning #3516.6 o8 Q- \4 Q$ ?" R
14. Basic Fibrob last Gro wth Factor, #45103P- 100.
4 R" G; S# `- S% g15. Beta-mercap toet anol, Sigm a #M-752 2.
3 B5 ~3 r m0 Y9 X. |% W3 d16. Collagen ase IV, Invit rogen #17104 -019." A$ w2 L+ W2 g' T9 V
17. DMEM medi um, Lonza #12-614 F.7 z1 S a' R' j% l% G( n/ z
18. DMSO, Sigma # D2650.3 {; q: b1 t/ Y q
19 . Fe tal bov in e serum (FB S) , Australia n origin Lo nza
" {& }7 B) F, a# 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
! J; o( B9 J) `4 ?+ y1 \) Lorigins a re US and US approved, followed by Au str a l i a n
0 \; I; T8 {2 F" @! J5 Q3 m& O8 {and Australian/USDA approved. Poor quality serum is a, I* e1 b! k( T
confirmed cause of feeder cell failure in hESC support.
X' R1 `4 C/ e; x- w5 `; t0 N7 y20. G1, Vitr olife #10128.
2 ~: F5 n8 q7 F9 Q21. G2, Vitr olife #10132.
! b V9 `9 i* G22. Gelatin, porci ne, Sigm a #G-1890.9 }, S4 ~: L- F; K: J
23. Knockout (KO) DM EM, Invitrogen #10829- 018.
, _+ T6 Q! s5 }24. Knockout serum replacement ( KO-SR), Invitrogen, i* u- {- x. u( z9 z
#10828- 028.
: D5 g) b( G% t- e, P0 u25. L-Glutam ine, Lonz a #17-605 E.5 V* R( W. T1 A" W' i( j! W
26. MEM Non-essential aminoacids (NEAA), L onza #
, L& T' [4 g O13-114E.1 I2 Z+ E( A0 Y) r$ ~
27. Mitomy cin C, Sigm a #M0503 . Hazard: this subst ance
' o* ]" t; w7 M# U3 h& }6 A9 bis very toxic and procedures for safe hand li ng and- @8 i$ b% g' ~ y" m- g
dispos al need to be in place. Do not brea the dust.0 c1 j& @1 {! Y' p& x5 l, U
Avoid contac t wi th eyes, on skin, on clot hing. Avoid
9 B2 K' {. W5 ^( R8 wprolonged or repeated exposur e.$ c; Q7 \ s6 k" | B, m) s g6 |
28. OVOIL, Vitrolife #10029.8 v+ n- Z1 t. s9 M
29. PBS with Ca3 V8 I! x5 e) j* t- C
+23 T& u& a! h) }: V# Z! u
/Mg! n+ e5 o% s& f$ x
+2
9 d# x" p$ f3 P" d8 p, Lonza #17-513 F.! O, j- [# w; i. a( j. f- i( d
30. PBS, without Ca) r" k' Q. [ }
+2' Y7 N+ h* f4 }9 Z( F& b
/Mg$ d/ l% U, k5 R
+2- c' u& m% X" d0 W" R; r2 G. D" q
, Lonza #BE17 -516F.
% V; f" l7 x, I/ l31. P re g na nt MF- 1, C F-1 o r MF-1 × CD- 1 fe ma le s, 1 2. 5 –13.55 D/ o' ?8 k$ O" y H" l9 ~( o
dp c, Charles River Laboratories.
0 _* Z/ [. ^8 o% w8 z W F32. Thawkit I, Vitrolife #10067.
5 Y9 V6 W) A6 w33. Trypsi n-EDTA, Cambre x # CC-5 012./ _- b) W9 m8 Q8 r2 B9 J
34. Tyrode ’s solut ion, acidic . Sigm a #T1788., s7 r% f( x- }1 }. @; u. L
Solut ions3 v0 M! ]2 k; A. m
1. Complete DMEM medium: DM EM plus 10 % v/v FBS,+ ]# B X3 I/ R. `" w4 _
1 % v/v L- Glutamine 200 mM, 1 % NEA A.6 `7 g7 t' a2 ~, r9 I8 I# h$ |
2. Freezing medium: 90 % v/v FBS plus 10 % v/v DMSO.
: T( |' j$ f/ p; y2 W1 f) g3. Gelatin solution: 0,1 % w/v gelatin in tissue culture grade
6 B, s& w3 k2 o; f5 [water. To avoid contaminants and residues (detergent,1 l7 M2 O% C* V; Q' D" s" z
traces of other chemical, etc.), use a new 500 ml Pyrex9 Z; M3 v% }* K, V8 ^
bottle. Autoclave the gelatin solution and store it at RT.
4 n9 w% N4 b" k; Q" Q4 K' R4. HE S m ed ium: K O - DME M supp lemen ted w ith 20 % K O-S R,
+ I0 g) s; I/ @: t2 mM L -Glutamine, 1× N EAA, 50 mMβ-Mercaptoethanol% g$ ] ~, C8 }* }
and 8 ng/ ml human basic fibroblast growth fact or." Y% \' |/ N, f) O6 s- z: l, ]
Methods& U" k& @3 v7 M
ESCs are derived from preimplantation stage embryos. The7 n3 F' j0 n v/ o2 ]7 x5 t
most common procedure involves culturing embryos to the5 d8 o3 ?+ C5 {% E, n0 j6 H
blastocyst stage (day five to seven after fertilization), isolating
- c7 ^1 g8 u) n, @: C8 @their ICMs, and culturing the latter on mitotically inactivated
# W1 H! M& X& c b: L: efibroblast cell layers. The resulting pluripotent cell popula-tions are selected by morphological and growth criteria. Novel
1 C/ H/ {+ H4 e% q* I3 }9 DhESC lines are then established after continuous subculture of$ L9 Z! Y: K: |
the initial outgrowth. A hESC population can be considered to
* x4 \9 O) [" k4 {3 J) i) q" sbe an established line when it reaches passage 8 with approx-imately 3 × 106 E0 G. N6 X, c) o0 @
79 O) l. e/ i, l/ F7 r$ @0 U; q2 l
cells, as defined by the UK National Clinical
* s* P1 d& P6 L0 @' T5 rhuman Embryonic Stem Cell Forum [ 14].: U, ]/ d# m6 @
Stem Cell Rev and Rep$ H, r: J6 r/ Q$ ~- G; n
After eight to ten co nsecutive p assages, the novel line is
/ Z- F! Z2 d2 l) r/ g! \5 ~' csufficiently expanded to check its viabi lity by freezi ng and4 N5 W' {) P* H
thawing .
- `6 \9 b+ J/ C+ _- s- r5 K5 BTa b l e1 defines the term‘one v olume’ for each of the culture
$ W, V0 N& R* g/ K$ V nv es se l s d es cr ib ed t hr o ug ho u t t he manuscript. All incubations are7 I4 \% z. f6 E! Z) z! |; U& u
performed in c ondition s o f 3 7° C, 95 % h um idity an d 5 % CO# N+ W/ g+ v. n' z) ^/ S+ U
2
6 r! h! H' i" |inair(SeeNote1).Figure 1 depi ct s t he expect ed time schedule
r: d+ h# K3 Q7 \8 T8 W8 Fto complet e a hESC d erivation p ro toco l, provide d that t he feeder9 F& {. g- \5 o
source has b ee n p revi ou sly v alidated.+ e: B0 V4 m6 M
Feeder Layer Preparat ion' I$ S' B+ t# V I: o- m
Primary cult ures of feeder cells have tended to be used for* |; s! w9 L; S$ e# Z, v. l
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
2 C8 B% F- c! wderivation can be performed on f eeders produced from
2 K. ], Q- P/ {estab lished cell lines, or prim ary feeder cells whi ch have' d: u- A, V7 |) S7 H
been immort alised. Altern atives to the use of prim ary mous e3 \7 q3 j2 V, O$ n
embr yonic fibr oblasts (pME Fs) as feeder layer s for hESC
: G2 K! q- Q' Q+ Y2 }6 Qculture are discussed in Not e 2.
+ V9 ]+ l( M: u5 R' O+ }0 nReg ardless of the type of feeders used, every feeder layer
4 Z; d9 h9 n! kmust be con ditioned before stem cells are seeded on top of9 q. p. \ @( N% @5 [
it. To do so, feeder cult ure medium is disca rded; feeders are
9 K. D9 k8 G% {: D9 K7 S' d0 C, b" Pthen washed three times with one volume of PBS each time9 f0 [) L0 P, j! t5 f$ V) @
and once with half a volume of stem cell medium (HES).
1 m0 r6 W5 n5 jPlates are ready for use, from 30 ′ to several hours afterwards.
7 P( y+ `. L" V9 LMEF Isol ation U: O' H$ Y4 x R( [
1. Sacrifice a pregnant female (12.5 –13.5 dpc) by cervical
" U3 P- T# A/ {4 W# T% w2 Odislocation. See Note 3 for tips on strain choice." b f, w% o3 |8 X `
2. Di ssect out the uterine horns and place them into a
( ?, Q7 f) `7 J: p" T. b10 0 mm Ø Petri dish containing one volume of ster ile/ C6 s) Z z2 k# j: c# i& P2 S, Z
PBS 1×. Transf er the plate to a laminar flow hood to; n5 d4 ?! H- S7 P
co ntinue with the procedu re.
7 S% s; s) ~# Z! r- [6 Z3. Cut open the whole length of the uteru s, so vite line: F( B8 \; H! |3 h/ b' c- ~9 m% Y$ x
sacs with embr yos and placentas are partially relea sed.( _: C1 @5 p* u; v. W9 X8 i4 ]0 k8 }
4. S e parate each embryo from its p lace nt a a nd out of the
% V" [8 S4 q3 W& e# ~ Gviteline sac and surrounding membranes, and transfer9 C3 V" G. w9 I2 e7 O) S2 _
them all to a ne w Petri dish with on e volume o f fresh PBS.
6 w2 u ^1 B5 w: W3 c M. T5. Wash the embryos and transfer them ag ain to a new
- i$ x8 U U; @/ zPe tri dish with one vo lume of fresh PBS.
, M3 s' ]! F/ `& n, i- t8 Z3 z6. Decapitate the embryos and separate the visceral tissues3 a: y( l6 x+ h0 p- M
from the bodies. Collect carcasses aside in the working
2 w; |; H" p: g, s# `2 R" Wplate. Aspirate and discard the PBS and the visceral
, t$ d& E* U6 F. R5 u2 \6 [, U" ]; @. a. \tissues carefully with a 10 ml serological pipette. Leave
3 g$ t4 I' A& _; N- N5 ?8 f7 R Tthe rest of the tissues in the plate.
: P% H) c ~) f" P7. Mince body walls into small pieces until a homogeneous* x) X+ a9 @) K! }* Z7 t0 L# [2 Z
suspension is formed." {4 R0 E3 G- s- h
8. Add 5 ml of trypsin-ED TA 1x to the plate and transfer
6 Y( k4 i0 _2 Eall contents to a conica l 50 ml tube.
& p' e4 \( e9 c# c. \/ p! z! V9. Incubat e diges tion at 37°C and 5 % CO7 S# P! D% F( w
2
( e- `6 s1 j* b. Zfor 30 min.
2 g* k0 k8 y4 ~Hom ogeniz e the cell suspension by adding 45 ml of U% ~! _3 i1 e. l
fresh cult ure medium and pipet ting up and down for
6 r* C/ F% o; \8 \. O, A, ?) S' Bten times, and cen trifuge to recover the cells (700 g for
: u+ d7 Q, @3 _( z2 [2 min )., d3 S# x1 w8 B' k! B8 p
10. Discard supern atant, ad d 50 ml of fresh culture medi a- C/ N( A! Z: V- v p9 l
and resus pend cells.
4 y( m8 [7 V& P# | w2 v11 . Cen trifuge the cells at 700 g for 2 min and disca rd+ a. \; G7 G7 \ g b
supern atant.
( l1 W8 B5 T- \3 K4 Y: N( p12. Resuspend t he cell s in 5 ml of complete D MEM
+ a& B) h! i, C' \4 U' Amedium.9 h4 p3 g; F' |9 R
13. Seed cell suspen sion at a rate of three embryos per9 J/ O5 I6 k+ [# H0 ~
F100 flask. (General ly four F100 will be set up at the+ r& | g+ q" e7 ]
be gi nn ing o f the procedure per dissec ted p regn ant+ k1 |( U i2 d& B {) V& j. b
female).
T5 k: d' k, s; }14. Incubate at 37°C and 5 % CO
0 |, Z" B0 [3 ]# E1 |26 Q- j* g# F4 _: E$ m
unti l 90 % confl uence0 d& {7 b1 ~& t; Y0 j
is reached (approx imately two to 4 days).
) ]9 ^; }' X3 t15. Split the cells twice by tryps inisati on, seeding them5 k+ V2 |/ {/ D5 _" e. m9 x4 i
onto n ew flasks/dish es be twee n 1:3 and 1:6 split
" W& |% y* B' S7 x: q% S/ Gaccording to growth rate.# U% s4 G: Q! o0 k5 j
After t wo passages , t he ce lls s hould be growing as
" r- `4 o! w1 M8 fmonolayers. Freeze 90 % of p assage 2 c el ls in vi als
/ a! V: {0 `1 F, L) Hcont aining four to ten millions units each. The frozen
, `: z1 V: x9 jcells wi ll const it u te a s tock of act ive pMEFs for fut ure
3 ~8 z" F# g* Z9 fuse.
' U$ F. _4 L, U2 }To continue with the production of inactivated feeder8 P i" j; o- N0 y
cells, expand the remaining 10 % of the original culture
2 J- J h& B1 x8 u$ K" Cup to pa ssa ge fou r. S ee d t he cel ls on to new f las ks/
c, g& P/ |+ _6 N2 {9 Xplates bet ween 1 :3 and 1:6 spli t according to grow th
$ z/ @! z2 M7 p F4 urate each pa ssage. Then inac tiva te an d freeze passag e* F' X& d+ g& S; e0 Y( j* A
four cells as explained below. See Note 4 for tips on
, n% H$ D# u4 S& x" a |. j. Dthese st eps.
# M* ]* N. f$ j) B4 i, [Gene ration of Batches of Frozen Feeder Cells by Mit omycin
l% `- j h$ w& G9 V9 T* o3 Y) ]C Inact ivation
( g) T$ H; U) a9 P* _: C1. Tr eat 80 % confluent, exponentially growing, L0 ~, p: c* U9 V, I2 i
pa ssage four pMEF populat ions (between 48 and
$ y- h- U$ a2 {- ^' N8 w% Q9 6 P150 plates), with 20 ml of 10 μg/ml Mitomycin C in
, U5 I Z, R/ X( |7 Ncomplete DMEM medi um, f or 2– 3 h at 37°C a nd5 Q3 `0 m! H* q/ o$ r; m
5% CO2" ^% _: ]+ Y$ A. ?% b1 M
.
; G+ v2 f% W- Z1 {Table 1 Definition of the vessels and volumes used throughout culture
% _, K7 i% w$ U' ^6 h' `+ Iprocedures1 s) i: e$ x- r/ R
Dish/plate Area cm
- C1 R' C# \- ^# _2
. h$ l3 u* M, }/well One Volume ml/well6 v4 \, a7 e p* s8 r+ f7 o- C6 g6 w
4-well plate 1.4 0,5& d! r( d: d7 `2 y
24 well plate 2.0 1
p% a/ p; W8 o, _+ POne-well 60 mm plate 2. 9 1
: Q* b& E9 @$ T) [9 V6-well plate 9.5 3- I6 D( }8 ~5 _, l8 ]' a
60 mm Ø dish 21.3 5% l; ~$ H }# X9 {0 S. Y7 e+ K
100 mm Ø dish 58.1 20
! a/ P$ ^8 h7 W; SF100 flask 100.0 25
, k8 A2 p8 y7 O6 z) I% w: d150 mm Ø dish 176.7 50
, s+ b0 b( C6 A W/ ?" ZStem Cell Rev and Rep& R2 Y1 m1 ?- n8 H4 O) _7 E
2. Aft er inactivation, disca rd the supern atant and wash
u. X4 \7 S2 v/ |3 b" a a. Zcell m onolayers 3 times w ith on e volume o f PBS
6 @6 W: Z5 G8 V0 u3 b& K4 r7 U5 @: Qwi thout Ca
6 Q' P: N9 o' J6 z" J1 b+2
" {3 s( p2 R0 r0 I9 x0 s/Mg4 c) U4 C D/ y( R6 Y
+2: ^/ v5 m+ ^% _% v
.4 A' t3 V& f0 w; L2 I3 E
3. Trypsi nise the cell s (2 ml per F100 flask or 3 ml per u1 d8 y7 A% |: r9 B
P1 50 p late) for 2 – 7 min at room temperat ure (RT) .
! ?' X3 M/ P: H% a. C( ?1 ?4. Tap the sides of the flasks/dishes to dislodge the cells,
) D3 c/ U( M( a- X Ban d harves t them into 50 ml conica l centr ifuge tubes .
( I, T% o. w; i# v, KPo ol the contents of tw o to three P150s in one tube.7 [) b1 {+ e8 l) N, y4 Q
5. Add 44 ml of compl ete medi um per tube.- o4 A7 T T: \- g/ m8 m# w$ T
6. Cen trifuge tubes at 700 g for 2 min .
2 l8 W4 A6 i8 Z4 }7. Br eak pellet manually by finger vortexing (see Note 5).
3 j% p% v2 X* D. V3 mResuspend each pellet in 30 ml of complete DMEM and
# D! r% L$ V9 b4 w" [count in a Neubauer chamber.
1 q2 ^0 L4 ~- h' S: V* u, N% I8. Co unt v ia ble ce lls and calcu la te the numbe r of
: B0 Q: ^0 l) M0 V/ xcryovi als needed, at a rate of 1– 4 million cells per vial.
9 W0 \! D; K4 D8 `2 A) ]- T9. Cen trifuge cell suspen sions at 700 g durin g 2 min ./ ^3 q) H% r6 |. X6 [7 ]
10. Label cryovi als with batch numbe r, amoun t of cells( Q* W; T9 `& ]7 e
and met hod of inact ivation, date and user ID.
0 ^: f) J0 q' k& G e% W8 ]11 . Discard s upernatants from s tep 9 above and break
$ h9 f* z1 R8 \pell ets carefully by finger vortexing.1 M; Y# C* A/ J# U/ f
12. A d d c ol d f reezing m edium to the pel let s a t 0,5 m l per v ial.. V( } H6 ~1 V$ w
Disp ense cell s uspension i nto labelled cryovials and# ?( e( r+ e5 g( t$ Y
transfer them immedi ately to a precooled Mr Frost y® con-tainer, and then into an − 80°C freezer (see Note 6).' X0 t* H0 D- d2 ^1 z
Feede r Layer Plating for IC M Culture3 h3 t% {' |5 l2 [4 x3 M! }
1. Dispense one volume of gelatin 0.1 % ont o the
2 k5 B" ?" r% U7 s8 L5 ?7 s" ?/ \) C: Qrequi red number of dishe s/wells, and incuba te them
6 V0 H, B* `. P6 Fat 37 °C for 30 min.# L. Q1 A8 q- ^/ j/ U# q# u" {* _
2. Equ ilibrat e 10 ml of fresh compl ete DM EM medium,
* m( k3 U7 k% f) U: P% i: ]in a 15 ml conical tube, in the CO. Q* o: s! K3 S2 i, `3 I4 b( b
2: }1 ~( I+ X# A5 Z$ i3 \0 R- j/ I( \0 A
incuba tor per each
+ f% x3 ]9 `; O1 – 2 vial (s) to be thawe d (see Note 7).6 _% j2 f _3 m0 t Y% y
3. Once the coated dishe s and the medi a are equil ibrated,
! B% Q ]+ N& q6 c& D1 i: s8 h6 uthaw the requi red number of cryovi als to plate 0.2– 0.36 g. U' ~$ R2 j# t( I. M7 c
mil lions of inactivat ed pMEFs per one-well 60 mm* F1 s6 F+ t) T1 r7 M2 ]! t
dish. Spray the frozen vials with 70 % ethano l. Roll
+ a5 J/ s* e# W/ [them in your hands, until only the last smal l piece of
1 B. O( v& p* Z+ O wice remains. Tr ansfer the c ontent of the vi als i nto: ~) n/ p; s+ ^+ Z8 \, V3 Z- H
the equilibrated compl ete D MEM. In order t o
3 X" \" l9 G$ Gavoid cell dam age i t is n ot advi sed t o thaw m or e* X3 {! ?' Y) }- h. y+ t, I
than four vials at a time.& g7 G7 ~: P2 R) O+ U+ f
4. Cen trifuge the tubes at 700 g for 2 min . Discard the
9 ~, |$ a9 }; v. Bs upern atant a nd gently break the pellet b y f in ger, r) R ^6 A) Z, C: N+ e6 o* |; ?
vo rtexing., z$ a" ^, N. d& x
5. In order to elimin ate resi dual DM SO, repeat step 4.
+ {" o0 m* p* ?' R H6. Dur ing the second centr ifuga tion, aspir ate the gelatin& _5 @3 x7 W8 b- M
solut ion from the plate(s) and dispe nse half a vo lume
& h5 G3 M: I& z8 @# Zof prew armed compl ete DM EM medium on each one.
- M q; N8 t" {7. Br eak pellets from step 5 above by finge r vortexing,1 K0 M) [' h) v6 D% d8 X
an d resus pend them in half a volum e of the wel l(s) to3 P/ k, N% R9 @
plat e.0 F) t; X# _3 i' p/ _- U
8. Di spense cells dropw ise onto the medium- containing
) s% v9 h) j+ t7 a* Rwel l(s).
# e; E0 N/ e: K, }! M9. Place plate(s) at 5 % CO* m) G& M- Z& _9 \9 {2 D
2, h! n3 _% d- ?5 c5 _
at 37°C, and c arefully shake3 ?2 w7 s4 w8 K; |) J3 }$ U5 z
them horizontal ly in all direction s to distrib ute the cells
4 H: Z0 H3 W# x; wev enly.; w e1 w! Q% e2 b) w
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
) m0 c$ _: k# R* k: M0 I- H6 Eattach. Optimal feeder density corresponds to a conflu-ent monolayer; higher and lower densities will result
3 j- N, Z$ v, J' seither in reduced hESC growth and increased differenti-ation, respectively.
- x8 Y: f8 M% t+ P1 i2 W( {/ Y, P7 pSe e Not e 8 for comments on feeder layer prepar ation.! ^8 H- W! y( o+ l1 m$ @, f
Embryo Culture
o8 s4 T- r+ uSurplu s embryos from clin ical in vitro fertilizati on (IVF)
/ S. w2 r$ I0 [: f" D5 Q Kprocedu res can be supplied, fresh or frozen , at any stage
' ~4 {6 w$ w1 U( b! gfrom day one to day six post- fertilizati on. It is firmly estab-lished now that low-grad e blast ocysts produce h ESC lines
( q2 w. _ R) C, |succes sfully [ 15, 16]. The refor e, the derivatio n of stem cell
% C' F( J( t `, y7 R& O& Ilines is almost guaranteed for a ny Re search Pr ogramme
4 r! ~& P/ U9 W" A( vestab lished in coll aboration with an IVF clin ical depart ment.- {# ]: r v: D- k
In this section of the proto col we will detai l the steps7 z! y2 V0 _/ R
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! W9 C' s% P) B# y
that are used in the clinical procedu res. Altern ativ ely, it is; y; n* e; N2 l3 V, ]
sim ple to p repa re the m from thei r compo nents [ 17]. Se e! g' Q' j: u5 O" E {5 @+ o; m& o
Note 9 for tips on embr yo cult ure.
$ L. p8 D5 P4 ^' B* K' @8 F$ SWe will detail Vitrolife® protocols, and advi se to fol low
% z7 K W6 y! @4 g& k) L6 zmanufacturer instructions in ca se of different brand reagents.
( Q) e/ |6 o# ?& P6 I$ z& cThe cul ture system us ed was a two-stage one, i .e. changing7 @5 ]' M, @) f: ~8 e: i- f
cul tur e m edia formulati on on day 3 or s ix-eight cell stage o f
: e$ T! `: K) H, s8 fdevelopment (from G1 to G2 series ), un til blastocyst formation .! b4 B! u0 ^% ?% \
Fig. 1 Time schedule for the
& X4 O) Y7 n( k% O- j1 S3 S- Gcompletion of a full derivation
3 g+ e, C: d8 h' ^, fexperiment/ [# Z$ z# E v+ l# y
Stem Cell Rev and Rep' j1 i3 v9 o/ a
Embryos are cultured in m icrodrops under oil, to: M' d! S6 W# O
prevent evaporation a nd to minimise pH changes. The
0 ` t. ^9 a( }8 r0 a3 Gfollowing protocol corresponds to t he culture of fresh or
7 ?' _* h# W) }& _2 ?: v Z# Ethawed cl eavage-stage embr yos. In the ca se of receiving
- Z: @5 \1 B+ Jfrozen embryos, it is crucial that the thaw ing i s per-formed in the reagents corresponding to their freezing.
7 F9 I" h5 j8 D" N# I& Q: jFollow manual instructions from the appropriate thaw-ing kit. After thaw ing, follow the protocol below for
! G- Y0 l7 b5 V4 R) k6 ]* Wembryo culture.
$ Q9 H: l2 f- P+ m) I+ R3 L0 ?1. Equilibrat e OVOIL® was hed wi th 1:10 volume of G1 or2 u1 V7 C. Y% Z1 \: j E
G2 for 6 h to overni ght at 37°C and 5 % CO2( w6 X2 Q7 A( `3 a
.$ X6 ^" x0 N# o
2. Equilibrat e G1 or G2 medi a as required (see Tab le 2 ) for# ^5 P0 t/ x8 `* H! [6 o- V! h
a maxi mum of 3 h.& l5 K9 |1 V0 B" X8 U
3. To set up embr yo cult ure plates, pipet te 25μ l of the1 d$ R' U! ]1 G% y( l
equilibra ted G1 or G2 media per embr yo on embr yo+ t7 J: M( q* H
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& A3 I+ h/ ]8 B! z! B8 O
formed drop(s).
4 |9 K2 y' H, \- j1 ]" B4. Assess embryo development daily (see Fig. 2 for a9 S& l6 \/ [) p/ C/ s3 C
di agram of g rowth pattern and c orresponding images7 ~" @4 P; U( W; N
of embryo development), and transfer them to fresh6 {5 M# m0 B1 N$ A
media d rops every secon d day (see Ta ble 2 for
$ N) K5 N8 ^/ b7 O9 h9 ?# u% kmedia c hange guideline). P re pare new drops as% Z, X/ S4 d2 B' _0 I
describedinsteps 1to 3above.If embryoculture
# }, c2 w3 E% W. Zstar ted i n G1, remember that once embryos r each
0 P) {' n* |% ~* zda y 3 o r the six-eigh t-cell stage, they must be# J' T7 p9 e3 ~: K
cultured in G2 medium., M+ v! E# @4 K; K
5. When embr yos reach da y six of develo pment , or the# ]! w" W! e J9 z b) u# i9 f
ICM of the blast ocyst is visible, it is time to set up the
& E# z! }6 w" l! r! x) ^' @2 w" e: vfeeder plat es to seed the former.! V. B, I( Z- S( e! t6 e
ICM Isolatio n an d Se eding
" _. y) L% _' p$ _0 C+ j- ?During denudation and ICM isolation i t is a dvised to5 P9 H$ M2 j" E* [! ]
process each em bryo indivi dually. Pulled glass Pasteur
4 C" e- X2 \" `4 epipettes 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
9 Y4 Y3 g& Q/ _5 h# CIVF p rocedur es.
7 H0 x5 }# B. h- @Con dition the feeders before the d enudation as follows:3 g- n0 G# {; y8 S" g# `
between 1 and 3 h before the seeding e liminate f eeder
+ {6 ?6 X& Y+ |2 {. omediu m. Wash three times wi th 1 ml of PBS with Ca+ Y1 q+ w* g& _5 T% y. d
+2# b" Z3 o+ C: z. @
/
3 p1 T1 e$ P& R# o5 E& VMg% `# {' H3 ^/ L) C9 l' F
+21 a8 ?9 W6 p) D- u- x
and once with 0.25 ml of HES medium. Add 0.5 ml of
; B/ a4 q1 @. R6 qHES medium and equilibra te for at least 20 min at 37°C and) `5 m. t1 u2 d4 ?! O+ j9 p
5%CO e8 s2 r9 ]% k: [
2
6 t; R+ I9 e) D$ x. At this point the new feeders are ready to recei ve
+ [2 h. {7 M; t+ n8 ~/ Q/ g/ hthe IC M.
6 T7 e/ b2 o D1 kZona Pelluc ida Remova l
. f: x' j& L, N6 `% [+ q/ R. x1. Equ ilibrat e separa tely ac id tyrod e’s (AT) 1×, G2 me-dium and HES medium at 37°C and 5 % CO
" v* [' T5 {; B' T. b2- v$ I7 z( M' i
, for a
& L5 o* g* \3 U4 c& X8 N* jmin imum of 20 min and a maximum of 6 h.
/ l' ^" L* E$ ^; C/ d; O6 {2. Bef ore any mani pulation is perfor med, images of the- F2 p+ R0 R4 s- {2 F7 w! M' [
blast ocysts at different planes (×40) should be taken. h x* i) b; N9 k8 E7 {, h
for further asses smen t of its grade. Afterw ards return it5 J- ^# N! }) y0 c
to the incuba tor.
- F( r! {1 A c ^ i3. Pu ll 2 long ster ile glass Pasteur pipet tes wi th the aid of
* S' `' L0 z C& Oa Mecker or Bunsen burner. Leave one of them opened7 d( A+ K2 c% R9 m
an d rounded , and close the other one leaving a small1 x) u# w# T8 @+ E
rounded ball at the tip (Fig.4 ). Check the thickness of
7 u( d! r6 T0 v( ^5 pthe tips under the s tereomicroscope and r epeat the# t5 q7 Q7 o3 \2 |2 L& H- E
procedure unt il a couple of pipettes whose sect ion3 F$ h8 t0 W% J+ P2 v' P( Z( O* c
mat ch the diam eter of the blastocyst are produce d.
, }! n' n& B) e ?: u$ x4. Se t three drops of AT forming a row in a 60 mm tissue
! B, I; c0 _8 z. jcu lture dish, an d mark thei r position at the rim of the
; p' |; s8 v- U9 md 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 a0 F7 b g, p: x G6 ]6 d
second row at the middle of the dish, and three drops
- _4 ]6 _# |# z t4 e% b5 Hof HES media at the bott om, again aligned in a row( ~$ {4 b: m6 t1 h
(S ee Fig. 3 for drop distrib ution).0 y `, n9 m0 B: u" }/ ~
5. Se t anothe r three drops of approximat ely 100 μ lof: u {' w$ g& E& M& f6 ~ p
HES medi um in a new 60 mm dish.* P3 X: U! k, J# J3 u9 A' `
6. PicktheembryoupinG2 mediumwitha 275 μ m
: l- Y* B( z2 k" z8 xcapil lary mi cropipette, a nd t ransfer it to the left AT
, A, ]5 J" M7 _6 C {0 A bdrop of the plate prepared in step 5 a bove. Stop' u$ G' i- l3 m" s1 l$ C
dispensing G2 medium into the AT drop a s soon
6 m W& }$ Z+ c$ d% F1 v& r# \as the embryo has been releas ed. Empty the pipette9 W" X+ P1 ^, d' j V* [
in the border of t he dish to discard r esidual G2& U1 G E/ J* H4 m' |% E
medi um.& [+ b, m+ u) ?) R% W3 h: F
7. Wash the pipette twice in the second AT drop.
5 s. F8 p0 a1 `3 D' b5 s8. Return to the fi rst drop, pi ck u p the embryo and `2 O: [* z1 t& h4 h9 q
trans fer it to the third d rop. Monitor unde r the micro-scope the dissolution of the ZP (0 – 2 min).% U( X) e! \' x" j
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$ d0 C- M7 c) i8 [
pre-w ash, dispe nsing the minim um amoun t of AT into3 s' r; `2 Q1 h4 R
the G2, the second to load the pipet te with fresh G2
8 e' M/ X6 j' G3 n4 ^; h2 ~medi um, and the third to was h the blastocyst .
1 A) h0 O6 U2 c9 R10. Tr ansfer the zona-free (denuded) blastocyst to the
4 r: Q7 j; q; I$ Fdrops of the HES medi a using the washing procedu re
: f( s4 }4 ?3 J7 u+ Tas described above for G2.
; v3 i2 z- v* b8 `3 \/ A+ K' Q11 . Immedi ately trans fer the blas tocyst to the drops of ~; l8 i/ }: w( i
the HES medi a prepared i n a sepa rate plate in s tep5 R" ^. ?6 c7 Z7 N3 @6 _3 x
2. Follow the s ame procedure of trans ferring to the( |( B( L' E3 W j/ |0 \8 E
left drop, washing t he pipette and loading i t in the
L' b# @& s7 x5 e0 Wsecond intermediate drop and transferring the9 z$ I0 q( m# J4 _- [% E+ N1 X
embryo t o the third drop. P l a ce the p lat e i n to the
. v* d o! I7 q1 O+ YCO
, ?1 o$ \6 Y6 n* i2
. V' U5 h; S. Iincubator.
0 o* f- k8 p$ s2 P- d( h8 d12. Usin g the ima ges take n in step 2 above, asses s the
) q: K6 h0 V9 @" jderiv ation grade of the denuded blastoc yst (see em-bryo grading s cheme in r eference [18]) and a ppl y
' Z/ u: e" f3 X4 H- hTable 3 for determin ing the best met hod of ICM iso-lation. Depen ding of the outcom e, g o to protocol A or
+ Z a) J6 p# rB be l o w. S e e No te 10 fo r c om me nt s on t h es e# g/ ?; H: K/ Y {) l
procedu res.7 P( e8 H! M. I4 A0 |5 m% B H
Stem Cell Rev and Rep% Y% g5 {! P' s0 D. B
ICM Seedin g by Pipett ing (A)) K2 q! s3 z- k5 X2 D" Q8 j- G* f, ~1 i
1. Choose an open pulled long Pasteur pipette of the same
: ]' b D$ j Q) V* _ g/ e9 w) Sdiameter as the denuded blastocyst (see shape in Fig. 4a ).' V/ F4 H+ r; b9 u& h
2. Load half of the tip with fresh war m HES medi um from
( I, e" v9 P$ Y% Sthe drops set up to equil ibrate before denudat ion (ste p 5" q: W5 [# L) {9 f$ i
on zona pell ucida remo val proto col).& ^) \$ {: b4 u! O) d1 H0 g' |0 E; }
3. Aspirate the denuded embr yo and pipette it in and out$ i* \7 ?! `) x" x& p- p% }9 F
while checki ng for loose ning of the TE cells.+ ^: i6 q, j" {# B2 d1 m
4. Aspirate the i solated cell clumps and dispense them
9 t" M& A; I: L2 g3 X" tsepara tely on the feeder plate.
* N( ?6 Y/ A! I5 k5. Ta ke an image of the seeded fragments of the bl as-tocyst. Pay attention to l ocate the ICM and record" d) z+ ~3 ^! E$ Z* s) s
its posi ti on.
4 F% p- W% \2 J9 [( y- u* FTable 2 Embryo culture: I6 q1 [3 A. ^: A8 z# O7 g! n1 w/ _
protocol3 K* @( Q4 H9 r1 t" o k
Day of development Stage Medium Image recording Media change
0 N& I4 G- v" b! B( U1 Zygote (2pn) G1 − G1; ]4 M! Z( H8 J1 u- R/ |0 {0 W6 `! b- l; e
22– 4 cells G1 Yes −
u$ [* N0 B/ P6 l36– 8 cells G2 Yes G1 to G23 K. B8 q3 S) R. b: F& Y/ g: B
4 Compacting morula G2 Yes −6 p7 K2 s) d5 Y0 z8 Y# O! a7 d
5 Expanding G2 − Fresh G2* d6 z- ?& Q& k$ b
6 Blastocyst G2 Yes −
; J+ p5 e& h) Q' |2 _6 e7 Hatching G2 Yes −
# B4 l6 H6 M$ Y! N3 g8 K+ YFig. 2 Dagram of early human4 T8 C- D+ p$ s& W+ C
embryo development. a-a’4 y$ ]6 d. b% ]) z$ g1 T
two-cell stage embryo, 1 day( H4 y& {- r7 d, ^: D2 O
after fertilization; b-b’ four-cell
+ ~+ P! |6 F2 `5 m/ s: xstage embryo, 2 days after
2 i( S6 b0 b7 b- |5 t- Ffertilization; c-c’ eight-cell
2 \) E3 R. R% p! f3 Ostage embryo, 3 days after f0 `3 p j9 z8 c& e
ertilization; d e a rl y e xp an di ng
# J) @1 M1 L! g; X9 T- R" k# iblastocyst;d’ morula containing8 o/ Z1 Q4 q V) N/ h5 r' f+ Q
m o re th an 1 6 ce ll s, 4 d ay s a f t er# D" ^% H {, N8 Z/ f
fertilization; e early blastocyst,
T. x' S! b" U' R2 q5 V3 d+ H$ m5 days after fertilization; f
i$ m" h+ y( I7 Hhatching blastocyst, around6 d8 I; w" ~6 x, o& N: ~; i$ p
7 days after fertilization. ZP:3 a& G/ c s& i/ {3 u
Zona Pellucida. ICM: Inner
5 n1 C! H+ o0 SCell Mass. TE:! N% e# c4 Q7 X4 V
Trophectoderm$ F: X& S; u) Y- h: a6 F8 I
Stem Cell Rev and Rep! y' _! ]2 x4 N8 g, r) m
6. Tr ansfer the plate to the incubator, avoiding abrupt8 T2 p) J$ t0 f% D9 G8 Q
movements t hat may separate the s eeded cells from
& ], c8 w0 Q1 R' e% ~5 P* B1 ?the feeders.% i) o- f8 k# w# |* f1 o
7. Inspect the plat es daily. Cha nge the medium every other# }4 P* e; M& d7 g/ m, b
day.
, \9 D! J- V4 }( F' f$ C! r( D8. Prepare new feeder cell plates as soon as pluripo tent1 X \4 T R& x( ~# m" z( _
growth is detected (see Not e 11 for deriva tion timelines q8 u# V0 U- s; ?
and Fig. 5 for ima ges on deriv ation progre ss).
/ ], P3 Q+ C& C6 w9. Split part of the emerg ing pluripotent colony when b1 M& N1 Y& H& f
resi dual TE cells begin detach ing from the bottom of- d* U4 i' e$ S1 r B
the plate. Follo w Secti on 4 below to do so.* C' C- E5 G/ n! o! ^- N9 l
ICM Seedin g by Mechan ical Cut ting (B)+ X' N. G! m) a1 u0 }7 H
1. Aspira te the denuded blastocyst w ith a pulled open
$ S8 Y1 G7 w4 n5 S, U- Jpipette (Fig. 4b) and dispe nse drops of approxi mately3 O- `, S5 T! U0 c2 K( D
20μ l on the dry area of the 60 mm dish containing fresh5 m9 z1 g( S0 a' u5 q
HES drops, until the blasto cyst is dispe nsed in one of% J( D3 l3 z+ b9 m! B3 U
them., y% u; T) r/ n( a5 f
2. Drag the denuded blastocy st across the dish, away from" Z+ e' h9 [5 q
the HES drops. The volume of the embr yo-conta ining
7 |. o4 A. x& G6 a7 D4 Edrop will diminish up to the point that the embryo& U6 x% Y' P; j+ D7 n0 U
flattens and sticks to the plastic. At this point you shoul d
1 D! M `; U1 v6 h" M1 pbe able to locat e the IC M: it is much brigh ter and less0 G: T3 n' q! ]* ]+ o
flat tened than the dim TE. Sa ve the pipett e without
* \. d! W4 V1 k" R' ?emptying it for follow ing cutt ing steps .
1 G5 g$ j7 \) J9 @6 t3. Pick a close d pulled (hold ing) pipette (Fig.4c ) with the
1 y9 B2 c5 I& P1 c7 d7 n5 e( ~$ [left hand (rig ht for left-hande d) and hold TE by pressing
5 H( k+ n; l) K- \/ U0 Iits border agains t the plate.
& a$ H% Z0 Q) B- {2 |; q6 F+ H. L4. Pick the open (cutting) pipet te still contain ing medium0 _3 \5 S& R% w& W0 u* A
with the right hand (left for left-hande d). With its edge,
, j6 d" w1 z2 d% \ |4 H0 @1 c0 ucut out as much transparent TE as possible. Imm ediately- C7 ]; U3 }9 B/ n
release the HES medi um to a void damag ing the ICM.: I8 |! A/ a7 k Q+ P9 y/ Z+ r) T- L
5. Scrape loose the ICM contai ning piece, which will be
$ w, J3 n8 h5 p5 o6 B" Qadhered t o t he plastic. M onito r the ICM w hile it
5 u' E9 r+ b0 @; Y4 mFig. 3 Shapes of long glass pipette tips for ICM and stem cell colony
1 R6 k- Q$ w: b8 d7 [) x7 Q; z$ o8 vprocessing. a transferring pipette with open rounded end; b cutting/ X8 ]/ s- U+ K- Y* l6 X* B, w; u
pipette with open bevelled end; c holding pipette with a fine rounded$ V- K9 Q& N2 i6 b
closed end; d curved hook with closed end for outgrowth scraping;e-g# d) A- r% H& p7 a1 \
flat hooks for colony cutting and scraping. h detail of the tip of the
, D) l' \ N1 R9 b, gholding pipette depicted in C
0 a. K0 s" v& O) t MTable 3 Choice of method for ICM isolation according to blastocyst7 f; E {! y# S' a& G9 }
grade
) I; q) r# S( Q4 s/ {6 [1 {. MICM0123$ J# W" e: v" e+ X5 o. ^. T4 C0 `
TE
4 _$ _) W% I" D$ }0AA 1 A A A/B
5 S4 ?3 S: i+ ?+ k2ABB 3 ABBB
- c, H, O! w9 T; v' K# |0 aTE score - 0: no cell layer formed. 1: few big cells forming a loose7 p$ c& T+ M3 f/ O/ v" P' M& E
layer. 2: cells forming a medium coherent layer. 3: many cells in a tight
# D2 e5 v+ X: N5 F% g R" Rcellular layer. ICM score - 0: no visible inner cell mass, inner to the TE
A9 o) F G, cin any plane of the blastocyst. 1: ICM with less than 10 compacted% k' l7 I l1 X+ w5 e
cells, or loosened mass with up to 20 cells in the central focused plane& p" b$ ~6 ]3 G4 z1 n
of the blastocyst. 2: ICM with more than 10 compacted cells, or more! {) s# e8 E! x; @5 F: |, u9 E4 u
than 20 loosened cells in the central focused plane of the blastocyst0 X9 M% S% r2 E0 f# g8 j# E9 O
under the stereomicroscope. 3: ICM with more than 20 compacted. t* o/ M; N& ~( d: G5 J3 m% X" w
cells, or more than 25 loosened cells in the central focused plane of the
# z. D; q: @, D: i8 r% F0 |/ Pblastocyst. Method A refers to pipetting for ICM isolation. Method B
7 z! V2 P3 X6 [consists of mechanical cutting of the ICM
# L' C$ e" D2 A. c4 H* mFig. 4 Derivation plate. Embryos are dispensed in AT in drop 1, and
" D1 P% b7 X k- @# M! mtr 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
; T+ l+ I) m. x/ ^Tyrodes. G2: embryo culture medium. HES: Human Embryonic Stem
5 l# m6 }( X/ o @' G4 Icell medium
+ A5 M# p* \. Z* W- PStem Cell Rev and Rep
. V5 ]9 {$ b' ]8 M, R( P/ {. q! u$ jdetaches and float s. Qui ckly aspir ate medium from the
1 ]( ?) z N uHES 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
2 h) e# i. O# l) s" T7 |/ a3 noccurred durin g cutting.! `$ s" Q9 U1 X
6. Aspirate the floating piece( s) and spread them onto the
7 C* V! W6 m/ v; c. h0 @feeder dish. Try to avoid pure TE fragments , recogni zed
& ?# R. ]! w3 J* O K$ j- _. pby thei r trans parency.3 O# r. u3 Z# Y& z! H$ ^
7. Take an ima ge of the fragments seeded, record ing the
; C% z% ?- D1 D: Fposition of the IC M piece on the dish.
. m2 }- y7 `& u! m8. Inspect plates dail y, feedi ng them each second day until' r2 [1 G4 q, A7 J% T6 z
pluripot ent cell grow th is obv ious.6 X% V0 _% u! T8 P8 M- O
9. Prepare new feeder cell plates as soon as pluripo tent) T9 p' D( v. t7 W: m: u+ R
growth is detected (see Not e 11 for deriva tion timelines
) o$ J% d# b2 j9 ^# ]and Fig. 5 for images on deriv ation progre ss). Go to2 E+ c$ M5 n: @- i. W! D
Section 4 below for the firs t spli t of the pluripot ent cells.
' G! q+ Q9 N* W0 L1 Z( wSplit of First Pluripoten t Out growth% p9 a; _% S( a2 H5 F* B: H
When the pluripot ent outgr owth(s) reach the stage de picted
4 T" e; p+ L/ D' i$ ]/ [in Fig. 5c , they are ready to b e split. Mechan ical cutting can
" M" x7 b3 O7 W ^0 L" ]+ Dbe perfor med wi th a glass pipette of any of the shapes A, D,2 @3 k1 w7 ]; Z4 a: V9 `
E or F depicted in Fig.4 . In a ny case, prepar e feeder plates8 u% _9 K4 ]8 |5 i7 C
the day before splitting. If t he pluripotent outgrowth is
; P) y; Z4 N) r7 t1 x# H( oisolated from the residual TE, a closed p ipette and a
~8 t, q, j: a2 cscrapin g met hod could be more effective. If stem cells are: q# k4 b6 w+ \4 A0 P' D; L: a
growing in clumps surro unded by TE, a n o pen pipette to cut" N: V5 S1 \+ K, i& ?- L. y
some of them will be preferred. It is wise to leave some- Y3 v, A1 _6 j3 ~% r% w
outgrowth(s) behind, u nt il split cells ar e s uccessfully
% U; H" {) C5 Q! }( l2 }. f; bgrowing in the new feeder dish.
- h, n% W- j" V' i9 F3 xNotes3 f, E/ {2 A6 Z: t9 S+ G5 p
Isolatio n and culture of mous e embr yonic fibroblas ts and2 f5 f! Z# o7 l3 W7 V
feeder preparatio n
. |% z' P. I$ T- A. z/ jNote 1. We describe here the deriv ation of hESC line s in+ y4 z9 [( W" f3 d: u% y' f
atmospher 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
! x. t e" H. p5 T' defficiency in stem cell deriv ation and lack of chro-mosome X inact ivation in hESC line s estab lished+ M4 Z# {6 c' S p$ C
under such condition s [19].
; M2 Q% v) t5 N" q' {Note 2. Pr imary MEFs a re the first cells to be us ed as
0 G. g( A' T I# ^% vfeeder layers for hESC culture. Alternatively,
: o, S! \( B( Qeither transfor med [ 20] or n aturally immortal-ised [ 21] M EFs, as well as human fibroblasts& p2 t4 A4 I! q( o
from several origins, includi ng foreskin [ 22]
' Z! M! z: C' h% tand placenta primary cells [8 ]andanimmor-talised placental f ibroblast cell l ine [23]have7 ^5 B2 f- `% u* k q6 C
be en u sed su cce ssfully as fee ders f or hESC* ]- a0 t3 V8 ?# T. u. W5 z+ x: g
deri vation and cul ture. Primary lines f rom hu-man f oreskin f ibroblasts (HFF-1) have al so
2 \' S3 h' e% {1 k( sbeen used reproducibl y to cult ure h ESC li n es.
: ]* X4 P7 i* `0 m8 `In the case o f HFF-1 cells, i rradi a tion i s the, _& L0 L" S# l) ]' ~
pref erred method of i nactivation. All m et hods; @4 d) U, i/ D! l
described i n this paper are equally valid r e-gardless of the t ype of feeder s used.
: b' f) a% ]- l4 tNote 3. Mice stra in has proven to be an importan t facto r4 b6 L5 x& u% H! }3 _
fo r th e q ua li ty o f th e fe ed e r ce lls d e riv ed fro m6 N7 g1 w; T. s9 k. a! ]
Fig. 5 Micrographs showing
$ b: h+ T+ p1 Z+ y2 B7 h. |9 Yevolution of the pluripotent and: J6 O; f5 v2 g9 B- ]; k
TE cells during a derivation7 {7 q! F0 Z7 N/ z( o! G# k# b5 L6 q
experiment. a ICM 1 day after
l, j: b& @# {8 ^9 bseeding. b ICM cell expansion' k p( b/ J6 k* j3 @' N
after 4 days in culture. Arrow' g% \1 P# s1 W# b8 @ k' j
points to the pluripotent
i, `: W; {$ \7 C7 e n. xoutgrowth, which looks like a
! B1 Z/ k! i) w+ i/ Bclump at this stage; c colony1 m" q# G: d9 ?! f
outgrowth 2 days after splitting" q) z; \% l. }" x& Z: w
the outgrowth in panel B: arrow
2 ~( ~* m& K4 c7 Y3 j& g& p7 a- @points to the expanding7 t1 j' A+ @" C! S
pluripotent cells out of the. U4 J* ~, m: ~$ @* T1 \; I
initial hESC clump; d residual9 k' M3 t8 V* l1 p' m' _
TE cells 3 days after ICM1 O0 H d7 M9 K' |+ G. h
seeding; e TE differentiated: K) \% R( [4 M$ T# L( a( _
cells 4 days after seeding; f TE/ N& K- [% O3 J4 a! |$ L* z" `3 y8 e
cells detached from the plate8 ? S, `) D! @* J# F# W
9 days after seeding; g detail of
, T: ]5 h' G2 H1 Cthe emerging pluripotent colony+ _# E8 \$ {/ u( O3 x0 Z: T# M3 N
of picture C;h early hESC( m3 x$ w' v, W& g7 K
colony at passage 4; i regular6 H) u6 |) V) q+ B
colony of an established line at- Z( B( m" u- D& z/ y7 j, q; N" t
passage six8 B# x8 y# W; ], z& h" ]
Stem Cell Rev and Rep+ ?# |3 o4 |4 q- v
them . In our hands, embryos from the CF- 1 and
) c) \& f* e% e2 R; ~1 k+ ~" u" mMF-1 line s, and from the cross between MF-1 and% ^! |. _& p1 R5 W; b4 g
CD-1, are much better than inbre d CD-1 embr yos.
D2 |6 u( \: e7 Q$ {3 H: DNote 4. Isolated pME Fs can be frozen at passag e zero, and
4 i" c. E, ~) j9 X8 |1 P6 z8 Hexpanded at a late r time to prepar e feeders . Nev-ertheless , the expansi on up to passag e tw o before6 Y: K7 z$ t N, f) S
freezing has the advantage of increa sing the num-ber of cells obtained at passage four. It has to be2 k% ?8 x6 ]; ~) P
noted that in the isolati on procedu re describ ed in
$ a9 X5 g J Athe Meth o ds secti o n, an d d ifferentl y fro m oth er4 E, z* Z2 B. ~, `! p
published protocols [ 24], tissue chunks are not6 z, e" `) m& @" U- B$ ]/ L, E
discarded before seedi ng after first trypsiniz ation.. k( ?& T2 J' r9 q. t8 |! a
This procedu re yiel ds signi ficantly higher numbe r
( l1 B) E, t- Yof cells. It is advis ed to culture chunk s a nd clumps
' i# e8 V( |" a; d0 @separately 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
3 M; j1 p$ w9 f* r6 Bnormally be suffici ent to produce enough numbe r. a7 S) g# r% K% v" t
of vials for cult uring up to six new stem cell lines0 W' k9 p, T- u+ i, y
during 1 year.) s4 e7 Z' M9 n. L& N1 Z1 j
Note 5. Finger vorte xing is a simple way to mix a solution; P+ H8 a1 m, M0 x0 @3 i
or disag gregate a pellet in a test tube. Hold the top
7 D$ k5 F" a5 z1 c7 ~- Lof the tube secure ly in one hand and draw the ring,
. H2 i1 x& d/ y# f" smiddle and index finge r of the other hand sequen-tially towards you, tappi ng the tube. This creat es a+ f2 @8 ?+ J6 W( X6 n: @
wh irlpo ol eff ect inside the t ube, w hic h can be, y; i- X3 ^- _) H- a8 S
adjusted in inte nsity by speed. It is milder than
% f) b, S0 c* H5 zmechanical vortexing and yields healthier cells
& h5 B+ T( f+ e/ y2 C5 }0 Fafter centr ifugation .9 y' S4 j: w" F
Note 6. Freez ing fibro blasts wi th a slow freezing met hod is
) }) s) R) L) @" \* Znot an issue , and they can be stor ed at − 80°C for+ o/ r# g& s& b7 M% x
months without a decreas e in subseq uent plating
& F7 b& j- V4 n8 w. `& Eefficiency. The proto col described herei n uses the8 |- Q# N- W7 h, C% H
slow rate freezi ng container Mr Frost y, which has4 O2 |! M# N( b
been vali dated and used for nearly 20 y ears i n4 D/ ?7 b! W6 R* d8 Y/ t& T2 ?
mammal ian cell freezing [ 25]. It is a cheap and/ s+ w' {* K: |3 v o
time savin g o ption when compa red to alte rnative
# G8 i; Q( y, H4 K5 lmethods such as contr olled-rate freezing and vitri-fication, which may be suitable for more delicate
4 x/ Z, j7 X: m5 }0 |3 l# h Smaterials like embr yos and hESCs .) [, z1 ^0 G8 ]' ^' M
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 avoid9 R) b! j1 }& D d) P4 v; ~
toxi city and different iation during cultu re.
1 Q& Q+ R+ w' i6 d& Z/ u8 BNote 8. MitomycinCis thepreferredmethodtoinac-t ivate M EFs, a nd detail ed st eps to p erform the
8 X" s7 F8 u& P1 d& [4 E0 J z- K dprocedur e are included in the text. Alternative-ly, γ - or X-ray irradiation can be used. In a ny
+ T# h6 i$ f8 k, ]' ^! Ocase, t he hESC supporting capaci ty of each
& C# ~: d2 U; lnew batch of feeders has to be tested on
4 N$ Q# e( w% i6 c0 H4 x$ q, [9 `1 dknown hESC lines bef ore c onsider ing t he m5 c$ z8 H& t6 j8 u1 v0 X' Y$ ]
valida ted f or use. Once the new batch has been) D' v5 t3 Y0 T0 ?7 G* ~0 f5 b& q. {
valida ted f or hESC cultur e, it ca n be used in
, f0 u) p3 Q: [9 u1 ssubsequent derivation e xperim ent s.: O5 S. r' W" ~* T! i' y
Embryo and ICM Cul ture, Q5 H3 A( `9 A' E8 p6 T
Note 9. The procedu res detai led here are based on the use
! G* a# @* L# xof high quali ty clin ical grade embr yos, but fre-quently, the quality of the embr yos donated for7 e* E, P3 o* p' i2 r, _. ]
research do es not correspon d to the standards C, l7 t, f1 K& W, c
presen ted in Fig. 2 . If by day 6 of development ,
$ D7 `+ i: V bcompa cted cell s as in Fig. 2e – e are not form ed,
| a0 a7 e `, e$ |" gextending embryo c ultu re i n complete HES! J+ r; V' X$ n; T- U: i& {! n
medium for 1– 2 additional days will selec tively
5 F# f5 I, B D! T. rfavour the growth of the ICM ver sus the TE.
, w3 {: |6 @. ?& T, P$ |This appr oach is supported in the most recent
8 |7 n3 W; o& ~ U: kli terat ure [ 15].' v% {( h* j7 s# ^
Note 10. Reg ar ding ICM isolation, s om e authors use a n7 x6 v K" K, X
extended acid t yrode’s treatment to weaken
) b, r% t1 Z' E+ \9 Lthe TE [26], thus facilitating the spreading2 t3 Y! b! l) B, f( b
of blastocyst onto the feeders. H owever, in
6 @( x. y, p0 a2 O) t( your hands, the efficiency of this method: ~5 o- ^4 v5 w) C8 j) [0 Z
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 g' [( f1 G. A7 m) q; L+ T
the same AT t reatment to all embryos usually" a/ s1 U# a! b4 M9 {9 X- O# w
results i n s evere damage to some of them.0 I; {4 h7 ^7 W3 c% I. v( g
Theref ore, fitting t he method for TE reduction0 M0 ?' H3 l, u, X
to the grade of each embryo (Table 2 )helps( L. U+ H' T, K1 U/ R
maxi mizing the r ecovery of viable cells f rom" B h, F. S4 j2 F* G# Q
the I CM. When TE is r el atively s mall com-pared t o the ICM, or when the ICM is not
% C" z6 I7 e$ B6 D& Tcompact, a pipetting method (Method A) is1 O6 U3 R2 A; p. w) p! S1 u
preferable. Alternatively, if the TE is robust! l& {" K& S* w4 `1 {- b
and t he ICM i s c om pact , mechanical cutting
. S* d- S2 D+ T: ^# ^(Met hod B) s hould be the method of choice.2 U2 D( F3 F2 |! w8 v+ ~! z
Speed during embryo denudation and I CM
% a; A- ]! f/ D \' Xisolat ion is criti cal, even if the m anipul ations$ h% Z# |- |; k0 S
are performed on a heated stage.0 _# ], U2 {" U$ X& s2 u
Note 11. If healt hy, the first seeding of the cell s from the! Z w. t" r4 v6 Z! X6 ^$ j
ICMs will att ach i n 6 h t o ove rn igh t . In o ur
0 B0 K5 p. Q# h& {/ e W( D/ a6 h4 Bexperi ence, those clum ps needin g more time to( a$ Y3 n$ d' F
attach are not robust enough to yiel d deriv ations.
# e" a2 X# W0 H X2 O4 ^" eThe first outgr owth after the attachmen t of the4 q. X& `4 @* o8 ^. X8 r
clump can be seen from days 3 to 16. At this4 H3 t* m5 S" j# c2 h: G* Y& z
point, the undifferentiated cells need the suppor t
" P8 l; G) A4 g4 j4 j; J" ^of fresh feeder layers to estab lish pluripot ent cell
1 Y, S/ F/ q: j% ?# e5 P+ n$ Egrowth. TE cell s evolve invar iably to form syn-cytial cells that invade the feeder layer and die
" S" d8 B7 D$ `# E. pleavin g g aps i n it. It is critical to have fresh
' O) W4 y: d i: ^* Lfeeders ready by the time the TE cells and deriv-atives detach from the plat e.
$ g5 h# T/ Y/ M3 P* V8 OAcknowledgments This protocol is the result of work funded by the
8 D* o, K/ r, c ~" W( O& ^3 VNorth West Development Agency (NWDA) i n t he UK and the
; Z% S _7 e# T' z2 m. N$ vMICINN-PLE2009-0091, IPT-20011-1402-900000 and FPI-CAIB
: E9 _8 W% V" j( {' ~Grant FPI10 grants in Spain.
2 @0 _6 \+ Y& \" NCon flic t of in terest Th e aut hors decl ar e no pot enti al con fl icts o f4 S' }7 z- w2 ]/ T
interest.& x, S' ^ X% Q4 P
Stem Cell Rev and Rep. P; `0 f2 }! `. K
References
2 W+ w- ^6 M3 `' g1 u1. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., et al. (1998).
$ [" @5 M$ ?/ p; S4 ZEmbry onic stem cell lines derived from human blastocysts. Sci-ence, 282(5391), 1145 – 114 7. Erratum in: Science 282(5395),
1 c4 o- {: @4 ?3 M- o7 S1827.& B! t9 c1 R+ T
2. Schwartz, S. D., Hubschman, J. P., Heilwell, G., Franco-Cardenas," Y' J6 G1 n$ e5 _
V., Pan, C. K., Ostrick, R. M., et al. (2012 ). Embryonic stem cell
$ u: w' P/ s( Ttrials for macular degeneration: a preliminary report. Lancet, 379
% h7 S) g" A& W* K* J(9817 ), 713 – 720.
) w6 O5 m& j3 x3. C a mara sa, M . V., Ke rr, R . W., S ned do n , S. F., et a l . (2 01 0) .+ t5 a" L4 i( C5 w
Derivation of Man-1 and Man-2 research grade human embryonic
4 Q8 a9 w: U9 B+ ]* Ms tem cel l l ines. In Vit ro C el lular & Deve lopm ental Bi ol ogy -Animal, 46(3 –4), 386 –394.& a$ l$ R3 r( Y+ ]7 m: N
4. Wang, Q., Fang, Z. F., Jin, F., et al. (2005). Derivation and growing! N% d& h+ r- w& a# I1 d
human embryonic stem cells on feeders derived from themselves.
5 g0 f4 w8 l. p: W+ l5 sStem Cells, 23 (9), 1221 – 1227.
! I, @1 i! m& Q" {$ P) t5. L ud wi g, T. E. , Le v en s te i n, M. E. , Jo n es , J . M. , e t al . ( 20 0 6) .
7 U8 t+ \" L& T' ^Derivation of human embryonic stem cells in defined conditions.& [: t7 `: B* J r! |6 \: S# [1 l
Nature Biotechnology, 24(2), 185– 187.
1 I2 T- ^4 S0 s* m+ t6. M ate ize l, I ., D e Temme rman, N ., U llm ann, U., et a l. (2006)." R N1 c2 a7 E* c2 X) T
D er i vat io n of hu ma n em br y o ni c s te m ce l l l in es fr o m em b ry os8 M9 B" r* f) c; r5 p z8 v
obtained after IVF and after PGD for monogenic disorders. Human9 _4 A# W4 o, j3 E; f f$ E$ }
Reproduction, 21 (2), 503– 511.' F! a" u; h, \* O! e
7. Chen, H., Qian, K., Hu, J., et al. (2005). The derivation of two
" V* @3 A2 S4 Q. M# \additional human embryonic stem cell lines from day 3 embryos# v* ^- e; {8 Y K3 e
w ith low m orphologic al s cores . Human Reproduction, 20 (8),1 a$ r/ Y+ o& ^: |
2201– 2206.
: L. p5 `) S3 P8. Genbacev, O., Krtolica, A., Zdravkovic, T., et al. (2005). Serum-free
' _5 i4 `- t5 Z" Pderivation of human embryonic stem cell lines on human placental
+ V% @* G. R/ B* {* dfibroblast feeders. Fertility and Sterility, 83(5), 1517 –1529.
9 z+ I- @7 U ^* i: T6 u* w9. Rajala, K., Lindroos, B., Hussein, S. M., et al. (2010). A defined
6 r; \1 f' p9 [ I; [/ F- La nd x en o-fr ee c ult ur e m et ho d e na bl in g t he es tab lis h m en t o f5 {- u, H# G; r" o, |
clinical-grade human embryonic, induced pluripotent and adipose
$ s' T$ }6 z" B4 S: |( V; Vstem cells. PLoS One, 5(4), 1024 –1026.
3 w! S2 ] {2 X* ?" `/ z e10. Hov atta , O. , & Sko tt ma n, H. (2 00 5) . Fe ede r-fr ee d eriv at ion o f
/ ~ ` m' ]; y' C) ?human embryonic stem-cell lines. Lancet, 365 (9471), 1601– 1603.
: }4 R' N8 D& s ^4 W$ B11. Aflatoonian, B., Ruban, L., Shamsuddin, S., Baker, D., Andrews," U% U n( M8 x
P., & Moore, H. (2010). Generation of Sheffield (Shef) human4 b/ @' J; a" y% `9 j: o+ L) A
embryonic stem cell lines using a microdrop culture system. Vitro
8 m# U9 N$ B9 o/ R4 v R/ mCellular & Developmental Biology - Animal, 46(3 – 4), 236 – 241.! |8 @6 C+ E2 |' @
12. St einer, D., Khaner, H. , Cohen, M. , e t a l. (2 010). De r iv ation,
7 ~ h% }9 }- e" I( Mpropa gat ion and con tr olle d differe ntia ti on of hum an em bryon ic
A* N$ l6 y. V' }: z$ \stem cells in suspension. Nature Biotechnology, 28(4), 361–364.
8 \* M" q& `( e4 S4 T1 l, y% H13. The International Stem Cell Initiative, Amps, K., Andrews, P. W.,
4 g6 j" Q" H2 M& X0 Q7 Qet al. (2011). Screening ethnic ally diverse human embryonic stem
5 B; L# q% i# j% x' A) [cell s ide nti fi es a chr om oso me 20 mi nim al am pli con con fe rri ng+ \$ m4 I; z. b9 ]6 V
growth advantage. Nature Biotechnology, 29(12), 1132– 1144. ~5 W; n$ n. E# C9 e9 t- V8 P
14. Murdoch, A., Braude, P., Cou rt ney, A. , Brison, D. , H unt, C.,
( @0 { ^5 ]* rLawford-Davies, J., et al. (2012). The procurement of cells for
* G2 i7 z' e' n& P X8 a! }the derivation of human embryonic stem cell lines for therapeutic( ~3 ]0 U0 F6 L- d* m! g* J
use: recommendations for good practice. Stem Cell Reviews, 8(1),& [8 M: M& Z8 W. A7 e0 q0 s0 n% \
91– 99.. H! V/ ~( T/ v5 y! Z/ H( m
15. Fan, Y., Luo, Y., Chen, X., & Sun, X. (2010). A modified culture
# G; b* `& {- `$ r* gmedium increases blastocyst formation and the efficiency of hu-man embryonic stem cell derivation from poor-quality embryos.
1 v7 w6 a0 O% B* GJournal of Reproduction and Development, 56 (5), 533–539.
8 T" B9 X8 \0 e4 X2 [- e; p16. Veiga, A., Camarasa, M. V., Aran, B., Raya, A. and Izpisúa, J. C.% n2 P2 ^* L- f* n! D
(2 0 0 7) . Se l ec t io n o f e mb r yo s f or s te m ce l l d er i va ti o n: ca n w e
1 G) C E- y% ]( p; doptimize the process? In Carlos Simón i Antonio Pellicer (Eds.),
. M! z% E6 E5 `+ wStem cells in human reproduction . Basic Science and Therapeutic
( x; N: a; ?, u8 \Potential. Informa: UK Ktd Chapter 11.
! q* N Z+ l* ], u, f5 C17. Pool, T. B. ( 2004). D evelopmen t of culture media f or h uma n
* X6 i2 @5 f, B( n5 v) s* M' ^8 Cassisted re produ ct iv e t echnolog y. Fertility and Sterility, 81 (2),$ d1 Q; Z. H0 a
287– 289.% _2 C* l% {( T4 m' h
18. Stephenson, E. L., Braude, P. R., & Mason, C. (2007). Interna-tional community consensus standard for reporting derivation of" a2 N) U3 L+ y
human embryonic stem cell lines. Regenerative Medicine, 2(4),
/ d h2 H# N1 M* r2 ]1 ~349–362.9 U" U- E u: k* \" V' e
19. Leng n er, C. J. , Gi mel br ant , A. A. , Erw in, J. A., et al . (2 010) .7 [$ F# U5 O% v1 x; V
Derivation of p re-X inactivation human embryonic s tem cells
6 ~2 i+ D3 C" m( e q4 ?7 m; Nunder physiological oxygen concentrations.Cell, 141(5), 872– 883.1 g8 ^! {% I: Y/ g
20. Choo, A., Padmanabhan, J., Chin, A., Fong, W. J., & Oh, S. K.7 x$ K" d$ K p: q3 n E+ [
(2006). Immortalized feeders for the scale-up of human embryonic
! c' s$ F$ R! Z; H! [stem cells in feeder and feeder-free conditions. Journal of Biotech-nology, 122 (1), 130– 141.7 T5 J/ c5 ]. t& t
21. Camarasa, M. V., Brison, D., Kimber, S. J., & Handyside, A. H.
7 L0 l: R# a1 s4 z+ d* v, ^(2009). Naturally immortalised mouse embryonic fibroblast lines/ t2 T! T8 W8 ]- P" J4 l* R
support human embryonic stem cell growth. Cloning and Stem6 f/ {' H4 Q( i
Cells, 11(3), 453– 462.
. b; N% X; A1 \22. Kibschull, M., Mileikovsky, M., Michael, I. P., Lye, S. J., & Nagy," J r/ W0 m/ P- O
A. (2011). Human embryonic fibroblasts support single cell enzy-matic expansion of h uman embryonic s tem cells i n xe no-free4 S! m' X, Y; c, g3 |
cultures. Stem Cell Research, 6 (1), 70 – 82.
: S3 v2 q* z; `# p) U23. McKay, T. R., Camarasa, M. V., Iskender, B., et al. (2011). Human( L( A& G$ I- i+ {6 Z- l
feeder cell line for derivation and culture of hESc/hiPSc. Stem Cell
2 S5 N% j6 o x; LResearch, 7 (2), 154– 162.
4 }' i6 G# a1 j4 `8 I1 {- P$ y4 C24. Schmidt, J. V. (2001). Embryonic Stem (ES) cell culture basics.: J5 {) f1 b4 `. {' z" P
Current Protocols in Toxicology, Chapter 15 , 15.1.1– 15.1.15.
) e: L7 G9 \# e0 s7 P) u$ V25. Venkatarama n, M. (1992). Effects of cryopreservation on immune
8 D) H$ \3 K3 p3 V- q$ Qresponses: VI, an inexpensive method for freezing human periph-eral blood mononuclear cells. Journal of Clinical & Laboratory
) a4 O6 {% G! }5 r2 uImmunology, 37 (3), 133– 143. \; f: z( o7 \; H0 {1 \
26. 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 ,
, j" z) |: n0 z# l: d! j" uC., L un din , K. , H y lln er, J., & Semb , H. (2 006 ). D e rivat ion o f
2 w! C0 C% O- W% S3 i3 Aa xeno-free h uman embryonic st em cell l ine. Stem C e lls , 248 b, u2 A5 J( F; {, H
(10), 2170 – 2 176 |
-
总评分: 威望 + 2
包包 + 5
查看全部评分
|
发表于 2013-1-31 11:18
