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Optimized Protocol for Deriva tion of Human Embry onic3 l3 J3 c0 `* Y/ T9 c9 J
Stem Cell Lines& `2 ] `9 ~# I4 g: H5 E! ?% L, e" k
María Vicenta Cam arasa & Víctor Miguel Galvez&" ?$ q; G0 ~+ d# W
Danie l Roy Brison & Dani el Bac hiller
& ?/ W0 [+ j. `7 e& ~7 Q: m#Springer Science+Business Media, LLC 2012% P( M# u+ c+ p9 i5 Q$ e7 `: n
Abstr act For t he pa st 12 ye ar s, th e b iolo gy an d a pplic atio ns
. n/ v3 C& K' m% A! E9 _of human embryonic stem cells (hESCs) have received great
5 ?# x9 q s5 }9 h1 V* Satte ntio n f rom t h e s cien tific comm unity. D e riva tive s of the first( m6 S3 w* m& A; L1 |# U$ J
hESC line obtained by J. Thomson ’s group (Science 282
# ~: g9 E$ ^9 o/ j(5391):1145 –11 4 7 , 19 98 ) have been used in clinical trials in# B7 a- \* @. ?: p/ j9 a
pa ti en ts with sp ina l co rd inju ry, an d othe r h ESC lin es ha ve' y7 _' K" ?" l3 y, \
no w b een u sed t o g en era te cell s f or use in tr eatin g blin dn ess
8 _ b! ?* W ~+ m( L a n ce t 3 7 9( 9 81 7) : 7 13 –72 0, 20 12 ). In addition to the classical8 \* N1 _9 Q+ N% K# x
pr ot oc ol b ase d on m ou se o r hu man f ee der l aye rs u si ng op en
2 e" s3 @% r+ w9 L+ ~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):1221–1227,0 Z. P) h8 e. @( Z, e! c4 j# H
2005 ; Nature Biotechnology 24(2):185 –187, 2006 ;Human
6 p( v( a7 H! {- z" ?8 ~, W3 a* ^Reproduction 21(2):503–511, 2006 ; Human Reproduction 20. k3 _0 G; ^6 ?- A) I3 g
(8):2201 –2206,2005 ; Fertility and Sterility 83(5):1517–1529,% w8 g1 ^7 Z& v# p
2005 ), novel hESC lines have been derived xeno-free (without
2 p8 U( e+ q( ^+ ]using animal derived reagents) (PLoS One 5 (4):1024–1026,
* r, e3 S3 u+ w- G o( R2010), feeder-free ( without supporting cell m onolayers)9 }- p8 n( C% R: Z9 [- w2 e* H
(Lancet 365(9471):1601 –1603, 2005 ), in microdrops under
% x7 ~0 b. U3 K# [+ Z% O! p( Doil (In Vitro Cellular & Developmental Biology - Animal 46) @/ B# K4 v+ v% U* T
(3 –4):236 –41,2010 ) and in suspension with ROCK inhibitor
$ u6 W8 m" W* t5 r I" p(Nature Biotechnology 28(4):361 –4, 2010 ). Regardless of the- p3 c$ f% m' D
culture system, successful hESC derivation usually requires
5 d. N) l5 f" H6 @- Goptimization 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. t. l" ]& j W W5 N9 g
hESC line derivation. Herein we address the crucial steps of
5 X9 T* a5 c' S" ^the hESC line derivation protocol, and provide tips to apply# w8 _" d) O: t/ V( {" Q
quality control to each step of the procedure.
$ ]& F( e# @4 \0 cKeyword sHuman embr yonic stem cell s. }2 E# }: c7 D6 E* q% s$ K1 n2 ]# y
.! K" O8 i$ E$ j s
Derivat ion* o% f1 X& E0 t# j5 z! N# l; _
.8 Z; S* q" p3 `
Blast ocyst! M/ B' b: w: w% F
.
; n p$ k. Y) x$ UCultur e o ptimizatio n
6 v: {7 X+ q8 l1 S5 ZIntroduction
" b1 j, I0 E% F7 K2 BTo da te, hundreds of human embryonic stem cell (hESC) line s
2 L/ }3 c" C6 K9 }; H4 |, D7 hhave been de rive d f rom surplus embryos after as sisted repro-duction techniques. Reported derivat ion e fficiencies range from, l3 w, V5 N$ q8 R* _
7 to 100 %. This wide span pr ob ably refl ects the di versit y of the* u( [3 d6 m/ I- J) e; X% |- s9 Y
methodologies involved. M any var iables of the procedure s uc h
4 l0 \+ [5 K# ^9 x$ T- E5 k0 \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)! I, |! E& a( ]& j
removal and inne r cell mass (ICM) isol at ion a nd type of feeder+ {- y5 y. p: ?% ?$ z* e
layer, ac count f or the d ifferences among laboratories. Neverthe-less, d espite the v ariabilit y, al l hESC li nes derived s hare growth
6 \- q9 K$ X, t3 }: sproperties 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
- ?: V/ J/ O9 z8 @4 Q2 e( I8 j' i/ tth e genetic stability of more than one hundred hESC lines4 E y; h2 b& \7 t t
using consensus protocols for karyotyping and genotyping.
( R* A, x v( I' gThis echoes the importance of defining robust and common
! s% n$ A% ~' B H- v. F2 _5 S9 Gcriteria for the assessment of both existing and newly derived
! q! z7 p' _: j: |: zcell lines, and for the validation of advances in culture con-ditions. [13].
4 G8 q1 x' k$ p3 Y- m) BM. V. Camarasa (*)
w2 n4 |( R; ^" P% p# U0 t:6 g1 Z K6 e. Q6 N5 p: ?
V. M. Galvez$ u8 p; E2 G3 H3 ^
:" l9 X" R$ W3 x2 I. }& Z) m
D. Bachiller
- N O5 t- d" f" uCaubet-Cimera Fundation, Centre for Advanced Respiratory/ b2 K4 N( ?# b8 ]0 J3 e; c
Medicine, Recinte Hospital Joan March,' S& T, V2 e: `; ^, p) d* m' S
Ctra Sóller km 12,
# l& ]4 C5 n) [07110 Bunyola, Illes Balears, Mallorca, Spain
9 T/ `& I# j# O0 y3 F% t8 ^e-mail: telomerasi@yahoo.es! [& G( p9 _3 u+ c) U" ^
M. V. Camarasa3 d8 Y, P# R5 ~& v
e-mail: mavi.camarasa@caubet-cime ra.es3 C s- ^& m" k# M m; H
D. R. Brison$ j1 y- ]8 i7 Y. g- R6 W& M1 [- q" V
North West Embryonic Stem Cell Centre, Faculty of Life Sciences,4 d! s x3 ]2 s& D5 K
Core Technology Facility, University of Manchester,, Q$ P5 h; a" F$ c7 ?
46 Grafton Street," ~* m$ O" I0 `% x) {" _
Manchester M13 9NT, UK" s: S$ Y4 l8 ^* Z
D. Bachiller
. S* S+ m* i: D1 O6 Y. V) ~' S! [Consejo Superior de Invest igaciones Científicas (CSIC),
$ F+ ?" L6 ~2 [$ yMallorca, Spain, t' X5 d w) ^/ E* B
Stem Cell Rev and Rep
; |8 Y9 D( y, E* t' I% L3 o1 N+ i2 LDOI 10.1007/s12015-012-9377-42 b+ J1 }" [* F. |: }0 {
This protocol focuses on t he main first steps of the- K% c0 [+ X6 a4 ]0 I
deriv ation of hESC lines, i.e. embryo culture and gradi ng,
* t: J; U5 U; A4 ?: R8 A% w- X3 ]and precise mani pulation of their ICMs and first outgr owths
7 W5 g8 N* ^3 K# v( v3 J! wof pluripot ent stem cell s. The Notes secti on includes alter-native procedu res as well as commenta ries and clarific ations
' A: C' O9 S8 v% t5 mon the techniques described in the Methods section. Assuming F2 j% A) C( H2 y' z; F0 G; p' G2 s
experience in mammalian basic tissue culture technique, this8 I; }! M0 }0 m2 k; Z
protocol should aid anyone wishing to derive and culture
- J1 L4 j7 P# q6 i3 D5 {* |hESCs to set up a rapid and reliable method. This protocol# n' {% H+ D I7 B1 A# r& q3 M0 |
reflects continuous work optimizing the derivation, culture
* v$ V; q& i) F# K( _0 Hand characterization of hESC lines over more than 10 years,1 i0 L4 N' L* v- c0 i, u x
comprising activities carried out in four different laboratories.
: z8 t2 J, ?0 ]2 Y# t3 @. Z) e/ nAdva ntages of the present p rotocol over others previous-ly pub lished in clude: optimiz ation of f eede r cell yield,; i0 m7 C' d' ?4 y6 C
increase of I CM cell production and adjustment of the0 l# H. y5 u3 S9 ] K, K
deriv ation protoc ol accordi ng to embry o grade.# r% Y; c; p( o2 t8 r7 c5 n# S# x
Feeder cell yield is optimised by culturing tissue aggregates) n1 w& N; @: u7 q
until they grow as monolayers. Embryo culture is optimized+ m8 H1 F5 L" s( n
so that growth of the pluripotent cell population is favoured
: C+ x L1 h8 Aover that of the non-pluripotent trophectoderm (TE) cells.
. k- t) A( w8 z9 |. {- K3 o% l" BFinally, the fire pulling of glass Pasteur pipettes to form thin. g4 N, F1 ]5 a' A0 a, L+ x
open and closed ends that match the size of the ICM or initial0 b5 S$ f; t1 R; _8 m
outgrowth to isolate, increase effectiveness of mechanical
- h- N9 a# Z5 N0 X3 a! Q9 r, Ysplitting techniques.1 P- W# }( S, A, a
Materi als
3 o8 a8 D3 i0 j/ n8 C& IMate rials a nd Rea gents/ ?7 g2 w2 p, X% J! |3 h
1. Bacteriological Petri dishes, 100 mm Ø, Fisher Scientific
6 Q, ~5 ^* s* J2 s) [#09-720-500.
/ M: n0 H5 F6 U2 ^2. Cen tre-wel l Organ Cultur e Di sh, 60 mm tissue culture% p4 r9 d6 Y0 l
treat ed, BD #353037.
# G2 g, {) i& T! c3. Lon g glass Pasteu r pipet tes, Fisher # 1367820C .
7 d3 k: s* u9 J$ ]/ F8 W( o4. Mr Frost y® Nalge ne, Sigm a #C1562-1E A.# O# \5 V+ X9 s
5. Se rological p ipette 1 ml, Cor ning #4485.8 C6 N& X& g! {
6. Se rological p ipette 5 ml, Cor ning #4487.4 P- N$ [$ Q; q
7. Se rological p ipette, 10 ml, Cor ning #4488.# j# f9 p, A7 ~1 X# [" O
8. Se rological p ipette, 25 ml, Cor ning #4489.
, @0 H' F) i1 t/ J2 R9. Se rological p ipette, 50 ml, Cor ning #4490.# V$ o* L h' v" Q! y' R
10. Tissu e culture dishes, 150 mm Ø, Cor ning #430599.& X% g' l4 M2 @0 q4 D7 S
11 . Tissue cult ure flas ks F1 00, 100 cm, a/ ^4 v7 t4 G8 \3 C* w0 H
2' N7 t) |4 U% G0 c! g N) R) u
Corning #3816 .
1 N4 c; j& t$ P* C/ u12. Tissu e culture plates, 4-well , BD #353654.
0 y5 C5 h0 y# x: l, ?: L: h, a13. Tissu e culture plates, 6-well , Cor ning #3516.2 `7 F+ w6 G0 m' g x2 m
14. Basic Fibrob last Gro wth Factor, #45103P- 100.
: \& g8 c4 E! P15. Beta-mercap toet anol, Sigm a #M-752 2.- h: E: U8 I5 l" W; c3 A7 V
16. Collagen ase IV, Invit rogen #17104 -019.4 z4 G( p x/ E
17. DMEM medi um, Lonza #12-614 F.
& [2 f$ ?2 e1 b1 u0 k18. DMSO, Sigma # D2650.: m" L% T1 Z# @3 F+ i( u
19 . Fe tal bov in e serum (FB S) , Australia n origin Lo nza8 `8 U7 `) ~1 @8 \9 Z
# 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; s1 n: A o) ?
origins a re US and US approved, followed by Au str a l i a n
- t* g& g# J2 ^5 {0 C$ B0 Nand Australian/USDA approved. Poor quality serum is a
1 R& Q0 B5 u; `7 `7 Z# z- rconfirmed cause of feeder cell failure in hESC support." p( h0 I; S: J& X5 f' h G
20. G1, Vitr olife #10128.
a9 J9 e% P1 a( }$ m21. G2, Vitr olife #10132.$ k& X( Z& y7 @1 M. _" e; L4 k
22. Gelatin, porci ne, Sigm a #G-1890. H' J# L2 ~! i- q+ R
23. Knockout (KO) DM EM, Invitrogen #10829- 018.
: x3 \8 g' `0 T- u* m24. Knockout serum replacement ( KO-SR), Invitrogen0 c3 u6 e/ @, Z" w. f7 J
#10828- 028.
1 g. D5 c$ W- B/ h, R' a' ]& A2 m25. L-Glutam ine, Lonz a #17-605 E.- z" l3 I: I2 s c6 @: a+ A P
26. MEM Non-essential aminoacids (NEAA), L onza #: j6 X v/ F2 f4 n: ?( M6 H
13-114E.! m( Q- A- t% H6 K
27. Mitomy cin C, Sigm a #M0503 . Hazard: this subst ance
" w5 A. N) F9 q- r' Iis very toxic and procedures for safe hand li ng and
' ^" v1 c7 |4 g0 Z$ C7 wdispos al need to be in place. Do not brea the dust.
U0 [7 R _$ i4 r! _6 ^Avoid contac t wi th eyes, on skin, on clot hing. Avoid
# y- j# `1 M/ @4 f2 ]7 T3 h7 Lprolonged or repeated exposur e.5 o. l0 z* v! N6 }
28. OVOIL, Vitrolife #10029.
2 l3 c% N" s5 }9 u! P29. PBS with Ca. @/ I# v( H( B7 g$ D3 d
+2
# Q* [' k! g( s9 S( [/Mg
O% ?& c: n9 p4 s+2! _( O: A" Q I6 R$ R
, Lonza #17-513 F.) K; h) c; X B
30. PBS, without Ca+ y) P0 `6 A+ q) E$ ?5 t2 u
+2( M. R2 m" E$ Z) e5 i7 i) S% S- u
/Mg
2 P% P1 R/ g' {+ k) Q+2
6 d, c7 Z1 W' W! M, a" A8 s, Lonza #BE17 -516F.9 \5 k0 \, l+ f) t' s! S! k) P/ i
31. P re g na nt MF- 1, C F-1 o r MF-1 × CD- 1 fe ma le s, 1 2. 5 –13.5
: i; D2 q4 d( pdp c, Charles River Laboratories.4 j/ \! V( z$ t
32. Thawkit I, Vitrolife #10067.
# e: b0 v# D: d/ [4 b8 f33. Trypsi n-EDTA, Cambre x # CC-5 012.- m% Q& W2 T' z; p
34. Tyrode ’s solut ion, acidic . Sigm a #T1788.
8 m8 N9 u6 q$ R- lSolut ions
7 ^4 H8 g6 N0 C3 \1. Complete DMEM medium: DM EM plus 10 % v/v FBS,
2 g5 D$ T9 h. S0 T0 o, l2 J1 % v/v L- Glutamine 200 mM, 1 % NEA A.
: z) Q# J4 k. Z9 ?7 |: Z- [2. Freezing medium: 90 % v/v FBS plus 10 % v/v DMSO.1 M1 n! J7 H, ?+ _7 g) k$ X3 j
3. Gelatin solution: 0,1 % w/v gelatin in tissue culture grade
& I5 w" T4 e" D& Bwater. To avoid contaminants and residues (detergent,
* r& D' P, R1 ~4 S0 X( M' q$ ctraces of other chemical, etc.), use a new 500 ml Pyrex
' b( X- |3 E) t abottle. Autoclave the gelatin solution and store it at RT.8 u- [) x+ W# L) Z" p# K" s, Q+ ^
4. HE S m ed ium: K O - DME M supp lemen ted w ith 20 % K O-S R,
1 Y% }& E9 ~* h6 o: u2 mM L -Glutamine, 1× N EAA, 50 mMβ-Mercaptoethanol
7 Q/ Q1 V0 u W* j2 ~" N8 ^and 8 ng/ ml human basic fibroblast growth fact or." O! H; N' x* ?* ]- C8 o2 O
Methods
# Y5 g5 K1 l4 M: RESCs are derived from preimplantation stage embryos. The
+ a' j& Q# V+ G+ G$ g" L$ F5 Umost common procedure involves culturing embryos to the9 [3 p0 e7 G7 K5 Q
blastocyst stage (day five to seven after fertilization), isolating
9 }( x" @9 }3 Etheir ICMs, and culturing the latter on mitotically inactivated' D2 c* ~/ l- r' n* \
fibroblast cell layers. The resulting pluripotent cell popula-tions are selected by morphological and growth criteria. Novel
% X: U) z9 {& ^4 r! F% u: dhESC lines are then established after continuous subculture of
! r9 ~4 y5 X7 K! k& B- fthe initial outgrowth. A hESC population can be considered to
/ c! ^, \: a5 ]1 n' ~be an established line when it reaches passage 8 with approx-imately 3 × 10
1 U$ W7 d' ^# K2 J1 I& f" _7
9 E& e, B" j: A% i; r3 J, W( xcells, as defined by the UK National Clinical! Z3 d4 A% i/ H) S8 @0 N
human Embryonic Stem Cell Forum [ 14].
7 N) U* V" c; O) U/ X3 RStem Cell Rev and Rep: u. ] j& G: t
After eight to ten co nsecutive p assages, the novel line is: L3 b) I: T) d7 } c% y
sufficiently expanded to check its viabi lity by freezi ng and( z- B& o0 y$ R
thawing .. ~2 }8 r! J1 c. ~/ w8 n/ E. u3 o
Ta b l e1 defines the term‘one v olume’ for each of the culture
' H9 p- L( U; ov es se l s d es cr ib ed t hr o ug ho u t t he manuscript. All incubations are6 N w& h' t9 V% \
performed in c ondition s o f 3 7° C, 95 % h um idity an d 5 % CO
: N" ^6 ^) e7 ?. \# M5 R1 r27 R+ J' T: m4 O- ~5 h$ f
inair(SeeNote1).Figure 1 depi ct s t he expect ed time schedule1 t, Q9 i' F8 [4 f0 a# s$ B
to complet e a hESC d erivation p ro toco l, provide d that t he feeder
9 E4 E& o# D+ bsource has b ee n p revi ou sly v alidated.
6 f; L& Y6 q0 ]0 DFeeder Layer Preparat ion: t% f' O, p1 j& t$ m
Primary cult ures of feeder cells have tended to be used for
$ @# {+ [0 e, ]' d- C# Qd 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
! H8 _/ `! K; f O# o# D+ @' Qderivation can be performed on f eeders produced from9 U/ J/ T9 @0 M. [
estab lished cell lines, or prim ary feeder cells whi ch have: v! L+ o( k$ J
been immort alised. Altern atives to the use of prim ary mous e8 S/ \- y* \3 v9 \; l4 H2 Q+ ]+ o* T: j
embr yonic fibr oblasts (pME Fs) as feeder layer s for hESC/ u6 j4 p% H+ d% s! w- m! R
culture are discussed in Not e 2.: Y- B& Q2 K k: V3 h2 v
Reg ardless of the type of feeders used, every feeder layer
- m c( v O1 q1 ~: ^% rmust be con ditioned before stem cells are seeded on top of
4 V! O r& F+ w+ x1 Ait. To do so, feeder cult ure medium is disca rded; feeders are. d) q* o( v `
then washed three times with one volume of PBS each time# a( A i. i8 b" L( l* ?
and once with half a volume of stem cell medium (HES).
" k' d; C4 P$ e. _/ SPlates are ready for use, from 30 ′ to several hours afterwards.' v/ i9 I3 ]1 |' d% ?! { B& [
MEF Isol ation
) E, I3 K& A! \! Q. Q0 q0 ?1. Sacrifice a pregnant female (12.5 –13.5 dpc) by cervical% O! D3 k- h4 K% _
dislocation. See Note 3 for tips on strain choice.
5 u6 I" R$ g5 m, p6 n6 ~, f+ ~6 V# o2. Di ssect out the uterine horns and place them into a; X8 K6 g* a2 _! k" q+ f0 @3 _) s
10 0 mm Ø Petri dish containing one volume of ster ile+ p. s2 c- A; u) t t- ?
PBS 1×. Transf er the plate to a laminar flow hood to
- w) A3 I" Q. K, Bco ntinue with the procedu re.# z4 n7 T8 T7 P- K
3. Cut open the whole length of the uteru s, so vite line
) }2 T. Z- U- C: bsacs with embr yos and placentas are partially relea sed.+ d: W# i$ r* d
4. S e parate each embryo from its p lace nt a a nd out of the$ ], }3 w( t1 k4 C) u
viteline sac and surrounding membranes, and transfer
% S1 b- h' T! F! M. E- B- R, hthem all to a ne w Petri dish with on e volume o f fresh PBS." `1 Q( {% O, Q2 i5 X* B" G
5. Wash the embryos and transfer them ag ain to a new
0 d1 M; _% s+ Q+ k gPe tri dish with one vo lume of fresh PBS.
+ ^/ R& [: ~8 g6. Decapitate the embryos and separate the visceral tissues
. u7 g( i1 f* [/ s$ e, A: w4 ofrom the bodies. Collect carcasses aside in the working7 |2 Z3 y. T' b" K4 }: K" z
plate. Aspirate and discard the PBS and the visceral
3 F) {( R# r- t/ ~, r) itissues carefully with a 10 ml serological pipette. Leave
1 U$ ^4 O5 b$ `; {the rest of the tissues in the plate.
# E5 S6 u! o6 y1 W& O5 [( A7 j7. Mince body walls into small pieces until a homogeneous# K; G7 J% [; Z& |4 L6 j
suspension is formed.; f: j# F$ W: z( r
8. Add 5 ml of trypsin-ED TA 1x to the plate and transfer- O- p+ q3 s9 c: d
all contents to a conica l 50 ml tube.2 z8 P8 k# Q6 C5 r
9. Incubat e diges tion at 37°C and 5 % CO8 L* w. A% @. S
2
3 c# [$ t4 Z) v7 t1 ufor 30 min.; G' H, i5 G/ A8 J7 A: Z
Hom ogeniz e the cell suspension by adding 45 ml of0 J k8 Y8 B. D" t
fresh cult ure medium and pipet ting up and down for
: Y( ?% I! t0 b' A" ^* `( Y8 Zten times, and cen trifuge to recover the cells (700 g for6 G0 ~2 I: Q& B$ `, o6 O( T4 I
2 min ).% H& W6 \; |, j& b
10. Discard supern atant, ad d 50 ml of fresh culture medi a
4 V8 ]' V2 T. D# Pand resus pend cells.4 C! z+ z" X+ S5 m, E) Y( I+ U% r
11 . Cen trifuge the cells at 700 g for 2 min and disca rd
/ O4 z! R* ^& R) o+ b) ]! Jsupern atant.' w- x, H4 V# O7 j1 l+ P% j: j) j
12. Resuspend t he cell s in 5 ml of complete D MEM
$ I. I D1 \0 Lmedium." T# Z4 P2 ]6 h1 k+ j9 R) f0 g
13. Seed cell suspen sion at a rate of three embryos per4 h& Y3 J+ \4 ?% Q) ], W& Y
F100 flask. (General ly four F100 will be set up at the8 B3 D( a5 [# B4 @0 y
be gi nn ing o f the procedure per dissec ted p regn ant
`* H4 i& e+ B2 Ifemale).% ^) i/ i$ I1 Q. v# [( j
14. Incubate at 37°C and 5 % CO
9 c$ {4 ?. J0 B: B2 I% ?& h1 F+ {1 ~2 S
unti l 90 % confl uence
/ C) f' I+ N0 @' R. H" V# m3 xis reached (approx imately two to 4 days).4 @7 d3 \/ i4 x0 n3 L5 m
15. Split the cells twice by tryps inisati on, seeding them
" D0 S; ?8 a$ b; K+ gonto n ew flasks/dish es be twee n 1:3 and 1:6 split
2 d8 _+ ^: \ S! i9 haccording to growth rate.
+ f9 z0 b! l: |After t wo passages , t he ce lls s hould be growing as
1 c6 A$ d* q0 C5 q: Z1 X2 Mmonolayers. Freeze 90 % of p assage 2 c el ls in vi als) ~" F. V/ p" v# z, S2 c. C) z
cont aining four to ten millions units each. The frozen
( P- U5 G8 K' {, ^7 E6 T! m2 vcells wi ll const it u te a s tock of act ive pMEFs for fut ure# ]+ I) |% A7 P6 k( Z {" w
use.
L, c+ q4 [+ k% c' [$ M+ STo continue with the production of inactivated feeder
5 H2 O; z7 m( u$ w- Z. `cells, expand the remaining 10 % of the original culture ^ `. D: d& l" S5 R7 D
up to pa ssa ge fou r. S ee d t he cel ls on to new f las ks/
9 b3 r3 `- d% e |plates bet ween 1 :3 and 1:6 spli t according to grow th
, l3 h3 f6 ]4 f r+ I9 Y Rrate each pa ssage. Then inac tiva te an d freeze passag e
) R! U+ m7 {1 |! U* [. hfour cells as explained below. See Note 4 for tips on
! k- p& U; {1 H! B% F/ nthese st eps.
+ _; e3 y2 d n. gGene ration of Batches of Frozen Feeder Cells by Mit omycin- o& ~, H% L/ g6 T, j- N+ G
C Inact ivation
- h$ w* i7 u3 c8 m9 J1. Tr eat 80 % confluent, exponentially growing,
" o h2 S6 ~! B! I! Cpa ssage four pMEF populat ions (between 48 and: g. t1 x7 y' C6 q
9 6 P150 plates), with 20 ml of 10 μg/ml Mitomycin C in6 S9 [4 E' m% h2 y
complete DMEM medi um, f or 2– 3 h at 37°C a nd
9 z# Y6 j% I( l! y5 q; o8 q5% CO2
3 Y/ U6 o, S& L( l.
6 [2 k+ F) f- l6 bTable 1 Definition of the vessels and volumes used throughout culture
; m( ]4 C; e# B/ _1 g8 L3 X9 Fprocedures
9 f$ C, k0 W8 z9 e6 }% [8 i8 w) TDish/plate Area cm5 Y8 {# p- q- t9 f, I2 H6 r1 I2 S
29 M/ H! `! {- k: I$ K
/well One Volume ml/well
/ {+ L9 N/ P; q2 S. W4-well plate 1.4 0,5
6 f0 W4 U) V+ w9 P i4 V) E24 well plate 2.0 1$ e) L T2 w6 e+ Z& } K+ C
One-well 60 mm plate 2. 9 1; a( v" B. ?4 m+ A S5 W! i: X5 }
6-well plate 9.5 33 d1 z" @1 v4 c, z0 g# J) K
60 mm Ø dish 21.3 50 }' k; B5 [, Y& Z+ s3 Z% T& l: m/ Y, W0 s
100 mm Ø dish 58.1 20& J8 L* i# _& x G5 w
F100 flask 100.0 25
; C- v+ [3 @2 s5 e150 mm Ø dish 176.7 50
3 Y+ n+ b: f+ K6 n/ |' cStem Cell Rev and Rep2 U0 \, _% |0 _$ q8 b, {
2. Aft er inactivation, disca rd the supern atant and wash
) U0 _! M. B- P; W- [/ Jcell m onolayers 3 times w ith on e volume o f PBS
5 H, |' X7 H( Iwi thout Ca8 ^( W1 m) c' |. i" v2 R
+2' n+ w. I% F4 s0 Z8 [
/Mg' L& T ~! Y" e% X/ z
+2
0 r9 x0 v K% T# x4 |6 J+ W.* D' D% t+ h3 l& j1 W+ ~; M
3. Trypsi nise the cell s (2 ml per F100 flask or 3 ml per" z: W9 s1 P; E8 H ?1 T5 z+ k
P1 50 p late) for 2 – 7 min at room temperat ure (RT) .& \! Q/ C/ [+ s3 {% `5 Y' Q8 O
4. Tap the sides of the flasks/dishes to dislodge the cells,
7 X4 l1 y6 g6 U" R! V0 ^an d harves t them into 50 ml conica l centr ifuge tubes .- J) f" E- _0 m/ |+ z
Po ol the contents of tw o to three P150s in one tube.' f5 n" F `- ^3 n+ F" W
5. Add 44 ml of compl ete medi um per tube.
' J/ E3 X( c+ L f. ?6 w% `* i6. Cen trifuge tubes at 700 g for 2 min .
. T+ [1 z D. H7. Br eak pellet manually by finger vortexing (see Note 5).( t7 g: s8 s# k6 a
Resuspend each pellet in 30 ml of complete DMEM and& L, V- _/ I" f' @
count in a Neubauer chamber.: O) z8 k# h e; G0 ~* _6 K6 i
8. Co unt v ia ble ce lls and calcu la te the numbe r of, ~- q* _( R( ?! B* e! K6 y
cryovi als needed, at a rate of 1– 4 million cells per vial.* t! q7 V# N% P( s* }, @
9. Cen trifuge cell suspen sions at 700 g durin g 2 min .5 d5 }1 Q, m) O/ g9 C7 V; U
10. Label cryovi als with batch numbe r, amoun t of cells
& C: Q( k3 p7 _4 W/ P1 M7 kand met hod of inact ivation, date and user ID.& w6 D9 v/ ]' a3 N) O& E# Q
11 . Discard s upernatants from s tep 9 above and break
* l+ E2 A8 o) dpell ets carefully by finger vortexing.! k- H3 J2 W' N, u
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 `; H7 K. n/ zDisp ense cell s uspension i nto labelled cryovials and) @$ c5 N* V. F+ ^, O. _( F
transfer them immedi ately to a precooled Mr Frost y® con-tainer, and then into an − 80°C freezer (see Note 6).
2 M( K; m+ P, X$ UFeede r Layer Plating for IC M Culture7 _7 g/ X" L# R, X: t8 D+ [
1. Dispense one volume of gelatin 0.1 % ont o the
+ {. K' e3 d; orequi red number of dishe s/wells, and incuba te them
+ ?( ^$ A. x$ n5 O8 ^2 Gat 37 °C for 30 min." X$ H* a' Z- H% g
2. Equ ilibrat e 10 ml of fresh compl ete DM EM medium,
& H# T0 s8 Y) |! q* M: ein a 15 ml conical tube, in the CO$ v! ~- N f1 V1 @9 i: y- D
22 N$ m$ ` \. M* Y3 ~
incuba tor per each$ p1 j- z% A5 G1 n; r
1 – 2 vial (s) to be thawe d (see Note 7).& I) j0 d" V! B; M
3. Once the coated dishe s and the medi a are equil ibrated,
8 O; R! J4 G7 |2 _/ A! x; K/ Qthaw the requi red number of cryovi als to plate 0.2– 0.3. \0 a3 A" g( Q8 k; @) u
mil lions of inactivat ed pMEFs per one-well 60 mm
8 T" ?4 c) `2 H# L( E+ ndish. Spray the frozen vials with 70 % ethano l. Roll9 r& c2 c6 {" B& C
them in your hands, until only the last smal l piece of
9 Y# { ?% \, _$ h2 E5 g0 ^$ y7 H4 \ice remains. Tr ansfer the c ontent of the vi als i nto
# _0 S) g; ^2 a7 C. {1 o' uthe equilibrated compl ete D MEM. In order t o
- z& D! S2 N* G$ o. X- `avoid cell dam age i t is n ot advi sed t o thaw m or e& y5 E; i+ G' g- f( ^
than four vials at a time.
I. F' e* L* l! s; D4. Cen trifuge the tubes at 700 g for 2 min . Discard the
" t; h8 j" F3 N# H H+ Ms upern atant a nd gently break the pellet b y f in ger
7 b# o, B. I# S7 t v) \ b! bvo rtexing.
7 B2 s# R Y/ M/ ~7 @4 _3 G5. In order to elimin ate resi dual DM SO, repeat step 4.( \# s- g. N% K" x* h6 u
6. Dur ing the second centr ifuga tion, aspir ate the gelatin& g& k% b2 D* M( b) P
solut ion from the plate(s) and dispe nse half a vo lume6 v+ ?4 M7 V$ S# A; f/ S: b
of prew armed compl ete DM EM medium on each one.
Z; F) R" t9 V7 W2 C% ~% e7. Br eak pellets from step 5 above by finge r vortexing,
- J9 ]" _( @( B: S3 ]2 w; Y7 man d resus pend them in half a volum e of the wel l(s) to
* t9 d# u! p9 N: E6 jplat e.
' O4 B( _5 r% j. w+ f: F8. Di spense cells dropw ise onto the medium- containing* @, N' C6 H* `4 b7 R% [- T
wel l(s).7 m! ]- m" c% D) h D- H) @
9. Place plate(s) at 5 % CO
5 X: E4 v' Z3 w0 W7 j2: ^0 m! a5 }% Z- L3 y) I% H/ F) C
at 37°C, and c arefully shake
& D: \: `! I& L3 Q+ v# Othem horizontal ly in all direction s to distrib ute the cells
, S' y3 L* i" }ev enly.( ]- n2 }5 p" D8 l5 C4 ]5 I3 Y
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' k+ f6 v5 [. H' g4 D6 D
attach. Optimal feeder density corresponds to a conflu-ent monolayer; higher and lower densities will result0 Y9 q* C2 k' J. l/ l4 z
either in reduced hESC growth and increased differenti-ation, respectively.
( z' C$ ]: `8 {, D: E3 x& i3 bSe e Not e 8 for comments on feeder layer prepar ation.
, y" C) p5 g3 B% a, XEmbryo Culture+ T" A) D. ~) X( f6 Y
Surplu s embryos from clin ical in vitro fertilizati on (IVF)7 U$ f$ ^% ~+ F8 p0 g4 O$ |- b, ^, y
procedu res can be supplied, fresh or frozen , at any stage9 M0 l' N1 A0 Q u' s* y1 o8 E# 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 lines
+ U! s( E; z5 v k+ lsucces sfully [ 15, 16]. The refor e, the derivatio n of stem cell
/ b4 m9 V4 ?4 p: f$ \lines is almost guaranteed for a ny Re search Pr ogramme
% ^% p# | Z1 Z- ^estab lished in coll aboration with an IVF clin ical depart ment." m5 z9 L/ E2 z
In this section of the proto col we will detai l the steps0 m5 \0 k8 W% |; K
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; y+ ~! O' z P9 I0 A6 f. i( i
that are used in the clinical procedu res. Altern ativ ely, it is/ g t! H3 U9 P: T* e: v* o
sim ple to p repa re the m from thei r compo nents [ 17]. Se e& F( t+ [) w/ A8 `# G
Note 9 for tips on embr yo cult ure.4 O- a) [) W: [/ Y2 N1 T% b" _
We will detail Vitrolife® protocols, and advi se to fol low- f9 f( ]. p, l m" k+ o5 t
manufacturer instructions in ca se of different brand reagents.
( s D' l5 o$ W, OThe cul ture system us ed was a two-stage one, i .e. changing( N: z% [2 \: v2 Q) t. }
cul tur e m edia formulati on on day 3 or s ix-eight cell stage o f
0 p2 @6 ?, C3 O. V/ b7 rdevelopment (from G1 to G2 series ), un til blastocyst formation .: H* {% R) X- Y5 M& O
Fig. 1 Time schedule for the3 G6 E4 @# M4 h" Q& R. m4 q
completion of a full derivation
) G w ]; X3 _6 |9 w# ?experiment
- {( z: z' Y8 I% g( |/ V& BStem Cell Rev and Rep
$ @! l# e3 M' r& e2 a2 HEmbryos are cultured in m icrodrops under oil, to5 i a9 g+ N7 ~: h5 \* Z: g
prevent evaporation a nd to minimise pH changes. The
& e* t# L! N: ffollowing protocol corresponds to t he culture of fresh or
8 c5 B8 f. e4 W2 o. U" Hthawed cl eavage-stage embr yos. In the ca se of receiving
/ q5 r+ L& _% @& ~2 |; H+ Ufrozen embryos, it is crucial that the thaw ing i s per-formed in the reagents corresponding to their freezing.; V% V9 r. h# W M/ ]
Follow manual instructions from the appropriate thaw-ing kit. After thaw ing, follow the protocol below for. r. F" b: d; k( q
embryo culture.
1 h! D7 z S' d* F8 B4 c. P1. Equilibrat e OVOIL® was hed wi th 1:10 volume of G1 or
8 f) I0 R) T9 ]G2 for 6 h to overni ght at 37°C and 5 % CO2
x+ ^- U q$ y5 N* j.# |) o8 o2 G1 l+ I3 F+ c" e9 u
2. Equilibrat e G1 or G2 medi a as required (see Tab le 2 ) for
1 b' k; G& ~, w6 L8 L& ? Oa maxi mum of 3 h.
+ ~0 n, V7 i5 J& ]$ V" H3. To set up embr yo cult ure plates, pipet te 25μ l of the
- N8 V4 i. `8 |& u/ g9 \, K* _: pequilibra ted G1 or G2 media per embr yo on embr yo
# k, K2 G, y5 n% Lculture d ishes/plates . Cover the drop(s) with equil ibrat-ed OVO IL®, and add 25 μ l more of the medium to the. Z8 p+ `* b0 Y) x }% x
formed drop(s).! r. S$ b4 `4 ^# t
4. Assess embryo development daily (see Fig. 2 for a3 P. w x) F8 a$ `0 k$ @
di agram of g rowth pattern and c orresponding images
% \6 i; t; J: _& ]. @of embryo development), and transfer them to fresh1 A! X5 A* i: v, {- y2 l
media d rops every secon d day (see Ta ble 2 for2 a1 H! V) D8 p- B. v/ k/ X
media c hange guideline). P re pare new drops as
' Y6 B5 S& m3 P8 _describedinsteps 1to 3above.If embryoculture" k+ Z7 h# V. q n1 S, f
star ted i n G1, remember that once embryos r each5 m" k/ A4 I3 _' h
da y 3 o r the six-eigh t-cell stage, they must be
( _5 P, |% c; Z: qcultured in G2 medium.
8 a! v2 @" y; T5. When embr yos reach da y six of develo pment , or the% H% v2 M8 J% q" ^ p4 n
ICM of the blast ocyst is visible, it is time to set up the
8 Z0 p$ z# z0 v: R2 H) m) _feeder plat es to seed the former.8 ]0 Z& p! x' h
ICM Isolatio n an d Se eding! k+ c' C* a( q* @) B$ E2 g: k
During denudation and ICM isolation i t is a dvised to% }! F, K, ?# w6 n
process each em bryo indivi dually. Pulled glass Pasteur$ `8 b$ k! Q5 P/ R9 }
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) B( b! g( I4 E( n
IVF p rocedur es.
7 }& _' b `; r7 V: O( _Con dition the feeders before the d enudation as follows:
4 L& d% h! s" p% jbetween 1 and 3 h before the seeding e liminate f eeder: t7 w. n. r9 u
mediu m. Wash three times wi th 1 ml of PBS with Ca
* y+ n9 v3 I7 |( x9 [2 S+24 U( K( P, e( P% W+ s7 t' P9 k& }" p# T
/
9 ~% @1 B3 B6 H; B7 E8 M- \Mg
( d5 G5 ~ \$ h8 M- C H+23 p! S8 B# ]* U& U6 \0 K* f0 T
and once with 0.25 ml of HES medium. Add 0.5 ml of( F) X( p' o( j4 i
HES medium and equilibra te for at least 20 min at 37°C and4 o% T+ }3 C% h) `$ \
5%CO
( a2 \4 }; X" s% Q' X Z2' y N7 [& V2 w- b( e& I
. At this point the new feeders are ready to recei ve! R& {; \! o) m9 M/ [
the IC M.
. \* @: u0 Z' {# G2 a( x3 p. F; T1 dZona Pelluc ida Remova l
0 R) E4 V; s$ a% @1 k. P1 d0 B) L1. Equ ilibrat e separa tely ac id tyrod e’s (AT) 1×, G2 me-dium and HES medium at 37°C and 5 % CO
/ ]/ `1 b' x% h+ q N; `# \28 B0 X8 y: R6 { N" P9 y5 X% A D
, for a* Q9 h; S8 U# E/ Y$ A4 c* B
min imum of 20 min and a maximum of 6 h.
/ s% Q3 d) A( T) ?/ R& Q" ^2. Bef ore any mani pulation is perfor med, images of the
8 j! w/ f8 w7 v3 ~' S1 jblast ocysts at different planes (×40) should be taken
3 w) @- [% L3 Q5 ~; jfor further asses smen t of its grade. Afterw ards return it
3 W, v+ R8 E% Y" q$ ~to the incuba tor.3 h' C; y9 i/ }
3. Pu ll 2 long ster ile glass Pasteur pipet tes wi th the aid of; F: Z" H( E, v1 I4 Y7 L. e' l
a Mecker or Bunsen burner. Leave one of them opened
- n6 c, w3 \5 ^4 p9 O7 ]! Y5 yan d rounded , and close the other one leaving a small0 n9 w0 c( h- m3 X! b
rounded ball at the tip (Fig.4 ). Check the thickness of
: F7 H$ L3 q0 l! }) a% z+ B+ {the tips under the s tereomicroscope and r epeat the
$ v4 U4 N) W. I ], h. aprocedure unt il a couple of pipettes whose sect ion
" n; n4 [$ l1 I2 [' T0 cmat ch the diam eter of the blastocyst are produce d.
7 {2 n& s+ \/ H, k- `4. Se t three drops of AT forming a row in a 60 mm tissue
" o: C+ @& Y' r; }& i: lcu lture dish, an d mark thei r position at the rim of the* r4 j5 [! ]" S/ I
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
7 Y" C0 Y3 ^7 B) }6 Osecond row at the middle of the dish, and three drops
6 c4 ]8 }0 w" ^6 G. kof HES media at the bott om, again aligned in a row
4 @1 T0 w" A1 ^0 H: _5 k6 l+ ?0 r(S ee Fig. 3 for drop distrib ution).
5 G8 A$ n) q- k; u5. Se t anothe r three drops of approximat ely 100 μ lof2 N9 M+ N0 P( Z/ y; Y$ a) g
HES medi um in a new 60 mm dish.; E* f d. @0 J9 T& N7 Y
6. PicktheembryoupinG2 mediumwitha 275 μ m
, ^' ^2 t ^2 C4 f- O% [capil lary mi cropipette, a nd t ransfer it to the left AT
+ e! n$ Y" _+ L+ r6 C3 Z0 i4 rdrop of the plate prepared in step 5 a bove. Stop* ?7 g# t1 D( ]( {1 k
dispensing G2 medium into the AT drop a s soon6 q' t- O5 B9 ]5 G
as the embryo has been releas ed. Empty the pipette
, H$ @0 y6 M! V: w/ ?in the border of t he dish to discard r esidual G2* i2 |" t! }" [2 f
medi um.6 E& N8 C( i& |
7. Wash the pipette twice in the second AT drop.9 s3 y1 Q, M) }' t/ R
8. Return to the fi rst drop, pi ck u p the embryo and
7 ?: m# [! U- h6 ]+ N- Z5 K" ?trans fer it to the third d rop. Monitor unde r the micro-scope the dissolution of the ZP (0 – 2 min).9 _+ Y( m8 Y( ]' V% e$ f
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
' z! x: F: e% T. a- kpre-w ash, dispe nsing the minim um amoun t of AT into8 N% m+ G! Y m! x5 F; g% {1 x
the G2, the second to load the pipet te with fresh G2 r) s% s6 V" t9 Q
medi um, and the third to was h the blastocyst .% n: a- Z1 l1 D$ {
10. Tr ansfer the zona-free (denuded) blastocyst to the
" g6 L5 |6 T5 E- \ tdrops of the HES medi a using the washing procedu re9 K3 c2 P u$ E( V' s5 z; B2 T0 S
as described above for G2.
8 ]/ Y+ o9 l5 x, l( m11 . Immedi ately trans fer the blas tocyst to the drops of
5 Z4 U% h, F" H+ [" J0 dthe HES medi a prepared i n a sepa rate plate in s tep) M2 L: T1 f2 g9 O T: g
2. Follow the s ame procedure of trans ferring to the7 L, g6 A1 \/ d: ^& Q7 K% l# {2 j2 U
left drop, washing t he pipette and loading i t in the
' P4 M% m0 z8 I0 W# h$ k7 Nsecond intermediate drop and transferring the
6 f6 W3 o7 }6 q3 q" Z2 E$ lembryo t o the third drop. P l a ce the p lat e i n to the
/ a" I) ?9 k; {* N% F' B& _CO
' C2 u& A1 G, `2 W% k/ W2
! d7 h7 p3 q" A0 k, U) C( `incubator.% T c9 X* D5 N5 v+ h; r- _
12. Usin g the ima ges take n in step 2 above, asses s the
2 q& _5 m5 E( K cderiv ation grade of the denuded blastoc yst (see em-bryo grading s cheme in r eference [18]) and a ppl y( |+ V6 a* f) W+ L
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
" E0 }. {7 a$ w; f# QB be l o w. S e e No te 10 fo r c om me nt s on t h es e
4 X8 w( n8 }! uprocedu res.
* N8 ?3 }! d* K4 `: Q! HStem Cell Rev and Rep; k+ g* i6 ]9 L9 ]5 j; M6 F
ICM Seedin g by Pipett ing (A)2 `- _% J: z: E: g% K- E- H8 N* ?
1. Choose an open pulled long Pasteur pipette of the same
/ L) A+ n: e( I% bdiameter as the denuded blastocyst (see shape in Fig. 4a ).0 J# U: C6 Z7 [" U! C7 W1 B
2. Load half of the tip with fresh war m HES medi um from
$ }/ v. L: [5 o6 Z' W) ^the drops set up to equil ibrate before denudat ion (ste p 5. j* E/ I% D' E9 x& H8 p# Q; o$ S2 I4 U) ]
on zona pell ucida remo val proto col).2 S) Q) N# m8 R! N
3. Aspirate the denuded embr yo and pipette it in and out: F8 C- N* ]. ?4 ~3 Q9 g% t
while checki ng for loose ning of the TE cells.
) o: M& ~3 ]" r7 O: e4. Aspirate the i solated cell clumps and dispense them( L: {0 @% @1 e% @: W; Y
separa tely on the feeder plate. d, m9 d$ ~+ m. f/ ~
5. Ta ke an image of the seeded fragments of the bl as-tocyst. Pay attention to l ocate the ICM and record
# u# c+ s/ c, u! Q$ w* uits posi ti on.$ {$ Y' @5 D: ^, }* M/ r
Table 2 Embryo culture" X6 v9 W2 B4 @: r& D
protocol
5 A$ e" a# y2 T5 V- d3 Y& PDay of development Stage Medium Image recording Media change
# N, }8 s" e+ b8 J: U0 g3 g1 Zygote (2pn) G1 − G1
* p3 ]/ l+ {0 t# o% q2 a! a22– 4 cells G1 Yes −% R* \$ s" F" h6 B9 b
36– 8 cells G2 Yes G1 to G2
. h, V5 V( J/ ]6 A! O4 Compacting morula G2 Yes −2 t, y4 Y! o( n2 T+ H
5 Expanding G2 − Fresh G2 M3 M" K) i# ]# z
6 Blastocyst G2 Yes −
+ {* f( r2 W! j* ]7 I7 Hatching G2 Yes −$ U. D. B; q1 y. d/ |) A* O& E" `
Fig. 2 Dagram of early human& F9 X' x4 e5 E; Y8 `- ~$ g6 [
embryo development. a-a’6 P% N6 A0 [7 h. F: }/ C
two-cell stage embryo, 1 day) ?) m) M1 X" S6 R6 N* t
after fertilization; b-b’ four-cell8 e4 Q& ~6 f: ?0 H. ]
stage embryo, 2 days after1 Y& o8 I( ~2 \4 K. J
fertilization; c-c’ eight-cell; O2 f8 k) x2 ?
stage embryo, 3 days after f
7 h' Z4 Y$ c$ I* Q, M9 ^, Jertilization; d e a rl y e xp an di ng
$ A& i% G, V e( T% x: a6 Jblastocyst;d’ morula containing
X& ?5 X9 p/ s0 h$ R6 cm o re th an 1 6 ce ll s, 4 d ay s a f t er0 w- ~0 E0 [2 L% w- i
fertilization; e early blastocyst,
% ?3 L. z4 f( Q5 days after fertilization; f$ U, p$ {( I! G& M; n% i
hatching blastocyst, around
2 E% v4 c% Q- l8 ?/ P* Z3 }8 |: ]7 days after fertilization. ZP:, A2 g( C1 \8 l% O* I9 Q
Zona Pellucida. ICM: Inner% a* b4 h7 l1 C
Cell Mass. TE:
/ N9 `; V! A9 ~3 C& Y: `& e% UTrophectoderm/ r4 [# V/ p b
Stem Cell Rev and Rep
7 v& [ ?3 {# p# Q& q6. Tr ansfer the plate to the incubator, avoiding abrupt
, n- b8 {/ A5 h* r# b+ dmovements t hat may separate the s eeded cells from* z' b5 O# U2 n I: B
the feeders.
$ @# ~0 N, |% l+ d0 v4 D1 M4 n8 y7. Inspect the plat es daily. Cha nge the medium every other
5 j3 k. H* [$ n5 S, H0 Nday.. } y( D5 L3 c- O f
8. Prepare new feeder cell plates as soon as pluripo tent7 [+ `# x4 b% @/ Q' T; e- Z: `3 T
growth is detected (see Not e 11 for deriva tion timelines
) b$ M5 L+ N! ^0 h; D3 Mand Fig. 5 for ima ges on deriv ation progre ss)., u" n, z0 i' [; g( A9 ]* {
9. Split part of the emerg ing pluripotent colony when+ g8 }: H& f0 p6 S$ c0 ?
resi dual TE cells begin detach ing from the bottom of6 W: I* M$ K8 U6 G7 Z
the plate. Follo w Secti on 4 below to do so.
( m) ~) e* P( D( }+ U; hICM Seedin g by Mechan ical Cut ting (B)4 @7 Q) f5 v# n8 e) k
1. Aspira te the denuded blastocyst w ith a pulled open, o5 B# [; J* F
pipette (Fig. 4b) and dispe nse drops of approxi mately& r) k% [6 n" A- f& [7 k
20μ l on the dry area of the 60 mm dish containing fresh) a2 N, Z1 S6 S6 j# q- F
HES drops, until the blasto cyst is dispe nsed in one of
* B/ f. K4 @1 Q6 N# U, nthem.- n: l+ ` y; A: p3 D2 l, R. d
2. Drag the denuded blastocy st across the dish, away from' \0 D, i; W% U) p6 q' B0 X( F
the HES drops. The volume of the embr yo-conta ining# U: i9 t% R3 t, S2 U
drop will diminish up to the point that the embryo
3 N1 L! R A t, p& Bflattens and sticks to the plastic. At this point you shoul d
* W" s' W* W4 N, Ybe able to locat e the IC M: it is much brigh ter and less
9 M) f) A0 A( ~/ I0 p, R( y& hflat tened than the dim TE. Sa ve the pipett e without
5 T. G- f& i" _( B+ e, j' u! iemptying it for follow ing cutt ing steps .) g: L* }9 L, E, {
3. Pick a close d pulled (hold ing) pipette (Fig.4c ) with the
) F' h3 g2 Y6 A8 }% B7 P: M% Oleft hand (rig ht for left-hande d) and hold TE by pressing7 N% @" o' I. B+ U, @
its border agains t the plate.
9 y" |! H, w# }" V8 w4. Pick the open (cutting) pipet te still contain ing medium
$ S1 I% G( k5 Z6 Cwith the right hand (left for left-hande d). With its edge,& S/ g3 c) S& W" O; r! P5 J) Y
cut out as much transparent TE as possible. Imm ediately/ ?! K- d: D5 A+ Y2 C% ^4 L
release the HES medi um to a void damag ing the ICM.8 s2 c8 C6 |" \+ A
5. Scrape loose the ICM contai ning piece, which will be
7 c3 ?8 ?4 p, g; Badhered t o t he plastic. M onito r the ICM w hile it: V" S3 K( w' U' u9 W
Fig. 3 Shapes of long glass pipette tips for ICM and stem cell colony
8 I/ ?! {8 U4 Q6 \2 Xprocessing. a transferring pipette with open rounded end; b cutting
% j) ]; R; ]) B# G# g7 b8 apipette with open bevelled end; c holding pipette with a fine rounded
. n3 Q* `( f7 j ~+ x2 Bclosed end; d curved hook with closed end for outgrowth scraping;e-g" h0 Y: E2 g6 w- [. w2 b6 |6 i2 ?# q
flat hooks for colony cutting and scraping. h detail of the tip of the0 p; S7 a N2 i
holding pipette depicted in C# `3 x+ Z: m! C, O
Table 3 Choice of method for ICM isolation according to blastocyst' E' K& S; p! ? _4 ~/ `8 x! F2 a3 W
grade
q# @6 j' ~7 i6 [5 j9 ]! WICM01231 a5 g c. N1 F2 Q: H! P
TE
& y' a# }& W% Z4 e& y. H0AA 1 A A A/B! f O9 \8 d' h
2ABB 3 ABBB
/ F) s n, i/ NTE score - 0: no cell layer formed. 1: few big cells forming a loose
# E+ k S0 D7 d4 G/ l% Llayer. 2: cells forming a medium coherent layer. 3: many cells in a tight% Z4 E; W8 B/ C3 e
cellular layer. ICM score - 0: no visible inner cell mass, inner to the TE- K" g9 q: z E, I7 C, i5 B, K4 m
in any plane of the blastocyst. 1: ICM with less than 10 compacted
7 b1 |. b7 v7 w k* Ocells, or loosened mass with up to 20 cells in the central focused plane
1 X% P5 y T, D+ Y+ r; M. lof the blastocyst. 2: ICM with more than 10 compacted cells, or more1 I6 U, z& Z4 Z0 A. R
than 20 loosened cells in the central focused plane of the blastocyst6 r( a1 W; s9 \" |% l
under the stereomicroscope. 3: ICM with more than 20 compacted
/ C3 e7 D9 K2 w3 v5 p+ vcells, or more than 25 loosened cells in the central focused plane of the% p/ o l( C: X) s
blastocyst. Method A refers to pipetting for ICM isolation. Method B# I$ _7 w1 @. ~( d- b8 `
consists of mechanical cutting of the ICM6 W. j4 |8 Q/ ~1 n
Fig. 4 Derivation plate. Embryos are dispensed in AT in drop 1, and1 ^; F) o; y& O9 P6 N1 }3 `7 i
tr an sf e rr ed s er i al l y ac c or d in g to t he fl o w o f th e ar ro w s. AT: Ac id: k8 a# q0 O/ D$ R# |* k) G
Tyrodes. G2: embryo culture medium. HES: Human Embryonic Stem
7 g4 [; u) r3 ^/ @cell medium
! u# H0 a7 c) {: N6 oStem Cell Rev and Rep" J# ~ _: T, C5 Y: V
detaches and float s. Qui ckly aspir ate medium from the3 E( H4 B/ z4 D. K0 |. 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 evaporation
* l1 ]) K0 R% p G" doccurred durin g cutting.
3 A! s" @; ^! Q1 [% R( j/ {. X6. Aspirate the floating piece( s) and spread them onto the
; [- }0 C7 o6 b7 Qfeeder dish. Try to avoid pure TE fragments , recogni zed( R- R# p3 N/ {; L
by thei r trans parency.
1 o, |8 x2 u2 V7. Take an ima ge of the fragments seeded, record ing the
# J4 i! y! `+ t5 T6 r' ]/ oposition of the IC M piece on the dish. ~; v4 B$ `# I: ^. {- R, |
8. Inspect plates dail y, feedi ng them each second day until
L: Y$ l' E0 i% Xpluripot ent cell grow th is obv ious. r3 L1 _* u) ^4 q
9. Prepare new feeder cell plates as soon as pluripo tent
$ p2 w- n% t0 P' f. Z) Igrowth is detected (see Not e 11 for deriva tion timelines' I p! b. I3 T5 \. t3 G+ P
and Fig. 5 for images on deriv ation progre ss). Go to5 H, P* k9 d) r2 x0 V
Section 4 below for the firs t spli t of the pluripot ent cells.
1 `* I0 e# l- n- k$ z fSplit of First Pluripoten t Out growth
" K( N( G* I& n r2 h; x& k% j& cWhen the pluripot ent outgr owth(s) reach the stage de picted
5 b8 D/ h- J! Nin Fig. 5c , they are ready to b e split. Mechan ical cutting can
4 Q5 V! N/ r- _( [# F5 nbe perfor med wi th a glass pipette of any of the shapes A, D,3 g q9 _% ?( w( u4 F$ R/ a
E or F depicted in Fig.4 . In a ny case, prepar e feeder plates
" O/ L9 \$ d5 c" ?the day before splitting. If t he pluripotent outgrowth is
* T* O$ y3 U: @# M Oisolated from the residual TE, a closed p ipette and a
. C; F. ]2 X0 n0 o$ |scrapin g met hod could be more effective. If stem cells are, ?* Q0 a f! w) f! G* ?5 E
growing in clumps surro unded by TE, a n o pen pipette to cut) S) U8 ]0 }: z3 J
some of them will be preferred. It is wise to leave some( c& B2 [- s3 b/ H% M4 u6 k7 `
outgrowth(s) behind, u nt il split cells ar e s uccessfully4 v# D0 o9 ?2 x. P1 ~- ^' d4 }8 h. a
growing in the new feeder dish.
% Q* I% D# T- s6 V! qNotes, i1 _$ z, _$ j8 ~
Isolatio n and culture of mous e embr yonic fibroblas ts and
1 [* K* \' m7 j+ y) c6 }9 `6 K9 h' lfeeder preparatio n
9 H( n, d& ?) c$ I7 k& P4 p# B$ [Note 1. We describe here the deriv ation of hESC line s in5 i! ]; }& ]0 u4 m$ x% d% P0 G
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
1 @4 w, l$ y5 u- c8 mefficiency in stem cell deriv ation and lack of chro-mosome X inact ivation in hESC line s estab lished0 P% }9 P& v/ L4 L4 ]* Y
under such condition s [19].
! m. _3 G" D6 D- `1 mNote 2. Pr imary MEFs a re the first cells to be us ed as
, L! V3 i/ \' s3 P& d6 i; b' zfeeder layers for hESC culture. Alternatively,3 a0 c: W8 L) m4 C4 O% h. u
either transfor med [ 20] or n aturally immortal-ised [ 21] M EFs, as well as human fibroblasts
' C" X5 x2 x: Q7 vfrom several origins, includi ng foreskin [ 22]
' U* v) s. K" B$ ~- i2 s6 n7 Jand placenta primary cells [8 ]andanimmor-talised placental f ibroblast cell l ine [23]have
$ _! @' @* q. N( V4 a* n! ybe en u sed su cce ssfully as fee ders f or hESC
/ I6 z( b9 ]7 X. [+ [; yderi vation and cul ture. Primary lines f rom hu-man f oreskin f ibroblasts (HFF-1) have al so
9 ]7 e2 l; U: n4 W; Zbeen used reproducibl y to cult ure h ESC li n es.
% T, U/ o: H; U3 S" A0 g* iIn the case o f HFF-1 cells, i rradi a tion i s the
( [8 d+ E0 W* W7 z& ~pref erred method of i nactivation. All m et hods
6 k3 X; [& V( c% S: @, qdescribed i n this paper are equally valid r e-gardless of the t ype of feeder s used.* O/ M" Q- p. F/ _$ {# `& O. Y$ j
Note 3. Mice stra in has proven to be an importan t facto r
# e y7 S1 s c+ `8 V: R( Nfo r th e q ua li ty o f th e fe ed e r ce lls d e riv ed fro m n9 f4 [ B, b# A$ H3 q6 o6 ?
Fig. 5 Micrographs showing" A' C, a( U% F3 P3 R; o9 B/ b, |
evolution of the pluripotent and
4 M9 T3 Q% \' I' F/ S) P- hTE cells during a derivation
4 [1 W+ e# Q/ |; z4 Xexperiment. a ICM 1 day after4 @4 b, o# z; e5 e& k0 I. Z
seeding. b ICM cell expansion1 ?3 j9 c3 A$ r- C2 A
after 4 days in culture. Arrow
9 T2 X: U6 B! O! d$ Upoints to the pluripotent$ `! ]: G7 ^0 W
outgrowth, which looks like a
9 c* P/ U/ A/ f/ ?9 @clump at this stage; c colony, r |" u4 b& \$ X T {
outgrowth 2 days after splitting# l0 G9 j5 x5 K7 D$ m
the outgrowth in panel B: arrow& {$ y% \$ n6 u6 {& l% w
points to the expanding# B8 s' S9 A6 D9 z/ D. J5 Z
pluripotent cells out of the: m: ^" B2 g/ _% |- n6 M
initial hESC clump; d residual
; b" q- n; I- X" J, G0 k9 jTE cells 3 days after ICM
$ a: ?7 }( @7 d0 |seeding; e TE differentiated2 H, w3 {* S. `' c
cells 4 days after seeding; f TE$ _: N9 K, Q: P. ~/ q" y. L5 M& I' x
cells detached from the plate4 }0 L1 P1 d9 j7 ?6 W& c! `$ c
9 days after seeding; g detail of6 N5 T) G6 s/ h& f3 ^& I
the emerging pluripotent colony
/ s/ N# D- I3 ~$ ^/ M3 lof picture C;h early hESC5 z) s) N: C# t9 n' p% J9 `
colony at passage 4; i regular
9 ^# O. W6 ?# r3 A$ ]: b; k1 fcolony of an established line at
7 x: Z& H5 `% @( ^passage six
( Y- a' v# g& {* H' xStem Cell Rev and Rep
' [8 v! \8 o/ v4 `. Pthem . In our hands, embryos from the CF- 1 and5 Z K. O# f3 G1 c) L$ t
MF-1 line s, and from the cross between MF-1 and& y6 T `1 k, `8 F) E
CD-1, are much better than inbre d CD-1 embr yos.
! w+ f" g& }5 r1 h i; R% A8 aNote 4. Isolated pME Fs can be frozen at passag e zero, and% l% k* t ?& ~7 W- r+ W" ?
expanded at a late r time to prepar e feeders . Nev-ertheless , the expansi on up to passag e tw o before! e, t3 m4 b8 F; l/ t' E
freezing has the advantage of increa sing the num-ber of cells obtained at passage four. It has to be
9 }: j. Q; Y7 H% {* W" g+ cnoted that in the isolati on procedu re describ ed in0 J; K! G8 y8 R
the Meth o ds secti o n, an d d ifferentl y fro m oth er( l {8 k `5 |, K' T
published protocols [ 24], tissue chunks are not
8 j& ^% o" ]5 I2 }- bdiscarded before seedi ng after first trypsiniz ation.
: C+ R3 o! ?) p, @2 n- b; b9 BThis procedu re yiel ds signi ficantly higher numbe r
1 ]/ `# f# ?6 a, J5 z! gof cells. It is advis ed to culture chunk s a nd clumps N5 Y$ n: E* D# a/ a% i0 N
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! `8 K/ c2 H7 ]' L/ s2 e. x
normally be suffici ent to produce enough numbe r
! C8 y! d2 \: D$ \4 pof vials for cult uring up to six new stem cell lines9 w- k! \8 Y( [
during 1 year.) n# K' K$ E; O0 O. {; J5 l+ [, A
Note 5. Finger vorte xing is a simple way to mix a solution. f/ y' y2 ]- R/ R4 o
or disag gregate a pellet in a test tube. Hold the top" b Q( Z; B9 J3 [9 `
of the tube secure ly in one hand and draw the ring,
$ I% s0 h9 K! ]& Omiddle and index finge r of the other hand sequen-tially towards you, tappi ng the tube. This creat es a8 A0 w+ X6 d6 {
wh irlpo ol eff ect inside the t ube, w hic h can be4 _% j' |- V+ ]' v) E
adjusted in inte nsity by speed. It is milder than
/ d& A$ V! Z+ L2 Qmechanical vortexing and yields healthier cells
* J6 B" k5 {! n% vafter centr ifugation .: h1 ?% |: m) A" `. }( |
Note 6. Freez ing fibro blasts wi th a slow freezing met hod is
4 ]4 x9 P5 o: z2 Inot an issue , and they can be stor ed at − 80°C for9 w$ c0 p5 T7 Q$ n& Y' a
months without a decreas e in subseq uent plating' Z D/ i! T+ t) I- L. r
efficiency. The proto col described herei n uses the
+ m! V2 ?- g1 t3 O y8 Wslow rate freezi ng container Mr Frost y, which has" b( ?* j- ^ b( r8 P6 j8 q6 B! K
been vali dated and used for nearly 20 y ears i n [: N& D, j4 A+ o
mammal ian cell freezing [ 25]. It is a cheap and
% q3 W4 Y- ~- Rtime savin g o ption when compa red to alte rnative9 Y% H+ s) h+ ]7 Y2 b
methods such as contr olled-rate freezing and vitri-fication, which may be suitable for more delicate
4 ?. t! Z J* Z: ~! Omaterials like embr yos and hESCs .
. f# x+ w1 }5 Z, a0 N6 W# T4 h( m- lNote 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
- [$ X# E6 T$ ptoxi city and different iation during cultu re.
% v$ h$ f: r& x0 a& @Note 8. MitomycinCis thepreferredmethodtoinac-t ivate M EFs, a nd detail ed st eps to p erform the5 T/ A9 w! `9 j% O2 u$ @" ?- O) B% ^
procedur e are included in the text. Alternative-ly, γ - or X-ray irradiation can be used. In a ny; t$ `/ R; J( z1 g" W: I5 u
case, t he hESC supporting capaci ty of each
3 v0 o+ r5 f9 inew batch of feeders has to be tested on7 g+ @; s+ k3 e
known hESC lines bef ore c onsider ing t he m2 c z! O6 t: G5 m3 J2 V
valida ted f or use. Once the new batch has been3 L; W# r8 [+ P1 [6 d0 W. f- w# O
valida ted f or hESC cultur e, it ca n be used in! g$ W" c* x% }' u/ @$ f
subsequent derivation e xperim ent s.
/ G( f* G% \1 e" v- LEmbryo and ICM Cul ture" Z' S, `5 Z& _
Note 9. The procedu res detai led here are based on the use
4 p' s3 p$ i% m. R6 t. @of high quali ty clin ical grade embr yos, but fre-quently, the quality of the embr yos donated for( X. ]; K- d( |9 F( G6 G) Z# @
research do es not correspon d to the standards
J: v- X# c" p1 e8 upresen ted in Fig. 2 . If by day 6 of development ,. {" q. j6 {( M
compa cted cell s as in Fig. 2e – e are not form ed,5 r) }& j( ]4 a4 T7 E5 n: E
extending embryo c ultu re i n complete HES
% x U, e! M& ^, ^4 Xmedium for 1– 2 additional days will selec tively) x' j @* F: ^- x
favour the growth of the ICM ver sus the TE.
2 _8 L6 P! V' J/ `This appr oach is supported in the most recent+ Y- E3 S9 i) X2 j+ R2 v/ `
li terat ure [ 15].
3 J4 ?' s6 k. xNote 10. Reg ar ding ICM isolation, s om e authors use a n+ x8 \$ o$ P' ]; F
extended acid t yrode’s treatment to weaken7 a' k$ `4 U' y; ], Q, ~1 l7 \
the TE [26], thus facilitating the spreading
+ h6 F" _& j7 O& h. m' o Q" Kof blastocyst onto the feeders. H owever, in/ V5 a+ Y/ `! `. j1 y1 A9 p
our hands, the efficiency of this method
( M8 x! z; R o' dd 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
. b' O C8 r6 T: \the same AT t reatment to all embryos usually# I7 g2 z8 ~9 q C3 _, ]9 P' P
results i n s evere damage to some of them.
" ^# X9 g% g4 ?1 g2 [Theref ore, fitting t he method for TE reduction
( L% l, m3 R3 gto the grade of each embryo (Table 2 )helps
! S! } W: M$ f5 Z% m. fmaxi mizing the r ecovery of viable cells f rom
% t( C0 j- ~6 N @; x5 Wthe I CM. When TE is r el atively s mall com-pared t o the ICM, or when the ICM is not3 B1 E# A" D) |
compact, a pipetting method (Method A) is
2 V8 J- ^0 a- k) `' n- Z) b4 w5 lpreferable. Alternatively, if the TE is robust
& B, W) F% V8 n9 `; T Fand t he ICM i s c om pact , mechanical cutting
( `: A$ U+ `! F" S+ P6 V(Met hod B) s hould be the method of choice.; w2 ]8 a& y; u8 K1 X- E- V
Speed during embryo denudation and I CM
0 u$ I' Q9 h0 N. ]. risolat ion is criti cal, even if the m anipul ations
* M% l+ K1 e& E& v" v, H" aare performed on a heated stage.. K/ x. } M0 [. M* b, M& {
Note 11. If healt hy, the first seeding of the cell s from the& f: S4 [2 o2 c# d
ICMs will att ach i n 6 h t o ove rn igh t . In o ur! a% k- Y* ]- \# W' w2 D5 m/ d9 D
experi ence, those clum ps needin g more time to, S8 R% C1 t$ H p, F3 `2 w3 b: B% z
attach are not robust enough to yiel d deriv ations.3 {$ w& x0 P% C% ^
The first outgr owth after the attachmen t of the
7 |0 p' v' I- j0 O' I% zclump can be seen from days 3 to 16. At this
/ V% F" n4 q* v# J8 {point, the undifferentiated cells need the suppor t
& T7 S9 @# Y+ @5 g' lof fresh feeder layers to estab lish pluripot ent cell% w2 l/ `7 d' v2 W5 _8 R) P, o
growth. TE cell s evolve invar iably to form syn-cytial cells that invade the feeder layer and die
6 S8 ^' K L5 ?: Wleavin g g aps i n it. It is critical to have fresh
7 E1 k- i! A) T* Rfeeders ready by the time the TE cells and deriv-atives detach from the plat e.6 j! X5 v! K- l
Acknowledgments This protocol is the result of work funded by the
* B# l4 G4 E) UNorth West Development Agency (NWDA) i n t he UK and the
4 q0 F9 [. f' H) e6 U7 RMICINN-PLE2009-0091, IPT-20011-1402-900000 and FPI-CAIB+ I7 g y6 b4 m0 q" c4 M! Q4 e
Grant FPI10 grants in Spain.
8 u! F( X: I6 ^* M6 YCon flic t of in terest Th e aut hors decl ar e no pot enti al con fl icts o f/ c' m& f- z( Q& A
interest.7 P+ M- C0 P, q% ~* R
Stem Cell Rev and Rep8 J2 T: ~3 I; t3 s5 f) j8 M
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