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Optimized Protocol for Deriva tion of Human Embry onic, Y' x0 Z& G6 Y$ W$ ~* j% ~
Stem Cell Lines/ ^ r5 R6 G3 x: m! N3 w4 h
María Vicenta Cam arasa & Víctor Miguel Galvez&- H) Y& R/ L$ B+ l, C# x) Q: T4 J/ V
Danie l Roy Brison & Dani el Bac hiller8 g! }# r8 T8 O( L- ~: _
#Springer Science+Business Media, LLC 2012& Z/ o+ h' k5 R& c6 V) a( ^4 \
Abstr act For t he pa st 12 ye ar s, th e b iolo gy an d a pplic atio ns
- @/ `$ N: u5 s; qof human embryonic stem cells (hESCs) have received great
7 }/ k) B! P9 v* Catte ntio n f rom t h e s cien tific comm unity. D e riva tive s of the first5 l7 E' r9 D% B* _6 b. _
hESC line obtained by J. Thomson ’s group (Science 282! Z5 y3 X: |2 x7 d; l3 B' k
(5391):1145 –11 4 7 , 19 98 ) have been used in clinical trials in* [$ _. T! u9 }) n& |9 D0 d
pa ti en ts with sp ina l co rd inju ry, an d othe r h ESC lin es ha ve, j- A+ z# u( `
no w b een u sed t o g en era te cell s f or use in tr eatin g blin dn ess
. \0 ~) A" K' k* V4 q/ k( L a n ce t 3 7 9( 9 81 7) : 7 13 –72 0, 20 12 ). In addition to the classical
( N$ w( M8 }4 K E7 Y! k1 j/ G2 mpr 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
" y2 L7 D' N/ A; B$ 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,/ h) S9 X n- X! Z' V' N6 _! z
2005 ; Nature Biotechnology 24(2):185 –187, 2006 ;Human
8 s, ^, s$ A# J0 i) @. e" PReproduction 21(2):503–511, 2006 ; Human Reproduction 20) P. e \7 q- i- p$ u4 o
(8):2201 –2206,2005 ; Fertility and Sterility 83(5):1517–1529,0 E6 U+ f; I7 R# E4 h
2005 ), novel hESC lines have been derived xeno-free (without
! }+ x+ E+ J, m1 cusing animal derived reagents) (PLoS One 5 (4):1024–1026,: e' D2 ^1 I$ [- Y: d& B' I3 O
2010), feeder-free ( without supporting cell m onolayers)
6 Y* `3 q" t+ T/ g7 }9 J: L y(Lancet 365(9471):1601 –1603, 2005 ), in microdrops under
4 J6 D" c# o- }1 h' Ooil (In Vitro Cellular & Developmental Biology - Animal 46' a( u" D0 h* \
(3 –4):236 –41,2010 ) and in suspension with ROCK inhibitor
4 H3 C* J2 l# a1 W( T, e(Nature Biotechnology 28(4):361 –4, 2010 ). Regardless of the
% t8 ?6 x( Z) v2 e( ?6 k% S3 `culture system, successful hESC derivation usually requires2 u0 _0 `* |- F+ L6 d! x7 Y+ d. l
optimization of embryo culture, the careful and timely isola-tion of its inner cell mass (ICM), and precise culture condi-tions up to the establishment of pluripotent cell growth during/ H) Q. k9 u q3 ~! B
hESC line derivation. Herein we address the crucial steps of
+ `" r' r$ T% K- lthe hESC line derivation protocol, and provide tips to apply# \$ b5 {7 X5 q: i6 i6 V9 h, g
quality control to each step of the procedure.1 I3 y' A: h9 Y+ _. ?; |' n& }
Keyword sHuman embr yonic stem cell s5 c. l) b* g( U* U* B
.
" S+ u# J) ^2 K9 k% c2 ^4 X) f: NDerivat ion
2 B' z2 v4 O( A2 g) y) N.9 C4 D6 T4 F( e. w
Blast ocyst; O$ |; Y. h, c
./ b( ^8 E8 A% B. Z
Cultur e o ptimizatio n' i" @/ K% A6 X2 A4 X& i' h: U
Introduction, N$ Z. f8 Q4 ?
To da te, hundreds of human embryonic stem cell (hESC) line s
D3 L$ S5 f1 o3 F+ uhave been de rive d f rom surplus embryos after as sisted repro-duction techniques. Reported derivat ion e fficiencies range from$ p: M$ L) w2 e9 u. V
7 to 100 %. This wide span pr ob ably refl ects the di versit y of the) v4 @2 R* w& n- Z1 t& [: @
methodologies involved. M any var iables of the procedure s uc h
/ x4 Z- V y( u8 l0 ]0 Ha 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 H, S# T: Y9 ~, M6 k
removal and inne r cell mass (ICM) isol at ion a nd type of feeder
+ } Z, Y8 [1 J' _" }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; h8 K* X c9 z
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. x# I) P; ^( j3 M5 ]3 U
th e genetic stability of more than one hundred hESC lines& L- h- e) ?3 c! f
using consensus protocols for karyotyping and genotyping., [; M6 E7 k9 H Y; B
This echoes the importance of defining robust and common
! I' e" s/ U" e4 H4 {% rcriteria for the assessment of both existing and newly derived( ]$ S0 g) p# k& j" D7 J* s" k- V
cell lines, and for the validation of advances in culture con-ditions. [13].1 T$ i- u1 O) V) d, @$ q
M. V. Camarasa (*)4 g+ f" M5 [5 b* N
:2 E. P% t. Y1 M+ e6 x6 X+ W
V. M. Galvez
$ d5 X: i& I/ b8 v( g:
3 ^9 p1 @8 y! z9 w7 MD. Bachiller: \% H, { T+ {/ _& ~' }
Caubet-Cimera Fundation, Centre for Advanced Respiratory) V0 \& L# s/ x* X
Medicine, Recinte Hospital Joan March,& D+ b/ M" T, E* M& M
Ctra Sóller km 12,
, z9 S" `' o2 k" H' t( [1 u07110 Bunyola, Illes Balears, Mallorca, Spain
0 P& S. c: ~' s- }( W7 C* Te-mail: telomerasi@yahoo.es
& M& o* u% A6 v" s# Q+ T9 i1 ]M. V. Camarasa
9 r: @: U m( O" m y# T+ q( t% Ne-mail: mavi.camarasa@caubet-cime ra.es& w0 U9 g. T( ?/ n
D. R. Brison7 P* e! `, n% J+ _
North West Embryonic Stem Cell Centre, Faculty of Life Sciences,
% I( E/ G" L' O, p3 q8 O$ @ qCore Technology Facility, University of Manchester,
0 [/ a& V% w; h5 a# B! N46 Grafton Street,9 @- C( K/ ]8 `
Manchester M13 9NT, UK
0 S& J, t" C. f# ]. FD. Bachiller
5 l* n1 h0 a" Q% }; A7 GConsejo Superior de Invest igaciones Científicas (CSIC),
* r" {6 r* A9 u4 _Mallorca, Spain
: P3 q9 u% i. q8 ], _Stem Cell Rev and Rep
5 O7 L- K# k1 j# KDOI 10.1007/s12015-012-9377-4
- Z& M) p9 j# R3 JThis protocol focuses on t he main first steps of the* ?5 Y8 o3 H" M, d! ?8 Y" i G
deriv ation of hESC lines, i.e. embryo culture and gradi ng,: D( A/ x- [2 }0 B) | [
and precise mani pulation of their ICMs and first outgr owths( E; B2 ]0 U1 A, d7 T4 Q3 X ^) B
of pluripot ent stem cell s. The Notes secti on includes alter-native procedu res as well as commenta ries and clarific ations9 }1 x" T( B8 ^% v J
on the techniques described in the Methods section. Assuming
! t c2 Q; |! z3 vexperience in mammalian basic tissue culture technique, this
- q/ j( c6 Y6 S/ l; mprotocol should aid anyone wishing to derive and culture4 n: G9 W9 n! m
hESCs to set up a rapid and reliable method. This protocol
& \- J/ X% j- t; Hreflects continuous work optimizing the derivation, culture# _9 x$ s" E6 q. s+ b$ C3 U' t+ q
and characterization of hESC lines over more than 10 years,
# U- X8 {: E' x- ?8 F* Q6 k$ Wcomprising activities carried out in four different laboratories.7 z5 A8 X f' H1 w, k
Adva ntages of the present p rotocol over others previous-ly pub lished in clude: optimiz ation of f eede r cell yield,+ h! ]6 ], D% E( D+ ~. ?
increase of I CM cell production and adjustment of the9 C9 [4 Y; N! Q2 _
deriv ation protoc ol accordi ng to embry o grade.3 F* W6 Y2 ?/ p# k4 S5 u6 s
Feeder cell yield is optimised by culturing tissue aggregates, v, S) u2 Z% u
until they grow as monolayers. Embryo culture is optimized+ L" G4 G Z4 U3 y5 j
so that growth of the pluripotent cell population is favoured
" i& b" R$ Y# mover that of the non-pluripotent trophectoderm (TE) cells.
+ Y# S R& h+ f# p- KFinally, the fire pulling of glass Pasteur pipettes to form thin6 j' ^# u3 K5 A& n! y7 H! l1 C
open and closed ends that match the size of the ICM or initial
, s3 }4 u6 P* s, uoutgrowth to isolate, increase effectiveness of mechanical9 q- Z' U% ]! B
splitting techniques.
6 p3 s( [8 V1 e3 n+ b9 k5 ^Materi als
7 a, k; j$ e/ T1 _0 {Mate rials a nd Rea gents
' h) R3 P7 k: n1. Bacteriological Petri dishes, 100 mm Ø, Fisher Scientific
6 L5 i& i7 Z9 X#09-720-500.
7 v& p$ M) p; F0 h! w2. Cen tre-wel l Organ Cultur e Di sh, 60 mm tissue culture
E# @7 ]: L$ b, y- Mtreat ed, BD #353037.
6 x- R+ _9 r: R; e: T3. Lon g glass Pasteu r pipet tes, Fisher # 1367820C .# `9 K7 |; O# A
4. Mr Frost y® Nalge ne, Sigm a #C1562-1E A.4 O: a6 J1 \+ ~& ^, S% K7 ?
5. Se rological p ipette 1 ml, Cor ning #4485., F7 D! J8 [: m7 L1 [ z
6. Se rological p ipette 5 ml, Cor ning #4487.& N6 W5 T1 ]3 ^" S3 B
7. Se rological p ipette, 10 ml, Cor ning #4488.) K/ @4 T" J5 c1 q2 _
8. Se rological p ipette, 25 ml, Cor ning #4489.2 S0 @6 ~7 r7 a
9. Se rological p ipette, 50 ml, Cor ning #4490. S6 x0 \4 j2 ^& E0 r- o7 T
10. Tissu e culture dishes, 150 mm Ø, Cor ning #430599.0 g: E; A+ C; k, n ^
11 . Tissue cult ure flas ks F1 00, 100 cm; j m3 N5 I: T0 ^
2* U$ ~% q s4 ^- f/ j8 d- \
Corning #3816 .! g( H3 c- e$ P g* {
12. Tissu e culture plates, 4-well , BD #353654.
: G+ X8 \; ^) D: V: @5 g, N/ D/ s9 v13. Tissu e culture plates, 6-well , Cor ning #3516.
& Q2 c- C s2 S# P14. Basic Fibrob last Gro wth Factor, #45103P- 100.0 i1 n) ^7 f' C& o1 |# B
15. Beta-mercap toet anol, Sigm a #M-752 2.
5 p# Y! f% i( l4 [# p: Y5 Q9 r16. Collagen ase IV, Invit rogen #17104 -019.' H- K( j5 m+ Y6 }6 h! Z# o4 s2 i
17. DMEM medi um, Lonza #12-614 F.
* ?8 g7 b# y8 x2 |* u18. DMSO, Sigma # D2650. N; L- V+ L4 q4 C% m
19 . Fe tal bov in e serum (FB S) , Australia n origin Lo nza
r( ^4 }- }9 y5 n) C% X9 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' |) e* O& y; y/ O* S6 ^+ C7 `$ h
origins a re US and US approved, followed by Au str a l i a n
3 p6 z; I' Y5 x, K7 \, ?and Australian/USDA approved. Poor quality serum is a
# d: I! ?3 ^( e* z% V6 ?$ M; Wconfirmed cause of feeder cell failure in hESC support.
3 x4 R7 r4 I) H9 @0 H1 y20. G1, Vitr olife #10128.& X# e3 T6 r: Z
21. G2, Vitr olife #10132.
& Z3 S7 f' ~9 \6 t; Z22. Gelatin, porci ne, Sigm a #G-1890.; @$ q$ r% C/ X0 l
23. Knockout (KO) DM EM, Invitrogen #10829- 018.0 S3 {+ x5 x1 _1 ~5 w* f: U
24. Knockout serum replacement ( KO-SR), Invitrogen
9 W# G' u5 r" d7 I; @#10828- 028.8 Z/ V( _. F' G; v) D9 `1 k/ i2 n
25. L-Glutam ine, Lonz a #17-605 E.
& N- z. X7 s* W# N26. MEM Non-essential aminoacids (NEAA), L onza #! M- e2 X' v) ^. B+ q
13-114E.
, K" B+ q4 J5 ?27. Mitomy cin C, Sigm a #M0503 . Hazard: this subst ance+ v$ P. L1 r, \9 O
is very toxic and procedures for safe hand li ng and. Q5 K5 w) b$ W+ j' X5 @% q
dispos al need to be in place. Do not brea the dust.
: k- u, J: E: wAvoid contac t wi th eyes, on skin, on clot hing. Avoid
6 e9 o4 X% B! ~, T( \2 iprolonged or repeated exposur e.6 T' W9 q) Z0 N( b
28. OVOIL, Vitrolife #10029.
9 v* A Q5 \1 j. }/ D2 n8 ^+ }29. PBS with Ca. d6 j/ P6 _$ k5 D4 f
+2
@6 X" U$ B8 y1 T& z. k# s/Mg
% B1 q% J$ \6 |* G+2 S! P2 N+ m p0 e0 `, I; T
, Lonza #17-513 F.- [9 a0 h& q' @0 r. P, N+ T4 C* w
30. PBS, without Ca% |) ^$ |% I v8 M, E& k: {
+2% S) @1 Z- C$ H* }0 _
/Mg
7 K$ S9 K$ K( }7 _1 g" |9 j+2
" ~! C6 D& t$ w! R, Lonza #BE17 -516F.
1 ]" E9 g# |) x31. 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
5 o, h9 M5 I4 c1 V) c) J0 c7 [dp c, Charles River Laboratories.
$ r: g! X& G$ K32. Thawkit I, Vitrolife #10067.1 l+ u3 ~2 y: f3 n
33. Trypsi n-EDTA, Cambre x # CC-5 012.! R+ H' Q: ^% [' x
34. Tyrode ’s solut ion, acidic . Sigm a #T1788.
2 v0 q' y6 r( Y+ b7 Y! Y CSolut ions
' ^0 }0 [! V- N1. Complete DMEM medium: DM EM plus 10 % v/v FBS,( g$ i G8 V0 W6 p. s4 r
1 % v/v L- Glutamine 200 mM, 1 % NEA A.
5 Y5 A1 _3 s% x3 b; i! `2. Freezing medium: 90 % v/v FBS plus 10 % v/v DMSO.
2 d, L6 d) z1 g7 L/ D" K3. Gelatin solution: 0,1 % w/v gelatin in tissue culture grade. j- y, ?8 X7 ]+ ], S' Y" N! T
water. To avoid contaminants and residues (detergent," R# |2 X$ z$ S3 @) P
traces of other chemical, etc.), use a new 500 ml Pyrex
( N' T) k4 Y8 obottle. Autoclave the gelatin solution and store it at RT.
# n; ~- s! X1 q q; w0 F6 x# g' U( G4. HE S m ed ium: K O - DME M supp lemen ted w ith 20 % K O-S R,
; {8 R7 @' o9 l. V7 H6 d8 u; O2 mM L -Glutamine, 1× N EAA, 50 mMβ-Mercaptoethanol8 h" d7 C" J$ j) N0 J; U
and 8 ng/ ml human basic fibroblast growth fact or.
# d4 N4 V. e6 H9 L1 ~Methods
# s$ E9 o, C+ V1 L% D3 KESCs are derived from preimplantation stage embryos. The3 Z8 x; P- [" U- _& n/ }
most common procedure involves culturing embryos to the
9 x" x( T4 c2 s' ?5 U, ^+ b, {blastocyst stage (day five to seven after fertilization), isolating# r0 {) N5 O8 ^
their ICMs, and culturing the latter on mitotically inactivated
1 A0 T5 J- Y. ~/ A, ^ Bfibroblast cell layers. The resulting pluripotent cell popula-tions are selected by morphological and growth criteria. Novel. q3 y o# t: S3 K, z
hESC lines are then established after continuous subculture of
& ^1 f6 p! e6 s# Nthe initial outgrowth. A hESC population can be considered to
2 \; A$ a" A0 a: r* hbe an established line when it reaches passage 8 with approx-imately 3 × 10* R0 E0 O) L' A: V, }
7. {- D% E# o$ p
cells, as defined by the UK National Clinical
' V8 Y/ P) P1 o' Yhuman Embryonic Stem Cell Forum [ 14].' t8 R5 D0 e, s( o
Stem Cell Rev and Rep
$ E D, v5 ^" M' TAfter eight to ten co nsecutive p assages, the novel line is( ~* j1 _4 q0 ~! M. P# s8 z3 F
sufficiently expanded to check its viabi lity by freezi ng and
! ?* q7 A. D% }( |thawing .5 k& ]3 a2 P+ F& p
Ta b l e1 defines the term‘one v olume’ for each of the culture& w" `! B0 P0 N- ~% }
v es se l s d es cr ib ed t hr o ug ho u t t he manuscript. All incubations are7 s! o: ]+ l3 s" _
performed in c ondition s o f 3 7° C, 95 % h um idity an d 5 % CO
a3 q+ R% t0 p; n' H# ~2* t+ } A$ ~$ D
inair(SeeNote1).Figure 1 depi ct s t he expect ed time schedule
- F7 x- |! e7 _- T! R3 Cto complet e a hESC d erivation p ro toco l, provide d that t he feeder
# _7 \) l: c$ \source has b ee n p revi ou sly v alidated.
5 B" ~0 E! H( ?1 GFeeder Layer Preparat ion% b8 d, q4 p4 |, l5 e2 ]
Primary cult ures of feeder cells have tended to be used for4 {( B, n4 W0 F
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; N/ m1 W4 l9 ?, d
derivation can be performed on f eeders produced from
: U+ j; H* _+ q i' `* R8 Oestab lished cell lines, or prim ary feeder cells whi ch have
* R' a# x9 R/ E7 T9 g9 rbeen immort alised. Altern atives to the use of prim ary mous e
, J% E2 V( ^4 r8 I0 Cembr yonic fibr oblasts (pME Fs) as feeder layer s for hESC
" z6 d+ e4 ?0 h6 Fculture are discussed in Not e 2.0 s! _ W4 M$ R9 M# p
Reg ardless of the type of feeders used, every feeder layer
+ }) @- K7 v( ~4 F* omust be con ditioned before stem cells are seeded on top of
) l% X$ ]3 y, H4 mit. To do so, feeder cult ure medium is disca rded; feeders are
: E/ H/ E% O4 Y: ^- rthen washed three times with one volume of PBS each time; y( }" n; [' H" P/ d# L
and once with half a volume of stem cell medium (HES).' M6 O7 ^9 S7 I
Plates are ready for use, from 30 ′ to several hours afterwards.% d+ r; O0 h! L. H- Z$ `4 H
MEF Isol ation
2 D# X; s3 x1 f' \! T! a9 [1. Sacrifice a pregnant female (12.5 –13.5 dpc) by cervical* I, y0 B9 L: n
dislocation. See Note 3 for tips on strain choice.
. ~8 c, A. V# P) U2. Di ssect out the uterine horns and place them into a) |) t, G" O6 x7 f; _* V; c
10 0 mm Ø Petri dish containing one volume of ster ile
0 s, l1 ^. H! B% UPBS 1×. Transf er the plate to a laminar flow hood to
. V& S: w* X1 a% h& S! S) qco ntinue with the procedu re.
5 o) D+ `- _' l$ g3. Cut open the whole length of the uteru s, so vite line7 g- T$ r: j O% a: p2 p# K7 w
sacs with embr yos and placentas are partially relea sed.
- R- x4 d6 H5 g9 ^' [- H4. S e parate each embryo from its p lace nt a a nd out of the
; P E7 v2 S* T) s2 `6 ~$ zviteline sac and surrounding membranes, and transfer; z7 z( m! }9 c9 a4 a
them all to a ne w Petri dish with on e volume o f fresh PBS.
& w9 P8 _' q/ d: V, `5. Wash the embryos and transfer them ag ain to a new$ P, n. `& p. H& m/ m* y( s
Pe tri dish with one vo lume of fresh PBS.
& L8 |" ^3 I4 ^! i) }6. Decapitate the embryos and separate the visceral tissues: d: O& Z5 E2 p( ^3 h8 F% ]. G
from the bodies. Collect carcasses aside in the working' M3 S& h$ ?) q/ w+ I, g& t
plate. Aspirate and discard the PBS and the visceral0 X( }! P" I, d' |6 F& N1 U6 n
tissues carefully with a 10 ml serological pipette. Leave; l/ t7 l- f4 T2 r! P" D2 L! Q
the rest of the tissues in the plate./ t* F# E1 G0 N$ G0 u0 T& R
7. Mince body walls into small pieces until a homogeneous
. Z, N1 M9 [2 S' J1 _, a: u$ x, ssuspension is formed.( Z" P" ~/ P4 O% Y$ q2 m
8. Add 5 ml of trypsin-ED TA 1x to the plate and transfer
7 b9 h% e# W/ P: Wall contents to a conica l 50 ml tube.- F5 `7 h; r$ A1 k; }8 C/ |3 m
9. Incubat e diges tion at 37°C and 5 % CO
; M% ~ V- ]+ N5 y( V2
. K" _, f6 \1 G3 efor 30 min.: N7 k- S# f" n. \. S
Hom ogeniz e the cell suspension by adding 45 ml of
8 |# a* ~7 a4 yfresh cult ure medium and pipet ting up and down for
, U8 Z9 j, W. a6 x5 oten times, and cen trifuge to recover the cells (700 g for: H( {2 i$ U% {7 ^
2 min ).6 _2 G! c* x! {* z% ^
10. Discard supern atant, ad d 50 ml of fresh culture medi a9 k# A: Y2 Q0 b/ Y+ I5 h+ ?6 _
and resus pend cells., L; B) ]* ~6 R2 j6 y
11 . Cen trifuge the cells at 700 g for 2 min and disca rd2 q3 @# A9 g3 q3 \$ s; m
supern atant.2 [$ q$ e! t6 S7 k
12. Resuspend t he cell s in 5 ml of complete D MEM6 Z5 \' w' {5 L# [- Y% t: r
medium.
" B& e: g" A0 x6 B9 n13. Seed cell suspen sion at a rate of three embryos per+ j; C# n6 E4 B. u& X, B
F100 flask. (General ly four F100 will be set up at the
$ ^3 @& f% G. Q/ ?be gi nn ing o f the procedure per dissec ted p regn ant( |" |* n2 \1 w7 u# A3 N
female).( Y. ]4 g9 N+ H! G! X
14. Incubate at 37°C and 5 % CO
8 o' `. i7 R3 Q. O. b, Z: \2: ~' R" U- g9 o/ c: \" |( i- X
unti l 90 % confl uence3 i# P4 u8 `/ S
is reached (approx imately two to 4 days).
3 x+ T: b5 j4 e/ b! D4 Y- j15. Split the cells twice by tryps inisati on, seeding them
0 A- w; S X8 W. `7 yonto n ew flasks/dish es be twee n 1:3 and 1:6 split
4 q5 p7 U$ t( U, Z& T! _/ }according to growth rate.
i" T; q+ C. c. m* I- O1 ZAfter t wo passages , t he ce lls s hould be growing as
% n; v8 f* B; {- z# C! w4 Q6 qmonolayers. Freeze 90 % of p assage 2 c el ls in vi als
9 ~# p& p) F0 y& \cont aining four to ten millions units each. The frozen+ Q& e$ ~& x+ @9 w5 G, h1 P7 Y7 z
cells wi ll const it u te a s tock of act ive pMEFs for fut ure5 ?6 H9 T4 O3 x4 @. M* {
use.- N q# k: V5 M' [ W: ^
To continue with the production of inactivated feeder2 r, H) a w- D3 { @
cells, expand the remaining 10 % of the original culture6 G( Z( T _8 T$ n1 c+ d
up to pa ssa ge fou r. S ee d t he cel ls on to new f las ks/
3 ?- s8 O5 e- F' h. N9 \$ M9 p9 Gplates bet ween 1 :3 and 1:6 spli t according to grow th
2 ]. M8 K; r1 G1 |. orate each pa ssage. Then inac tiva te an d freeze passag e( b; N8 z6 Z1 x& {" g, `
four cells as explained below. See Note 4 for tips on M1 H( a8 f) ^( r* ~ W) u
these st eps.
8 f4 F& }( P; hGene ration of Batches of Frozen Feeder Cells by Mit omycin1 Q( x- o7 a% s4 W1 |
C Inact ivation
O' G9 N* e- g. V. c! ]1. Tr eat 80 % confluent, exponentially growing,* Z# c+ Q" P' t( A* V. e# v0 O9 T
pa ssage four pMEF populat ions (between 48 and
( d$ u1 ]. a& U5 S9 6 P150 plates), with 20 ml of 10 μg/ml Mitomycin C in1 v* L# M; U- F8 t
complete DMEM medi um, f or 2– 3 h at 37°C a nd, Q: ] o7 f0 N4 n( O s* f: S
5% CO2# y8 b6 Y; T! Z( V; _) }2 |
.
0 @, S% ]/ d; m" G7 y: z1 C. S& nTable 1 Definition of the vessels and volumes used throughout culture
! ]6 U4 S- j, c9 X4 h- O2 K! Eprocedures$ q! x! F ], q* `( Y2 G$ l6 y2 m5 D
Dish/plate Area cm
( q; }1 O' c$ R# A2
+ i5 C! T$ v$ M* }: g9 a9 W/well One Volume ml/well
& G( u- l) ^2 d( T2 Z5 b$ D4-well plate 1.4 0,5: N, Z8 p7 c' q/ G1 u/ P1 o4 H
24 well plate 2.0 1
2 k) y' u0 N5 U9 d& zOne-well 60 mm plate 2. 9 1% h* ~1 ], r5 J, E3 I; U" h
6-well plate 9.5 3: i+ \3 \* U3 K# x
60 mm Ø dish 21.3 56 D% m8 g- t' r7 T4 `$ r/ z
100 mm Ø dish 58.1 20# C i, T" z3 f- v, ?3 S( s5 y$ o
F100 flask 100.0 25& J" D; m" U( h3 O; X
150 mm Ø dish 176.7 50% ~4 ?* P! [' k+ T/ j7 O/ Q
Stem Cell Rev and Rep
* Z" z% ^) ^4 E0 x( i# v" l2. Aft er inactivation, disca rd the supern atant and wash- q _/ s. @: ?- L. ]4 D
cell m onolayers 3 times w ith on e volume o f PBS
& }) ^' c$ H4 X, R0 n6 z }wi thout Ca# W8 Y' [. F2 D# L+ H: H9 f
+2
0 u; W$ o; ~; ^* e$ ]. P7 A1 R/Mg7 z4 W) Q4 G/ L. X3 @2 G1 r# `6 c1 I
+2
: Q9 k. D- W. P* C, \) A. p.
: O6 f% a% U% ~! \9 H# j# D- h" B3. Trypsi nise the cell s (2 ml per F100 flask or 3 ml per
6 D5 r' U; ~2 }% oP1 50 p late) for 2 – 7 min at room temperat ure (RT) .
( a4 @( @$ ?- I4 E* ~0 f4. Tap the sides of the flasks/dishes to dislodge the cells,
& O4 k6 y; e; }an d harves t them into 50 ml conica l centr ifuge tubes .2 F% R( b% m0 x" h
Po ol the contents of tw o to three P150s in one tube.' s/ W8 [2 A0 X) r, B' t* x" E2 F
5. Add 44 ml of compl ete medi um per tube.3 P: ]0 \% q N
6. Cen trifuge tubes at 700 g for 2 min .8 ^1 n) W% O' L) U
7. Br eak pellet manually by finger vortexing (see Note 5).; Q8 P+ ~1 w% ^" B# |8 _' ~
Resuspend each pellet in 30 ml of complete DMEM and
! D! V0 f, i( _8 ]- ]9 ^count in a Neubauer chamber.5 w$ `; I- M# K7 T. j" q
8. Co unt v ia ble ce lls and calcu la te the numbe r of
7 l% ]) _9 |8 ^6 Zcryovi als needed, at a rate of 1– 4 million cells per vial.: R0 R. v5 F8 z3 X# Z, ^
9. Cen trifuge cell suspen sions at 700 g durin g 2 min .
1 j5 Q$ N# v5 A2 n0 E10. Label cryovi als with batch numbe r, amoun t of cells4 ^. C% p1 a! Q" a* ]; J* Q
and met hod of inact ivation, date and user ID.
, w( t; K4 f* O+ ]" @5 q11 . Discard s upernatants from s tep 9 above and break
! n" W* C# v* m" [! r$ V- h: \4 {pell ets carefully by finger vortexing.0 @! ^) x7 _4 S, Y5 O) J1 \# X. d' V
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.
7 a8 Q m# h2 e; w/ K, mDisp ense cell s uspension i nto labelled cryovials and( F0 Y0 o. Y d7 e! @8 u
transfer them immedi ately to a precooled Mr Frost y® con-tainer, and then into an − 80°C freezer (see Note 6).3 @% o- E/ f& ]/ y2 p* Q
Feede r Layer Plating for IC M Culture$ L' P* Z) L' M- j6 J- d
1. Dispense one volume of gelatin 0.1 % ont o the' I% }) ^. }, c( X$ M
requi red number of dishe s/wells, and incuba te them2 r2 C$ ]( W4 U; O5 R( c N, G
at 37 °C for 30 min.
! g. j! k K+ ?+ j* q1 ~+ K2. Equ ilibrat e 10 ml of fresh compl ete DM EM medium," W) j9 i5 X& E7 M
in a 15 ml conical tube, in the CO
; m; H$ C/ y* @+ ?4 w! o% t2
I1 V5 u' T& z8 V: ^- L' q$ Tincuba tor per each+ Q" P6 g# s( W E
1 – 2 vial (s) to be thawe d (see Note 7)." d. V7 c# n7 n1 @
3. Once the coated dishe s and the medi a are equil ibrated,* B3 \& P3 k. u
thaw the requi red number of cryovi als to plate 0.2– 0.3# f' D/ N) t3 f, u% c
mil lions of inactivat ed pMEFs per one-well 60 mm3 g! \6 ^! X. v1 T9 y4 j
dish. Spray the frozen vials with 70 % ethano l. Roll- X% M& o- E( z( Q" K/ M
them in your hands, until only the last smal l piece of3 p9 w5 L% M4 |8 I+ [9 D
ice remains. Tr ansfer the c ontent of the vi als i nto- s0 O$ `8 A; Y* i1 ^
the equilibrated compl ete D MEM. In order t o
, b) O" C6 g% u4 ~, l1 Zavoid cell dam age i t is n ot advi sed t o thaw m or e8 B( ?. j7 j5 v- U5 t1 X& N
than four vials at a time./ c6 Q7 J1 P& t$ ]! B r3 m
4. Cen trifuge the tubes at 700 g for 2 min . Discard the8 H0 g( L0 h' k# A8 n/ b
s upern atant a nd gently break the pellet b y f in ger
( }: r3 X3 Y6 q6 avo rtexing.
$ z$ Q) u$ }/ l- t P6 ~9 \5. In order to elimin ate resi dual DM SO, repeat step 4.
4 Q' W4 a3 a9 I. u& G# M: W6. Dur ing the second centr ifuga tion, aspir ate the gelatin$ B. j2 f% H! E
solut ion from the plate(s) and dispe nse half a vo lume
+ {. t: m; b) l- a, j+ qof prew armed compl ete DM EM medium on each one.0 F+ Q3 l! @* p2 {
7. Br eak pellets from step 5 above by finge r vortexing,
+ _- {5 r: l! Ian d resus pend them in half a volum e of the wel l(s) to3 p- F3 Y* e5 j9 g3 n
plat e.+ q* ?$ W( d( y
8. Di spense cells dropw ise onto the medium- containing
6 ]4 J- N6 S U" B* lwel l(s).9 M7 n e, a* F! H) m
9. Place plate(s) at 5 % CO' c# ]/ A, N3 c" _( a
2# [% W0 H7 {6 g9 J
at 37°C, and c arefully shake7 l$ `4 x" l7 v3 V1 T( M. H* o
them horizontal ly in all direction s to distrib ute the cells
$ n# O+ H8 x( P7 C& tev enly.0 \8 B+ a( k3 G1 d, Q, }; |
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
/ c3 P H( Q! J3 aattach. Optimal feeder density corresponds to a conflu-ent monolayer; higher and lower densities will result
. L: f8 v$ Y# P( qeither in reduced hESC growth and increased differenti-ation, respectively.
+ M# K' O! n) n. S9 s* SSe e Not e 8 for comments on feeder layer prepar ation.
4 |! F- h8 M) N, O& \' f7 ^Embryo Culture
/ b( a2 n ` Z' K) D; }2 r1 F; J2 `; zSurplu s embryos from clin ical in vitro fertilizati on (IVF)$ M# l. W! W. s% C1 p
procedu res can be supplied, fresh or frozen , at any stage
& ~8 F: r5 S( |% J: x. |from day one to day six post- fertilizati on. It is firmly estab-lished now that low-grad e blast ocysts produce h ESC lines
, m8 K% X8 z/ ksucces sfully [ 15, 16]. The refor e, the derivatio n of stem cell
% r8 _" e+ B% }! t, s$ p$ }* ?lines is almost guaranteed for a ny Re search Pr ogramme
* E% I' i- _# g6 _estab lished in coll aboration with an IVF clin ical depart ment., A3 e" Q0 i$ \! ~ q9 N
In this section of the proto col we will detai l the steps
4 V2 {/ B1 u: F; t+ [( Pinvolv ed in embryo cult ure for hESC deriv ation. It is advis-able to use the same clinical grade embr yo culture reagent s
# E1 P9 R& h& K$ E* e0 r- e' d8 Lthat are used in the clinical procedu res. Altern ativ ely, it is: k7 f3 C8 x' g
sim ple to p repa re the m from thei r compo nents [ 17]. Se e! K9 I( _) l* L( K9 f$ G
Note 9 for tips on embr yo cult ure.) q0 g- L, d# Q/ ~! |
We will detail Vitrolife® protocols, and advi se to fol low# j) C: M% R' W# z, I7 q& c
manufacturer instructions in ca se of different brand reagents.
1 z: ]+ S: _' t4 ^9 rThe cul ture system us ed was a two-stage one, i .e. changing
* g; K- A3 u5 acul tur e m edia formulati on on day 3 or s ix-eight cell stage o f0 u, t2 y' i, Y9 u) U
development (from G1 to G2 series ), un til blastocyst formation .
5 _; f2 o X% iFig. 1 Time schedule for the
6 W/ h" p5 D3 b- Pcompletion of a full derivation
" M% u0 H7 o* n. zexperiment
$ _3 D+ R( B$ i! L" C" ~5 LStem Cell Rev and Rep
~* u; I. Z4 R; r4 BEmbryos are cultured in m icrodrops under oil, to
* [+ Y# J; X' V* X( fprevent evaporation a nd to minimise pH changes. The2 T! O& ~6 t" N% I$ w( _
following protocol corresponds to t he culture of fresh or
1 d8 D2 F) q6 V: ]9 Jthawed cl eavage-stage embr yos. In the ca se of receiving& L; I5 O6 @) A, W$ d1 N
frozen embryos, it is crucial that the thaw ing i s per-formed in the reagents corresponding to their freezing.
/ O/ S# w( B6 iFollow manual instructions from the appropriate thaw-ing kit. After thaw ing, follow the protocol below for' h* |/ q1 | {# H) M$ ?! F8 x. E
embryo culture.6 \5 U' f2 T" G" ?8 x# v1 j R! ~
1. Equilibrat e OVOIL® was hed wi th 1:10 volume of G1 or7 B3 A: i6 y! D% y3 @% `
G2 for 6 h to overni ght at 37°C and 5 % CO2
. I$ u8 i* u2 D5 d.
1 r0 g$ ^: O/ N% A9 b, g2. Equilibrat e G1 or G2 medi a as required (see Tab le 2 ) for( f m6 Q5 `" z$ y, h2 T
a maxi mum of 3 h.5 d2 b: P; L3 E7 I" k. d- m
3. To set up embr yo cult ure plates, pipet te 25μ l of the9 s. n t5 J, U1 `/ v& n. f
equilibra ted G1 or G2 media per embr yo on embr yo
" }6 x+ X( `# ~5 f3 v+ M: ^6 Aculture d ishes/plates . Cover the drop(s) with equil ibrat-ed OVO IL®, and add 25 μ l more of the medium to the
9 j& l, b5 n n2 i/ {# zformed drop(s).
) |. v8 d3 V/ H8 S* [% c. ?" i* \4. Assess embryo development daily (see Fig. 2 for a% U! R- D, _7 G; O) l" M3 j$ k' a
di agram of g rowth pattern and c orresponding images# y8 ?* S5 e! A! J6 J w" [
of embryo development), and transfer them to fresh
0 ~7 R3 D7 q) j$ s: @media d rops every secon d day (see Ta ble 2 for
4 Z0 ?: r4 s) G4 s, Xmedia c hange guideline). P re pare new drops as: X3 ~- n+ ? R% x" g
describedinsteps 1to 3above.If embryoculture- I! H( i3 h; h) Z8 n1 w i( j. z, Z
star ted i n G1, remember that once embryos r each
& \! a, n: c I# T8 {da y 3 o r the six-eigh t-cell stage, they must be
$ G; f! p% D1 y* N* mcultured in G2 medium.0 T+ b9 @* q# P$ ^
5. When embr yos reach da y six of develo pment , or the5 o) j& o/ b- r# A' ~
ICM of the blast ocyst is visible, it is time to set up the; Y3 S8 Y/ i- n4 D( P9 |
feeder plat es to seed the former.
- q+ O5 E8 h8 j& XICM Isolatio n an d Se eding( x7 l" @+ ^9 o0 R8 C8 I: c
During denudation and ICM isolation i t is a dvised to, Z( q6 ~) o. d
process each em bryo indivi dually. Pulled glass Pasteur
8 s5 ?7 R( X7 m3 o( ^2 Ipipettes 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
5 N6 R9 Z7 ^7 i, a) k) A; ZIVF p rocedur es.6 Y1 w7 Q$ Y& N( ?- F5 v
Con dition the feeders before the d enudation as follows:
( E# _/ s5 t1 C$ E5 k1 rbetween 1 and 3 h before the seeding e liminate f eeder
& N/ S) W# e9 M9 R! j8 xmediu m. Wash three times wi th 1 ml of PBS with Ca$ Z( N" ?( s$ w( q$ q9 n5 d
+2" Q/ o( U" [# m; I0 X/ J
/
. x+ _" c8 O g# _ CMg& o, r; X5 Z- q2 E
+2
8 T0 m+ D: I' S5 g; Uand once with 0.25 ml of HES medium. Add 0.5 ml of
, w/ B+ O) z' s- P; q: WHES medium and equilibra te for at least 20 min at 37°C and; ~$ H& f5 M2 n
5%CO
* N7 \4 k: S! l2 p, R! W2 L2
( u" P+ x9 V/ Z( ]) n( F. At this point the new feeders are ready to recei ve
( Q5 Q6 r7 L1 J& ithe IC M.
5 N9 ^0 U1 ~$ ?3 T6 q; h9 v) M& F. |Zona Pelluc ida Remova l
" c% ^- i% a* i% R4 _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
- j3 {9 L( l( V6 g) j1 A2
" B' h) E4 v8 U' x, for a; G+ ^6 ~+ G5 D: A' ]
min imum of 20 min and a maximum of 6 h.$ k- Q+ ^$ t# ^
2. Bef ore any mani pulation is perfor med, images of the
: V \9 r: A5 ablast ocysts at different planes (×40) should be taken
3 s3 e2 k' h% Y, Q3 ~1 Y8 r. ]% T' ]for further asses smen t of its grade. Afterw ards return it( G, d3 T1 _% k/ l
to the incuba tor.
2 Y" Z) t* N4 [; g G) U- G3. Pu ll 2 long ster ile glass Pasteur pipet tes wi th the aid of$ K8 \( I5 l+ H0 Z- X$ u" n3 ?2 t" c
a Mecker or Bunsen burner. Leave one of them opened
. A/ F j5 N. q. man d rounded , and close the other one leaving a small
* H. y0 B: [; ^! [% Q+ ]rounded ball at the tip (Fig.4 ). Check the thickness of
0 z' W5 f2 e' V: j% U* f3 Ethe tips under the s tereomicroscope and r epeat the9 e/ H' _) ?0 i a5 m
procedure unt il a couple of pipettes whose sect ion3 _* N0 S/ f9 M1 Z* {8 H0 }& v
mat ch the diam eter of the blastocyst are produce d.
0 L6 ~+ {" m* y0 w! n' `4. Se t three drops of AT forming a row in a 60 mm tissue. d4 z+ F4 k( `' W
cu lture dish, an d mark thei r position at the rim of the
: k1 ]& l& h A7 y/ P% }2 V' L, @3 vd i s h. A dd a n ot he r t h r e e dr op s o f G 2 m e d ium i n a
9 j, d m' {+ k: D \second row at the middle of the dish, and three drops
6 P( Z y8 G; q# K j/ m7 O7 @5 Xof HES media at the bott om, again aligned in a row
9 d) }0 s& w( e7 [4 k(S ee Fig. 3 for drop distrib ution).
B. ~; M6 q* `4 N1 d' q5. Se t anothe r three drops of approximat ely 100 μ lof4 j9 W9 t3 S$ q, r8 {4 g
HES medi um in a new 60 mm dish.4 w% z4 v" ~ J( G( [6 n
6. PicktheembryoupinG2 mediumwitha 275 μ m
* m, x) e% J4 y5 D7 |2 Q( Ucapil lary mi cropipette, a nd t ransfer it to the left AT3 o) n0 @+ D, s( a1 F
drop of the plate prepared in step 5 a bove. Stop, s" k# e% r( ~! r9 ~! f
dispensing G2 medium into the AT drop a s soon
' b g+ \2 T% M$ r1 D! {3 qas the embryo has been releas ed. Empty the pipette$ F( i+ Q$ j Y+ R" k# O4 J8 ?# _2 {
in the border of t he dish to discard r esidual G21 U1 Z, O& e8 X1 B( T* Q. N7 }7 t
medi um.6 P- G6 G) t, ]! m) h3 F
7. Wash the pipette twice in the second AT drop.: v* ~6 b8 g7 \) W5 |* W
8. Return to the fi rst drop, pi ck u p the embryo and
5 J7 ?$ ~4 s) {6 L& itrans fer it to the third d rop. Monitor unde r the micro-scope the dissolution of the ZP (0 – 2 min).. P* a" I5 c( E3 N p) h0 _ F. A
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
6 \) [& W7 s% f+ y- xpre-w ash, dispe nsing the minim um amoun t of AT into) u- r2 _% ^- j/ h0 O5 A, Y: h
the G2, the second to load the pipet te with fresh G2/ O$ ]: I2 k3 w, `. Q1 j5 [7 b/ B, |
medi um, and the third to was h the blastocyst .
2 e2 o' `: {* X7 x0 v# ^/ F10. Tr ansfer the zona-free (denuded) blastocyst to the7 b9 E# }7 P. g- D# _
drops of the HES medi a using the washing procedu re' C6 T" Q& q. K o
as described above for G2.
4 d" T( x% |3 I11 . Immedi ately trans fer the blas tocyst to the drops of3 d- q0 U# o. W% [1 i) J% `
the HES medi a prepared i n a sepa rate plate in s tep0 x$ J& d+ m, `: F
2. Follow the s ame procedure of trans ferring to the
; Z6 D; V& U! B$ ^left drop, washing t he pipette and loading i t in the1 ~: X( [+ H' T1 v0 e/ \ W: y
second intermediate drop and transferring the
. ?: q- c( D( R# c; G5 q- fembryo t o the third drop. P l a ce the p lat e i n to the9 f/ Q) F+ n5 F) X! C
CO5 i+ |% d2 p2 N7 E$ a4 V. \9 w
2
) S/ E# x) m- {8 \0 N H* T3 p9 Zincubator.; U- w3 l7 _( f' b0 w
12. Usin g the ima ges take n in step 2 above, asses s the
i* g8 k& {( @. G# Vderiv ation grade of the denuded blastoc yst (see em-bryo grading s cheme in r eference [18]) and a ppl y
+ K; c: e4 z1 ^Table 3 for determin ing the best met hod of ICM iso-lation. Depen ding of the outcom e, g o to protocol A or
" C) m7 q, E2 P6 U- V0 YB be l o w. S e e No te 10 fo r c om me nt s on t h es e1 B6 U3 o$ B* Y5 \- C' _! Y* {4 C
procedu res.( {7 _8 K* W3 x6 p8 S/ z. H. Z) l2 k& w
Stem Cell Rev and Rep) s3 p6 F4 u1 X& H
ICM Seedin g by Pipett ing (A)
' o! T, K& Y9 L6 k. M3 n$ x1. Choose an open pulled long Pasteur pipette of the same
" D, ^" |4 y2 u5 Fdiameter as the denuded blastocyst (see shape in Fig. 4a ).; u; U6 @9 t$ n0 K5 m# W4 M4 h
2. Load half of the tip with fresh war m HES medi um from
5 z+ l% ~; I8 v g' o: B/ vthe drops set up to equil ibrate before denudat ion (ste p 51 n2 B2 {5 c! {" q9 t% S2 f$ i
on zona pell ucida remo val proto col).
/ `& b( I' ^8 b2 a9 r+ O3. Aspirate the denuded embr yo and pipette it in and out# B- g/ N) b7 }# [3 Z
while checki ng for loose ning of the TE cells.
9 A( T+ a$ a4 n* _% r. e7 |/ C4. Aspirate the i solated cell clumps and dispense them
% s) C5 M6 J' a2 K7 Gsepara tely on the feeder plate.
9 H3 D$ l9 `9 l$ P2 _5. Ta ke an image of the seeded fragments of the bl as-tocyst. Pay attention to l ocate the ICM and record5 m$ f& M' H( a8 r) C' ^
its posi ti on.. ^8 u, Q d) U
Table 2 Embryo culture! f; B' f+ r9 P: h
protocol
) f) x5 N L9 Q' ?- p# RDay of development Stage Medium Image recording Media change: }/ P1 |* b, A- g' j+ @0 q
1 Zygote (2pn) G1 − G1& `: `5 i* W1 ^' K( C/ F! p
22– 4 cells G1 Yes −
# y) R, Z, }$ U6 F/ j' Y36– 8 cells G2 Yes G1 to G2
' _1 ^: |8 y6 C- e% l/ A, t4 Compacting morula G2 Yes −
( K) p/ N# |8 `: }* C% f- j% }5 Expanding G2 − Fresh G2
$ \9 D4 k. R5 x7 R6 Blastocyst G2 Yes −
$ b$ w, c5 _/ Q- F7 Hatching G2 Yes −
8 R$ @4 [+ Z) ~: d9 X3 j2 m0 O xFig. 2 Dagram of early human/ w& B1 @( C! T' E5 _
embryo development. a-a’
* m4 r# K) e5 ^two-cell stage embryo, 1 day: g$ J2 n" X6 d4 c4 V
after fertilization; b-b’ four-cell
* w, U" S% T, H7 ^- |, mstage embryo, 2 days after
: @- n2 S+ I! U G# mfertilization; c-c’ eight-cell
9 R# Z1 U8 R( ]; g! D$ o6 g) U, mstage embryo, 3 days after f
7 m% |+ C7 c" a9 t7 Iertilization; d e a rl y e xp an di ng5 u" N4 c' a3 Z
blastocyst;d’ morula containing
% Q3 a+ v% w& L+ Bm o re th an 1 6 ce ll s, 4 d ay s a f t er
0 _ w1 ^! h+ wfertilization; e early blastocyst,
% m& C' ?/ R0 Y+ C7 o# \; m0 C5 days after fertilization; f
2 ^; T1 R3 k* ]# `hatching blastocyst, around' {) z, e3 J& l( K
7 days after fertilization. ZP:
. R+ S; {1 E6 |Zona Pellucida. ICM: Inner
# z8 f9 ^6 q8 Z- s# e. R% F# qCell Mass. TE:$ G0 h4 T) y' V, ^% c9 F# f
Trophectoderm
~5 T6 a/ c: g& {: G. p$ pStem Cell Rev and Rep& k# n/ e7 E; c4 d( @% V5 j2 y
6. Tr ansfer the plate to the incubator, avoiding abrupt
' U3 i% [3 \3 Ymovements t hat may separate the s eeded cells from) p, E7 f9 J2 z$ z. e: Y( T7 \# ~5 b4 p
the feeders.1 }% l6 _8 G N: s0 g" D1 g" }
7. Inspect the plat es daily. Cha nge the medium every other. |* f3 _/ z$ h; K+ S1 k( {2 f6 S% e; `
day.
' q7 g+ T5 D( L" a9 d8 @! h8. Prepare new feeder cell plates as soon as pluripo tent
: ]7 f2 s6 s1 ~, Z( W( ]" e5 W# Jgrowth is detected (see Not e 11 for deriva tion timelines7 r# b( }/ ^0 K, V
and Fig. 5 for ima ges on deriv ation progre ss).
5 W$ F: q1 |' F# P* X* u9. Split part of the emerg ing pluripotent colony when; n& [+ b1 i8 ]! G. d
resi dual TE cells begin detach ing from the bottom of+ X7 [/ T% q4 o% d, a7 Y+ r
the plate. Follo w Secti on 4 below to do so.
3 m- l. p9 D9 q6 }, ?9 ]( |7 P3 TICM Seedin g by Mechan ical Cut ting (B)
/ q7 }9 Q4 |- @' o1. Aspira te the denuded blastocyst w ith a pulled open4 `/ y: a8 e$ t r
pipette (Fig. 4b) and dispe nse drops of approxi mately
, Q& b0 X' R4 ]; u20μ l on the dry area of the 60 mm dish containing fresh1 g6 W$ n5 ?. G) K% p0 H2 j* f
HES drops, until the blasto cyst is dispe nsed in one of1 Z2 {& m: O5 ~$ z
them.
' k$ E6 S1 W; ~! j5 R! }2. Drag the denuded blastocy st across the dish, away from. t$ ?" d) v% S1 e
the HES drops. The volume of the embr yo-conta ining1 @, {; R% i0 v: W
drop will diminish up to the point that the embryo
" Q5 H0 R' v! J( j$ E7 }3 ]flattens and sticks to the plastic. At this point you shoul d4 m( Y. i# \2 c# H7 S s( M
be able to locat e the IC M: it is much brigh ter and less; M% k5 G. Z' a! U: n; H' [( @
flat tened than the dim TE. Sa ve the pipett e without& c: G4 n' F6 {+ X9 I" I, u* |
emptying it for follow ing cutt ing steps .
% A* z: O+ S' E C R3. Pick a close d pulled (hold ing) pipette (Fig.4c ) with the
3 }; H9 C$ ^" ]7 o" Zleft hand (rig ht for left-hande d) and hold TE by pressing
/ y% Z- J) X2 @% Sits border agains t the plate.
, O7 _1 ]! `9 r C; k H/ X6 ~4. Pick the open (cutting) pipet te still contain ing medium
8 t' z4 Q' L1 ]% gwith the right hand (left for left-hande d). With its edge,
5 s. u4 @1 t# O* P0 D# ycut out as much transparent TE as possible. Imm ediately
0 z1 ^! _: C+ hrelease the HES medi um to a void damag ing the ICM." v- k7 s; N& J% R) I j3 Q
5. Scrape loose the ICM contai ning piece, which will be7 e: y1 X s) e3 K7 {
adhered t o t he plastic. M onito r the ICM w hile it6 r# q. b7 v, G$ D
Fig. 3 Shapes of long glass pipette tips for ICM and stem cell colony' E$ S& X" m" {: C, I) @
processing. a transferring pipette with open rounded end; b cutting" N6 s( v- R+ T% C5 Y/ Q; [; k
pipette with open bevelled end; c holding pipette with a fine rounded4 R! M l" B1 a( \' F; b$ j. M2 B7 b
closed end; d curved hook with closed end for outgrowth scraping;e-g% j3 N* k+ \! S
flat hooks for colony cutting and scraping. h detail of the tip of the i7 ], L+ { e1 H
holding pipette depicted in C
# ^, {6 D# ~" y" G! a7 vTable 3 Choice of method for ICM isolation according to blastocyst& v4 @* v. A1 n6 V
grade
, `( J0 e* V5 q! aICM0123
1 l- ]* f9 a2 u' dTE
" p; B" Q! N7 ]; G- g* I0AA 1 A A A/B
& F/ B D2 K' H/ m9 W2ABB 3 ABBB
2 e. x! I/ J! } sTE score - 0: no cell layer formed. 1: few big cells forming a loose; Z. Y' @; f( m) O" }1 }7 a
layer. 2: cells forming a medium coherent layer. 3: many cells in a tight& m* H1 @ h4 _7 V
cellular layer. ICM score - 0: no visible inner cell mass, inner to the TE( d! d0 @6 T5 J* u4 m
in any plane of the blastocyst. 1: ICM with less than 10 compacted& [' _. } h G
cells, or loosened mass with up to 20 cells in the central focused plane3 B* E$ d1 M( k5 R
of the blastocyst. 2: ICM with more than 10 compacted cells, or more
y$ ?1 m* i9 [7 b! m* A7 Z/ u. Kthan 20 loosened cells in the central focused plane of the blastocyst5 Q$ F7 v0 N* e
under the stereomicroscope. 3: ICM with more than 20 compacted
! z, b# p D7 V- ?; E/ ~1 lcells, or more than 25 loosened cells in the central focused plane of the5 ?' }! p' D! S! l: Z2 G$ V+ s2 B
blastocyst. Method A refers to pipetting for ICM isolation. Method B% R( c+ _" \( c G
consists of mechanical cutting of the ICM: [% }0 M& c. n1 D* t& K& f: O
Fig. 4 Derivation plate. Embryos are dispensed in AT in drop 1, and$ q. l8 {4 b) j
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 id3 M b( @+ h' q( D: Y. K
Tyrodes. G2: embryo culture medium. HES: Human Embryonic Stem. V3 F( E1 a% {4 L2 ^
cell medium* v; D1 k# ?9 B& e( ]# Q* l; F
Stem Cell Rev and Rep. m- @0 H2 c* A4 F; P7 c
detaches and float s. Qui ckly aspir ate medium from the5 f5 l: ~" I+ D: A. ^7 `& D
HES 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 evaporation6 x* L* k8 W4 \# ~3 a8 H& E
occurred durin g cutting.; X0 M8 @ v# x- U
6. Aspirate the floating piece( s) and spread them onto the
8 |- v* J9 _/ E) l( U$ mfeeder dish. Try to avoid pure TE fragments , recogni zed
# F; s s9 Y& k% o1 j2 M& F+ mby thei r trans parency.: @2 w" P" x' b8 v) _9 ^- R% P8 Z
7. Take an ima ge of the fragments seeded, record ing the$ f/ n) y6 S3 Q2 N" |/ L
position of the IC M piece on the dish.9 C% m+ B; x. d+ ?0 W; i: _
8. Inspect plates dail y, feedi ng them each second day until7 h# N- V7 `* O- E0 B
pluripot ent cell grow th is obv ious.
* |1 G& F% f8 j8 n$ j2 z9. Prepare new feeder cell plates as soon as pluripo tent) @) Q* C, x2 f6 f
growth is detected (see Not e 11 for deriva tion timelines3 g# A% N' r( T1 m6 S8 |4 k
and Fig. 5 for images on deriv ation progre ss). Go to0 s& [7 D) ~ y; s% X8 [8 j
Section 4 below for the firs t spli t of the pluripot ent cells.
3 J. H5 w' G& f9 h+ y+ cSplit of First Pluripoten t Out growth
) O1 l; ~! @0 JWhen the pluripot ent outgr owth(s) reach the stage de picted
) E8 S$ F8 B9 y* v) n! L7 Yin Fig. 5c , they are ready to b e split. Mechan ical cutting can
8 E8 d) m/ s: b+ r& J" R( }( Ibe perfor med wi th a glass pipette of any of the shapes A, D,5 B8 d8 t& q; W
E or F depicted in Fig.4 . In a ny case, prepar e feeder plates3 P* O3 T3 y% ], y
the day before splitting. If t he pluripotent outgrowth is6 O3 x& A! w; o
isolated from the residual TE, a closed p ipette and a
: r' D2 C9 U3 E: c: A, u/ ascrapin g met hod could be more effective. If stem cells are' o/ D$ D X9 B2 x1 W
growing in clumps surro unded by TE, a n o pen pipette to cut
]; f" a4 w! J2 [some of them will be preferred. It is wise to leave some1 y4 w) c* G3 d9 g
outgrowth(s) behind, u nt il split cells ar e s uccessfully
$ d% J" x, ]/ x7 p( Pgrowing in the new feeder dish.7 G$ r3 h" g& v, T8 t7 c6 k
Notes
) C% D+ m1 O+ y8 |7 I2 XIsolatio n and culture of mous e embr yonic fibroblas ts and. N& C0 e3 O' N2 j
feeder preparatio n
$ s: ^$ p! `5 c/ n2 KNote 1. We describe here the deriv ation of hESC line s in9 _7 x/ R4 R$ s: O4 Y0 ~
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 higher0 [% |6 K& A U- G8 g$ f: p
efficiency in stem cell deriv ation and lack of chro-mosome X inact ivation in hESC line s estab lished
' m. Q4 F9 f# a+ f7 z/ e1 ounder such condition s [19].
6 W" f* h% _8 O# ]/ Z( j$ pNote 2. Pr imary MEFs a re the first cells to be us ed as
6 `0 I6 E- i1 f) N4 vfeeder layers for hESC culture. Alternatively,
/ P* J0 _. [/ |either transfor med [ 20] or n aturally immortal-ised [ 21] M EFs, as well as human fibroblasts
- J9 ]( p1 p- T( f! ?9 ofrom several origins, includi ng foreskin [ 22]/ d4 ?- M$ m% E: h0 D
and placenta primary cells [8 ]andanimmor-talised placental f ibroblast cell l ine [23]have1 H+ z$ t$ W# K/ ]" }
be en u sed su cce ssfully as fee ders f or hESC
$ N$ r0 ]' O f; b# X. bderi vation and cul ture. Primary lines f rom hu-man f oreskin f ibroblasts (HFF-1) have al so
+ H. X. q- b8 @been used reproducibl y to cult ure h ESC li n es.% N, l# D" P# M! A$ n
In the case o f HFF-1 cells, i rradi a tion i s the; M$ T9 _: ^0 v/ ?$ [0 N2 o) N
pref erred method of i nactivation. All m et hods
2 \; h3 y) y( j1 l! T, b2 }described i n this paper are equally valid r e-gardless of the t ype of feeder s used.
4 V0 Z9 n+ M+ h7 E0 O( t& C4 QNote 3. Mice stra in has proven to be an importan t facto r
, S. l( k: O7 s- C5 |7 Hfo r th e q ua li ty o f th e fe ed e r ce lls d e riv ed fro m
& u; u) L% ]* ^& K8 o- L$ LFig. 5 Micrographs showing8 ]# r# M" A! B* j6 o' f+ E
evolution of the pluripotent and( b. z) {. ]: X( W4 [
TE cells during a derivation
, ]5 q6 H/ ?& rexperiment. a ICM 1 day after
# V g/ {- x& F: o' X& zseeding. b ICM cell expansion( C. M% w, R( D6 z% a( G
after 4 days in culture. Arrow
, ]0 L& a" b! ?5 Z: O' _points to the pluripotent/ ]8 i L- Q Z$ @* n
outgrowth, which looks like a
% v. R& M) X9 I7 \- Gclump at this stage; c colony
6 d* r$ p$ `4 w- Y3 f, Joutgrowth 2 days after splitting3 g0 ^/ u0 t& }5 L8 o
the outgrowth in panel B: arrow7 H3 j D) m* ^: G R; f
points to the expanding+ M4 X) r! I; g0 e+ G2 C3 W
pluripotent cells out of the4 X' h% y6 S, M$ I
initial hESC clump; d residual: y* ]* m Z; O4 b6 g; \5 O
TE cells 3 days after ICM
% s! M/ c. w j1 Fseeding; e TE differentiated
3 w; `5 D4 p, u) l' pcells 4 days after seeding; f TE8 v0 o2 `$ ~/ D, J: G7 a5 W. ^
cells detached from the plate
; M: ?7 b; J9 v8 @7 J/ l& G6 N9 days after seeding; g detail of
0 V7 L6 w+ K7 U' m4 Rthe emerging pluripotent colony
2 e" H; q! X5 q! m2 h7 Jof picture C;h early hESC
4 J( |/ \6 \. b1 K' u6 Hcolony at passage 4; i regular
) {! A0 W5 U/ X( T8 z, Rcolony of an established line at- w: }) k- @0 h+ M6 r3 p
passage six
7 @" |, F3 V6 v- N( ^) ?Stem Cell Rev and Rep: X2 @8 o3 u- k' J" V3 H! v
them . In our hands, embryos from the CF- 1 and* w1 F* q$ T' S H# t
MF-1 line s, and from the cross between MF-1 and
7 @$ u6 G2 n- F* L0 XCD-1, are much better than inbre d CD-1 embr yos.
$ K! |% }8 h0 f; l( [0 y0 lNote 4. Isolated pME Fs can be frozen at passag e zero, and
4 H7 [6 e* z$ |5 ~" O8 _/ Pexpanded at a late r time to prepar e feeders . Nev-ertheless , the expansi on up to passag e tw o before: Z& U3 Z$ z# {* q/ s+ G5 ? G
freezing has the advantage of increa sing the num-ber of cells obtained at passage four. It has to be2 ]& E7 N0 h6 l4 Z1 d
noted that in the isolati on procedu re describ ed in; Y9 R9 J0 _ a+ u
the Meth o ds secti o n, an d d ifferentl y fro m oth er; A$ A& @! `5 u: C' k3 v
published protocols [ 24], tissue chunks are not
/ P6 @# j* }6 P0 Fdiscarded before seedi ng after first trypsiniz ation.
( X- y& `4 \# }! F' O; ZThis procedu re yiel ds signi ficantly higher numbe r
# I. o+ f. y; ^' qof cells. It is advis ed to culture chunk s a nd clumps8 o- ?9 k! f0 g9 v Z$ c/ U
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
5 J, r7 q1 P% H* Tnormally be suffici ent to produce enough numbe r9 {8 ?) x' ^7 u4 \. Q( l4 O1 R
of vials for cult uring up to six new stem cell lines# Z! q0 G# F) h& G+ k
during 1 year.
# y& F7 W! a. dNote 5. Finger vorte xing is a simple way to mix a solution. f* y5 h2 l9 h$ C2 B& y
or disag gregate a pellet in a test tube. Hold the top' x8 g& v) ^0 E& }, p8 C6 r
of the tube secure ly in one hand and draw the ring," b. o4 D* t l, Z& H3 A
middle and index finge r of the other hand sequen-tially towards you, tappi ng the tube. This creat es a
( K3 t. l6 v0 U: swh irlpo ol eff ect inside the t ube, w hic h can be! j; k6 R3 j; V1 B+ V# h; w
adjusted in inte nsity by speed. It is milder than
1 Q6 T! ]+ A) }, I' Zmechanical vortexing and yields healthier cells. M) g; l' H2 A. j$ e8 ?
after centr ifugation .5 t3 B( Z+ l! l0 u9 t" X
Note 6. Freez ing fibro blasts wi th a slow freezing met hod is9 C, u$ ]& C; Z& \" r
not an issue , and they can be stor ed at − 80°C for
, _" O( V+ E8 a( w1 c# L7 Rmonths without a decreas e in subseq uent plating0 Y: o9 F* {/ }4 H3 _9 G( P3 |
efficiency. The proto col described herei n uses the4 D6 e* w( z3 Q# M/ p$ e+ Z
slow rate freezi ng container Mr Frost y, which has
3 k0 A) E# L3 d6 V' Pbeen vali dated and used for nearly 20 y ears i n7 @5 k. ?) `$ g- [" E
mammal ian cell freezing [ 25]. It is a cheap and" O+ y, w8 r1 K
time savin g o ption when compa red to alte rnative! a, S4 W [: S8 J) H
methods such as contr olled-rate freezing and vitri-fication, which may be suitable for more delicate
) [0 X, E- V- e+ Mmaterials like embr yos and hESCs .
$ b E0 g& [. N! V' p4 H u0 E: \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+ C: I% q& ?- {. G- `
toxi city and different iation during cultu re.
- T' e; n- L) r; w6 zNote 8. MitomycinCis thepreferredmethodtoinac-t ivate M EFs, a nd detail ed st eps to p erform the
$ A3 @: O, m) b# b3 P9 O7 D9 Aprocedur e are included in the text. Alternative-ly, γ - or X-ray irradiation can be used. In a ny2 Y2 i& R) }3 x7 K, M. o! K6 l* r" U
case, t he hESC supporting capaci ty of each
7 B" J6 `! t4 P! b3 X, Bnew batch of feeders has to be tested on
7 ?2 r- z0 \) q: _& O: Kknown hESC lines bef ore c onsider ing t he m
0 v7 I2 r0 [' x# K! x2 fvalida ted f or use. Once the new batch has been3 u, Y4 U& _& E8 C
valida ted f or hESC cultur e, it ca n be used in
! [# ` Z5 R9 _; U0 ?% Zsubsequent derivation e xperim ent s.% l D" \$ E( m1 m4 f
Embryo and ICM Cul ture! R* R* o& \! g" U) @1 p) z @
Note 9. The procedu res detai led here are based on the use9 Y7 ?7 y: r* w1 \0 w9 \
of high quali ty clin ical grade embr yos, but fre-quently, the quality of the embr yos donated for
% G% w2 x6 [4 presearch do es not correspon d to the standards3 @% r# x% J+ b
presen ted in Fig. 2 . If by day 6 of development ,
: X0 s6 i5 F) Mcompa cted cell s as in Fig. 2e – e are not form ed,2 G7 h$ h. w8 P4 @
extending embryo c ultu re i n complete HES
6 {3 i- q, ?4 h" o5 Z) r5 u7 omedium for 1– 2 additional days will selec tively/ P; A! r( P+ u) q* T
favour the growth of the ICM ver sus the TE.
) U2 F" f! [+ ~/ rThis appr oach is supported in the most recent
* N2 h/ N/ }0 hli terat ure [ 15].6 T o0 s6 o- i5 i9 z6 X: k, B
Note 10. Reg ar ding ICM isolation, s om e authors use a n
8 g5 Z" H) J; f2 b X, Xextended acid t yrode’s treatment to weaken# K3 M6 X) D9 `( d' A2 u
the TE [26], thus facilitating the spreading
) B) w; d& c7 k# g/ K* qof blastocyst onto the feeders. H owever, in8 y/ Z) t" ?5 A, I- W4 I/ N+ p* I
our hands, the efficiency of this method# I9 ~' K1 z r/ |
d e p e nd s g r e at ly on th e T E g r ad i ng . A p pl y in g) R Y6 Z V, j
the same AT t reatment to all embryos usually
9 P2 C1 I+ `7 n. J) S gresults i n s evere damage to some of them.' u: j5 @# _4 b
Theref ore, fitting t he method for TE reduction" W. @6 l' _/ Y7 d* X" v
to the grade of each embryo (Table 2 )helps$ W( i/ ^1 I$ ~# s1 V! r
maxi mizing the r ecovery of viable cells f rom" }3 u2 S- w" G$ f6 v# L3 ~
the I CM. When TE is r el atively s mall com-pared t o the ICM, or when the ICM is not
% f- ~7 W" R) D: M3 L! xcompact, a pipetting method (Method A) is! v9 A" H# R3 ?+ o. j; `
preferable. Alternatively, if the TE is robust
* @* P' _$ z/ j' s! s5 i* I* ~4 {and t he ICM i s c om pact , mechanical cutting' v, z J0 R: I
(Met hod B) s hould be the method of choice.( y. X0 n7 U1 I4 x( S7 o
Speed during embryo denudation and I CM
5 [0 k+ q' E% ~6 uisolat ion is criti cal, even if the m anipul ations) n$ j" O' Q `+ k3 g$ x5 A
are performed on a heated stage.
( U, W& m5 s4 {# h! q0 e' r8 T/ kNote 11. If healt hy, the first seeding of the cell s from the
* O8 o, z) E- i( k4 Y5 GICMs will att ach i n 6 h t o ove rn igh t . In o ur
% |" M( e5 v. [; p* E5 W9 t B8 yexperi ence, those clum ps needin g more time to
4 |( A0 j% S) H; K6 ?attach are not robust enough to yiel d deriv ations./ e2 {% J! A: U
The first outgr owth after the attachmen t of the D. x6 J, |9 ], f
clump can be seen from days 3 to 16. At this
8 d! A7 A) X3 k* d' x; Xpoint, the undifferentiated cells need the suppor t! X7 u& f6 _7 Y( K
of fresh feeder layers to estab lish pluripot ent cell
/ c& n9 T/ E( k A/ }6 C( jgrowth. TE cell s evolve invar iably to form syn-cytial cells that invade the feeder layer and die- w1 V% X0 @9 c# ^/ U) x+ T8 ~
leavin g g aps i n it. It is critical to have fresh" |$ T& _8 V/ r) L. X
feeders ready by the time the TE cells and deriv-atives detach from the plat e.
5 H4 K: o; a& ~; T7 _+ kAcknowledgments This protocol is the result of work funded by the
0 [4 I8 ?4 c* U. t6 C7 l( x) q9 yNorth West Development Agency (NWDA) i n t he UK and the$ \) |- b& \2 M, W% k0 i
MICINN-PLE2009-0091, IPT-20011-1402-900000 and FPI-CAIB
% c' [$ @ N [. GGrant FPI10 grants in Spain.
+ C) \: @: s, ^3 w! A% g4 dCon flic t of in terest Th e aut hors decl ar e no pot enti al con fl icts o f. N0 z+ y( n: C S0 g/ y5 }# u
interest.2 E* A' Z; o1 s9 {: a* i
Stem Cell Rev and Rep# d& P2 q. Y2 A# i, o
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