General ES Cell ProtocolsÅßÌ¥¸Éϸ°û»ù±¾²Ù×÷£¨Baylor College of Medicine£©

Ф°®»ª

EMBRYONIC STEM CELLS PROTOCOL1 _( K0 X- J" y( A0 I+ A7 ~: J2 F. f
Allan Bradley's Lab, Baylor College of Medicine, Houston, Texas ÅßÌ¥¸Éϸ°û»ù±¾²Ù×÷ ' f, _! Z2 |! ~4 m+ E

! |$ k6 F2 F5 e2 d5 GEMBRYONIC  STEM  CELLS  PROTOCOL
$ e. U" K# Q; w" U& G! @" n8 A, T
) Y* L9 A7 l7 Z
5 ]+ P% _7 i9 }) H( P
, T7 M  Y  J3 N9 f2 d$ c: x        If you are embarking in growing ES cells, be prepared to refeed 9 L! N: O3 f) L  p
them DAILY.  All procedures should be carried out using sterile
) q! Q2 y8 w3 Atechniques.  The growth and maintenance media for ES cells is M15MEM
% d9 |* p8 F# O4 ^: R(no pyruvate, high glucose) 15% FBS, 1X GPS, 1XBME.  Handling ES cells:  
; ?* l1 Z& P/ E2 tgrowth, maintenance, passing, freezing and thawing is conducted in a / H$ \. N0 W; t7 ^
manner to protect and maintain the quality of the cells and keep them in
; |8 c) k$ L- K9 ca pluripotential state.  Serum quality is critical for successful growth
" V3 t+ h* d4 X8 L2 S2 Cof ES cells and especially true for blastocysts.  The quality of the
$ C% i4 |. u5 [feeders is very instrumental.  Remember also that in passing, freezing, # E( L6 V" ?" H8 b
and electroporating ES cells; it is best that the cells are still at 2 A7 K4 f# s) u. k
exponential growth (80% confluence) for optimal results.
9 J! l/ J0 v9 P' V
5 x3 i- Q* @% ^! |1 h! v8 v6 y& O
9 e# j# `- C# Y2 {' FTHAWING                (QUICK THAW)+ \' W$ e6 U+ f7 m& @, L6 u
" D/ K. ?/ C- O
1.        Remove cells from the freezer and quickly thaw in a 37oC waterbath.
, p" m2 t0 F# ^0 M- P/ |4 w
6 x# r: d3 G. D" A" ?2.        Transfer the cell suspension to a sterile 15 ml tube.  Add 10 -
" h% I7 H7 }  _7 h/ x' `12  mls of M15 media to 1 ml of cell suspension.
  i2 u4 c# _1 f& s/ d  i% S4 l( j$ P* v+ D" A; z/ \
3.        Gently mix and pellet the cells by centrifuging @ 1000 rpm for 7 minutes.5 m+ i0 C; T4 {& H( T

2 }/ N! V  ?4 c0 P/ M4 m; D1 {4.        Aspirate off supernatant and resuspend cells into 6 mls of M15,
" K! T. V7 u! X; X" x; rand plate out cells in a 6-cm feeder plate.
+ p! G$ Y3 f2 ^
) i; u" B1 a' k3 }' o2 U5.        Refeed cells daily with fresh M15.  Upon 80-85% confluence, cells
' ]! U  n4 L  J, }# u: Y& xneed to be passage or freeze.  (M15 media:  DMEM, 15% FBS, 1X GPS, 1XBME)3 P* K5 q9 T; S9 j3 a3 T

- O, Z3 N+ i- b9 r; a9 Z: l, S1 x- t0 i, S$ k
2 D: v0 b: Y2 P! ^
PASSAGE OF ES CELLS
" q7 E, |  p* D( r
% i2 a; d' t& L/ |# NES cells typically should be passaged every 2-4 days (apart from colonies
) E0 `$ R% a8 Q, c6 U. [under selection).  If passaging is neglected the cells will differentiate % e! [, r9 g5 M0 j
and you will select for variants that might have lost totipotency.  Cells
( j" G$ y- m7 q2 Z  x- V3 o  tmust be fed when media begins to turn orange.  Yellow media (acid pH) is . u( _  A6 ^: i# ^
very bad for ES cells and should be avoided at all costs.  If you are , W) m7 v" z% J0 Q, f2 g  d* {% _
planning to passage and believe that the cells might turn yellow
/ w, p0 Z! j) ]7 eovernight feed last thing in the evening and again the next morning , J9 f4 r9 h/ a; J" O( X
before passaging.  DO NOT PASSAGE CELLS WHEN MEDIA IS YELLOW.0 ^7 j  m; e: U2 q3 |

- y$ D4 ]& }3 F( q& M/ B1.        Check cells under the microscope for 80-85% confluence.
) }0 z# o+ V$ {, _1 S( K3 A2 j  h( r7 }' d2 a% V& e9 z
2.        Refeed cells 3 - 4 hours before passing them.  (VERY IMPORTANT)
$ o' V1 `+ c4 J9 g! g! V5 E
4 p4 c. Y+ L$ {  k
- U( h" a! R3 ^  D7 F0 }8 P' O3.        Aspirate media off.  Wash one time with PBS.  Add 500 µl of
/ M8 |5 g. ^( [# @3 I8 p* Q5 [trypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.
) K) E5 G+ e5 ], p5 A! g& [9 `
" H3 q5 X  F# L9 y9 |4.        Incubate @ 37 oC  for 15 minutes.0 M5 c0 z8 Y( \+ \! c) C
! [. d5 L8 Q( j  K& a; @
5.        Add media, M15 to inactivate the trypsin.  About 2 mls to 1 x ; Q- v) E9 R  f7 c- q
6-cm dish or 4 - 5 mls to 1 x 10-cm dish.- R6 m7 k% }7 {" e
6 `8 E) p" l# Y
6.        With a transfer pipette, pipet up and down several times to
1 R/ P! n4 D0 Sseparate the cells and break any colonies.
* E0 w. o" F. y+ A# c9 R# h
0 i' @5 b5 _/ B. g4 n! z. U+ d7.        Determine the number of feeder plates you need, depending upon
# m. r0 o& H& Lthe passage you are doing.  Add fresh media, M15 to the feeder plates (to
) E+ v0 Q1 F5 o: \" T; j  @1 x 6-cm feeder dish: 6 mls of media; 1 x 10-cm feeder:  12 mls of
3 l- k. h  G% B1 Q4 nmedia).  Split ratios for ES cells can vary from 1:1 to 1:10.  Do not " f' x: t% z6 w6 i! i$ ]
exceed 1:10.  7 A& e  g! y" v0 v: A
        The area relationships for the various dishes are as follows:
0 `2 _# N! c- y5 z( Z6 J7 V/ u# P# V8 \) @8 {: C6 \; C" V- r" E4 R

7 \0 x$ ]. F, M' e; f7 _& T# o% ~4 V! x# N8 b( \0 g2 t% g# E" S
Dish                 Media                   Trypsin        Area (cm2)        Diameter (actual)5 f. O3 {% X# W  C$ R
(6 mm)  96 well    200 µl/well      30-50 µl           0.3           0.6 cm& I+ a1 c# L$ a3 N
(10 mm) 24 well          1.0 ml            200 µl        1.8           1.5 cm
9 m+ k( ^8 A) K. Y1 i# @6 H(30 mm) 6-well plate        3-4 mls        400 µl        9.6           3.5 cm
% e% _& Y. W3 U* N9 R9 j(6-cm)  dish          6 mls             0.6 ml        21.2          5.2 cm
$ W3 |* W7 b6 Z! |(10-cm) dish  12 mls        1.5 ml        60            8.7 cm
- Z- G5 p2 j. P, `! Q) a% r(15-cm) dish 30 mls        2.5 ml        154           14 cm
3 t% J! H9 ]+ {- B& k, t1 e, L
' b3 l+ L- T, q/ y: k! o& ~3 @/ b2 _9 @4 q/ b. D

/ C1 S0 P5 j5 s* ?/ G; zSome typical passaging ratios:
  ~9 T6 h( x( f. i  o- [, N# Z5 o/ @/ r/ |; w- I8 c
1:6 = 1 x 60 mm to 2 x 90 mm               
$ D! B" e5 J" s1:6 = 1 x 30 mm to 1 x 90 mm3 F8 A' h9 D$ I$ G6 T: D6 u* i  B
1:4 = 1 x 30 mm to 2 x 60 mm                    0 q2 ]7 h, ?: `2 ^# U$ h& S
1:5 = 1 x 24 well to 1 x 30 mm (6-well plate)                        9 i5 F; ^- C( Q" V) n0 }
1:6 = 1 x 96 well to 1 x 24 well; q0 v5 t% G8 R. }

% U% A7 {* ]6 O. }% E+ j6 j+ `, |2 E& O
5 z; a( K5 S0 i; a
8.        Aliquot the cell suspension into plates in the volume specified
! x2 Q, @  U6 H8 t4 ~4 Q+ pfor each plate.  Remember to use Feeder plates.  Always check the feeders
8 [  s7 X7 M6 K( ]) l* Q; W/ Q1 ibefore using them.  They should be confluent, no gaps, not contaminated
% D" |4 a5 w$ X; X5 Q  f4 Z1 kand not dividing.  Use feeders that are older, (1-2 weeks old), the 2 S6 C- R7 c. \3 {% Q) `6 Q  q
advantages are many:  any contamination is assessed, also any dividing 2 v- k; T$ D6 u, x/ b8 z
run-away cells can be detected, and the passage will be earlier.  Also,
6 i7 b5 I* Y# C* R9 V. }5 aolder feeders have settled nicely and flattened.6 V0 O! \' f9 n! c0 z* q

1 t- i6 R0 s2 {4 I: _9.        Mix to have a uniform cell distribution. Return plates to the TC / [  `8 |4 @5 b+ T+ s
37 oC  incubator.1 d, Y- h% a- q7 b- u* F

4 s" d1 Q  L% `% _' ?& j) ~FREEZING ES CELLS                (SLOW FREEZE)# s6 x7 z$ m$ P( X+ m# g
+ @, F+ ]; f; b; [
1.        Check cells under the microscope for 80-85% confluence./ S7 E4 e$ B; n+ Z' P$ K% ?

' a! J6 e+ U% a* l6 C2.        Refeed cells 3 - 4 hours before passing them.
' f. _+ P1 z( q0 O
" D: C0 T5 ^! Y3 |8 j3.        Aspirate media off.  Wash one time with PBS.  Add 500 µl of
$ T! m3 x/ c  ?9 s! l7 Jtrypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.3 `" q3 I$ d' s8 w! _4 l, U: O

; f0 \$ I) s7 ^. B& L4.        Incubate @ 37oC  for 15 minutes.
- A' r1 o. f; g# _& q! }( u) A) A7 y/ K
5.        Add media, M15 (M15 media:  DMEM, 15% FBS, 1XGPS, 1XBME) to + G1 M0 o* c9 ?6 L. U  _
inactivate the trypsin.  About 2 mls to 1 x 6-cm dish or 4 - 5 mls to 1 x ; H" _8 H! T- s$ _5 j; Z6 }' ~
10-cm dish.5 H8 `" z. d5 [$ ^8 g7 X; s) A2 L  E
9 ?5 U0 o, \' F" e
6.        With a transfer pipette, pipet up and down several times to
3 t) O/ p' I) h) K# Tseparate the cells and break any colonies.  Collect cell suspension in a ! R7 k4 k# c( e* y8 U1 r+ w# I/ M) u; [
centrifuge tube and add more media to count.7 s" X) @. q7 j( Z
$ I, c% z9 u7 A
7.        Count a 200 µl aliquot and calculate the total cell number.  From
& X6 V/ U7 H( W7 q; S' P: @this, calculate the volume of media required to give a final density of 3 l" k' A1 M+ }; w9 v9 P
3.0 x 107 cells/ml.  This density is very important, do not deviate from
- z# g2 P1 C6 X0 M- U2 Q0 rit. 2 ~& |. Z9 M. B/ q  h( |0 J

7 {/ W0 r/ u+ d2 J4 Y& x8 k8.        Pellet cells by centrifuging @ 1000 rpm for 7 minutes.
7 ?9 ]0 s3 Y; b) e% u& ^5 x$ `/ T/ V0 M4 R1 U, [  |
9.        Aspirate off supernatant and resuspend the pellet in 1/2 the
! }* a) f; {/ V4 N; N7 u  Zvolume calculated in Step 7 above, with M15 media.+ c1 K0 t# Y" s3 X2 v$ @
# E& }1 C3 h) g/ v# a# d- C  m$ I
10.        Add 1/2 the volume with 2X Freezing Media (60% DMEM, 20% FBS, 20%
2 v% ]/ q' {, T  EDMSO, freshly prepared); the cell suspension is diluted as a result:  10% * S" g* r( O8 O5 o, p
DMSO is the final conc.  Add the freezing media dropwise, mixing well 4 c. n. V6 U/ r
after each addition.2 ^7 n! ?7 c* P& i5 F

/ A0 \  C( C' j6 U11.        Aliquot the suspension into sterile freezing vials, pre-labeled 8 z0 C5 ?- V" g) \: B- _/ c6 q
with the cell type (AB2.2, AB1, etc.), clone number, passage number and ) ?1 p) Z, U3 F" K
date.  A typical aliquot would have 0.3 ml - 0.4 ml of ES cells (@ a ( `" ^5 x' Z, n% I7 \
density =  3.0 x 107 cells/ml)  this is about 9 x 106 cells - 12 x 106
  ^% X, V6 M8 v( M0 P7 `) z8 pcells total/vial.; r0 s! O: p, P0 w; ~7 f

3 Y$ |5 o6 [6 ~, G1 T/ e- B. V1 e12.        Place vials into a freezing container.  It is critical that the
9 |. Q! D3 S5 C) zfreezing rate is not faster than 1¡C/minute.  Do not use any untested " ^+ B. o2 N  ?2 u" t
styrofoam container since freezing rates vary greatly and this will most + l& R1 v# D7 d: b: ~. l2 w7 L
likely result in death of most of your cells.  Freeze cells overnight at & t( v0 Z4 U0 A" w/ y* }
-70oC, (24 hours).' x, l6 x4 C8 j) m) Z/ J! Y& P. L9 i

) a$ d2 [7 `/ t$ [13.        Next day, transfer cells to the Liquid Nitrogen freezer (or
# j/ z: K+ P+ K' A( d2 G. @-135oC freezer).