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# M9 B5 X }6 J6 `' W8 T
Allan Bradley's Lab, Baylor College of Medicine, Houston, Texas 胚胎干细胞基本操作 0 q. u2 n( E) o2 N3 J$ N7 Z n5 |/ b9 _) m& x
EMBRYONIC STEM CELLS PROTOCOL 3 D% ^( m6 j2 a) j5 _& w+ |2 r6 {- K
. i( e2 E9 ^& E7 U2 N0 [ ' C3 m' `6 G$ B6 t: |$ n If you are embarking in growing ES cells, be prepared to refeed e7 L6 k+ } T# |
them DAILY. All procedures should be carried out using sterile " {! ^( x( [4 q8 I8 etechniques. The growth and maintenance media for ES cells is M15MEM # _' ]! f' P* Q% F3 [ C- M
(no pyruvate, high glucose) 15% FBS, 1X GPS, 1XBME. Handling ES cells: $ Q3 c+ |% c- ?0 ~growth, maintenance, passing, freezing and thawing is conducted in a 7 e; H2 ]* C- [manner to protect and maintain the quality of the cells and keep them in ' i/ O! S. \3 `) H9 T/ R2 p
a pluripotential state. Serum quality is critical for successful growth + O- d1 W# L0 e; ]2 f! Q8 D! J& \of ES cells and especially true for blastocysts. The quality of the / }5 X% i9 {+ F! ?feeders is very instrumental. Remember also that in passing, freezing, : T8 m& z7 \) I9 n. o4 u# O. dand electroporating ES cells; it is best that the cells are still at " d6 \3 M1 D. G! I8 u3 l& kexponential growth (80% confluence) for optimal results.4 V, E) F' E& `" g
8 U+ t; y) s$ N" u6 e& m ' ]' s; `) p j4 u; rTHAWING (QUICK THAW) 2 N- G# m6 o1 v" b4 W % f# v- }: v& T/ Y! U% i3 {2 }+ ?* q1. Remove cells from the freezer and quickly thaw in a 37oC waterbath.1 m, u+ Q0 U7 Y4 L6 o! h u4 T6 x7 k
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2. Transfer the cell suspension to a sterile 15 ml tube. Add 10 - ! w- _+ ?4 ^ [5 O" j12 mls of M15 media to 1 ml of cell suspension.) r$ G1 ~+ t/ l
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3. Gently mix and pellet the cells by centrifuging @ 1000 rpm for 7 minutes. % x( E# [# L" z @& b2 V0 [* C/ Q# W Z) r6 w5 b4 K; q
4. Aspirate off supernatant and resuspend cells into 6 mls of M15, 5 v, [8 p: w& C% ]3 |and plate out cells in a 6-cm feeder plate. - H; W. n7 @) k, } 6 F- I9 A% W$ @4 Q$ h5. Refeed cells daily with fresh M15. Upon 80-85% confluence, cells * n5 t7 ]" [* q* g6 @+ ]need to be passage or freeze. (M15 media: DMEM, 15% FBS, 1X GPS, 1XBME)9 u; s! d1 }- H$ j6 o( C4 U$ w
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J4 c9 J5 }/ V6 B! E$ ~" EPASSAGE OF ES CELLS 7 q. u+ M3 A9 c # W: |4 R4 i4 R7 N2 s7 sES cells typically should be passaged every 2-4 days (apart from colonies & I( Z- ~8 c6 r* J( Junder selection). If passaging is neglected the cells will differentiate $ R* O7 c5 c$ P/ F4 K0 jand you will select for variants that might have lost totipotency. Cells 4 v% T$ {# V/ l. l4 J6 f* ~
must be fed when media begins to turn orange. Yellow media (acid pH) is 9 s& a5 ~% H" N& f; Hvery bad for ES cells and should be avoided at all costs. If you are % L) J/ G; }% c4 ]planning to passage and believe that the cells might turn yellow ' h/ }0 q4 Z3 y8 Governight feed last thing in the evening and again the next morning 7 ^0 u# s' }. ?4 I" P! t
before passaging. DO NOT PASSAGE CELLS WHEN MEDIA IS YELLOW. 8 k: u; D) B' z p m : `! k' r: n# }1. Check cells under the microscope for 80-85% confluence. , g# ~* ?& G6 R! B ; B" }2 |# i! J/ R2. Refeed cells 3 - 4 hours before passing them. (VERY IMPORTANT) ' \- u: t5 k& r* a0 d2 X# a # M) t( O' c3 v! Q5 Z ) ~; @) |5 H; F- F2 V2 v3. Aspirate media off. Wash one time with PBS. Add 500 祃 of 6 w1 F0 Y$ f' E6 ^% j5 \( V" h
trypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate. ( b7 B& q2 ]3 h- d. c3 B8 s1 L0 D( s - Y! Y, z+ V$ [* @, }5 O2 T4. Incubate @ 37 oC for 15 minutes.: o4 [/ u7 e" }" p& i
% s4 i5 N6 X6 B* z" l. c1 j4 s5 L0 t5. Add media, M15 to inactivate the trypsin. About 2 mls to 1 x % s! u7 o+ O I* q! |# i8 [/ B$ {
6-cm dish or 4 - 5 mls to 1 x 10-cm dish. + U% X& [ b2 O- X' q; _) \* l8 X" @2 a$ d3 M7 H. y
6. With a transfer pipette, pipet up and down several times to , C' O4 A# ~7 H/ S3 ?# S1 Mseparate the cells and break any colonies. " G. ]0 X/ K8 P! x0 f( z 0 W, x- D8 J& p5 n8 f7. Determine the number of feeder plates you need, depending upon ( P" s# A2 F) k: g" T9 M: D ythe passage you are doing. Add fresh media, M15 to the feeder plates (to 5 d0 m* p! X8 Z. S) o/ S% D
1 x 6-cm feeder dish: 6 mls of media; 1 x 10-cm feeder: 12 mls of ( m$ z* @7 y( \- H) \: Cmedia). Split ratios for ES cells can vary from 1:1 to 1:10. Do not ! i3 r6 i" Z6 \; ]+ Iexceed 1:10. ) f1 D5 r* t1 F2 o% n
The area relationships for the various dishes are as follows:9 C$ _( V' P" j7 D% E4 o# ^+ h
8 Q# d- H9 c" |; @5 c" `+ t" H - o" p' q3 \5 p& s: f* ] 5 ? n% q" }+ V( K! Z+ h1 iDish Media Trypsin Area (cm2) Diameter (actual) * D9 R5 H) b' p& M(6 mm) 96 well 200 祃/well 30-50 祃 0.3 0.6 cm , t4 a l& n7 q4 ](10 mm) 24 well 1.0 ml 200 祃 1.8 1.5 cm2 g. C& t- g+ V' K8 X6 T
(30 mm) 6-well plate 3-4 mls 400 祃 9.6 3.5 cm( Z( D/ }- V# K( f
(6-cm) dish 6 mls 0.6 ml 21.2 5.2 cm 1 g" g3 ]5 F8 f {8 |(10-cm) dish 12 mls 1.5 ml 60 8.7 cm, t( w, s0 ]- ]- h2 w8 O
(15-cm) dish 30 mls 2.5 ml 154 14 cm: G) B" x$ M p; u/ i1 A
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Some typical passaging ratios: ( D+ p( q7 X9 f9 E8 F( Z1 _. l1 `: J& T- K
1:6 = 1 x 60 mm to 2 x 90 mm ' E! W, m! ~9 O* H d/ Z
1:6 = 1 x 30 mm to 1 x 90 mm# R5 D$ G) c- D. ]
1:4 = 1 x 30 mm to 2 x 60 mm 6 H0 p7 [7 D, V. g( ~/ R
1:5 = 1 x 24 well to 1 x 30 mm (6-well plate) : A( j2 E; G; R/ G. b9 A1:6 = 1 x 96 well to 1 x 24 well3 z" m. f* m4 D
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8. Aliquot the cell suspension into plates in the volume specified 0 l& }& p0 K6 \$ s; l
for each plate. Remember to use Feeder plates. Always check the feeders * ^+ u! w2 j2 I7 B$ {before using them. They should be confluent, no gaps, not contaminated . c# t3 ]* S* x# \# q0 p1 Uand not dividing. Use feeders that are older, (1-2 weeks old), the & F0 C& ~; [6 L3 k8 C2 R4 R8 m- g
advantages are many: any contamination is assessed, also any dividing + n& d: U/ ?) Trun-away cells can be detected, and the passage will be earlier. Also, + }* T3 [( `4 \
older feeders have settled nicely and flattened. 1 B$ J, b2 a2 ~* O' Z$ h. Z ; |3 ]; l: I/ J3 {; m/ }1 g% K! v9. Mix to have a uniform cell distribution. Return plates to the TC 8 q) v: d8 U; _8 p3 L
37 oC incubator.2 i! e( w. F+ U1 J1 }. u
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FREEZING ES CELLS (SLOW FREEZE) 8 ^$ }' O0 A# h$ O+ }! w+ Z7 ]( j' V3 U, F# M1 U! i
1. Check cells under the microscope for 80-85% confluence.' ?5 D7 g& L8 ~2 L* C. i! ^' L
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2. Refeed cells 3 - 4 hours before passing them. 5 \& V; G" l7 }1 c7 t; e! o8 ?4 `; K: P, ^. _+ W
3. Aspirate media off. Wash one time with PBS. Add 500 祃 of - d7 e1 l! _) ztrypsin to a 6-cm plate, or 1 - 1.5 ml of trypsin to one 10-cm plate. 2 b' w: l& ^* T) F: x 2 E" {6 K( S e& U: S" {. C4. Incubate @ 37oC for 15 minutes.5 P, n/ Q k& D( z4 C
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5. Add media, M15 (M15 media: DMEM, 15% FBS, 1XGPS, 1XBME) to 2 T; Q" S. @$ x$ Zinactivate the trypsin. About 2 mls to 1 x 6-cm dish or 4 - 5 mls to 1 x 2 l7 I/ o0 G# U4 g
10-cm dish.& h. o H2 j+ P |& Q+ l6 |) W3 V# M
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6. With a transfer pipette, pipet up and down several times to / G4 k! X8 i) i+ {
separate the cells and break any colonies. Collect cell suspension in a - Z9 m4 E8 ?9 y4 G
centrifuge tube and add more media to count. 9 P M2 D. t+ n4 p' R8 H8 v6 l% s0 Y; F- D% x
7. Count a 200 祃 aliquot and calculate the total cell number. From 0 H" L: a- d+ L+ z
this, calculate the volume of media required to give a final density of 7 E! L' C V: ~. h4 v! H. ^ H# c3.0 x 107 cells/ml. This density is very important, do not deviate from 8 i) O O; @, r9 C r( m
it. $ c5 ^% N: _0 T' N& b, z! q" e' l) O8 Q8 N. v6 q
8. Pellet cells by centrifuging @ 1000 rpm for 7 minutes.* g! J7 H ]4 `5 S% u% N' y
$ r0 x1 @ X5 ~' `9 k9. Aspirate off supernatant and resuspend the pellet in 1/2 the % \- A N, M& U2 h+ ?
volume calculated in Step 7 above, with M15 media. ) A( W* }" V" X+ H' k& k0 T4 L+ j6 y
10. Add 1/2 the volume with 2X Freezing Media (60% DMEM, 20% FBS, 20% ; H* A; y5 u( a5 lDMSO, freshly prepared); the cell suspension is diluted as a result: 10% ' g" J; ^/ E* }) nDMSO is the final conc. Add the freezing media dropwise, mixing well % U2 K) D6 c0 Q! t7 y2 u& Q" vafter each addition. $ G8 Y/ Q. q" R" ~8 ^# P* V/ X; S$ k7 T
11. Aliquot the suspension into sterile freezing vials, pre-labeled : y, X9 t& Q* y9 g, ^with the cell type (AB2.2, AB1, etc.), clone number, passage number and * `6 F% T9 |3 v( t5 Q. Q
date. A typical aliquot would have 0.3 ml - 0.4 ml of ES cells (@ a ) H5 ?: E3 e+ O8 C# [
density = 3.0 x 107 cells/ml) this is about 9 x 106 cells - 12 x 106 F! ]: b8 d6 B1 hcells total/vial.& p- _4 ^. j4 e* e" k, U1 @: n0 ?
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12. Place vials into a freezing container. It is critical that the ' F8 k j9 ~$ M) o
freezing rate is not faster than 1/minute. Do not use any untested 8 O" @9 R0 o$ Q# S" o, m/ f& r' b0 Jstyrofoam container since freezing rates vary greatly and this will most 7 w4 j5 f. m" ]likely result in death of most of your cells. Freeze cells overnight at % t: |# m, m& q- A2 T* H& D3 Z
-70oC, (24 hours). 6 o+ w2 Z" r: ?- T8 h q2 i: T0 o! O5 h13. Next day, transfer cells to the Liquid Nitrogen freezer (or 6 m5 A/ R5 [) Y0 D( [
-135oC freezer).作者: 眼镜蛇 时间: 2009-3-9 09:01
MEM # _' ]! f' P* Q% F3 [ C- M