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Optimized Protocol for Deriva tion of Human Embry onic
6 O4 O0 S! e5 C c8 l' iStem Cell Lines
% m* L3 D& F" w0 O/ g2 zMaría Vicenta Cam arasa & Víctor Miguel Galvez&
1 K! x. t6 L Y. e, X- P' KDanie l Roy Brison & Dani el Bac hiller) g! X' m1 [. u; F; t& ^
#Springer Science+Business Media, LLC 20121 ~0 g/ T' I. L
Abstr act For t he pa st 12 ye ar s, th e b iolo gy an d a pplic atio ns7 N9 S2 ~8 H' D n
of human embryonic stem cells (hESCs) have received great2 D: } u. r' t+ T4 @
atte ntio n f rom t h e s cien tific comm unity. D e riva tive s of the first& Z5 S, x, u |2 ] ~
hESC line obtained by J. Thomson ’s group (Science 282
; ~) E5 I) r- x- r8 c(5391):1145 –11 4 7 , 19 98 ) have been used in clinical trials in
5 _1 q) _6 b3 s5 ~1 l% V6 N# upa ti en ts with sp ina l co rd inju ry, an d othe r h ESC lin es ha ve
2 R' Z* A! ^ [& w* [* Fno w b een u sed t o g en era te cell s f or use in tr eatin g blin dn ess& f2 H8 l) S1 ~2 R4 ~
( L a n ce t 3 7 9( 9 81 7) : 7 13 –72 0, 20 12 ). In addition to the classical
' K i8 v$ a& |) z( y8 gpr ot oc ol b ase d on m ou se o r hu man f ee der l aye rs u si ng op en7 {' t4 V/ ]+ ~. [
cu lt ur e m eth ods ( In Vitro C ellu lar & Dev elop men tal B iolo gy -Animal 46(3 –4):386 –394 , 20 10 ; Stem Cells 23(9):1221–1227,
( E( D- J0 u' n) F) r0 R2005 ; Nature Biotechnology 24(2):185 –187, 2006 ;Human
" v- a8 ?& F3 i) C cReproduction 21(2):503–511, 2006 ; Human Reproduction 20. c, b9 x8 P; L1 N3 m3 l$ |' U
(8):2201 –2206,2005 ; Fertility and Sterility 83(5):1517–1529,: {7 I2 A+ y" a2 w( T
2005 ), novel hESC lines have been derived xeno-free (without
- I8 V0 n, d. a& o5 jusing animal derived reagents) (PLoS One 5 (4):1024–1026,
$ v8 F% q2 s# Q. h4 N' c- @# Y2010), feeder-free ( without supporting cell m onolayers)
; I9 c' |# N; P8 d1 c3 S g(Lancet 365(9471):1601 –1603, 2005 ), in microdrops under
( r# |" H4 B) F. i# N% woil (In Vitro Cellular & Developmental Biology - Animal 46
0 d# @* T+ [& ]- ^# r/ O(3 –4):236 –41,2010 ) and in suspension with ROCK inhibitor
$ |2 d- Q. E) g( X' A(Nature Biotechnology 28(4):361 –4, 2010 ). Regardless of the
. t" R- m9 k; ]0 oculture system, successful hESC derivation usually requires4 E% Q7 ?% h) `2 R# G" S8 `
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 during1 s) c9 ~( g' A/ H2 Y0 f, }4 @% {
hESC line derivation. Herein we address the crucial steps of( B6 V p* ^/ C5 b! Y/ b1 m7 u" z/ t
the hESC line derivation protocol, and provide tips to apply2 q2 q8 [6 o" o0 |
quality control to each step of the procedure., D: |9 T! u3 d
Keyword sHuman embr yonic stem cell s
9 N# A. I# ?& i: M* B# R.
6 @% y4 C3 B1 l3 \* j: @( ]6 j9 FDerivat ion
4 S' b F/ E" S/ h) e.1 S4 G, t' a" e
Blast ocyst
x7 c9 o }5 }' _1 V0 F! S- [.
( \1 |6 P5 d* \# jCultur e o ptimizatio n
$ e9 h+ e5 c) @ X/ ]Introduction% }& [( V/ g- }% L. P" G
To da te, hundreds of human embryonic stem cell (hESC) line s7 P- u4 b" x$ k( k% ~5 m, x& l4 i
have been de rive d f rom surplus embryos after as sisted repro-duction techniques. Reported derivat ion e fficiencies range from3 n2 P/ C0 R+ f9 R5 Q
7 to 100 %. This wide span pr ob ably refl ects the di versit y of the k6 @* m4 ~# @& B
methodologies involved. M any var iables of the procedure s uc h3 o0 y6 _. g! a$ s* |' t8 W3 o
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)
: t( @) ?) {+ s3 O+ f3 L mremoval and inne r cell mass (ICM) isol at ion a nd type of feeder
0 s: d& v8 W! v- f }1 Jlayer, 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( X' |" E$ \4 G# e
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 analysed3 A7 F/ W. Y( n
th e genetic stability of more than one hundred hESC lines; y- V0 Q C6 c! {. z
using consensus protocols for karyotyping and genotyping.( b8 l0 B$ G* j, W" B) ?
This echoes the importance of defining robust and common0 q0 D* q9 v0 K: C
criteria for the assessment of both existing and newly derived' o$ k5 D- {# x2 H
cell lines, and for the validation of advances in culture con-ditions. [13].
, N$ [$ i# _( q5 ]M. V. Camarasa (*)
, T' s) y1 k. H' Q) h; H- e:, \( r% N8 J* S$ _0 r
V. M. Galvez
& L, r; y! z* }0 J1 {+ _+ b6 }:( n @ o ~9 Y7 Y) H" {/ ]7 G
D. Bachiller
6 ?% o* a5 f6 E) E8 UCaubet-Cimera Fundation, Centre for Advanced Respiratory
* W& r7 z' h5 @6 MMedicine, Recinte Hospital Joan March, m! ^, E! Q1 e9 \3 f# E
Ctra Sóller km 12,, D& ]" }2 I! i
07110 Bunyola, Illes Balears, Mallorca, Spain
( g( a7 | e8 X5 ye-mail: telomerasi@yahoo.es0 e4 I6 ^ j" G7 ?
M. V. Camarasa
; P: x6 `7 X, \" j# D/ K4 X% te-mail: mavi.camarasa@caubet-cime ra.es
2 m! I6 D( n9 F" [, qD. R. Brison- _* x# `2 a. |
North West Embryonic Stem Cell Centre, Faculty of Life Sciences, a3 w2 g) d' H
Core Technology Facility, University of Manchester,
7 J$ p& P4 t5 d4 N: r: g46 Grafton Street,
% U- s) R8 b$ X0 x/ lManchester M13 9NT, UK2 P+ z+ |) I/ J) E! G
D. Bachiller
( ?5 R8 g% s! m0 EConsejo Superior de Invest igaciones Científicas (CSIC),# R+ [ X9 F$ Y+ K n9 h
Mallorca, Spain
, k! Y# Q- n7 |, E4 H8 }Stem Cell Rev and Rep
, \" H5 G# P4 q0 ZDOI 10.1007/s12015-012-9377-46 B. F: n7 M% O
This protocol focuses on t he main first steps of the% d. w: V! Y6 q, i# n
deriv ation of hESC lines, i.e. embryo culture and gradi ng,
1 ?$ I2 ^: E. h6 W8 k8 @. Nand precise mani pulation of their ICMs and first outgr owths
6 ?5 [6 k3 n% u* y6 b$ Rof pluripot ent stem cell s. The Notes secti on includes alter-native procedu res as well as commenta ries and clarific ations
7 s: V. T: U0 f, g+ i- Lon the techniques described in the Methods section. Assuming
. w0 L' }: V) \7 a4 I5 B! z5 k5 j1 Texperience in mammalian basic tissue culture technique, this, C; t/ f' M' F% }
protocol should aid anyone wishing to derive and culture
( J, [+ C1 e0 ]) d+ x. I$ uhESCs to set up a rapid and reliable method. This protocol
7 g( w9 X) a# U$ lreflects continuous work optimizing the derivation, culture" l7 O0 S$ m! s: w9 }1 g# V
and characterization of hESC lines over more than 10 years,
6 ~: k7 L( @) zcomprising activities carried out in four different laboratories.
8 _0 F; b3 Z0 lAdva ntages of the present p rotocol over others previous-ly pub lished in clude: optimiz ation of f eede r cell yield," |) l$ ^" v, t
increase of I CM cell production and adjustment of the5 ~$ L7 d m/ F& A
deriv ation protoc ol accordi ng to embry o grade.
6 V7 ?6 f1 [% y0 u! ?0 wFeeder cell yield is optimised by culturing tissue aggregates
: [) U0 S) @7 Iuntil they grow as monolayers. Embryo culture is optimized
7 ^! i- H9 ?+ q( s" C$ S" M7 Bso that growth of the pluripotent cell population is favoured
7 j2 L, E) |6 P3 wover that of the non-pluripotent trophectoderm (TE) cells.0 p( ~7 W" f- g$ T- ]8 m. Y1 e1 k
Finally, the fire pulling of glass Pasteur pipettes to form thin; S$ \% B- I" ^1 T
open and closed ends that match the size of the ICM or initial
+ Y# i( o- u! i2 `- y7 _1 ooutgrowth to isolate, increase effectiveness of mechanical& t, ^. S3 F' R; I0 c
splitting techniques.! M6 W. g) Q2 l& f
Materi als
]/ l# |9 M1 t- h C" @Mate rials a nd Rea gents
1 _! @% {5 I# j1. Bacteriological Petri dishes, 100 mm Ø, Fisher Scientific T3 w- {5 r% b% L! f5 E
#09-720-500.# b6 M( ]" V+ o2 h" S+ j) b/ p" s" t+ {
2. Cen tre-wel l Organ Cultur e Di sh, 60 mm tissue culture
6 }, O8 x" e1 }" _7 y( ztreat ed, BD #353037.
: K4 C/ g0 ^$ R/ i; a; W3. Lon g glass Pasteu r pipet tes, Fisher # 1367820C .# b& m+ d9 V, P! ?
4. Mr Frost y® Nalge ne, Sigm a #C1562-1E A.
$ |) U; X6 P, U- m8 N5. Se rological p ipette 1 ml, Cor ning #4485.. |3 l+ L8 p9 J/ S. C/ e; `& H
6. Se rological p ipette 5 ml, Cor ning #4487.
# Z$ ~7 k* O5 M0 I% E# w+ L6 a7. Se rological p ipette, 10 ml, Cor ning #4488.
4 T* A g! f% S3 i8. Se rological p ipette, 25 ml, Cor ning #4489.
+ ^' q4 H) O$ h9 \# m# J9. Se rological p ipette, 50 ml, Cor ning #4490.. e! B; J# y, R
10. Tissu e culture dishes, 150 mm Ø, Cor ning #430599.
2 g! B; J' @2 D) l11 . Tissue cult ure flas ks F1 00, 100 cm; m# w+ }5 R/ r( f, y
21 a* q/ W4 q& o6 e
Corning #3816 .
# a4 {& s- Q3 l* S$ a X6 z/ t12. Tissu e culture plates, 4-well , BD #353654.
" l' U( y7 s3 }13. Tissu e culture plates, 6-well , Cor ning #3516.
% ]" X) u2 r5 b, Y14. Basic Fibrob last Gro wth Factor, #45103P- 100.
3 l, {8 b# W) o* c% R# t8 e% Y7 y- U6 C15. Beta-mercap toet anol, Sigm a #M-752 2./ h+ B2 w4 ]3 M, y0 h
16. Collagen ase IV, Invit rogen #17104 -019.. T, ?( i8 G* A" M0 p: y# W+ ?
17. DMEM medi um, Lonza #12-614 F.; ~7 y h0 e+ ~: H
18. DMSO, Sigma # D2650.8 ~# v! B7 n7 t5 d, u$ l
19 . Fe tal bov in e serum (FB S) , Australia n origin Lo nza
8 h7 ~) u9 }8 T3 Q# 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
) {( }& X+ }7 g2 M' E# W* @origins a re US and US approved, followed by Au str a l i a n
. |9 ]" B& n8 F6 Gand Australian/USDA approved. Poor quality serum is a
' H2 k$ l: O' \) }; X" w) Tconfirmed cause of feeder cell failure in hESC support.2 P# }) D }. a) g. S
20. G1, Vitr olife #10128." A4 i3 `3 z1 X! J/ S1 @
21. G2, Vitr olife #10132." U' A' [0 R0 n, v3 F, I+ Y
22. Gelatin, porci ne, Sigm a #G-1890.
: V; D! C- g, O' |23. Knockout (KO) DM EM, Invitrogen #10829- 018." h" N7 J1 i: @6 J6 w0 V
24. Knockout serum replacement ( KO-SR), Invitrogen% q& U) i( _4 ^7 u; b& R( ^
#10828- 028.
- \6 d% H9 C9 K7 ~" n25. L-Glutam ine, Lonz a #17-605 E.6 u; K5 u5 s* a
26. MEM Non-essential aminoacids (NEAA), L onza #
8 d0 F t7 a1 i( H# a2 [# U13-114E.
0 f0 ^6 W+ x( D) C8 [' D27. Mitomy cin C, Sigm a #M0503 . Hazard: this subst ance5 E8 f* b" _; R+ O- N/ e
is very toxic and procedures for safe hand li ng and9 Z9 D2 {3 M+ j- b
dispos al need to be in place. Do not brea the dust. s7 S! ? Q) R8 ]% {
Avoid contac t wi th eyes, on skin, on clot hing. Avoid0 f' Z9 u* U8 l' U! h
prolonged or repeated exposur e.
0 R1 Z5 x9 q1 |% R; \+ }2 W28. OVOIL, Vitrolife #10029.
! m( t! A$ M' T3 |6 d& D& g29. PBS with Ca
7 ]6 k/ R* B' {0 F* m( P+20 x; T5 B% g5 c$ y! @4 N5 S
/Mg
/ j, e6 ]* d6 n/ d$ q+2
, F9 j& ]$ h) k$ S, R+ {, Lonza #17-513 F.
* D* g7 v; X( o) @30. PBS, without Ca
3 x$ ~4 Z5 a- Z+2
: {* Q/ y0 Y8 C7 J/Mg
0 k* v3 G7 O7 \. `+2" }+ X- u1 V! a1 u) h
, Lonza #BE17 -516F.
! p' C. O3 O6 @) m1 T2 q5 n31. 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
3 \5 w8 i( z Q8 {dp c, Charles River Laboratories.
+ I- q! F" h3 e' z, x' h32. Thawkit I, Vitrolife #10067. ~3 u2 h% }4 a' J M, n
33. Trypsi n-EDTA, Cambre x # CC-5 012.' H; c! O" v; Y- C
34. Tyrode ’s solut ion, acidic . Sigm a #T1788.) S( h8 ^! P: Z7 Z
Solut ions
4 P6 c/ u. w+ }7 u9 H {% ^1. Complete DMEM medium: DM EM plus 10 % v/v FBS,
' [" X/ y: d8 L1 % v/v L- Glutamine 200 mM, 1 % NEA A.
' b( i: L% F# j3 _2. Freezing medium: 90 % v/v FBS plus 10 % v/v DMSO.
7 Q( H& s/ Q# T: w$ E2 T, |3. Gelatin solution: 0,1 % w/v gelatin in tissue culture grade; F" M1 S! E6 A7 `! f$ t2 e
water. To avoid contaminants and residues (detergent,/ Z4 e7 u5 q; [& A
traces of other chemical, etc.), use a new 500 ml Pyrex
, U. e, R6 L. k* F( _bottle. Autoclave the gelatin solution and store it at RT.) c y3 ], Z7 t- m K* M# D, t
4. HE S m ed ium: K O - DME M supp lemen ted w ith 20 % K O-S R,' @" y0 i1 m x8 a+ U
2 mM L -Glutamine, 1× N EAA, 50 mMβ-Mercaptoethanol
' _- U! \. h! Xand 8 ng/ ml human basic fibroblast growth fact or.9 `" O) ?* V) t2 D
Methods
8 V& R" V4 E6 Y2 JESCs are derived from preimplantation stage embryos. The
& V' w7 ?$ Q# xmost common procedure involves culturing embryos to the7 Y- g$ W7 W1 o9 M" B2 u# b' p G
blastocyst stage (day five to seven after fertilization), isolating
( M- T {6 }+ \their ICMs, and culturing the latter on mitotically inactivated, w; U, B9 v! \6 z6 r3 k
fibroblast cell layers. The resulting pluripotent cell popula-tions are selected by morphological and growth criteria. Novel
3 |+ b4 \% W: C( l1 EhESC lines are then established after continuous subculture of
8 _1 T# [. @6 k7 U. L2 g& d! S) bthe initial outgrowth. A hESC population can be considered to b1 m& H1 ~2 h# a7 a; c; q
be an established line when it reaches passage 8 with approx-imately 3 × 10) ]: R' V9 Y% x" U6 `$ ^, h+ v
7
6 ^8 ?7 h2 ?# s) n I: y5 {' Q2 Icells, as defined by the UK National Clinical1 b: O! C5 H% g" Z! x" W7 R
human Embryonic Stem Cell Forum [ 14].
. o3 M: q$ ~2 h) ?) c7 v- {Stem Cell Rev and Rep
0 C7 Z2 p$ O6 Z' I7 M" f; j0 yAfter eight to ten co nsecutive p assages, the novel line is
; H5 X5 ~5 }8 N7 w6 Osufficiently expanded to check its viabi lity by freezi ng and
) N8 r9 @" v* K3 c$ @2 {+ Qthawing ./ E" n6 x: |, i1 H
Ta b l e1 defines the term‘one v olume’ for each of the culture9 g' H, \ X+ p$ r
v es se l s d es cr ib ed t hr o ug ho u t t he manuscript. All incubations are& c. E. W6 _( Q( [6 i2 q$ c8 t
performed in c ondition s o f 3 7° C, 95 % h um idity an d 5 % CO2 w6 i# z/ b* M/ b, Q
2
& U5 f0 t9 A5 l" k9 Y, ainair(SeeNote1).Figure 1 depi ct s t he expect ed time schedule4 {) V8 E6 o2 e* u! R2 k4 [
to complet e a hESC d erivation p ro toco l, provide d that t he feeder
# }3 C" ]. I4 c l7 S$ M9 Hsource has b ee n p revi ou sly v alidated.: Q3 i3 k- Q, p* T G, r, A0 P! W
Feeder Layer Preparat ion0 N" G7 j' {! Y r. M& C S n, h8 f
Primary cult ures of feeder cells have tended to be used for6 d: g- a% O5 Q
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/ ^: t& L; s. R
derivation can be performed on f eeders produced from6 ~1 R& B5 B5 p+ J3 ]2 U
estab lished cell lines, or prim ary feeder cells whi ch have
! J4 e a" b B6 jbeen immort alised. Altern atives to the use of prim ary mous e
$ L4 a8 q+ _# e+ \. e+ |embr yonic fibr oblasts (pME Fs) as feeder layer s for hESC& Z! E3 C6 w! ], a
culture are discussed in Not e 2.
5 d, x. S( ]# f, s) \0 U: F! I; y. K# XReg ardless of the type of feeders used, every feeder layer
7 A: d5 |( ]2 |5 emust be con ditioned before stem cells are seeded on top of
) E; J. J8 ~$ D4 ~# _7 v' W' d6 Wit. To do so, feeder cult ure medium is disca rded; feeders are7 y3 _1 m' B' s/ \( q- H _. S9 N
then washed three times with one volume of PBS each time" h4 c' r& g* d* u5 }
and once with half a volume of stem cell medium (HES).
6 @1 B* g, P* A( fPlates are ready for use, from 30 ′ to several hours afterwards.1 |% E I8 P% N) m2 T2 r; k( c
MEF Isol ation8 w: c! ~* e O' D
1. Sacrifice a pregnant female (12.5 –13.5 dpc) by cervical. N! ^2 {5 N$ R- B7 n* u7 I( _3 {
dislocation. See Note 3 for tips on strain choice.
- p7 M0 w& {2 m4 ]* r2. Di ssect out the uterine horns and place them into a
2 ~( G8 N, K; z0 j0 n7 J5 I6 ^10 0 mm Ø Petri dish containing one volume of ster ile
8 x! p" z0 f! l0 g9 v: Q6 }+ CPBS 1×. Transf er the plate to a laminar flow hood to
; V- I9 u' @/ Cco ntinue with the procedu re.) A' x# ]: b2 e
3. Cut open the whole length of the uteru s, so vite line. S, C0 @7 T# }- |& j
sacs with embr yos and placentas are partially relea sed.
0 M2 O, J" e" B+ E$ T& {' \4. S e parate each embryo from its p lace nt a a nd out of the* v3 k+ p/ M; Z9 t
viteline sac and surrounding membranes, and transfer
3 a. g( K: I) Ethem all to a ne w Petri dish with on e volume o f fresh PBS.) \& N; }* I. [, l8 [# n. H( o
5. Wash the embryos and transfer them ag ain to a new
. l* q' }) ~1 VPe tri dish with one vo lume of fresh PBS.+ K" i4 N: M( [4 N
6. Decapitate the embryos and separate the visceral tissues
) {4 |1 z) w! K1 {4 Q2 \2 k+ Hfrom the bodies. Collect carcasses aside in the working
; b& v7 r2 X) Z* _plate. Aspirate and discard the PBS and the visceral
* Q8 D9 U7 a( t6 L, c7 h5 Q; z. }: ^tissues carefully with a 10 ml serological pipette. Leave1 @. |' p& A' S* c: k9 K
the rest of the tissues in the plate.
! h$ ~/ j/ e( u5 v7. Mince body walls into small pieces until a homogeneous
& B' A2 L) e" Y Y: s$ _ {suspension is formed.
+ y9 q1 D/ ~* v( J8. Add 5 ml of trypsin-ED TA 1x to the plate and transfer2 S- R+ g1 L; f" H! h ^
all contents to a conica l 50 ml tube.( P- g/ G+ ? o3 y2 r! }
9. Incubat e diges tion at 37°C and 5 % CO
0 ?4 D' {6 s i2 ~/ ?; {. |2
9 G, s( @& X( n S6 wfor 30 min.
- U2 b+ `" X, I' VHom ogeniz e the cell suspension by adding 45 ml of
4 ]- ?$ `& s0 Q0 ]. afresh cult ure medium and pipet ting up and down for. v( y- {$ z% m) N
ten times, and cen trifuge to recover the cells (700 g for
2 U& o a. V! d1 c7 V& O2 min ).
3 Y- z: p9 M. l10. Discard supern atant, ad d 50 ml of fresh culture medi a+ _6 W0 ^( _; k9 i4 b+ @$ b7 m- E
and resus pend cells.
8 l. s4 V' Z1 G; t! Y/ G2 _( h11 . Cen trifuge the cells at 700 g for 2 min and disca rd
( u. o0 ?, p4 w- Vsupern atant.) j; @ h J* W( }
12. Resuspend t he cell s in 5 ml of complete D MEM( ^, T z$ E6 P) c6 F0 n: N7 Y4 v
medium. s; N- n9 p+ _3 M
13. Seed cell suspen sion at a rate of three embryos per) ~) Y: D$ _6 B! J3 m1 \7 \! J
F100 flask. (General ly four F100 will be set up at the
9 e2 H- `7 {% f9 Z. z4 ^be gi nn ing o f the procedure per dissec ted p regn ant
" M1 ?& X3 _3 t+ ffemale).
2 D8 T {! }+ I; }! ?0 a14. Incubate at 37°C and 5 % CO
5 J4 n+ e3 N% j2# c$ I" r" b7 R! s5 Z
unti l 90 % confl uence4 j8 h/ t3 e* i- _1 C2 P
is reached (approx imately two to 4 days).
6 @. h" R, ?4 Y T15. Split the cells twice by tryps inisati on, seeding them4 I: n6 b Y3 R
onto n ew flasks/dish es be twee n 1:3 and 1:6 split
( m( j& }, E" L# o" Q! i7 w6 Caccording to growth rate.
3 i* e( W" h. u) V+ [# l% ?After t wo passages , t he ce lls s hould be growing as
+ U* V2 s9 h1 `( |7 |( W ?& Umonolayers. Freeze 90 % of p assage 2 c el ls in vi als
0 C; r8 y0 B, I) e$ v/ vcont aining four to ten millions units each. The frozen
, a, \" b+ ~4 o' @: R( hcells wi ll const it u te a s tock of act ive pMEFs for fut ure) V9 E, ^1 m, f! {: ~8 p
use.
# ~3 Y: M% Y" u6 _" P( R# rTo continue with the production of inactivated feeder) E6 B8 \4 H8 {$ d% m3 N, ^" a
cells, expand the remaining 10 % of the original culture
4 J- R0 V9 h9 x4 \" Bup to pa ssa ge fou r. S ee d t he cel ls on to new f las ks/3 T& o. j4 Y/ b& T+ m: [
plates bet ween 1 :3 and 1:6 spli t according to grow th8 V) Z( ~% K' D& B! L
rate each pa ssage. Then inac tiva te an d freeze passag e
- z2 P/ g% s8 ~7 S0 [9 U5 Ffour cells as explained below. See Note 4 for tips on; `2 n* b- K! @3 Q0 e4 @9 y
these st eps.
5 W; h( s, C1 R5 U" s- Z$ N' KGene ration of Batches of Frozen Feeder Cells by Mit omycin/ j& O. ~+ H9 ]" v! G' g S/ X
C Inact ivation2 m, [! ^+ J. B m
1. Tr eat 80 % confluent, exponentially growing,
/ M7 g5 U" }+ e6 V3 T1 u" `pa ssage four pMEF populat ions (between 48 and
/ Z2 g+ Q1 C, e9 6 P150 plates), with 20 ml of 10 μg/ml Mitomycin C in
0 L" G( t) K6 n; d& k* @# I; jcomplete DMEM medi um, f or 2– 3 h at 37°C a nd
" |# k. s. {' D+ ~( j8 N# p5% CO20 @* b) [# F7 E3 Z; O: ^4 i6 H
.
, r6 I1 ?9 K/ F6 m4 B2 _Table 1 Definition of the vessels and volumes used throughout culture
6 t! D4 R- X$ nprocedures( v" l0 i7 t4 E/ e+ {# o3 l
Dish/plate Area cm v& ^/ S0 ~3 I. b
2
; ^8 e; l. V6 {( k, Z3 O7 v/well One Volume ml/well
& J1 e% s2 y& j0 s4-well plate 1.4 0,51 h5 b- _. c+ r; Q( f( G
24 well plate 2.0 1
+ n/ \' X9 N2 S9 I [( _7 l" yOne-well 60 mm plate 2. 9 1
h; y" d: a& v7 f/ N8 k6-well plate 9.5 3
- W, ~* D0 Z5 ?$ e! ]& g60 mm Ø dish 21.3 5
3 h3 s: K: w0 Y, h0 q100 mm Ø dish 58.1 20
$ T! n* w+ F# S- @, |. i& Z( fF100 flask 100.0 250 d8 ^4 G3 T. N
150 mm Ø dish 176.7 50' W. n; X) @' U7 C
Stem Cell Rev and Rep
3 v4 X: ~# k j& q" V, l1 P2. Aft er inactivation, disca rd the supern atant and wash
/ N; h9 c/ k# A" mcell m onolayers 3 times w ith on e volume o f PBS- s3 z5 l& B, C; W( I. W0 F
wi thout Ca
: J$ M: M+ h0 D+2
- |$ t3 D& l" P, x* O W/Mg! N4 w+ l. W+ t' y+ Q
+2
* W- C6 u ~8 u- x. h.% e: W o0 H; |: {8 P
3. Trypsi nise the cell s (2 ml per F100 flask or 3 ml per9 G$ A# Z% ]# K6 W4 L
P1 50 p late) for 2 – 7 min at room temperat ure (RT) .
2 X( E) ~8 ~5 |' F4. Tap the sides of the flasks/dishes to dislodge the cells,
, C9 B: h c1 e( @' }4 San d harves t them into 50 ml conica l centr ifuge tubes .
4 O S7 `) v% \8 n- LPo ol the contents of tw o to three P150s in one tube.
* N J7 V+ E6 @. _6 n2 a1 u* z: A5. Add 44 ml of compl ete medi um per tube.
+ t2 D* ^7 Z# |* I6. Cen trifuge tubes at 700 g for 2 min .1 d: y. r2 X6 K) f
7. Br eak pellet manually by finger vortexing (see Note 5). W( q7 ~' f3 u* E# l1 }
Resuspend each pellet in 30 ml of complete DMEM and5 @# A2 v; T, z! o0 q0 d6 N- o$ r
count in a Neubauer chamber.5 x+ j9 N5 Q7 }) Z4 h0 O
8. Co unt v ia ble ce lls and calcu la te the numbe r of
! y$ O3 J$ G- d$ D8 O9 T/ acryovi als needed, at a rate of 1– 4 million cells per vial.
+ ^9 E1 W+ m( G9 P9. Cen trifuge cell suspen sions at 700 g durin g 2 min .: a& ~3 L4 X% }. b* w) w, Y; x `
10. Label cryovi als with batch numbe r, amoun t of cells% H$ O" m4 s/ i( L5 h! U% L) y% `8 P0 p
and met hod of inact ivation, date and user ID.- G8 R( v4 j/ K1 F4 }
11 . Discard s upernatants from s tep 9 above and break
/ g* K" f! `1 r" Epell ets carefully by finger vortexing.# B4 m0 _( h7 u% j
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.; C& I# [- ~6 n- ^! Z
Disp ense cell s uspension i nto labelled cryovials and
. t; G' ?1 O, W d9 Btransfer them immedi ately to a precooled Mr Frost y® con-tainer, and then into an − 80°C freezer (see Note 6)." B. I, c8 @7 c$ M# L
Feede r Layer Plating for IC M Culture9 S0 k9 x# V1 Z2 R8 G$ |
1. Dispense one volume of gelatin 0.1 % ont o the/ v7 u# J" _8 z% S$ M* u! K$ | A, {
requi red number of dishe s/wells, and incuba te them
' z. d. g2 ]8 ^" mat 37 °C for 30 min.- K$ ~2 e4 G ^& Y9 S; b
2. Equ ilibrat e 10 ml of fresh compl ete DM EM medium,
. _! D& u" A' M3 c4 Z% Z/ Yin a 15 ml conical tube, in the CO
, {9 @3 b3 u+ E9 U8 ~: q7 N* h6 Z2& S6 [8 |+ I6 W$ A
incuba tor per each
3 U: X8 `4 c" o# L2 q. `1 – 2 vial (s) to be thawe d (see Note 7).
% C0 _! a/ R% U* X* O# W x) F; _3 X1 m3. Once the coated dishe s and the medi a are equil ibrated,$ p+ C" o. @0 f$ H2 Q3 O/ w l
thaw the requi red number of cryovi als to plate 0.2– 0.3
% }% E9 K' M* }4 q$ N1 Zmil lions of inactivat ed pMEFs per one-well 60 mm. q2 \; G8 T8 ?1 u! A
dish. Spray the frozen vials with 70 % ethano l. Roll2 h+ j0 F4 s( I& O! _
them in your hands, until only the last smal l piece of
# E( \% h% R( Rice remains. Tr ansfer the c ontent of the vi als i nto' a: W: S" ~5 I, X
the equilibrated compl ete D MEM. In order t o
" M6 y. |, w$ f& n# [avoid cell dam age i t is n ot advi sed t o thaw m or e0 N6 E' z/ L* w% y$ @% L8 k* [' \
than four vials at a time., X+ U! s" n( [; \! @. V% S
4. Cen trifuge the tubes at 700 g for 2 min . Discard the
5 c5 j' e' a% p5 Q' X( Y. @/ B) ]s upern atant a nd gently break the pellet b y f in ger0 R6 s; Q/ @+ s* r/ m* _
vo rtexing.
* r, j* b% F3 W; _1 x5. In order to elimin ate resi dual DM SO, repeat step 4.* D* v( R1 P3 ^" H; y
6. Dur ing the second centr ifuga tion, aspir ate the gelatin
9 [+ g6 p5 A2 `. U T# _" A1 ~/ J6 U7 bsolut ion from the plate(s) and dispe nse half a vo lume" }+ Q: e8 ^0 B0 i
of prew armed compl ete DM EM medium on each one.
* q0 |8 P6 @" Q7. Br eak pellets from step 5 above by finge r vortexing,- m4 C) k* R) a4 P+ R$ J
an d resus pend them in half a volum e of the wel l(s) to) C4 o' W1 Q& `3 F1 A; W! }
plat e./ F Z) k' k1 c1 H& l5 ^
8. Di spense cells dropw ise onto the medium- containing
1 P7 P# x0 d3 P) p iwel l(s).
7 ]0 p- P- ^1 T' X+ T* w, S+ s* C9. Place plate(s) at 5 % CO8 A0 P4 P C1 M0 N. c7 S" x
2: [; Y& S: ~ C2 e
at 37°C, and c arefully shake
6 f4 O8 {" j( m: A, N; i Ithem horizontal ly in all direction s to distrib ute the cells$ N; G) [: M8 J; n# z# S
ev enly.
% `+ _9 e% z' @# _- S" S4 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
" R' e7 o0 j+ r; J0 Zattach. Optimal feeder density corresponds to a conflu-ent monolayer; higher and lower densities will result
" R0 S' _$ L, c; y- Q) C' ?" z. ]either in reduced hESC growth and increased differenti-ation, respectively.
* w* Q, a# ?# O% i& f& b/ USe e Not e 8 for comments on feeder layer prepar ation.8 T# b2 k2 |4 m2 |* d$ e) t5 `
Embryo Culture
; I2 T6 \6 S5 g; S% U. iSurplu s embryos from clin ical in vitro fertilizati on (IVF), ^" s0 i. I4 Q' K: U
procedu res can be supplied, fresh or frozen , at any stage. W1 r# j# j$ ^- M8 S" `% s# C
from day one to day six post- fertilizati on. It is firmly estab-lished now that low-grad e blast ocysts produce h ESC lines9 }7 D+ Y. O3 k
succes sfully [ 15, 16]. The refor e, the derivatio n of stem cell
' q( {3 W7 h- n8 P' m7 H- k1 Hlines is almost guaranteed for a ny Re search Pr ogramme. R, n, p3 n/ y' N7 B+ Q$ A
estab lished in coll aboration with an IVF clin ical depart ment.
" d; `# E$ v( d2 ^In this section of the proto col we will detai l the steps9 n T y% S1 v3 [% |3 t
involv ed in embryo cult ure for hESC deriv ation. It is advis-able to use the same clinical grade embr yo culture reagent s
# Z1 T- q1 u% g) g# }% e- ~that are used in the clinical procedu res. Altern ativ ely, it is1 k# ^% U0 H5 Y2 Y1 D/ O
sim ple to p repa re the m from thei r compo nents [ 17]. Se e
% D( s7 e7 r1 u! \, a7 u" p: P7 A* QNote 9 for tips on embr yo cult ure.
' w8 ?, N0 ^9 l7 zWe will detail Vitrolife® protocols, and advi se to fol low
8 Y2 `0 r+ v/ d: Ymanufacturer instructions in ca se of different brand reagents.& h) n5 \% \( Q5 g' j
The cul ture system us ed was a two-stage one, i .e. changing
. _0 [( Q. l- rcul tur e m edia formulati on on day 3 or s ix-eight cell stage o f
5 l3 ]6 f7 G1 ]) \development (from G1 to G2 series ), un til blastocyst formation .
2 D- P' y' ^. z q: T- w1 ?& E: @Fig. 1 Time schedule for the
0 P+ K4 D( o2 A' Q) J6 hcompletion of a full derivation
3 @& I% s0 t) ?, t& b# jexperiment- F0 [3 ^, H" g4 N" B' ^
Stem Cell Rev and Rep
, _( [! y& K+ s- HEmbryos are cultured in m icrodrops under oil, to7 n; I: Q# s! T" C
prevent evaporation a nd to minimise pH changes. The
5 @, B' _2 j! ~8 Ifollowing protocol corresponds to t he culture of fresh or
+ b) z8 e7 I5 Lthawed cl eavage-stage embr yos. In the ca se of receiving
. J( _$ ^ E# v3 g2 vfrozen embryos, it is crucial that the thaw ing i s per-formed in the reagents corresponding to their freezing.2 Y6 L, e( Y" p
Follow manual instructions from the appropriate thaw-ing kit. After thaw ing, follow the protocol below for
, S9 A9 w3 H* G4 qembryo culture.: @ o% Z, i/ s- n( D9 W& e7 ~
1. Equilibrat e OVOIL® was hed wi th 1:10 volume of G1 or
; w7 z+ D3 j, A9 f, |3 `% GG2 for 6 h to overni ght at 37°C and 5 % CO2
7 y5 z9 P5 m$ ].7 n" p7 \% g6 ` l1 x( g
2. Equilibrat e G1 or G2 medi a as required (see Tab le 2 ) for
) m. C. F& Y( R$ V( [. {6 q. ya maxi mum of 3 h.& D6 n6 w: x: w% ^3 N) C
3. To set up embr yo cult ure plates, pipet te 25μ l of the
% `, i2 ~2 I3 yequilibra ted G1 or G2 media per embr yo on embr yo
6 f7 _! `' z. ?$ J0 L* F: Uculture d ishes/plates . Cover the drop(s) with equil ibrat-ed OVO IL®, and add 25 μ l more of the medium to the
2 w3 N& f" c6 D0 z Kformed drop(s).% t- Q( T0 ? i0 C# I
4. Assess embryo development daily (see Fig. 2 for a
$ h; I( X0 f" m5 p: mdi agram of g rowth pattern and c orresponding images! K$ O/ B& U2 \* H
of embryo development), and transfer them to fresh/ _) L j; y1 X$ I# U
media d rops every secon d day (see Ta ble 2 for2 P* @0 ^ c8 Q4 ^9 v9 y9 N8 T) Q
media c hange guideline). P re pare new drops as0 U3 h8 \3 d( P$ H- I
describedinsteps 1to 3above.If embryoculture7 |, y5 U: X" Y
star ted i n G1, remember that once embryos r each
- V+ A- c+ C" U: T7 w0 t6 fda y 3 o r the six-eigh t-cell stage, they must be4 w% {# e1 n Y* y/ u
cultured in G2 medium.
+ Y0 Z- x& T& R% h5. When embr yos reach da y six of develo pment , or the8 w- b! @# e% g! O0 V! o
ICM of the blast ocyst is visible, it is time to set up the9 b# Y6 @" \2 H! T! t& Y( l1 T
feeder plat es to seed the former.5 ]9 K- f" {( z/ j1 F
ICM Isolatio n an d Se eding8 X3 Q8 \: S+ Y! W2 X
During denudation and ICM isolation i t is a dvised to
+ G& e/ V- \4 g6 yprocess each em bryo indivi dually. Pulled glass Pasteur6 v: w! J& T9 o6 N$ J2 j* Q
pipettes or ca pillaries a re recommended, or commercial-ly avai lable em b ryo h andli ng p ipettes used f or clini c al
+ N6 m& Z) r" }8 z; JIVF p rocedur es.
Q- A, p: b5 T6 a8 p, }Con dition the feeders before the d enudation as follows:
+ ~" Q$ e1 `$ E/ h* Dbetween 1 and 3 h before the seeding e liminate f eeder
( o# d5 X9 i* Q, jmediu m. Wash three times wi th 1 ml of PBS with Ca1 p' d5 O6 A7 x9 l
+2
1 v |. t. f7 r0 T6 _' a. e: n7 y2 ~/
; m, G7 K* B& p3 yMg0 J& e* } p$ B7 w9 Z
+2
# X$ R, Z9 [; R& _/ Kand once with 0.25 ml of HES medium. Add 0.5 ml of
) y, w- ^; H: ~9 m) G& p0 KHES medium and equilibra te for at least 20 min at 37°C and
6 J R+ x: D, I5%CO0 b$ @! ^! R. e% S% z P
2( a, b# A! {0 g& o) n6 N6 b' j
. At this point the new feeders are ready to recei ve
) K+ c% F; ^! u6 i" a$ r0 y% ^the IC M.
$ p' O+ ~$ |% c4 KZona Pelluc ida Remova l8 G a/ b9 U' R
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
# |8 Q0 M* H: A3 v& Z$ D! R* b2& c$ n( X) |+ v$ {) x' W4 k
, for a
- z9 C" `: s* V, i7 O8 Q0 _min imum of 20 min and a maximum of 6 h.# M+ R5 P4 k6 Q, n4 m5 v
2. Bef ore any mani pulation is perfor med, images of the
; i2 \" g6 V$ V0 T; W/ nblast ocysts at different planes (×40) should be taken
8 h0 r. f9 x1 E# I2 n' _for further asses smen t of its grade. Afterw ards return it
+ h9 N, L w* }. V& |- {/ I Vto the incuba tor.
* P& A' _5 x& o5 g; b. d5 o3. Pu ll 2 long ster ile glass Pasteur pipet tes wi th the aid of
& |5 e% b s' ~0 Aa Mecker or Bunsen burner. Leave one of them opened1 n5 @. z2 X C/ q3 {. j
an d rounded , and close the other one leaving a small3 y9 X1 q; N& X& s
rounded ball at the tip (Fig.4 ). Check the thickness of/ m& d7 x$ k5 Q! X# D
the tips under the s tereomicroscope and r epeat the/ i2 F. B8 B. ]7 m# l: @7 K* h
procedure unt il a couple of pipettes whose sect ion: L+ U* z9 C6 Z6 c7 ~8 m I q9 U
mat ch the diam eter of the blastocyst are produce d.# P" x/ m" H2 j
4. Se t three drops of AT forming a row in a 60 mm tissue
% p' {- t) e$ D" c; tcu lture dish, an d mark thei r position at the rim of the& W$ s" u. x5 S& N7 W
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
* K I& }0 C; l" k8 Gsecond row at the middle of the dish, and three drops
$ c1 `$ v! ~1 z# S Zof HES media at the bott om, again aligned in a row3 a+ R5 L. `$ q! w' v/ ~! A
(S ee Fig. 3 for drop distrib ution).: p: @: d; a o5 o
5. Se t anothe r three drops of approximat ely 100 μ lof; X( m3 K) _3 k9 u
HES medi um in a new 60 mm dish.$ J/ s* c" c% C' Y
6. PicktheembryoupinG2 mediumwitha 275 μ m0 w, l! E+ N' b6 s; O
capil lary mi cropipette, a nd t ransfer it to the left AT( B6 M! G/ m% P: _# h8 j* r, r8 L
drop of the plate prepared in step 5 a bove. Stop' }7 `9 z7 W0 z ~2 ?! C/ y
dispensing G2 medium into the AT drop a s soon
* p% N+ o/ |% {3 Z/ I! m6 [% ~as the embryo has been releas ed. Empty the pipette. L4 R" E1 ]8 T, d0 a3 n6 [0 A
in the border of t he dish to discard r esidual G21 z0 i* I+ m$ s: Z
medi um.
$ u4 I; _* v! g- Q) s" k+ Y7. Wash the pipette twice in the second AT drop.
3 c# ?8 _& \2 r4 {8 h5 j# F0 l8. Return to the fi rst drop, pi ck u p the embryo and
3 o/ @$ O6 r) v$ t( W3 ~6 i+ D! {trans fer it to the third d rop. Monitor unde r the micro-scope the dissolution of the ZP (0 – 2 min). d% V# n( ]$ d6 ?% z) z
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. Y" r, @: q4 D4 j( f c
pre-w ash, dispe nsing the minim um amoun t of AT into
+ [% A" ^& y# f, n# e* bthe G2, the second to load the pipet te with fresh G28 A4 v ^6 Y, g b- u7 d- Q
medi um, and the third to was h the blastocyst .
. h+ Q* H/ Z7 |6 R" F) l% z/ s10. Tr ansfer the zona-free (denuded) blastocyst to the$ K* T" J" r4 `; G2 {
drops of the HES medi a using the washing procedu re
2 q0 l: q6 u) R9 zas described above for G2.
, J9 n' J. U1 V8 ]; S( y11 . Immedi ately trans fer the blas tocyst to the drops of; }5 M }, L# w8 u' @% p- |. G D
the HES medi a prepared i n a sepa rate plate in s tep2 Y ?7 t2 ]- n/ `) i# T, S% r
2. Follow the s ame procedure of trans ferring to the6 J& R ?1 O: }2 R
left drop, washing t he pipette and loading i t in the2 t2 J. x) X& s' q7 `8 n+ K9 E
second intermediate drop and transferring the8 t5 v7 X1 f1 q2 m- y
embryo t o the third drop. P l a ce the p lat e i n to the. O8 y8 ~6 S- c
CO0 I* z/ \1 ^' d. M+ c% `
2$ l7 z2 ^! b' V9 K; }$ F2 P
incubator.) R+ l- ?- I! e/ I) _6 S
12. Usin g the ima ges take n in step 2 above, asses s the6 u" V; k) b' g3 N5 t
deriv ation grade of the denuded blastoc yst (see em-bryo grading s cheme in r eference [18]) and a ppl y# p# Z$ f; C3 k
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
: O& d) [& l% P% F! pB be l o w. S e e No te 10 fo r c om me nt s on t h es e, U+ b5 N B- F: g) m/ e
procedu res.9 a/ w2 [. N0 A
Stem Cell Rev and Rep
% i9 _/ ?/ r+ ]* lICM Seedin g by Pipett ing (A)- N5 _2 H) j7 [
1. Choose an open pulled long Pasteur pipette of the same* D* f4 Y" B4 p7 V4 h6 {3 ]
diameter as the denuded blastocyst (see shape in Fig. 4a ).
* S4 p; O# x) t# |+ J/ L2. Load half of the tip with fresh war m HES medi um from3 O G7 h$ P" K5 L: X! w7 `0 G" X7 E
the drops set up to equil ibrate before denudat ion (ste p 59 S: B# M: b/ [4 e. q% ` t
on zona pell ucida remo val proto col).
7 T2 G4 N4 U L, J! f3 G* ]- e: O% K3. Aspirate the denuded embr yo and pipette it in and out Z. ^% ?: `) ]$ F. |* _# R. s" Q
while checki ng for loose ning of the TE cells.
1 S0 l- B0 Z1 E4. Aspirate the i solated cell clumps and dispense them# ~% `5 G5 H( \/ W: d
separa tely on the feeder plate.4 a4 ?7 o" e7 n; A- A# c- E
5. Ta ke an image of the seeded fragments of the bl as-tocyst. Pay attention to l ocate the ICM and record9 Q$ k$ M( d3 x1 C+ W
its posi ti on.
+ X9 A6 u+ T( \/ i8 X9 _9 E! cTable 2 Embryo culture
: R9 N* ~0 P9 y! i; }+ Nprotocol
7 s+ F9 v$ v* UDay of development Stage Medium Image recording Media change
9 [7 n) q/ N& `! d8 V4 |3 a. I- B m1 Zygote (2pn) G1 − G16 V/ _) x1 u; u# \0 v, g" K
22– 4 cells G1 Yes −
: }. u$ l( @2 ^3 p' ?; @+ U0 u36– 8 cells G2 Yes G1 to G2
9 Q3 _+ {- C: G: J4 Compacting morula G2 Yes −4 N( O* }9 s7 j' ?
5 Expanding G2 − Fresh G2
6 s( l: l9 T! f' g: l+ d" z6 Blastocyst G2 Yes −
" e6 `% a+ u9 L4 i7 Hatching G2 Yes −2 h7 @$ {+ E% C/ W
Fig. 2 Dagram of early human
( L0 J8 r0 [" e Bembryo development. a-a’! w% M; m/ b; f8 [% t
two-cell stage embryo, 1 day
z6 i' a+ A7 g( G% X& tafter fertilization; b-b’ four-cell
" |5 ?' i) u/ i: V* Z$ Wstage embryo, 2 days after
- i8 S: x" e* i) W6 Q9 Afertilization; c-c’ eight-cell8 m' A! r: [: E3 u& o/ J$ u+ M
stage embryo, 3 days after f" x" d& z6 m( Y6 i. g. y, }+ _* Y
ertilization; d e a rl y e xp an di ng
, u' W h$ [; d- {3 P0 {, Eblastocyst;d’ morula containing
) l1 r9 z! F/ fm o re th an 1 6 ce ll s, 4 d ay s a f t er
3 B2 l: N7 B2 D0 x: yfertilization; e early blastocyst,
/ G! J4 G; K0 f. v5 days after fertilization; f- A* `% ~% `: M& x, i0 Y& z" g
hatching blastocyst, around7 `* q6 a" N' E. @
7 days after fertilization. ZP:! n) z. o( p- w7 S; i' [9 X0 H+ b
Zona Pellucida. ICM: Inner w- S+ Z& p; @6 P a1 ]/ g
Cell Mass. TE:
4 J! B3 R$ F7 [: `1 zTrophectoderm. H# r! [( m% t% \1 ?
Stem Cell Rev and Rep' }7 h$ @7 p$ _( Y1 r
6. Tr ansfer the plate to the incubator, avoiding abrupt" R5 U2 P, I) E/ ?
movements t hat may separate the s eeded cells from
8 e R$ A, s6 M6 X8 K7 T! vthe feeders./ f* v% u9 j! T; K V, x4 e
7. Inspect the plat es daily. Cha nge the medium every other
) [1 p6 Z1 q, L9 `! Oday. ^1 v) _7 p- d k. ^# ~
8. Prepare new feeder cell plates as soon as pluripo tent: z- q# ~7 ]6 X* w8 G
growth is detected (see Not e 11 for deriva tion timelines- Z4 N* r* n7 l0 e* B
and Fig. 5 for ima ges on deriv ation progre ss).
7 I" |4 k" f0 f* [" |9. Split part of the emerg ing pluripotent colony when
" M6 n; r( ]$ T# ^( M% j7 W5 Jresi dual TE cells begin detach ing from the bottom of6 M0 I+ B1 n( s; I3 b# U- N
the plate. Follo w Secti on 4 below to do so.; @# }; M# s5 {* F; A0 T
ICM Seedin g by Mechan ical Cut ting (B)
' W1 k& L/ C0 H& ?1. Aspira te the denuded blastocyst w ith a pulled open0 O h3 _7 A# P% W4 |) Z* A
pipette (Fig. 4b) and dispe nse drops of approxi mately( w( o8 l$ D! {' D0 E, M$ ~
20μ l on the dry area of the 60 mm dish containing fresh
; P7 M) D: k' }% KHES drops, until the blasto cyst is dispe nsed in one of
8 m) Y/ O; Q9 qthem.
' i1 [5 L8 X& K' k2. Drag the denuded blastocy st across the dish, away from3 A) T( b, w% T0 \
the HES drops. The volume of the embr yo-conta ining
0 i' a, \* @7 B$ I( Vdrop will diminish up to the point that the embryo
# k( ]8 o; D5 ?! _flattens and sticks to the plastic. At this point you shoul d
. w9 X# f5 _4 e. G! L" Y) X" xbe able to locat e the IC M: it is much brigh ter and less
$ m. ]9 y$ R3 B( R- ] q$ f; Aflat tened than the dim TE. Sa ve the pipett e without
( h+ i; G3 T- H6 @emptying it for follow ing cutt ing steps .7 Y. x0 D9 [. j! S- e
3. Pick a close d pulled (hold ing) pipette (Fig.4c ) with the
?9 [/ w+ D1 wleft hand (rig ht for left-hande d) and hold TE by pressing
( l r8 ?3 |" u bits border agains t the plate.
0 i1 s1 T3 j0 s* ? F: I4. Pick the open (cutting) pipet te still contain ing medium
8 O% o/ z1 F0 m: y8 V3 [2 W: Pwith the right hand (left for left-hande d). With its edge,% v2 [ T& [1 k; i! Z! i4 F
cut out as much transparent TE as possible. Imm ediately: c) T+ e) z3 c
release the HES medi um to a void damag ing the ICM.
; P0 n5 f( P0 Q6 j+ X5. Scrape loose the ICM contai ning piece, which will be+ z5 r$ \5 f" f+ V0 y
adhered t o t he plastic. M onito r the ICM w hile it
}* `) d8 p9 z3 k4 XFig. 3 Shapes of long glass pipette tips for ICM and stem cell colony
$ l2 D& z3 [1 R& yprocessing. a transferring pipette with open rounded end; b cutting
8 Z/ j! V. {& m3 I& @pipette with open bevelled end; c holding pipette with a fine rounded
1 ?- p/ Y( e2 ?# nclosed end; d curved hook with closed end for outgrowth scraping;e-g
# H! w' ~! V, x! D- uflat hooks for colony cutting and scraping. h detail of the tip of the
9 D& \# s9 b! q# ^( Yholding pipette depicted in C! L& P1 J8 e, c1 f5 u
Table 3 Choice of method for ICM isolation according to blastocyst! \- s" Y( t* C) L0 g
grade
& f3 g/ i: u3 i- A9 R( ^ICM0123+ n2 |8 }+ b. @2 |1 k& ^' q
TE
( Q' l- C! Z7 {: ~+ V1 T, _0AA 1 A A A/B
( `+ ^9 C; V% @& C* T; P! f% b2ABB 3 ABBB
9 I- u9 Y; K) E" l: oTE score - 0: no cell layer formed. 1: few big cells forming a loose
8 n( U: A+ `( b& K* |& g2 e) Clayer. 2: cells forming a medium coherent layer. 3: many cells in a tight
( R# u4 U& O" e! _cellular layer. ICM score - 0: no visible inner cell mass, inner to the TE
h9 P6 B J- ^; pin any plane of the blastocyst. 1: ICM with less than 10 compacted2 G+ v& z7 v& v% U- {4 H
cells, or loosened mass with up to 20 cells in the central focused plane: A- [2 J1 j7 y* D/ l, x: t
of the blastocyst. 2: ICM with more than 10 compacted cells, or more
- D8 j" W% L/ n5 f0 jthan 20 loosened cells in the central focused plane of the blastocyst
0 G, _+ n: K( O, u) iunder the stereomicroscope. 3: ICM with more than 20 compacted3 p, E& i/ ?" f0 q, g! V
cells, or more than 25 loosened cells in the central focused plane of the6 y/ ~* ]% L, v* j$ w7 N% d
blastocyst. Method A refers to pipetting for ICM isolation. Method B5 e1 @& `9 Y9 J. I9 `# R
consists of mechanical cutting of the ICM
, o: t- R Y) z* t' @% NFig. 4 Derivation plate. Embryos are dispensed in AT in drop 1, and
: R" \1 X9 Z1 \: etr 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
6 F) q+ p) ]$ ]$ a0 z4 tTyrodes. G2: embryo culture medium. HES: Human Embryonic Stem+ b) q C/ R7 ~6 u) f3 p# e1 d9 y
cell medium* s- G4 |# ?0 p% [; q
Stem Cell Rev and Rep
f7 i Z& ~- B6 E1 Odetaches and float s. Qui ckly aspir ate medium from the
2 M6 `) X9 w. t- hHES 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/ X' l( T( p7 \, u
occurred durin g cutting. K/ B# q" x" U) Y, R% m
6. Aspirate the floating piece( s) and spread them onto the9 c6 \- _& G* i+ e8 L
feeder dish. Try to avoid pure TE fragments , recogni zed- \3 j9 ~3 C9 }" E( G t8 m: ^% Y) f
by thei r trans parency.- Z! n9 t& Q6 n4 R$ o. S
7. Take an ima ge of the fragments seeded, record ing the$ L S9 X# ?1 b9 ^- p( [
position of the IC M piece on the dish.
6 t7 u& J" _/ L, c. `0 [; m6 ^8. Inspect plates dail y, feedi ng them each second day until
2 Z f$ Y7 v7 Gpluripot ent cell grow th is obv ious.8 V, @& u# f* V$ \( z
9. Prepare new feeder cell plates as soon as pluripo tent
1 N( ?5 w$ a9 e7 K0 i% ngrowth is detected (see Not e 11 for deriva tion timelines
, y: j" P' t1 a) G5 dand Fig. 5 for images on deriv ation progre ss). Go to4 r4 v7 H* {5 ?0 ~* o6 T
Section 4 below for the firs t spli t of the pluripot ent cells.1 R2 e5 U! X$ Z! o% W$ H4 y) _- O
Split of First Pluripoten t Out growth ~0 m. a: F @$ O: L
When the pluripot ent outgr owth(s) reach the stage de picted
; r6 g& z$ z3 Q# ain Fig. 5c , they are ready to b e split. Mechan ical cutting can
+ N( J3 `+ [( q2 W6 c7 Ibe perfor med wi th a glass pipette of any of the shapes A, D,
+ | b4 ^- s# @9 w bE or F depicted in Fig.4 . In a ny case, prepar e feeder plates2 Z& s; |7 M9 F+ N. y& L
the day before splitting. If t he pluripotent outgrowth is( ^0 B( x; v8 Q! T; I% Q- [
isolated from the residual TE, a closed p ipette and a
4 X# \; f( T% i' d3 R( |) p5 Lscrapin g met hod could be more effective. If stem cells are
1 ?4 ?3 g/ Z* {; s( Tgrowing in clumps surro unded by TE, a n o pen pipette to cut5 i2 [6 ^% I; d5 w
some of them will be preferred. It is wise to leave some
! w5 p& ^8 d [" i) x9 j& Noutgrowth(s) behind, u nt il split cells ar e s uccessfully) K. V2 g) H# O* ~7 \0 X2 \
growing in the new feeder dish.* q+ K) m; ~# n- J
Notes
+ |) z5 Z8 ^9 F9 j QIsolatio n and culture of mous e embr yonic fibroblas ts and9 ?& C G# g5 V8 @% a2 h
feeder preparatio n/ C, v- ^- o( U# x% R
Note 1. We describe here the deriv ation of hESC line s in# c8 B$ x: y- f( I2 {
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" g+ d" M- m% ?) W6 F; I3 [
efficiency in stem cell deriv ation and lack of chro-mosome X inact ivation in hESC line s estab lished! y* |* |" Q& u: d/ v% V K' M/ s
under such condition s [19].
7 _: |, D/ e4 E+ X% w6 ]% h; VNote 2. Pr imary MEFs a re the first cells to be us ed as
0 Z# C( {. r6 `4 ~/ A% xfeeder layers for hESC culture. Alternatively,
8 k1 L7 N$ \: `either transfor med [ 20] or n aturally immortal-ised [ 21] M EFs, as well as human fibroblasts% |* C3 n1 C$ B. y+ Q
from several origins, includi ng foreskin [ 22]
5 T2 `$ G2 ?' S) q. F7 W8 ^9 l I7 \and placenta primary cells [8 ]andanimmor-talised placental f ibroblast cell l ine [23]have/ d1 q6 n0 b- w" O# f% r
be en u sed su cce ssfully as fee ders f or hESC1 u, k6 m4 a @! _2 \5 ~
deri vation and cul ture. Primary lines f rom hu-man f oreskin f ibroblasts (HFF-1) have al so. b1 c& i; _6 {% M( l" n0 {1 d( p( l
been used reproducibl y to cult ure h ESC li n es.. n, d: f/ F- U i' e# h- l! S) u
In the case o f HFF-1 cells, i rradi a tion i s the
# t/ U3 ~! z) i N) \( C4 Spref erred method of i nactivation. All m et hods
4 n" w9 }* M" W* h& {7 Kdescribed i n this paper are equally valid r e-gardless of the t ype of feeder s used.9 ]2 W5 `) J k0 f
Note 3. Mice stra in has proven to be an importan t facto r
( E, Z% g0 m( O. r5 dfo r th e q ua li ty o f th e fe ed e r ce lls d e riv ed fro m# V; @) }0 m3 |7 L; I& u; z
Fig. 5 Micrographs showing
4 |& ^! F( t. Z% |! u+ @+ Zevolution of the pluripotent and, _& |) X4 x4 o4 p3 H; Z/ S6 `. b
TE cells during a derivation
( b) i) X. Z/ S( C, G T; |experiment. a ICM 1 day after
8 E- |# h3 T! u1 p0 Eseeding. b ICM cell expansion
- U0 G6 E9 [) j# m; \1 I1 \after 4 days in culture. Arrow
) Z9 ^0 j! j G- ?4 y8 v. ipoints to the pluripotent) m& h+ N! l! f) ?1 z1 F
outgrowth, which looks like a
3 b7 i- r# f; U% M0 v; g4 I$ E" hclump at this stage; c colony
8 {& e) X* \) o! K* poutgrowth 2 days after splitting
- ~" c2 Y6 \) \8 i( ythe outgrowth in panel B: arrow6 L3 O3 q2 N) z5 A, b) Q
points to the expanding+ p# e' v* D4 [7 [
pluripotent cells out of the* V. \& r5 P X @ @4 X' [6 w
initial hESC clump; d residual& {# r# ]) e/ R" C" o3 K6 j9 D
TE cells 3 days after ICM) @7 ?* k- t& l2 d4 z
seeding; e TE differentiated* B. m# s8 l. {2 s2 L
cells 4 days after seeding; f TE
4 e9 S2 n. B7 M( j l. R" |( qcells detached from the plate
$ }7 ^3 [7 Y6 a" Z7 d- l6 {# U" J9 days after seeding; g detail of; O9 H; Q% l" }" H. q8 j
the emerging pluripotent colony+ k+ Y0 a' k( l8 ^- {
of picture C;h early hESC
! ^+ i. M# w: p- @# A+ hcolony at passage 4; i regular4 P* m$ ^6 ^( W# a4 E6 R
colony of an established line at
- L4 q: m- E ~5 rpassage six8 G4 ^6 R6 [" T( e, o
Stem Cell Rev and Rep
. s6 E r7 t- \# nthem . In our hands, embryos from the CF- 1 and
, L d; d& q3 [MF-1 line s, and from the cross between MF-1 and" _+ Q+ X3 X2 A0 P; o+ e
CD-1, are much better than inbre d CD-1 embr yos.
+ J! V* t0 Y% z2 i' p7 Z* lNote 4. Isolated pME Fs can be frozen at passag e zero, and: ~& L, s6 m6 K+ q; b& U5 `
expanded at a late r time to prepar e feeders . Nev-ertheless , the expansi on up to passag e tw o before
, ^& U' k+ f6 N$ Sfreezing has the advantage of increa sing the num-ber of cells obtained at passage four. It has to be
% m/ ~; R i8 g5 s. F, Y/ |' Tnoted that in the isolati on procedu re describ ed in
1 M) u! C$ e1 S' o% athe Meth o ds secti o n, an d d ifferentl y fro m oth er
6 B8 G6 l! `4 G: g+ T, H) U; }published protocols [ 24], tissue chunks are not
. R* z7 `4 B* {/ |& ^discarded before seedi ng after first trypsiniz ation.
! q/ M6 `7 G8 g1 Y- @# m: GThis procedu re yiel ds signi ficantly higher numbe r! N, ^9 W0 W7 z* C, h5 b
of cells. It is advis ed to culture chunk s a nd clumps; e0 e) G) G+ N6 x b$ H
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
6 h3 h- {: I& c! ~9 w" E. Tnormally be suffici ent to produce enough numbe r- F" y- N: X' f7 b0 F2 s% C* q
of vials for cult uring up to six new stem cell lines
4 L) g2 H; M- i) ]% E4 Oduring 1 year.
: n* w8 m1 ~7 D e* _: Q+ v) iNote 5. Finger vorte xing is a simple way to mix a solution
) c( E4 W# Y/ d# E8 _+ cor disag gregate a pellet in a test tube. Hold the top5 d7 n! F6 `4 w% D- v
of the tube secure ly in one hand and draw the ring,
! O/ n7 V2 F R- R. y' |middle and index finge r of the other hand sequen-tially towards you, tappi ng the tube. This creat es a
/ Z9 h8 s6 T+ ^1 G# v5 Ewh irlpo ol eff ect inside the t ube, w hic h can be0 L1 l) w" @& S
adjusted in inte nsity by speed. It is milder than! `# m' C/ j( Y2 V3 Q9 r
mechanical vortexing and yields healthier cells, P1 H# H! l$ s- Z' o$ g3 {
after centr ifugation .
( F+ T. i0 i, a& r2 GNote 6. Freez ing fibro blasts wi th a slow freezing met hod is& S' C( @8 [* R) n- F
not an issue , and they can be stor ed at − 80°C for, k3 F1 V) j* G' i9 d$ Q
months without a decreas e in subseq uent plating
, H* n0 R1 f3 o& @% |4 X5 uefficiency. The proto col described herei n uses the+ u4 C1 w2 D X' U' Z1 b6 _; C
slow rate freezi ng container Mr Frost y, which has
$ q* U" j6 {8 h/ N( X6 \been vali dated and used for nearly 20 y ears i n' k9 h( m3 G& ?2 @
mammal ian cell freezing [ 25]. It is a cheap and
8 H# ^* w6 z5 u0 I% \' F: Stime savin g o ption when compa red to alte rnative9 ]! c4 d/ {& C N8 w1 c
methods such as contr olled-rate freezing and vitri-fication, which may be suitable for more delicate$ e& S# d1 F0 a! _# j, w" A& m
materials like embr yos and hESCs .2 v! Y- s; A" [" i* @* ~
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
% ?' \4 [. h8 E# S: U9 g# jtoxi city and different iation during cultu re.1 g3 V& o+ o0 T5 | m. I- B- e
Note 8. MitomycinCis thepreferredmethodtoinac-t ivate M EFs, a nd detail ed st eps to p erform the
) {: G1 B& u+ s3 A3 Mprocedur e are included in the text. Alternative-ly, γ - or X-ray irradiation can be used. In a ny
: p. Q% i+ S1 \" e O$ }# q: P9 }case, t he hESC supporting capaci ty of each
- B5 r+ [1 f6 lnew batch of feeders has to be tested on- k( P/ s' [4 f% y+ j, }) J4 [* m
known hESC lines bef ore c onsider ing t he m
& O4 T$ u! h1 j& Dvalida ted f or use. Once the new batch has been
3 l- i( i+ z) Z" ^8 t* xvalida ted f or hESC cultur e, it ca n be used in9 ~ T8 S& L! z; v6 t
subsequent derivation e xperim ent s.
- \+ n3 c7 K# |& uEmbryo and ICM Cul ture
/ x0 E( q' W: x2 g4 o! |& kNote 9. The procedu res detai led here are based on the use
/ N/ W. \, ^1 J* _7 Sof high quali ty clin ical grade embr yos, but fre-quently, the quality of the embr yos donated for
/ e0 M: c. y/ n; ~research do es not correspon d to the standards
% V! K( X2 D% ~1 R4 ?$ ]' zpresen ted in Fig. 2 . If by day 6 of development ," E7 u. x+ \, I) ~% H2 ~" A
compa cted cell s as in Fig. 2e – e are not form ed,1 p0 c8 G) [2 W" x) _
extending embryo c ultu re i n complete HES. p% d1 Y0 O7 E
medium for 1– 2 additional days will selec tively
9 {( [7 A4 _5 \3 rfavour the growth of the ICM ver sus the TE.1 X4 F, u" R6 h# ]9 h# E8 P6 _
This appr oach is supported in the most recent% J+ Y& X" H- s/ F% U- {+ i
li terat ure [ 15].' {9 M9 l1 E8 m% x O5 k( t( S8 _
Note 10. Reg ar ding ICM isolation, s om e authors use a n
2 B+ h7 N- G g3 Qextended acid t yrode’s treatment to weaken6 A Q- W: {* S. T
the TE [26], thus facilitating the spreading
1 h; }8 B7 P5 ~- o2 \of blastocyst onto the feeders. H owever, in* d% z X5 g. |" n1 `# N
our hands, the efficiency of this method
+ L6 t1 D1 W6 P" _3 Ud 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 g7 r" H" Y' a% n+ [
the same AT t reatment to all embryos usually
3 X$ P( ?% M# W: @2 Kresults i n s evere damage to some of them.
" F% t( y+ S R3 G- d1 iTheref ore, fitting t he method for TE reduction
* W% t' @% k. v8 g9 X9 Nto the grade of each embryo (Table 2 )helps
2 N3 u" w/ ~9 }7 C- M4 @) Pmaxi mizing the r ecovery of viable cells f rom
3 M& d0 w4 E4 W4 g u- Tthe I CM. When TE is r el atively s mall com-pared t o the ICM, or when the ICM is not3 |. r' ]) J8 n* U/ t4 w% E
compact, a pipetting method (Method A) is
1 {9 F. w# t0 H7 Apreferable. Alternatively, if the TE is robust% `" Q. n( m: W$ ^, a1 F: S3 f' |' g
and t he ICM i s c om pact , mechanical cutting1 j3 |2 Q6 d) K& t0 y) N9 m
(Met hod B) s hould be the method of choice.& M _) y2 r. G' `7 l, {( J) y1 k
Speed during embryo denudation and I CM/ f- T0 T3 q4 B( |, Z! Y
isolat ion is criti cal, even if the m anipul ations
$ u2 i3 u! [3 R5 T; Y5 w" nare performed on a heated stage.
% g/ q" U4 V1 y" J( PNote 11. If healt hy, the first seeding of the cell s from the
3 |% o6 \7 Y. z5 sICMs will att ach i n 6 h t o ove rn igh t . In o ur
g" m, \ Y% d# uexperi ence, those clum ps needin g more time to
2 w2 P* E6 o0 P. O, ?8 ~attach are not robust enough to yiel d deriv ations.
3 ?! |' j q ?; zThe first outgr owth after the attachmen t of the
( [ a" u5 H7 q4 J' y, wclump can be seen from days 3 to 16. At this$ ~2 e9 _3 Z" j, |
point, the undifferentiated cells need the suppor t
9 W, B; M* h1 Y, eof fresh feeder layers to estab lish pluripot ent cell
G' i; @8 h7 v h1 m! z1 J3 Rgrowth. TE cell s evolve invar iably to form syn-cytial cells that invade the feeder layer and die3 ?# o( U6 E; T
leavin g g aps i n it. It is critical to have fresh2 ~' M& c% O( o7 G* h* @6 f1 y8 ?
feeders ready by the time the TE cells and deriv-atives detach from the plat e.5 R% |# H+ U" N( w: D0 k
Acknowledgments This protocol is the result of work funded by the! Y8 E+ K* m# k3 p- F7 [
North West Development Agency (NWDA) i n t he UK and the u0 f% j5 s! k4 }4 H; N( [2 H+ N
MICINN-PLE2009-0091, IPT-20011-1402-900000 and FPI-CAIB; g, J2 b# p7 H! V8 X& c6 V) L
Grant FPI10 grants in Spain.. i! a* e" u7 H' Q
Con flic t of in terest Th e aut hors decl ar e no pot enti al con fl icts o f
. f- Z; p* T! |0 b. ]% w& g1 Pinterest.* F9 ~% L7 v( L" D2 R8 \# H
Stem Cell Rev and Rep
+ }' ]3 v4 d- b+ k! x* GReferences9 l$ ]' v8 f) e; W2 F1 a% K
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