标题: General ES Cell Protocols胚胎干细胞基本操作（Baylor College of Medicine） [打印本页] 作者: 肖爱华 时间: 2009-3-3 14:28 标题: General ES Cell Protocols胚胎干细胞基本操作（Baylor College of Medicine）
EMBRYONIC STEM CELLS PROTOCOL 6 J; x* L6 S1 ?Allan Bradley's Lab, Baylor College of Medicine, Houston, Texas 胚胎干细胞基本操作 , i) {! p/ j b7 }6 n1 h9 m ~( g, v$ q$ m
EMBRYONIC STEM CELLS PROTOCOL( [# K* x- D7 X0 ?; k! B
" @1 j2 D# s9 { E6 Y' c; U9 `* e9 h9 Y% s
! A0 \+ Q% O0 b9 {) m5 P If you are embarking in growing ES cells, be prepared to refeed 6 S, G5 Z$ V$ }& Y7 M V2 J* O* B
them DAILY. All procedures should be carried out using sterile % `! c0 a2 T" f% D( e4 B
techniques. The growth and maintenance media for ES cells is M15MEM # J4 l$ I! U! { y
(no pyruvate, high glucose) 15% FBS, 1X GPS, 1XBME. Handling ES cells: & F2 g: r* M/ O' X/ Y
growth, maintenance, passing, freezing and thawing is conducted in a $ f- }& G5 I% F# }- }: {
manner to protect and maintain the quality of the cells and keep them in 8 C' X4 a5 p: F0 h! v: W za pluripotential state. Serum quality is critical for successful growth # W+ j# |& m6 g1 X" W5 R
of ES cells and especially true for blastocysts. The quality of the 6 i: d4 _7 n5 ifeeders is very instrumental. Remember also that in passing, freezing, & z/ c9 [. M H7 b: g! i* A4 x
and electroporating ES cells; it is best that the cells are still at 5 v1 n- G) G6 B% k6 o( I) o. w5 B r
exponential growth (80% confluence) for optimal results. 8 X8 @9 P2 w, p ! \1 G0 e0 D! O2 d+ L ' Y! J/ E% H: i# X# n/ A6 ?) yTHAWING (QUICK THAW) # _' P E5 A% f9 |: T & t- y7 T2 n4 H, e7 l1. Remove cells from the freezer and quickly thaw in a 37oC waterbath. * l1 U" [# C$ p" q: ?% o 6 I- n" D% _5 o6 b2. Transfer the cell suspension to a sterile 15 ml tube. Add 10 - 5 p/ \3 U) D6 u% G
12 mls of M15 media to 1 ml of cell suspension.; H) N: `5 g/ F$ i5 t
7 Q( W* J+ K$ T0 f2 e/ Z
3. Gently mix and pellet the cells by centrifuging @ 1000 rpm for 7 minutes.3 O6 w0 N5 F0 u l+ g- \
: C5 c8 z! s: v9 Z+ T
4. Aspirate off supernatant and resuspend cells into 6 mls of M15, ( Y; [, g& y w0 Y
and plate out cells in a 6-cm feeder plate.2 c8 \2 C1 F3 H q
9 k+ j+ I! m* D; O9 l+ m9 M
5. Refeed cells daily with fresh M15. Upon 80-85% confluence, cells - l$ U% B; m/ x
need to be passage or freeze. (M15 media: DMEM, 15% FBS, 1X GPS, 1XBME) 8 Q# K! d% U9 V' B5 V8 v( e R) u8 G) w' L$ R
5 V7 G* T' ^ \* C' P" g
+ w. |( V6 k$ j# LPASSAGE OF ES CELLS% s O! A9 d- b5 H8 x
# w: b5 _3 |% V4 N2 P" hES cells typically should be passaged every 2-4 days (apart from colonies 1 V0 T1 ^4 ^8 F' n
under selection). If passaging is neglected the cells will differentiate 7 v* U9 Z. n; v$ `
and you will select for variants that might have lost totipotency. Cells 6 [, q& n2 e, h# \# Y) a- l+ T9 Umust be fed when media begins to turn orange. Yellow media (acid pH) is ; m* t V- ?! j5 @ ^
very bad for ES cells and should be avoided at all costs. If you are - {% y, ?: Y" @& n, w3 o6 y
planning to passage and believe that the cells might turn yellow 0 v9 Z S4 i+ O7 n# S3 v5 Hovernight feed last thing in the evening and again the next morning # D+ k5 [1 R8 Z4 _; ^! X) p6 z
before passaging. DO NOT PASSAGE CELLS WHEN MEDIA IS YELLOW. " E7 ` @0 P4 D7 a8 I6 j" f# w& Z5 e9 L' R
1. Check cells under the microscope for 80-85% confluence.( [6 V( }6 u& o4 R d8 c1 k- m
* ~( ?. F. w/ C' @5 H2 |! P2. Refeed cells 3 - 4 hours before passing them. (VERY IMPORTANT) $ J. w+ G+ J+ V; b. L * w3 V% C/ N& a 5 Y2 `2 y/ A6 D: S( g {# i3. Aspirate media off. Wash one time with PBS. Add 500 祃 of 1 {( p! }; J/ c, C% r$ s7 i5 p+ j, t
trypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate.4 u, G& e7 ^- |* l
% V0 R; c# y: n5 W$ ` x3 P# L
4. Incubate @ 37 oC for 15 minutes. $ h4 R" p3 ~( z; c- q6 d 8 z0 a5 |4 A2 Q: t/ h3 F! ?5. Add media, M15 to inactivate the trypsin. About 2 mls to 1 x 1 x% ?5 K$ l# L! n: k3 b8 t6-cm dish or 4 - 5 mls to 1 x 10-cm dish.( s: y3 |+ k7 v
' P8 b* m; m3 C7 O6. With a transfer pipette, pipet up and down several times to h( K9 w) p) z' g, Tseparate the cells and break any colonies. - s( m8 d$ G7 q( b" y& e/ \6 q) \5 P% w+ Y0 p% p2 G& r8 K1 x
7. Determine the number of feeder plates you need, depending upon & L) v- I0 { u$ u+ N4 _0 F0 b
the passage you are doing. Add fresh media, M15 to the feeder plates (to $ G& \% V+ F' F7 u- a1 x 6-cm feeder dish: 6 mls of media; 1 x 10-cm feeder: 12 mls of 4 y5 M0 K% i/ I- @$ }1 u Q! x
media). Split ratios for ES cells can vary from 1:1 to 1:10. Do not : S$ p V/ e! Z# }- [3 e
exceed 1:10. 0 f& F* M/ e4 B: V' D! q The area relationships for the various dishes are as follows: 0 |3 c2 k$ B K# F( a/ M7 S- m. ]: x9 ]: Y/ y3 M) s
) q! z6 |* ?" h" z( h! C" t
) r7 b0 d8 @: d, fDish Media Trypsin Area (cm2) Diameter (actual) , G; n# i% U4 Z(6 mm) 96 well 200 祃/well 30-50 祃 0.3 0.6 cm 6 L0 q# f" l2 F" s5 _( H9 x(10 mm) 24 well 1.0 ml 200 祃 1.8 1.5 cm! N3 y! [4 q3 a6 E2 ^( M+ A
(30 mm) 6-well plate 3-4 mls 400 祃 9.6 3.5 cm9 R$ |1 b# H' u8 ^# l# m
(6-cm) dish 6 mls 0.6 ml 21.2 5.2 cm ; O- I/ a6 g* f* N; c8 Q& T8 q(10-cm) dish 12 mls 1.5 ml 60 8.7 cm6 r! _2 v3 N% a/ p
(15-cm) dish 30 mls 2.5 ml 154 14 cm 6 g2 W+ g- `& ]5 _7 B- p' d. g5 J3 y! T% r9 `! V5 w+ V
! u2 @" M9 ^0 w' P3 p
7 B. |* ?( z% F8 x0 ISome typical passaging ratios: * w- `" s: f2 E! A 3 Z. j- D* a( r1:6 = 1 x 60 mm to 2 x 90 mm 3 b, H% R+ y( T# a- o/ |6 H& S* D& H6 A
1:6 = 1 x 30 mm to 1 x 90 mm' I8 z* \2 H7 c: o% S# o
1:4 = 1 x 30 mm to 2 x 60 mm + w$ W9 F" r9 D# E7 T$ x: |" ^+ l
1:5 = 1 x 24 well to 1 x 30 mm (6-well plate) ?0 U8 V& R4 b; m% i2 O
1:6 = 1 x 96 well to 1 x 24 well# Y9 X, v) r; Q0 \8 @- D
2 b3 t m5 p1 c) o1 u; [& o6 W1 _0 i3 h. @+ e
' ^- n/ G, t& z. x z! C. @6 X
8. Aliquot the cell suspension into plates in the volume specified $ d# i( L& R% R- _( R Ifor each plate. Remember to use Feeder plates. Always check the feeders 7 I) N% G6 r4 h! k/ X& F+ p
before using them. They should be confluent, no gaps, not contaminated 2 {7 K+ N. C: v% w! F' K' uand not dividing. Use feeders that are older, (1-2 weeks old), the k% e N- }6 k. y+ @* f J
advantages are many: any contamination is assessed, also any dividing P7 r, G- {. R/ e( ]' F* U$ `run-away cells can be detected, and the passage will be earlier. Also, $ h! L) {8 M7 F- E* Golder feeders have settled nicely and flattened.! f( T1 t/ w6 Y/ e; e
( Y% x; Y- T& Y
9. Mix to have a uniform cell distribution. Return plates to the TC / i& M+ U0 |$ ]1 u5 W+ R x7 j37 oC incubator. $ y3 J% [9 x3 i2 ~ ' E) p$ e: j/ N% T: L- rFREEZING ES CELLS (SLOW FREEZE)- e: x1 l/ p" _; z" e
7 j/ u3 f- C- Q. @1 o+ Z
1. Check cells under the microscope for 80-85% confluence.2 J7 k5 _3 N7 y. x( j! H
# d! t+ ~' V- ~7 ^, H7 H5 V2 C
2. Refeed cells 3 - 4 hours before passing them.8 F: k- J" e. ~, x2 K2 k
3 W. N9 g9 T. b# h& F. J2 {% i7 O
3. Aspirate media off. Wash one time with PBS. Add 500 祃 of ) X5 m- f3 Y' s/ o6 m6 B$ {trypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate. - ^# [8 I' N" W; b) q u* \% s3 d0 ]2 Q8 v' {+ ~' V+ q A8 \6 A
4. Incubate @ 37oC for 15 minutes.* J" R5 I5 m/ J+ y+ x! f) `- x
$ \. `; z. a# w/ v8 g5. Add media, M15 (M15 media: DMEM, 15% FBS, 1XGPS, 1XBME) to 4 z, g. s9 L! D: finactivate the trypsin. About 2 mls to 1 x 6-cm dish or 4 - 5 mls to 1 x 8 U5 C: L: f0 W. D0 Y
10-cm dish. $ Q$ |: L2 P: B0 V1 M7 q4 k$ V9 l$ m- Z2 _+ Q
6. With a transfer pipette, pipet up and down several times to 6 V7 }3 v* U8 b$ T8 r. fseparate the cells and break any colonies. Collect cell suspension in a ' |- b: \6 _) C8 | t4 Y% n
centrifuge tube and add more media to count.4 w. |& j; V5 P- M
' w! H! {& h7 {* L8 z+ Q
7. Count a 200 祃 aliquot and calculate the total cell number. From ! [: u T6 U* q: o2 i
this, calculate the volume of media required to give a final density of 6 P0 `+ B9 d) l+ f3.0 x 107 cells/ml. This density is very important, do not deviate from + I2 j9 k3 {% hit. B! j; [7 e. ?$ `; v
5 S. S, P! ^# W% ` {, X' K8. Pellet cells by centrifuging @ 1000 rpm for 7 minutes.. r5 ?& G/ c( s1 Z; f7 t8 ~
- X% `0 @3 L/ m
9. Aspirate off supernatant and resuspend the pellet in 1/2 the ) y- Z8 Z# e& L2 B3 b- H4 Y4 O0 ]9 V
volume calculated in Step 7 above, with M15 media.0 F5 _" ]$ _/ \% l
; N! e0 z1 J0 v$ X4 N# E6 {10. Add 1/2 the volume with 2X Freezing Media (60% DMEM, 20% FBS, 20% * ^7 i, S; V6 S/ Z sDMSO, freshly prepared); the cell suspension is diluted as a result: 10% 2 Z1 ` ]" k# Z2 \
DMSO is the final conc. Add the freezing media dropwise, mixing well 9 K, O8 g4 W! o' P' j# {
after each addition.! u8 k$ ]1 q: a8 T' _5 ~9 |
" k( v2 B% z9 A6 c7 u6 l* s11. Aliquot the suspension into sterile freezing vials, pre-labeled ( d* u+ o1 Q c) Awith the cell type (AB2.2, AB1, etc.), clone number, passage number and - {- p1 C1 J0 K6 I
date. A typical aliquot would have 0.3 ml - 0.4 ml of ES cells (@ a 3 F' Q* \0 g$ M( l& n' cdensity = 3.0 x 107 cells/ml) this is about 9 x 106 cells - 12 x 106 & W/ r# Q) h1 e Q y# dcells total/vial. : E. E. F5 H+ V) q 6 x' {7 q7 t* C ?2 W! I" _3 S- m12. Place vials into a freezing container. It is critical that the " F' x! v! t( [8 r( N
freezing rate is not faster than 1/minute. Do not use any untested . K7 p+ c8 q% i. q
styrofoam container since freezing rates vary greatly and this will most 4 w% V3 X3 n% [; j9 Clikely result in death of most of your cells. Freeze cells overnight at 3 L/ R$ k4 N$ Y$ @$ _-70oC, (24 hours). 8 `3 q* x. ~3 e9 f; P3 ^" _ $ S$ z A& N) [13. Next day, transfer cells to the Liquid Nitrogen freezer (or + T/ I. Y) k4 L# r* f" D-135oC freezer).作者: 眼镜蛇 时间: 2009-3-9 09:01
MEM # J4 l$ I! U! { y