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Optimized Protocol for Deriva tion of Human Embry onic
- n5 m) n3 y2 y8 G+ [8 r4 sStem Cell Lines$ F. `: h6 W2 b4 P7 S
María Vicenta Cam arasa & Víctor Miguel Galvez&
( m% {: e# U" pDanie l Roy Brison & Dani el Bac hiller" G8 d+ f+ H! ]% Y
#Springer Science+Business Media, LLC 2012
0 p6 ?, [& L( w9 B5 QAbstr act For t he pa st 12 ye ar s, th e b iolo gy an d a pplic atio ns
! O& n0 S' ^6 T' Gof human embryonic stem cells (hESCs) have received great( g: V, p5 ?# J
atte ntio n f rom t h e s cien tific comm unity. D e riva tive s of the first
- J' A9 Y g+ `' s$ E' MhESC line obtained by J. Thomson ’s group (Science 282" [6 l* n/ B9 z2 m) X
(5391):1145 –11 4 7 , 19 98 ) have been used in clinical trials in5 k, U' F( k8 [! z5 m6 t0 V
pa ti en ts with sp ina l co rd inju ry, an d othe r h ESC lin es ha ve: ~/ I- x# q, Z! C% a8 R
no w b een u sed t o g en era te cell s f or use in tr eatin g blin dn ess
6 f# C; Q8 D+ ~/ e+ q( H }" Q( L a n ce t 3 7 9( 9 81 7) : 7 13 –72 0, 20 12 ). In addition to the classical
" ^; F$ B( j0 s7 v6 N( F( {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
& g: b. Q& G3 f: gcu 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,
- G7 w' x2 W1 x+ g$ G2005 ; Nature Biotechnology 24(2):185 –187, 2006 ;Human
) d& G; `; u- L! m/ lReproduction 21(2):503–511, 2006 ; Human Reproduction 20* w% q- `2 q0 m3 J# `
(8):2201 –2206,2005 ; Fertility and Sterility 83(5):1517–1529,% n$ `" Z5 N4 ]2 X. B) l
2005 ), novel hESC lines have been derived xeno-free (without
0 @8 d- q* o7 busing animal derived reagents) (PLoS One 5 (4):1024–1026,9 N x5 ]3 d; K3 {+ l" l
2010), feeder-free ( without supporting cell m onolayers)! ?! J) z2 G+ b' e" s3 L- N
(Lancet 365(9471):1601 –1603, 2005 ), in microdrops under' E5 k: {. H. B; r7 ]# |# E
oil (In Vitro Cellular & Developmental Biology - Animal 46
0 _8 x( c5 G( q1 w0 t. R(3 –4):236 –41,2010 ) and in suspension with ROCK inhibitor
+ g6 ?) g, E, S. Q/ }( E(Nature Biotechnology 28(4):361 –4, 2010 ). Regardless of the
6 |* P3 o$ V9 q% {! zculture system, successful hESC derivation usually requires
+ D' n6 f0 d/ ~2 c5 e# @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
! I0 J2 H- K' G" q5 xhESC line derivation. Herein we address the crucial steps of! o& t% Q" @! ]" ]6 ?/ Y' [
the hESC line derivation protocol, and provide tips to apply
" c. ~$ V: @1 P4 p% equality control to each step of the procedure.3 p- V2 J, F9 b( `# p* j
Keyword sHuman embr yonic stem cell s
3 c) y2 Z# p$ T) ].
/ f* V& s6 @( zDerivat ion
" X7 B' ~% A3 p9 {' ~" L, Q7 ~9 Z.4 G& ?7 G4 O% u2 ~2 a+ f, F
Blast ocyst
7 b* `- G3 M) g3 L9 w.
: v2 K# i- ?+ O. xCultur e o ptimizatio n
7 `$ Z0 m0 S( e$ U. G- B$ U' @! tIntroduction, i1 w3 z+ I' q+ Z. D
To da te, hundreds of human embryonic stem cell (hESC) line s! j% v, L5 U4 G8 ^: u
have been de rive d f rom surplus embryos after as sisted repro-duction techniques. Reported derivat ion e fficiencies range from
' a: v( n2 H4 C! N: r* F7 to 100 %. This wide span pr ob ably refl ects the di versit y of the. D5 O$ I. b" L: _. A; n) f
methodologies involved. M any var iables of the procedure s uc h# A. G+ M& V$ h' N
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)
+ ^9 e) S4 F' H4 nremoval and inne r cell mass (ICM) isol at ion a nd type of feeder, n/ E, p q: K) N, u" F
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 growth3 V6 D3 _6 z) T9 W: ^, O6 Z# P
properties 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# \6 a: ?" U: P* v4 L$ a
th e genetic stability of more than one hundred hESC lines
- {; Z) O% ^7 u5 G! r, Gusing consensus protocols for karyotyping and genotyping.& C& J' l# ^0 O7 ^
This echoes the importance of defining robust and common
. G' d; B" f/ n$ L4 Q1 k. i8 `3 ucriteria for the assessment of both existing and newly derived
1 i3 m1 _0 ~; M G3 ?+ Z! X) n: Ecell lines, and for the validation of advances in culture con-ditions. [13].2 R) }: e2 `0 Q- H& ?
M. V. Camarasa (*)# p% A+ _% v; l" `7 o7 M! l
:( ^. I- r* w" n0 t' r Y. V
V. M. Galvez
! e1 v! [' }( {9 V& u:8 z7 i0 i# V' H I6 d
D. Bachiller& B# f$ s1 b5 ^$ U# _! @
Caubet-Cimera Fundation, Centre for Advanced Respiratory
' N" k" R) I9 m6 u3 \: X8 ^Medicine, Recinte Hospital Joan March,% H# H/ q, u( Z, W' [( ~- u5 G V4 c
Ctra Sóller km 12,
/ q% N$ o7 @/ [; i) Z5 U07110 Bunyola, Illes Balears, Mallorca, Spain. R+ t2 c; i0 Z# N
e-mail: telomerasi@yahoo.es
$ {% | v& o1 t$ Q/ m% L, d' MM. V. Camarasa
8 @2 ?! q4 y; N, h3 X# n0 n4 qe-mail: mavi.camarasa@caubet-cime ra.es
6 G0 Q4 c* U& |: A/ MD. R. Brison
4 n. v" K5 I% c1 ]# vNorth West Embryonic Stem Cell Centre, Faculty of Life Sciences,, P$ j3 u7 B, d& @0 M) F: y8 S8 Z
Core Technology Facility, University of Manchester,1 I7 Z# O" B2 R
46 Grafton Street,% g8 ~* c8 B/ D& o2 s
Manchester M13 9NT, UK
& _$ V! E, f8 rD. Bachiller% V# I6 b$ M7 V: r e6 {
Consejo Superior de Invest igaciones Científicas (CSIC),
7 ?6 ^, |/ _9 Y% G. g: pMallorca, Spain& F% E8 w" p5 C; s
Stem Cell Rev and Rep4 f* u! U) Y# f d# u: o
DOI 10.1007/s12015-012-9377-4) N% h) |9 @8 Q' H
This protocol focuses on t he main first steps of the( G! x/ v* t; a2 z5 i( I8 N
deriv ation of hESC lines, i.e. embryo culture and gradi ng,. S5 C3 O: q5 _5 H4 E4 f* n
and precise mani pulation of their ICMs and first outgr owths4 K+ I2 Y% D8 H/ R
of pluripot ent stem cell s. The Notes secti on includes alter-native procedu res as well as commenta ries and clarific ations
( k3 L' s9 Q5 Q! l( aon the techniques described in the Methods section. Assuming1 \; E Y- w5 k
experience in mammalian basic tissue culture technique, this
! }+ N0 d9 L& |# r- q3 n9 Sprotocol should aid anyone wishing to derive and culture' }! j/ ~ L; E9 |' F
hESCs to set up a rapid and reliable method. This protocol# [$ h: [& q B5 ?; r, |9 F
reflects continuous work optimizing the derivation, culture
3 l+ h- R8 T/ B( }: Yand characterization of hESC lines over more than 10 years,* V* U" t) D7 V0 a; p, N- e7 S
comprising activities carried out in four different laboratories.4 L' }6 A/ c2 {* ]
Adva ntages of the present p rotocol over others previous-ly pub lished in clude: optimiz ation of f eede r cell yield,: D2 v) N! k4 K) T) m
increase of I CM cell production and adjustment of the
' z7 C7 Q0 j/ xderiv ation protoc ol accordi ng to embry o grade.
: b4 b5 h4 G4 ~' T8 YFeeder cell yield is optimised by culturing tissue aggregates
+ b. J' p6 S! x9 buntil they grow as monolayers. Embryo culture is optimized
0 A8 |" D8 {0 f/ s( [/ S. fso that growth of the pluripotent cell population is favoured
1 X) ^2 Z% I; Z! z. P& n" D Qover that of the non-pluripotent trophectoderm (TE) cells." y" q V! C) Q1 Q, M
Finally, the fire pulling of glass Pasteur pipettes to form thin V; @, B5 H! H6 e- v/ ?+ @
open and closed ends that match the size of the ICM or initial9 m) F# a- T6 j6 i! |$ V
outgrowth to isolate, increase effectiveness of mechanical
: D5 |& `. u2 @* Z% ssplitting techniques.
l! [, l/ y' R, B V# @' XMateri als7 S" P1 J8 {; i$ ~" t$ S$ ^7 f
Mate rials a nd Rea gents
% S% \) i Y2 n% \! o7 w- z# f1. Bacteriological Petri dishes, 100 mm Ø, Fisher Scientific8 X8 w+ P$ N' U* T" a' Y; R
#09-720-500.
7 e0 ?: M( n$ |# x9 Y4 W2 l2. Cen tre-wel l Organ Cultur e Di sh, 60 mm tissue culture/ k0 K S0 l* e/ [8 t
treat ed, BD #353037.
; W$ {: |' T8 p3 z1 s3. Lon g glass Pasteu r pipet tes, Fisher # 1367820C .
" Z e5 w5 c0 N3 u% e4. Mr Frost y® Nalge ne, Sigm a #C1562-1E A.
# |% c1 O+ t% l: D5. Se rological p ipette 1 ml, Cor ning #4485.
9 |, X v) _6 i8 }6 v6. Se rological p ipette 5 ml, Cor ning #4487.
/ c# T. I9 R& a- x e7. Se rological p ipette, 10 ml, Cor ning #4488.
+ o5 T% X4 M: q/ i8. Se rological p ipette, 25 ml, Cor ning #4489.
0 G! @7 v8 ^( Z4 z6 Y8 [' P9. Se rological p ipette, 50 ml, Cor ning #4490.
9 q! l2 F- d1 E+ o7 \; o6 @10. Tissu e culture dishes, 150 mm Ø, Cor ning #430599.
! n F9 p5 P* m8 l" \ e11 . Tissue cult ure flas ks F1 00, 100 cm
% y8 E1 E/ n! R% ~6 m9 D; |* ~2
) H! T( |- G( a: K8 ~Corning #3816 .3 E/ W! ?! v$ ?4 \: Z
12. Tissu e culture plates, 4-well , BD #353654.
/ F$ I% q& h9 r% @. \. Y1 @13. Tissu e culture plates, 6-well , Cor ning #3516.
6 P* e X4 f( e( q( ^ O% j2 h6 e14. Basic Fibrob last Gro wth Factor, #45103P- 100.
+ v- T1 D0 l: F: V" a15. Beta-mercap toet anol, Sigm a #M-752 2.
- N/ ]' W$ B b: X7 J3 a4 g16. Collagen ase IV, Invit rogen #17104 -019. Z6 T* J& o" D9 @" H I+ U" x
17. DMEM medi um, Lonza #12-614 F.; p* ~& H8 O* K N
18. DMSO, Sigma # D2650.
$ a* j$ j6 G ]( \, v* |, k3 O% U; t19 . Fe tal bov in e serum (FB S) , Australia n origin Lo nza! `# ?) N7 V5 g4 w+ T3 T
# 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, F5 i" x4 K- Y4 [* M
origins a re US and US approved, followed by Au str a l i a n
6 y% f* [* r7 \" `6 Q' Gand Australian/USDA approved. Poor quality serum is a
+ k6 N1 u3 v" H8 R a. i5 Hconfirmed cause of feeder cell failure in hESC support.% p2 M3 c1 H2 F4 `' O% }- c
20. G1, Vitr olife #10128.) J/ J7 d, Q' g
21. G2, Vitr olife #10132.& l& F% @& W- Z2 O d
22. Gelatin, porci ne, Sigm a #G-1890.
5 Z) O: v, D. K+ u7 S) \0 \23. Knockout (KO) DM EM, Invitrogen #10829- 018.
+ w/ }) u$ [( L6 V4 I24. Knockout serum replacement ( KO-SR), Invitrogen7 `. i1 u# z; T/ F/ W" ~
#10828- 028.
+ [; ~+ r1 _5 H9 O2 N5 a25. L-Glutam ine, Lonz a #17-605 E.
! l; O2 o) b3 q& J' J26. MEM Non-essential aminoacids (NEAA), L onza #
: q* N" S8 `; o. \8 @13-114E.9 j' Z" N3 k0 e; X& S7 @8 {# g
27. Mitomy cin C, Sigm a #M0503 . Hazard: this subst ance
+ ^" d$ a+ A1 N5 A4 \- q( k5 Dis very toxic and procedures for safe hand li ng and
$ t6 i% D* q! t: n, ydispos al need to be in place. Do not brea the dust.; ~9 x8 r' j' d) Q- G4 Q' s; R
Avoid contac t wi th eyes, on skin, on clot hing. Avoid. A2 m* ~4 w7 b* q
prolonged or repeated exposur e.
! x& |, L+ q; ~. B0 V8 v28. OVOIL, Vitrolife #10029.* w7 u8 _/ B8 |( @* `( |
29. PBS with Ca
: l8 n* l) p4 A- d6 O" e+20 f( H u3 y+ D% H4 C8 r
/Mg* O# u& W6 v5 F6 D
+2
+ C' V" `0 G1 b4 x- q) T, Lonza #17-513 F.5 P* m6 u" Z8 g; k4 w: J8 ]9 C9 v: J
30. PBS, without Ca r3 W8 Z2 v4 j7 ~
+2% K& C9 L, ]$ k3 U1 w
/Mg* Z& i' Q' {5 {% x
+26 \' n8 R1 P9 a5 g) o6 q
, Lonza #BE17 -516F.2 d8 H$ x( C8 [. }: {
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
9 i5 F+ R1 O3 E) Hdp c, Charles River Laboratories.7 X# d& m G/ _
32. Thawkit I, Vitrolife #10067.
* u3 F1 @4 \ H. k33. Trypsi n-EDTA, Cambre x # CC-5 012. e& M' s p8 G
34. Tyrode ’s solut ion, acidic . Sigm a #T1788.5 t$ R$ t4 }9 g* j+ Z
Solut ions
L; \6 }: E+ q `6 f, a5 g, |1. Complete DMEM medium: DM EM plus 10 % v/v FBS,( S) e @/ I: x. B5 F3 s0 ~) a7 U
1 % v/v L- Glutamine 200 mM, 1 % NEA A.
V/ |: J/ D/ X: P/ o6 t2. Freezing medium: 90 % v/v FBS plus 10 % v/v DMSO.
( f! z5 |! O+ T3. Gelatin solution: 0,1 % w/v gelatin in tissue culture grade9 m8 B% L3 z0 W/ _
water. To avoid contaminants and residues (detergent,# H+ l+ o# j& J5 f; e
traces of other chemical, etc.), use a new 500 ml Pyrex) Y5 Z ?8 C9 `& N
bottle. Autoclave the gelatin solution and store it at RT.
% T% F r* X8 e* y* z: h2 N8 a7 ~4. HE S m ed ium: K O - DME M supp lemen ted w ith 20 % K O-S R,
2 g# H4 `& h8 R. i9 y* n9 {2 K7 a+ h2 mM L -Glutamine, 1× N EAA, 50 mMβ-Mercaptoethanol8 \( }$ l3 f& f
and 8 ng/ ml human basic fibroblast growth fact or.3 i3 x$ _! C8 a8 X- v6 S
Methods$ _) X6 Z9 f% ]4 Q( P
ESCs are derived from preimplantation stage embryos. The" h+ F1 c5 t9 H! w2 ]6 _) r
most common procedure involves culturing embryos to the
( O3 Y' d% e7 t# ~( |- tblastocyst stage (day five to seven after fertilization), isolating8 X2 s. w% ^% _' R% b$ Y
their ICMs, and culturing the latter on mitotically inactivated
" f& h2 z) G: _/ a4 _fibroblast cell layers. The resulting pluripotent cell popula-tions are selected by morphological and growth criteria. Novel
; B; S. C5 R5 f& h9 o- ghESC lines are then established after continuous subculture of
* V+ [+ ^+ v0 }the initial outgrowth. A hESC population can be considered to# @) ^! ?/ d+ r
be an established line when it reaches passage 8 with approx-imately 3 × 103 J- _/ D( f/ i6 Y
7# e- S+ r: B b0 I8 h+ ^
cells, as defined by the UK National Clinical
: ]( v3 s7 N' xhuman Embryonic Stem Cell Forum [ 14].
$ S; d1 T9 @7 K. B3 xStem Cell Rev and Rep& @; R( {/ d/ }' r; x( N% `7 l. Z
After eight to ten co nsecutive p assages, the novel line is
4 A) [! D3 [) J8 wsufficiently expanded to check its viabi lity by freezi ng and Q V/ N/ L' t$ }6 O$ y
thawing .
0 {6 e3 p; b0 P# Z( W, ~0 yTa b l e1 defines the term‘one v olume’ for each of the culture# W$ e1 P& S6 x0 \, m
v es se l s d es cr ib ed t hr o ug ho u t t he manuscript. All incubations are! s0 X' ]" V- P+ D/ @1 U7 o% d
performed in c ondition s o f 3 7° C, 95 % h um idity an d 5 % CO
& g- z$ F1 q& [- A0 F n, w29 D( K& ^. F: r) ]& k. b5 i' ]$ i
inair(SeeNote1).Figure 1 depi ct s t he expect ed time schedule
$ v3 v' r3 Q! ?; L. q/ Bto complet e a hESC d erivation p ro toco l, provide d that t he feeder& r$ v7 c/ y$ M0 \+ d" V3 d4 e
source has b ee n p revi ou sly v alidated.! Z; q" [; |% g5 S
Feeder Layer Preparat ion
4 H/ u, E- l: d3 @Primary cult ures of feeder cells have tended to be used for8 i% Y' p- Y7 `
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 E1 Q6 e. N3 v5 A8 nderivation can be performed on f eeders produced from f: m2 K2 ^8 C; O# A# {/ E
estab lished cell lines, or prim ary feeder cells whi ch have
( m: |" x" n0 L. N$ |0 Gbeen immort alised. Altern atives to the use of prim ary mous e
- r3 \: H& K( z3 E% [1 xembr yonic fibr oblasts (pME Fs) as feeder layer s for hESC
# D" |) y7 U0 @culture are discussed in Not e 2.* U6 ?( c+ h% T$ Y5 ^; x8 \
Reg ardless of the type of feeders used, every feeder layer0 Q! V! G9 t# @. z7 g
must be con ditioned before stem cells are seeded on top of! T- D1 [8 Z& |' ^( u
it. To do so, feeder cult ure medium is disca rded; feeders are! |& | K% S+ Q8 ^) h+ R
then washed three times with one volume of PBS each time& ^4 W& O3 o. Q- w w
and once with half a volume of stem cell medium (HES).
* b5 X' L1 y' E% |9 ]Plates are ready for use, from 30 ′ to several hours afterwards., j3 X' k8 w* l5 m$ z& F5 a0 R
MEF Isol ation/ b- _: C# S5 c6 A) u9 }6 z4 V
1. Sacrifice a pregnant female (12.5 –13.5 dpc) by cervical4 A/ ^1 [, o; ^1 ?
dislocation. See Note 3 for tips on strain choice.) O. F$ K/ _ f6 c
2. Di ssect out the uterine horns and place them into a
$ N4 }0 J4 C* r; P3 Y. y10 0 mm Ø Petri dish containing one volume of ster ile
% k' L3 C. x# S' s2 T3 S0 QPBS 1×. Transf er the plate to a laminar flow hood to7 j4 i y$ U/ z1 l3 v
co ntinue with the procedu re.
% }2 h- I/ T7 Z- d5 h! {2 a3. Cut open the whole length of the uteru s, so vite line
% C* U9 [; c& L3 G, O0 gsacs with embr yos and placentas are partially relea sed.
5 x |0 B: w2 X( A% B$ Z4. S e parate each embryo from its p lace nt a a nd out of the8 B# f8 T2 i, D7 D6 \$ T
viteline sac and surrounding membranes, and transfer1 `: P ~, q9 a/ ]; b: B. w! z
them all to a ne w Petri dish with on e volume o f fresh PBS./ M& A" p% d* Z1 X& @- |7 a
5. Wash the embryos and transfer them ag ain to a new' a6 F3 N# h7 m) g
Pe tri dish with one vo lume of fresh PBS., [7 ^2 \9 h( O# p1 r3 X
6. Decapitate the embryos and separate the visceral tissues" A, W' `4 S( Y( o; H
from the bodies. Collect carcasses aside in the working( v# y5 {8 T9 g. d5 _
plate. Aspirate and discard the PBS and the visceral; s* A, B2 ]$ N" `7 i+ [6 P+ E* T' G
tissues carefully with a 10 ml serological pipette. Leave
/ s; h4 J. C- p- R% Othe rest of the tissues in the plate.
: X; t$ s8 ?1 [5 |7. Mince body walls into small pieces until a homogeneous
+ o' C( G% r3 S( d) l$ S; i+ @suspension is formed.
' {$ m$ e: }5 w3 s- d5 X% X0 O8. Add 5 ml of trypsin-ED TA 1x to the plate and transfer3 a$ B$ f# G* p
all contents to a conica l 50 ml tube.
6 A) O1 f' u$ J4 k5 m9. Incubat e diges tion at 37°C and 5 % CO+ G" G- ?( b+ I4 t8 y
2
! A1 ?6 R( H) `: K1 L' ufor 30 min.
) L B. e% c, Q0 ~+ S) I t7 X2 aHom ogeniz e the cell suspension by adding 45 ml of3 M2 u; O& t, M( j
fresh cult ure medium and pipet ting up and down for
2 I/ n, p( u5 s1 `# H5 eten times, and cen trifuge to recover the cells (700 g for" m$ [8 ^( m. ^& O
2 min ).+ |, H* P( h8 V4 Z' y7 y
10. Discard supern atant, ad d 50 ml of fresh culture medi a
* t4 Y; q: `' m+ tand resus pend cells.9 \/ w) U. |4 ]2 l! }; y
11 . Cen trifuge the cells at 700 g for 2 min and disca rd) i/ s* c( q e$ F+ R
supern atant.2 d8 G! I9 U, I% n, S
12. Resuspend t he cell s in 5 ml of complete D MEM/ z7 m A* y7 h. ]0 |
medium.
) }) \$ b. }4 }' k13. Seed cell suspen sion at a rate of three embryos per
6 M4 r, C/ s0 jF100 flask. (General ly four F100 will be set up at the* e+ z$ }/ Y6 o; Z" j
be gi nn ing o f the procedure per dissec ted p regn ant
- R$ y# A4 G0 t" V: D7 y6 R, n1 Ifemale).* g0 j7 f9 t5 e4 [( x! F) I: g
14. Incubate at 37°C and 5 % CO
7 g* {8 T. U6 N' Y26 L6 {7 Z1 E1 |
unti l 90 % confl uence
8 \& f- T8 a% a- {is reached (approx imately two to 4 days).; f/ X9 n9 \9 R/ o" n1 e
15. Split the cells twice by tryps inisati on, seeding them
' v7 S0 b6 f0 I1 Y8 Fonto n ew flasks/dish es be twee n 1:3 and 1:6 split3 r# V! h" L+ [
according to growth rate.
* z. f: f, O3 c/ Y- V; q1 gAfter t wo passages , t he ce lls s hould be growing as4 @8 b1 Z) r! k) w
monolayers. Freeze 90 % of p assage 2 c el ls in vi als
8 V0 d2 S+ S Mcont aining four to ten millions units each. The frozen* P, y ?6 C4 ]" v& W J$ h" H6 s; B
cells wi ll const it u te a s tock of act ive pMEFs for fut ure5 s3 w# P8 n3 V
use.
' l6 k( ]+ c4 u3 g- i4 [0 u% T! B+ WTo continue with the production of inactivated feeder6 s3 L3 I6 {' O+ V" w
cells, expand the remaining 10 % of the original culture) l4 L5 `! G' L- w5 E
up to pa ssa ge fou r. S ee d t he cel ls on to new f las ks/3 |! M9 m1 {/ |0 q
plates bet ween 1 :3 and 1:6 spli t according to grow th
( a( }2 K+ s4 h W$ nrate each pa ssage. Then inac tiva te an d freeze passag e/ G y, t- B1 n. m2 W |% N
four cells as explained below. See Note 4 for tips on- w" w, ^! g9 V5 m
these st eps.
$ X8 ~7 C0 m q6 g/ c1 EGene ration of Batches of Frozen Feeder Cells by Mit omycin
$ A; ~- m& n' J4 yC Inact ivation) t) i( m* p# k( j6 }5 x* T
1. Tr eat 80 % confluent, exponentially growing," F" P! {9 l8 J+ K. Y
pa ssage four pMEF populat ions (between 48 and2 y1 U6 |3 T* k& D" V* N" \
9 6 P150 plates), with 20 ml of 10 μg/ml Mitomycin C in
0 v" J5 K1 a3 d% b/ `complete DMEM medi um, f or 2– 3 h at 37°C a nd
3 w8 e) B+ r, e. V' B5% CO22 r5 U$ s& h9 m2 r
." t5 K) i$ z+ D8 o2 [4 n0 H
Table 1 Definition of the vessels and volumes used throughout culture, a+ K0 f L( m: Z" {
procedures; v0 q/ M) m1 P+ _0 r' \& C
Dish/plate Area cm9 M5 [2 I8 X! I; f
28 A6 `) R) L7 y, b* R2 D2 f2 R
/well One Volume ml/well
; x$ A! R: x* a# b, x& j4-well plate 1.4 0,5- ~8 Z* Y4 i6 ~1 E+ d7 E
24 well plate 2.0 1
8 c: t5 W. f O* g3 F7 T0 _4 a. gOne-well 60 mm plate 2. 9 1) K0 \/ Z. T0 f
6-well plate 9.5 3! z& ?, e" l6 Z9 R Q2 @2 z
60 mm Ø dish 21.3 5
6 c1 \+ k, Y2 A; z3 t' ~2 a$ X9 U100 mm Ø dish 58.1 20
' m- {' Y1 r% z* w2 tF100 flask 100.0 25' o+ y4 S; t! I
150 mm Ø dish 176.7 50
5 n; W. D) ]6 ~0 Y- \3 M2 JStem Cell Rev and Rep4 P6 Y$ p5 W T9 S
2. Aft er inactivation, disca rd the supern atant and wash* m- S8 R7 h0 J r0 @
cell m onolayers 3 times w ith on e volume o f PBS
1 V, l* W+ u3 a# c7 _7 e- gwi thout Ca
9 X+ }) V; {- \ d+2/ o1 |' s+ G( Q6 W: i, |( ^
/Mg
6 a" V. O, G+ g, I) @+2
, q. r: s: _- |# Q.! S1 c( C3 \3 \" o. \
3. Trypsi nise the cell s (2 ml per F100 flask or 3 ml per
% r8 Y1 |: t7 n; d6 A4 ZP1 50 p late) for 2 – 7 min at room temperat ure (RT) .- X- e1 x, D( M, h
4. Tap the sides of the flasks/dishes to dislodge the cells,
) i) T' @% }* v/ H% S7 A& }an d harves t them into 50 ml conica l centr ifuge tubes .
( {+ r) L3 }& z8 o9 |( }Po ol the contents of tw o to three P150s in one tube.
7 n# X2 M! M1 r* J/ Y" O5 w5. Add 44 ml of compl ete medi um per tube.
3 \ k6 X% s8 j1 n5 r! a" B6. Cen trifuge tubes at 700 g for 2 min .
' f( w" u+ G: N* {# v7. Br eak pellet manually by finger vortexing (see Note 5).
9 o# O5 H% u1 q+ y+ c. p- [& F6 wResuspend each pellet in 30 ml of complete DMEM and ~8 d* Z k8 m4 r( P
count in a Neubauer chamber.: Y# {! |. A$ L9 ?& W2 ~
8. Co unt v ia ble ce lls and calcu la te the numbe r of
: F2 g! u) N8 V! Kcryovi als needed, at a rate of 1– 4 million cells per vial.5 h' M' s2 v6 _/ ~% u
9. Cen trifuge cell suspen sions at 700 g durin g 2 min .* s6 }, ~) L( C% y) f' W
10. Label cryovi als with batch numbe r, amoun t of cells
2 P" R/ `+ r6 S' A+ nand met hod of inact ivation, date and user ID.2 H& h7 T7 h" X4 ]$ P
11 . Discard s upernatants from s tep 9 above and break6 [% m* C/ F B( A1 \3 _: l
pell ets carefully by finger vortexing.; t% p* d4 H9 e8 U, r7 E/ E
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.
" J1 Z9 d/ I; r# G/ yDisp ense cell s uspension i nto labelled cryovials and
9 `0 [0 i- `! O# M/ Mtransfer them immedi ately to a precooled Mr Frost y® con-tainer, and then into an − 80°C freezer (see Note 6)." y7 g* I/ d3 L ^
Feede r Layer Plating for IC M Culture
( }2 h* W# ]7 x1 N8 m4 W5 {1. Dispense one volume of gelatin 0.1 % ont o the/ c( k. ~ |1 e5 S! E4 F
requi red number of dishe s/wells, and incuba te them
$ |5 h8 Y5 I& m- xat 37 °C for 30 min.
% k" w \: T* S2. Equ ilibrat e 10 ml of fresh compl ete DM EM medium,
3 m4 h# t& K* I+ C7 gin a 15 ml conical tube, in the CO& X9 [( c/ ]; x d5 O( W/ _
2
1 Q/ A% _7 e5 Z2 fincuba tor per each% M8 T/ f: {, P0 J0 p
1 – 2 vial (s) to be thawe d (see Note 7).( R# {& H' Q( V$ c' z! Q* q; Z0 d
3. Once the coated dishe s and the medi a are equil ibrated,1 [. g* ^4 Y5 T/ H& R0 ~
thaw the requi red number of cryovi als to plate 0.2– 0.3$ @6 ]2 _0 m7 M
mil lions of inactivat ed pMEFs per one-well 60 mm) W0 T% o- e% C! Q: k# ~4 z
dish. Spray the frozen vials with 70 % ethano l. Roll
* }6 F/ w8 O) M/ ~$ ?! H: n4 ^them in your hands, until only the last smal l piece of/ i/ O3 j; j) I$ r5 u5 v) A5 }
ice remains. Tr ansfer the c ontent of the vi als i nto
, k. D2 O5 U9 a; fthe equilibrated compl ete D MEM. In order t o
, d/ }( {6 S7 ?9 X8 Qavoid cell dam age i t is n ot advi sed t o thaw m or e
5 [- k" ~$ g) X2 Xthan four vials at a time.7 d E8 o/ L: e
4. Cen trifuge the tubes at 700 g for 2 min . Discard the/ B/ d2 d2 r) e% l" _0 M' J
s upern atant a nd gently break the pellet b y f in ger
* Q7 [$ y* \+ {. L2 k1 bvo rtexing.
* y3 y2 `9 A. L9 H' T5. In order to elimin ate resi dual DM SO, repeat step 4.3 b* i9 j! [- K8 W8 e/ u/ Q& q# _% Q
6. Dur ing the second centr ifuga tion, aspir ate the gelatin. d' g+ s ]- c% x: O
solut ion from the plate(s) and dispe nse half a vo lume
3 Y$ N! y5 F) \* T0 ~9 F' f) Lof prew armed compl ete DM EM medium on each one.
5 a- M8 N9 f3 Q* V& S3 ^3 ^$ B7. Br eak pellets from step 5 above by finge r vortexing,
! i# G: M4 W- X0 O7 Z, Oan d resus pend them in half a volum e of the wel l(s) to
7 w' x% v% i; k5 _5 l* Jplat e.4 b8 K4 y, _0 C, |$ n% R
8. Di spense cells dropw ise onto the medium- containing, G3 t/ a+ w. F6 a: u- Z) F6 }0 s
wel l(s).$ h1 B% n* G/ h( y) M: h2 N
9. Place plate(s) at 5 % CO; G/ k+ i5 Q' T* l- z" O5 Q
2
3 b7 ~' Y. P* s# c+ Tat 37°C, and c arefully shake7 P/ ~6 {( L& f) m! M. e
them horizontal ly in all direction s to distrib ute the cells5 g9 b4 v% |8 Z3 a6 M7 b, o9 N
ev enly.
/ t( @ l! y2 i10. 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, u7 [5 Z' u& O8 N i- s
attach. Optimal feeder density corresponds to a conflu-ent monolayer; higher and lower densities will result
$ T2 x+ K" f, }8 ~; _% n) u8 K$ G- M9 xeither in reduced hESC growth and increased differenti-ation, respectively.
1 c& L; }4 a* z( p/ C- bSe e Not e 8 for comments on feeder layer prepar ation.
' j f. o3 G' Q; H% t' @Embryo Culture
, B/ i$ K$ G; _' DSurplu s embryos from clin ical in vitro fertilizati on (IVF)
7 q/ w e5 P" d' R4 g; yprocedu res can be supplied, fresh or frozen , at any stage
, i. v( y4 Q8 Cfrom day one to day six post- fertilizati on. It is firmly estab-lished now that low-grad e blast ocysts produce h ESC lines$ k3 z+ K% E4 k4 D6 L( `
succes sfully [ 15, 16]. The refor e, the derivatio n of stem cell5 J* @% Y- V- U# w
lines is almost guaranteed for a ny Re search Pr ogramme$ V$ U" y) S6 ^- u! t( B, y' y V2 E4 e
estab lished in coll aboration with an IVF clin ical depart ment.
$ D. L. V! Z( C q9 Z, QIn this section of the proto col we will detai l the steps
3 L% z4 H m: L5 }* ]8 X* finvolv ed in embryo cult ure for hESC deriv ation. It is advis-able to use the same clinical grade embr yo culture reagent s( g, ^2 T) P# t8 L4 }
that are used in the clinical procedu res. Altern ativ ely, it is. {7 L: R2 B k. h
sim ple to p repa re the m from thei r compo nents [ 17]. Se e: U. G R d: S! A, a9 ~$ O% y
Note 9 for tips on embr yo cult ure.
8 {1 T' `& a* y3 e; J4 nWe will detail Vitrolife® protocols, and advi se to fol low [7 S) K. t3 Q9 @2 _
manufacturer instructions in ca se of different brand reagents.4 ]. |& p) D0 g3 Q$ j8 [4 o
The cul ture system us ed was a two-stage one, i .e. changing
/ e6 U! a% I% y( L4 r# e$ ?* T- Kcul tur e m edia formulati on on day 3 or s ix-eight cell stage o f( ]1 ? a. T- `4 _
development (from G1 to G2 series ), un til blastocyst formation .
1 T3 N8 g" }! N3 P) i I' dFig. 1 Time schedule for the
) H3 w, R8 o1 ]# i3 p0 x9 vcompletion of a full derivation
9 R/ @4 Q2 X! K, T9 Dexperiment+ ]8 u. \/ Z: X6 c Z# B
Stem Cell Rev and Rep
. Q7 r# _# A3 m1 K' @# aEmbryos are cultured in m icrodrops under oil, to, A' u! [1 Q4 B+ A2 b/ h8 d
prevent evaporation a nd to minimise pH changes. The
- _6 p' E! `9 m3 {following protocol corresponds to t he culture of fresh or
+ F) T6 d& V; X' @8 t3 p t* Bthawed cl eavage-stage embr yos. In the ca se of receiving
* X5 w4 c- X K8 @( a1 ?frozen embryos, it is crucial that the thaw ing i s per-formed in the reagents corresponding to their freezing., k: L+ i" Z v8 R+ r
Follow manual instructions from the appropriate thaw-ing kit. After thaw ing, follow the protocol below for
6 S( _8 _8 }) oembryo culture.- h7 H! r$ P2 B! q! a
1. Equilibrat e OVOIL® was hed wi th 1:10 volume of G1 or* C0 I5 Y3 i$ A! h0 B4 R2 g
G2 for 6 h to overni ght at 37°C and 5 % CO2
/ v/ [% ]- D& N. ^1 u5 N D.
% ]* g( K3 I( T/ J; P, s2. Equilibrat e G1 or G2 medi a as required (see Tab le 2 ) for. |9 S5 ?7 n4 S0 W1 n
a maxi mum of 3 h.
, S: Y9 K$ M5 j( b3. To set up embr yo cult ure plates, pipet te 25μ l of the' ?" H3 p$ {. k: i1 ^
equilibra ted G1 or G2 media per embr yo on embr yo
" { F5 r* J% P/ Gculture d ishes/plates . Cover the drop(s) with equil ibrat-ed OVO IL®, and add 25 μ l more of the medium to the
$ } ?+ s0 W% w; Wformed drop(s).
1 @$ N6 `- A3 P$ `4 p& u4. Assess embryo development daily (see Fig. 2 for a
6 w- |3 t& L' ?7 X! `9 ~di agram of g rowth pattern and c orresponding images7 u8 c) w% u. A8 J& ?- f4 f |
of embryo development), and transfer them to fresh
( K+ A; r3 m5 X5 x& T) v: ?media d rops every secon d day (see Ta ble 2 for
2 [' p% y! m* n0 m/ m/ N4 C/ Xmedia c hange guideline). P re pare new drops as- K/ y7 Z2 M* D) `. A& l
describedinsteps 1to 3above.If embryoculture
& U# z5 W; l: J9 q* Istar ted i n G1, remember that once embryos r each: K) g7 f; @, j- B
da y 3 o r the six-eigh t-cell stage, they must be$ F& m+ U: u* Z: D& n
cultured in G2 medium.
8 i u* o2 J2 o/ G7 [) l! v/ r5. When embr yos reach da y six of develo pment , or the
4 |* L6 |. q% zICM of the blast ocyst is visible, it is time to set up the
" T6 }# H$ A) [9 g: X2 ~feeder plat es to seed the former.! I6 g( S2 Q' A: [6 A+ J$ c4 ]
ICM Isolatio n an d Se eding& r1 C1 S) T4 o9 h* f
During denudation and ICM isolation i t is a dvised to, D+ ?' S. m. S! ?- Z* R& |
process each em bryo indivi dually. Pulled glass Pasteur
?/ R$ p; s" g* Q7 Y* Jpipettes 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" b$ C+ q# E$ |2 q: C+ _
IVF p rocedur es." J2 H( [ F" K5 u+ \
Con dition the feeders before the d enudation as follows:
$ |" b: @% v3 F6 h+ Dbetween 1 and 3 h before the seeding e liminate f eeder
1 h, O5 q9 g6 `" q9 x" dmediu m. Wash three times wi th 1 ml of PBS with Ca
+ r' Z6 a# }2 V" ]- w, C O" L4 V+2& w2 a ?8 y: @! |# }
/: K" h! `* f# b# P. W* z
Mg
& t$ C8 v+ q, K. ?6 ]. |+2
/ A/ Z0 _" ]- Band once with 0.25 ml of HES medium. Add 0.5 ml of
- X s' T, `1 r+ T% n- B5 [7 I/ yHES medium and equilibra te for at least 20 min at 37°C and
$ D; V' B1 w8 o, Z* B& l3 F& D5%CO% u' X; G* g \
2
5 t! }0 \" D" \9 u. At this point the new feeders are ready to recei ve! u' q9 p( |# o: O
the IC M.
) O( Y6 {5 b6 W0 y4 RZona Pelluc ida Remova l
5 j! P x9 O+ b9 F# t+ @1. Equ ilibrat e separa tely ac id tyrod e’s (AT) 1×, G2 me-dium and HES medium at 37°C and 5 % CO; ?- O# }6 C, O+ X. E0 \
2
' ^ G" ? X; \$ \& o* K4 P, for a1 D6 ^6 x" E. N% Q& V- I; G6 m, R
min imum of 20 min and a maximum of 6 h.! F- B: \5 Z* i; Q* e. ?, m9 w
2. Bef ore any mani pulation is perfor med, images of the
& u; A/ G k$ Z% \! p5 [blast ocysts at different planes (×40) should be taken
0 v% ]4 S5 H- gfor further asses smen t of its grade. Afterw ards return it, V' I; Y- W1 ?5 `6 B
to the incuba tor.+ v) S9 b' {" t) s$ b
3. Pu ll 2 long ster ile glass Pasteur pipet tes wi th the aid of. E) J+ a" v! R7 `4 S) L
a Mecker or Bunsen burner. Leave one of them opened. C6 ^: C0 a$ B, n( M v
an d rounded , and close the other one leaving a small% V; z; f0 Q8 ?8 [) Y
rounded ball at the tip (Fig.4 ). Check the thickness of" r# o3 `+ @8 ?+ g) C
the tips under the s tereomicroscope and r epeat the
6 |/ m" O. W; ?& \( r$ Pprocedure unt il a couple of pipettes whose sect ion- x* {- x: c' {
mat ch the diam eter of the blastocyst are produce d.' y' R; p- C& o5 F8 Y
4. Se t three drops of AT forming a row in a 60 mm tissue
0 I" |- ]4 B6 [& {. A2 V0 Ecu lture dish, an d mark thei r position at the rim of the6 a0 Z* }+ d: i( Q) g
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 J- M6 W. g/ w5 H+ f+ S
second row at the middle of the dish, and three drops- U4 z3 L1 }( Q9 R
of HES media at the bott om, again aligned in a row: T1 Q8 r; X5 w( w
(S ee Fig. 3 for drop distrib ution).
/ I! W/ O% W) D" R9 P& }1 {5. Se t anothe r three drops of approximat ely 100 μ lof
4 X; \; s7 c2 k4 s0 fHES medi um in a new 60 mm dish.$ x; ]/ o: ?* A/ x
6. PicktheembryoupinG2 mediumwitha 275 μ m$ n- a( H7 S0 g5 @9 a* R+ f& T, h
capil lary mi cropipette, a nd t ransfer it to the left AT
9 O! c, m! r4 }1 t$ @drop of the plate prepared in step 5 a bove. Stop
?; l! w! Q: R( g# m8 {dispensing G2 medium into the AT drop a s soon: f! R s8 m0 E$ x# T+ Q- u
as the embryo has been releas ed. Empty the pipette
* I, R" {) @: Z9 ~1 _( tin the border of t he dish to discard r esidual G2
: f0 p* o9 o9 b6 G) ^medi um.
0 {$ P* n: A3 P' a4 F7. Wash the pipette twice in the second AT drop.
( \+ D+ Y4 F2 _8. Return to the fi rst drop, pi ck u p the embryo and/ t$ i+ d$ |4 r/ a i" b2 d2 @- J
trans fer it to the third d rop. Monitor unde r the micro-scope the dissolution of the ZP (0 – 2 min).
( s; ^( H. _3 k- g9. 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! R1 L/ @! ]/ O' Q3 h( i
pre-w ash, dispe nsing the minim um amoun t of AT into
' a* \7 j( ?; Z* V: Gthe G2, the second to load the pipet te with fresh G2( D$ H. d8 A) Q$ j4 n" r3 P9 Q* D
medi um, and the third to was h the blastocyst .
- n% k% g! L7 _1 r' u9 k2 T6 v- k10. Tr ansfer the zona-free (denuded) blastocyst to the
" R) [: {- _+ m8 ?( U) z3 J9 \drops of the HES medi a using the washing procedu re
+ z" n* z2 l! Vas described above for G2.
7 b+ I6 H; Y' K8 w0 a$ k& K11 . Immedi ately trans fer the blas tocyst to the drops of
0 K/ `$ [0 ^& H5 Tthe HES medi a prepared i n a sepa rate plate in s tep
1 H- i, O8 @& T! p& s$ l( g2. Follow the s ame procedure of trans ferring to the2 q6 g V# `8 I' M! |; P3 g
left drop, washing t he pipette and loading i t in the
) j2 j$ ?& u2 [/ i6 }' o2 f& vsecond intermediate drop and transferring the! `3 I# N( ^8 r5 X
embryo t o the third drop. P l a ce the p lat e i n to the! w% |5 v9 M' ]+ ^5 p2 S5 B
CO$ X& h3 Z) M ^. a& C
2
8 l2 o7 ~- g6 k7 Q* p, A1 t9 U4 zincubator.) A( @- t- T0 e6 j) m7 m0 U
12. Usin g the ima ges take n in step 2 above, asses s the0 e A( ^% u& z+ t6 r4 d. a
deriv ation grade of the denuded blastoc yst (see em-bryo grading s cheme in r eference [18]) and a ppl y8 j2 G- u! ?) t
Table 3 for determin ing the best met hod of ICM iso-lation. Depen ding of the outcom e, g o to protocol A or2 T- l, o5 J$ E0 M- m: H
B be l o w. S e e No te 10 fo r c om me nt s on t h es e
, B+ }3 }8 S# m; t# J0 p, ^procedu res.
9 e6 i( H& C- M$ d$ }$ Z% D+ K% XStem Cell Rev and Rep
0 H6 s( v/ O0 D$ X8 BICM Seedin g by Pipett ing (A)
9 ]" A5 n) p% K) ]1. Choose an open pulled long Pasteur pipette of the same
% E1 R" e( y1 `" M5 wdiameter as the denuded blastocyst (see shape in Fig. 4a ).
5 _9 }* m5 N2 I9 {5 f& ?2. Load half of the tip with fresh war m HES medi um from. U9 ?) w; u. y* E, l4 `0 t0 ~
the drops set up to equil ibrate before denudat ion (ste p 59 ^ s) G s. w" \0 X
on zona pell ucida remo val proto col).
k3 S& d7 g& V3. Aspirate the denuded embr yo and pipette it in and out
. f5 e, M7 l5 a/ z" T; Vwhile checki ng for loose ning of the TE cells.
5 l* E: E8 ]" F, r$ n$ h0 X4. Aspirate the i solated cell clumps and dispense them/ a9 ?. [( v9 P0 {$ L) {
separa tely on the feeder plate.
& S0 z9 q0 d! a. R9 k+ d7 y: C5. Ta ke an image of the seeded fragments of the bl as-tocyst. Pay attention to l ocate the ICM and record
* Y" v4 A6 g) F {& k3 c* J$ sits posi ti on.
* U0 ^) w9 v9 P k' O' @. m" t3 vTable 2 Embryo culture1 X* Z- g# n8 `: a! a9 p$ t- n7 i3 J
protocol
% I5 F8 f; f |: Z( ~ kDay of development Stage Medium Image recording Media change
% |# O. D7 Y/ w) _% ~1 Zygote (2pn) G1 − G1
7 C; O3 i- N0 q! n! x2 u22– 4 cells G1 Yes −" U9 o6 l; j7 b8 u6 Y. y8 {
36– 8 cells G2 Yes G1 to G2 q o8 C S1 f* h( {. F; N
4 Compacting morula G2 Yes −: ^* w. U) @- b8 R
5 Expanding G2 − Fresh G2, s4 @8 L; O- c0 U. B
6 Blastocyst G2 Yes −
$ B3 T/ ^2 c* m4 Z% e7 Hatching G2 Yes −
3 N* m$ }9 D/ n% S0 a1 C; |+ o4 r. ]& ~Fig. 2 Dagram of early human+ |8 h; w; n6 Z# J3 G1 l8 |
embryo development. a-a’( {8 }: \9 |# b! r* {) v; ^$ q
two-cell stage embryo, 1 day
4 q" l3 u0 l5 Lafter fertilization; b-b’ four-cell( I: N( u/ r, N* h/ g, T3 [
stage embryo, 2 days after
$ X3 [ a j# G# P' Y7 X! Jfertilization; c-c’ eight-cell
3 M# G9 Y( m; u1 p2 ?* D: nstage embryo, 3 days after f- @* s8 O6 o5 Z. n& d
ertilization; d e a rl y e xp an di ng
* ]! M# k+ J# J" z- C/ w% O7 Vblastocyst;d’ morula containing; O6 o3 e5 N, @/ f1 \0 f
m o re th an 1 6 ce ll s, 4 d ay s a f t er
2 ?$ k, L9 `4 N3 h9 E$ yfertilization; e early blastocyst,
( s! P0 r/ J Y) W6 R7 }5 days after fertilization; f2 S: R, m L( b; G! l v
hatching blastocyst, around
1 m7 I$ Y' `7 I7 days after fertilization. ZP:/ u3 q( p2 m. N1 k/ u/ C
Zona Pellucida. ICM: Inner3 t1 X1 j2 v* ~
Cell Mass. TE:' T( S) f/ C. b2 D+ c o
Trophectoderm
4 d X1 M% p, t1 L2 O) {Stem Cell Rev and Rep( ? ?1 S1 h) S. A" K! O& M+ R
6. Tr ansfer the plate to the incubator, avoiding abrupt& _0 w3 Z j2 K: `
movements t hat may separate the s eeded cells from1 r- `& ^* `5 ]6 d) c! j
the feeders.
6 P/ \1 E# t5 q7. Inspect the plat es daily. Cha nge the medium every other
z7 N9 C: z- ]5 vday.
( W' k4 F/ b9 P8. Prepare new feeder cell plates as soon as pluripo tent
5 |3 r( q2 R5 Y, Z/ ugrowth is detected (see Not e 11 for deriva tion timelines' T9 I+ ~# A+ w6 ] W& V
and Fig. 5 for ima ges on deriv ation progre ss).
" ~" l; \, l7 l u1 I9. Split part of the emerg ing pluripotent colony when. s. q/ S4 B: U* F: W# J
resi dual TE cells begin detach ing from the bottom of h% J, R# e7 Q* w
the plate. Follo w Secti on 4 below to do so./ y2 T- J# D$ M4 Z, X- [" G+ y% q
ICM Seedin g by Mechan ical Cut ting (B)0 ]3 ^. T5 f+ m M8 Y
1. Aspira te the denuded blastocyst w ith a pulled open& o1 a2 b1 h0 l$ u5 G6 I7 v+ {
pipette (Fig. 4b) and dispe nse drops of approxi mately
" f' i- T$ `* x' S20μ l on the dry area of the 60 mm dish containing fresh
4 d. C4 i. c& E4 {! v. E6 R3 fHES drops, until the blasto cyst is dispe nsed in one of
7 g/ P4 D" }5 Z) M" [: J, K( Xthem., |3 i4 q" \4 Q C+ U" i
2. Drag the denuded blastocy st across the dish, away from
9 z7 b$ g* x q0 X0 I3 @the HES drops. The volume of the embr yo-conta ining
+ b2 ~% u0 G, e9 Y+ Edrop will diminish up to the point that the embryo
5 u6 Q+ D% o+ w. O7 xflattens and sticks to the plastic. At this point you shoul d
1 s9 H8 G7 f6 f/ W/ Vbe able to locat e the IC M: it is much brigh ter and less
6 P8 }! c! {$ L6 p* _2 zflat tened than the dim TE. Sa ve the pipett e without% ~# Z5 N4 v% E( \8 M& X
emptying it for follow ing cutt ing steps .
" C: i& g. O9 n3. Pick a close d pulled (hold ing) pipette (Fig.4c ) with the
% y- @! D- ~ y6 s) k7 x4 @left hand (rig ht for left-hande d) and hold TE by pressing% ?* x' @4 i( ^5 k4 B7 M
its border agains t the plate.7 b+ R! J/ W4 w6 p8 [# H
4. Pick the open (cutting) pipet te still contain ing medium
8 `* |: x$ v6 A0 Pwith the right hand (left for left-hande d). With its edge," [' G) R! A6 @! H: U/ _
cut out as much transparent TE as possible. Imm ediately
6 p: G/ |0 } j2 X, W5 qrelease the HES medi um to a void damag ing the ICM.
7 S7 D3 H a# I& o3 I5. Scrape loose the ICM contai ning piece, which will be: U% _( M a8 M6 |/ S! \2 {
adhered t o t he plastic. M onito r the ICM w hile it
& ~ \/ w% {& d! SFig. 3 Shapes of long glass pipette tips for ICM and stem cell colony" d- {! ~4 m2 P Y
processing. a transferring pipette with open rounded end; b cutting# b% S% D" s N$ v% O$ F( A
pipette with open bevelled end; c holding pipette with a fine rounded4 c8 g) t' S9 y. |
closed end; d curved hook with closed end for outgrowth scraping;e-g5 }, @& P( _, s1 k& O
flat hooks for colony cutting and scraping. h detail of the tip of the
0 ?0 Q" @& ?* l' v% {holding pipette depicted in C
2 C1 G* z' ]/ V7 I" RTable 3 Choice of method for ICM isolation according to blastocyst
0 W3 X! n) e. e; J, B- G7 Wgrade( [8 |: B9 T+ l; W1 z8 L, v, S H
ICM01234 r4 p9 W/ I* t9 G N; K5 ]
TE: ^/ H5 D2 u1 V/ {5 ^+ Z9 |
0AA 1 A A A/B+ X7 z5 ?' \5 {' F0 S/ t
2ABB 3 ABBB
( Q5 c- [9 {# ]+ M4 [TE score - 0: no cell layer formed. 1: few big cells forming a loose
' u6 ^* s& \& D+ {, x! D6 @ Z5 ^, blayer. 2: cells forming a medium coherent layer. 3: many cells in a tight1 k$ a+ ^: l% G8 G; o/ S
cellular layer. ICM score - 0: no visible inner cell mass, inner to the TE
R* k! t8 R, A1 Vin any plane of the blastocyst. 1: ICM with less than 10 compacted
% Z" [9 F. k" S4 O; @$ ^& u/ Vcells, or loosened mass with up to 20 cells in the central focused plane
4 b1 }: R. m& Xof the blastocyst. 2: ICM with more than 10 compacted cells, or more4 C+ q; f9 S! i' n6 J9 a6 z$ {
than 20 loosened cells in the central focused plane of the blastocyst
! q5 s& x _. b- u- Punder the stereomicroscope. 3: ICM with more than 20 compacted
7 L7 \" J9 {. O) Q& Q; l9 Pcells, or more than 25 loosened cells in the central focused plane of the; ]: F3 |# Y4 C$ _9 B$ }
blastocyst. Method A refers to pipetting for ICM isolation. Method B! D$ w+ X, B5 K5 N, _1 l' e8 I
consists of mechanical cutting of the ICM
O7 s! z! j' {% Y3 JFig. 4 Derivation plate. Embryos are dispensed in AT in drop 1, and5 Y7 w+ h/ g* o' F/ q* b+ [% }/ L
tr 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% c, q x6 y4 v3 T2 z: T8 `( F
Tyrodes. G2: embryo culture medium. HES: Human Embryonic Stem
" n8 N3 r. L4 B, f5 tcell medium
! w& J" b# R+ [) G; U+ V0 M7 w1 ~' mStem Cell Rev and Rep7 C& L, e" Q# M4 s% @
detaches and float s. Qui ckly aspir ate medium from the
1 ?8 S0 h( |9 M" cHES 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* y1 X8 r- k$ t1 H
occurred durin g cutting.
/ f9 @+ u2 ]" J$ V- f3 @6 T6. Aspirate the floating piece( s) and spread them onto the2 A8 h) ~' [- }% {0 ~
feeder dish. Try to avoid pure TE fragments , recogni zed! h% m' ^7 `* k+ H& ?: {9 X0 w
by thei r trans parency.
2 d. F5 B" t2 Q7. Take an ima ge of the fragments seeded, record ing the
! x" q* B4 s9 S( v; p/ jposition of the IC M piece on the dish.( y% z+ y! b4 s5 Z, c+ B$ U6 s! E2 }$ R
8. Inspect plates dail y, feedi ng them each second day until
( m2 @* O- V" J1 ^( H$ ]* Dpluripot ent cell grow th is obv ious.
7 M2 }; J5 ] e% P8 g9. Prepare new feeder cell plates as soon as pluripo tent
" y, d& F3 o! a8 E5 |( l+ }, ~: ggrowth is detected (see Not e 11 for deriva tion timelines9 M/ B1 b6 j. z9 q4 C
and Fig. 5 for images on deriv ation progre ss). Go to
6 X; [" B. {. E) ?8 z) v0 h- e5 iSection 4 below for the firs t spli t of the pluripot ent cells. ~; x! h; e1 }9 A$ x& }* I ]
Split of First Pluripoten t Out growth8 T- F5 b+ }! L0 [; k* \/ i. B
When the pluripot ent outgr owth(s) reach the stage de picted" }: N" g% r; \; I- b v; L
in Fig. 5c , they are ready to b e split. Mechan ical cutting can
5 n5 J0 G$ b; i* f; Gbe perfor med wi th a glass pipette of any of the shapes A, D,
) t1 l. @! x, X2 UE or F depicted in Fig.4 . In a ny case, prepar e feeder plates( D, m# n) p& [
the day before splitting. If t he pluripotent outgrowth is
# b+ N; v" w5 p, E# \. v, Pisolated from the residual TE, a closed p ipette and a
$ j3 W9 l2 p9 T: j3 Ascrapin g met hod could be more effective. If stem cells are4 A- P( u5 r1 [6 M: i0 W
growing in clumps surro unded by TE, a n o pen pipette to cut
5 g/ i' _8 z4 M+ H8 F1 C1 rsome of them will be preferred. It is wise to leave some
- _0 j$ j4 }& v# i7 A* U* C/ Poutgrowth(s) behind, u nt il split cells ar e s uccessfully$ ?7 C. x, R/ t# @
growing in the new feeder dish.) p$ [+ W3 u* S/ I- `, w5 P
Notes# J+ Y& A# w9 ^+ l6 m) F( O
Isolatio n and culture of mous e embr yonic fibroblas ts and
( o) ^/ v) D8 U: M# q6 g1 U) [feeder preparatio n
8 E" b5 U+ M, e9 A4 h6 fNote 1. We describe here the deriv ation of hESC line s in
4 v2 o k2 D ?6 _# ^, _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
2 w$ N6 S0 P0 Q) D: tefficiency in stem cell deriv ation and lack of chro-mosome X inact ivation in hESC line s estab lished, ?0 ^+ f( m4 j7 m
under such condition s [19].
; Z' w; e" k% ?, R/ PNote 2. Pr imary MEFs a re the first cells to be us ed as
" ^5 I! R/ ~) R6 bfeeder layers for hESC culture. Alternatively,& n# i4 z8 C! [- K. O9 T
either transfor med [ 20] or n aturally immortal-ised [ 21] M EFs, as well as human fibroblasts
! O$ B" ]4 D( A& dfrom several origins, includi ng foreskin [ 22]9 {! h$ u5 F& L. c: k
and placenta primary cells [8 ]andanimmor-talised placental f ibroblast cell l ine [23]have
) [. @) x* ?# l$ H; y5 ^ lbe en u sed su cce ssfully as fee ders f or hESC" g+ D# g" t! o, S
deri vation and cul ture. Primary lines f rom hu-man f oreskin f ibroblasts (HFF-1) have al so5 G- h4 w' H ~: A2 x) L) e! R
been used reproducibl y to cult ure h ESC li n es.
6 g2 P0 N+ k* R3 M9 iIn the case o f HFF-1 cells, i rradi a tion i s the
4 F3 w) @" C$ W" M! ?7 d" }: Fpref erred method of i nactivation. All m et hods
. ]$ o, B8 m* W8 W1 n4 N& adescribed i n this paper are equally valid r e-gardless of the t ype of feeder s used. O- F( O* E' ?# c8 N
Note 3. Mice stra in has proven to be an importan t facto r
1 o( n" W( F5 u$ M0 h6 Rfo r th e q ua li ty o f th e fe ed e r ce lls d e riv ed fro m e4 ~. e k( Z' A6 S( v& i( z
Fig. 5 Micrographs showing& |1 ]( i; i6 U% i% B x& }7 A% z
evolution of the pluripotent and2 X" M; C, C# D4 n0 ^* i# u
TE cells during a derivation$ H* v3 w2 J. w6 B' f+ ~$ X
experiment. a ICM 1 day after7 z7 O# t# G& @& P
seeding. b ICM cell expansion" h g7 h$ a4 H, E* o3 y
after 4 days in culture. Arrow
2 `! a0 _$ z) y6 g( d' q0 \9 Mpoints to the pluripotent- }% ~7 |* A; l7 u
outgrowth, which looks like a& K# _; U6 q( {! P! _& E2 T3 ^
clump at this stage; c colony3 h8 _) `: [8 O; N0 ^7 N
outgrowth 2 days after splitting
5 P0 f6 @4 b% _2 K' kthe outgrowth in panel B: arrow
, k0 Y9 r$ l3 y& ?points to the expanding
3 O5 C$ R9 X* G6 }: i1 Ppluripotent cells out of the
: C3 f$ [- A/ pinitial hESC clump; d residual# v* Q3 b F! G2 H# v1 P
TE cells 3 days after ICM) l/ k. s/ v) M5 n; N! K: E U
seeding; e TE differentiated
6 t# d2 \! m5 O! k8 k3 H# gcells 4 days after seeding; f TE
3 `8 Y( @2 r5 z: G5 Dcells detached from the plate4 N3 f. k/ U; J+ O2 ?& q
9 days after seeding; g detail of- ]% q& p! S# k* C4 ~' d' f5 n
the emerging pluripotent colony) x$ @( J& i; C3 T
of picture C;h early hESC
, V6 N" q2 q. q+ D* e3 t6 [$ r1 acolony at passage 4; i regular
3 U9 d! a' R3 ^) X' Jcolony of an established line at! {2 Q% }: j; R) q! d3 d% g
passage six
7 J5 e2 h' J% ?2 W- kStem Cell Rev and Rep
U- Y0 q) D5 t6 ^them . In our hands, embryos from the CF- 1 and( I+ v4 M- k* C- @) N$ w: r
MF-1 line s, and from the cross between MF-1 and
$ Z6 o. |" `% C8 Q" g1 HCD-1, are much better than inbre d CD-1 embr yos.
$ W5 {+ l2 F$ h" h" WNote 4. Isolated pME Fs can be frozen at passag e zero, and+ f# Y/ I5 m/ P) j/ ]: W1 _3 ]
expanded at a late r time to prepar e feeders . Nev-ertheless , the expansi on up to passag e tw o before
# r/ @. s/ g; o: {freezing has the advantage of increa sing the num-ber of cells obtained at passage four. It has to be
( [5 B6 i! ?: V v8 hnoted that in the isolati on procedu re describ ed in) b9 p5 r0 w. [# ~+ ^* w
the Meth o ds secti o n, an d d ifferentl y fro m oth er( R8 }# p- I8 y' K
published protocols [ 24], tissue chunks are not
( E; G6 E, P4 b2 }1 L; idiscarded before seedi ng after first trypsiniz ation.
/ \5 D5 F5 U1 T* ]* w+ u/ pThis procedu re yiel ds signi ficantly higher numbe r* d/ I$ I- ]* F
of cells. It is advis ed to culture chunk s a nd clumps# l- d2 k) v" s" g
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
) z, e, `/ R; p+ p/ A* Pnormally be suffici ent to produce enough numbe r. b! A0 z6 ]- x3 c# L9 s
of vials for cult uring up to six new stem cell lines, c+ `9 v+ `" I- }! L# }& z
during 1 year.
2 d; K- w2 i& G+ RNote 5. Finger vorte xing is a simple way to mix a solution2 ~" l; b, ~) y( D, I0 q
or disag gregate a pellet in a test tube. Hold the top) b) |; W" e' B; O s' M
of the tube secure ly in one hand and draw the ring,1 `9 a: Z* ~# Q& ?3 j
middle and index finge r of the other hand sequen-tially towards you, tappi ng the tube. This creat es a( n8 u/ k3 T2 E6 {
wh irlpo ol eff ect inside the t ube, w hic h can be
. h; E* A0 p! Yadjusted in inte nsity by speed. It is milder than
x7 J6 J8 H. w* F2 H7 D: umechanical vortexing and yields healthier cells
* k" T/ H. o6 N( e; q" x3 ?after centr ifugation .+ d Z4 j0 h$ H) Y
Note 6. Freez ing fibro blasts wi th a slow freezing met hod is# y. S# h9 @0 e" D: o
not an issue , and they can be stor ed at − 80°C for- |* `' U, i- i% l
months without a decreas e in subseq uent plating
. M! \# H W/ K' Y! K/ ]: Yefficiency. The proto col described herei n uses the# P$ s5 L; t( P! x4 e7 Q ]
slow rate freezi ng container Mr Frost y, which has" w/ F& G/ u5 S3 m$ n# H2 C u
been vali dated and used for nearly 20 y ears i n
5 z9 R8 z+ m" }mammal ian cell freezing [ 25]. It is a cheap and
: F7 o! U% T" ~1 p. f* I4 xtime savin g o ption when compa red to alte rnative
# u! n- {' }7 N$ l tmethods such as contr olled-rate freezing and vitri-fication, which may be suitable for more delicate/ g. \6 l7 f+ f5 @
materials like embr yos and hESCs .9 K, a+ h! E6 J- ?: b* j
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' K0 L$ M5 Z1 U8 q, p
toxi city and different iation during cultu re.
+ @8 {! N) Y+ T8 BNote 8. MitomycinCis thepreferredmethodtoinac-t ivate M EFs, a nd detail ed st eps to p erform the
( ~7 V& d: v- ?2 M1 b7 C/ K- t% c0 Iprocedur e are included in the text. Alternative-ly, γ - or X-ray irradiation can be used. In a ny3 W- J6 A. B7 m2 M. L% N
case, t he hESC supporting capaci ty of each
4 B, n6 `0 j/ b" [4 T: m- i) Pnew batch of feeders has to be tested on
* e) t' u* r1 w. Xknown hESC lines bef ore c onsider ing t he m
# G1 u- B7 k/ z1 W6 ?5 ]8 M% H. E& g# kvalida ted f or use. Once the new batch has been
; A: ^4 t2 M3 e& V& Fvalida ted f or hESC cultur e, it ca n be used in
* Q# x9 G9 k% o# u* ]! a1 |- ~subsequent derivation e xperim ent s.6 ]& b% I5 ~; H9 i
Embryo and ICM Cul ture! f7 `; }' I. R! N2 C7 B
Note 9. The procedu res detai led here are based on the use
, v* l$ J- d2 ]8 cof high quali ty clin ical grade embr yos, but fre-quently, the quality of the embr yos donated for
. V9 U, W0 {3 `research do es not correspon d to the standards
, V6 R1 Z+ l* c7 ]presen ted in Fig. 2 . If by day 6 of development ,6 m7 _+ i+ I3 r! G
compa cted cell s as in Fig. 2e – e are not form ed,
6 {% v! t; g' c$ z4 ~$ eextending embryo c ultu re i n complete HES
7 z0 y; a; s" Emedium for 1– 2 additional days will selec tively
: _2 m; C4 J: ~( i3 bfavour the growth of the ICM ver sus the TE.
! L4 V- Z( g/ v$ E4 XThis appr oach is supported in the most recent, G) n& y. j, {+ R8 G) B. T/ v
li terat ure [ 15].
: ]5 s2 [# ]* \9 z8 G1 i- nNote 10. Reg ar ding ICM isolation, s om e authors use a n
) F+ @) d8 L$ R, i1 Fextended acid t yrode’s treatment to weaken
: f! p+ B* m u9 I0 K! Z* vthe TE [26], thus facilitating the spreading9 c' B9 v( u X6 e( [1 F1 N5 _
of blastocyst onto the feeders. H owever, in
2 P' T7 v9 G/ I' F: oour hands, the efficiency of this method0 A9 s! W @* K, K
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 K0 M; U: x1 e' b
the same AT t reatment to all embryos usually$ N5 B$ X7 E5 @* Q# F5 v
results i n s evere damage to some of them.
! |6 ~- U3 o: R% o9 m5 rTheref ore, fitting t he method for TE reduction
# z6 U L9 E* x) lto the grade of each embryo (Table 2 )helps
) g: b1 L. p" Y T. I/ S) fmaxi mizing the r ecovery of viable cells f rom2 Q6 y4 j! r3 C% p% S& P4 L
the I CM. When TE is r el atively s mall com-pared t o the ICM, or when the ICM is not& o9 B F8 L8 K! z4 W
compact, a pipetting method (Method A) is2 b1 Q5 {5 @& [: |( p! l. t
preferable. Alternatively, if the TE is robust8 o c/ S. q5 u) k. o; ]3 P+ Q7 F0 H
and t he ICM i s c om pact , mechanical cutting# ]2 P+ v9 A# t! g' v
(Met hod B) s hould be the method of choice.
7 }" `6 w9 j2 y2 _/ U/ c( uSpeed during embryo denudation and I CM) S& S" p$ U. j8 H0 o
isolat ion is criti cal, even if the m anipul ations
+ G& P, W, A: Qare performed on a heated stage.
( Q8 S# ^% E. h* q2 n* ?0 o2 C8 iNote 11. If healt hy, the first seeding of the cell s from the/ w* {9 ?! m, G
ICMs will att ach i n 6 h t o ove rn igh t . In o ur
- {# E( B, \4 q7 u' A s4 Nexperi ence, those clum ps needin g more time to
Z9 j! ` L q# ?4 Battach are not robust enough to yiel d deriv ations.
& N- d9 ^( e4 fThe first outgr owth after the attachmen t of the
1 ^0 D/ m9 z+ `# S& x. X/ z- h' wclump can be seen from days 3 to 16. At this
% x: C5 n0 }: [$ v2 o% }6 ipoint, the undifferentiated cells need the suppor t
v$ M. {( C( Gof fresh feeder layers to estab lish pluripot ent cell
; Y& t' j# j4 T4 H, J0 xgrowth. TE cell s evolve invar iably to form syn-cytial cells that invade the feeder layer and die
8 D8 {4 F7 T, K# P2 Cleavin g g aps i n it. It is critical to have fresh
# u& [4 e# T+ V1 C7 I: [feeders ready by the time the TE cells and deriv-atives detach from the plat e.( M5 L9 H& w: z1 t/ [
Acknowledgments This protocol is the result of work funded by the) |% S o2 X1 B
North West Development Agency (NWDA) i n t he UK and the
7 @- x6 D ~) p. D; s. f/ rMICINN-PLE2009-0091, IPT-20011-1402-900000 and FPI-CAIB# V+ Z0 i' j% I" t- U+ p+ t/ c
Grant FPI10 grants in Spain.4 S# U: {; I4 u" k N4 q% t1 \
Con flic t of in terest Th e aut hors decl ar e no pot enti al con fl icts o f$ V/ ]0 k9 Z5 W* i$ z* G/ l
interest.
5 b/ b, Z" }% E7 y5 Y9 O" }- QStem Cell Rev and Rep( y* }; X; ^4 N5 H3 j# m
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