iPS 建系经验

细胞海洋

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" ?2 J+ i) f0 I* u! D本文系ｘｙｚｅｎｇｈ版主原创 非常感谢
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IPSC Generation by Lentiviral System Protocol& ~9 v, j, y  l7 l/ j$ x& g
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# C1 l  S2 z$ j+ U  WLentiviral Packaging
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MEDIUM( @- K# u4 |2 V
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.2 k. W2 e+ _) ?; C5 [& s3 q. U7 X
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE
5 K! L: Z* I1 n- e6 H5 R) S! eMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. & B, J$ z. k0 O: v, ^

0 @3 A! ]! x9 i- v5 i% P9 p- ?) cREPROGRAMMING FACTORS$ U: U3 R6 ~! R: n& G" w
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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) q! N3 m, ?0 i  g3 K6 rMaterials Each T75 flask
5 _, y4 O0 S; d- u, B2 s293FT cells    10-15x106
, _& {5 B( z- M* S1 N8 vMD.G (VSVG)   5 µg- J0 s4 T% K# S4 m. q& O! [: z6 [% m
PsPAX2   10 µg
1 L0 G$ Z. B9 I) e$ U- DPSIN vector (~10kb)   5 µg
1 m! D2 x8 @! iSuperfect (Qiagen)   40 µl 1 j( m2 e, p3 O+ M
IMDM   400 µl
( W8 C3 g$ u; P& c293FT medium    10ml
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.( v" K* z! x; `+ d9 t
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
) U3 t  h0 t- R: [; n3.  Add 10 ml of 293FT cell suspension to each T75 flask.
( A: B; u; v. J3 x, @& S. b' Y4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).9 Y& v  E6 A- p: U
5.  Add corresponding amount of Superfect to the DNA solution, and mix with brief vortex. Briefly spin down to collect liquid at the bottom of the tube.  m; e. r" [9 |. e. }$ V  I
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.; o+ ^0 l. y& H2 A8 \
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.$ W9 S! X$ W) o! v6 W2 ]. M
8.  Incubate the cells at 37ºC O/N.& z! ?+ @7 }6 W; I/ N* F5 X
9.  Day 2: The next morning (9-10 am), remove the medium, and add 8 ml of 293FT complete medium (with sodium pyruvate) to each T75 flask.
$ ?! h4 L3 F* f, l# w10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.: _1 f2 z! O% N# e( h
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
) q% V1 a& ~7 X: F, f& y1 Q12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
: _* L% P# H6 U% |5 H' s13.  Use filtered virus directly or store in 800 ul aliquots in cryovials at -80°C until use. Lentivirus can be stored at -80°C for several months without loss lot of infectivity.1 [" j% _+ X# u+ \

3 K  I7 h4 f9 |Preparation of human fibroblast cells (IMR-90)
2 B# M4 O6 g+ q0 U, Y0 u; c1. Day 4 (~ 10am): when fibroblast cells have reached 80% confluence, aspirate medium, wash once with PBS, cover cells with 0.05% trypsin, incubate for 5 min at 37 ºC.
' T: e6 e3 |8 Y" P" r* ]2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.; Q2 U( X7 {6 V: Z
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
4 w9 \  b5 N7 P) T/ i! ?8 j( y: }4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.  }" _: u- K, s" a4 p/ ]' E. I2 V5 G
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
1 F$ A% t% Q- T/ y6 V- K2 t) y6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
& Y$ H5 }! |2 K0 \7. Incubate at 37 ºC, 5% CO2, for 6 h.3 m8 F9 X; q/ G3 q" m
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Lentiviral infection) t% ]$ |1 J% G8 Q
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
# t* J& \9 C7 E6 x$ H4 T. a2. Day 4 (~ 6pm) Add equal volume (800ul) supernatant of each factor ( Yu1, Yu2, Yu3) to fibroblast dish in the presence of 10ug/ml polybrene, incubate at 37 ºC, 5% CO2, for 2h.4 N0 |5 B% z* S
3. Day 4 (~ 8pm) Add another 2ml fresh fibroblast medium and incubate at 37 ºC, 5% CO2 for overnight, then aspirate the virus medium and replace with 2ml fresh fibroblast medium.! `  [, D+ i8 K4 Q% C* X% V
4. Repeat transduction (including virus harvest) as described above.  O9 v6 g0 b' S4 |& ]- S4 |$ u1 ]
5. A 3rd transduction may be necessary.
6 B9 k- {) E$ Q8 H6. Day 8 (9~ 10am) Trypsinized fibroblast cells completely (4 days after 1st transduction), transfer to two 100-mm MEF dishes (3 x 10^6 MEF cells /dish).
* e# m# S# R  F, ~7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
4 e7 @. h8 j! e8. Day 18 (9~ 10am) Change to conditional ES medium. Observe any colony formed and pick them up when they grow up.

细胞海洋

IPSC Generation by Retroviral System Protocol
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6 V+ ]7 ^2 ?/ z' U, w7 l+ D. _Retroviral Packaging % i* H" t5 G+ O, @1 D; _
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MEDIUM3 I9 r$ b! l3 R. x
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.% r  h, t' D9 V# o& u1 A- u% Z7 `
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.# L: K1 N& W- n6 A) v
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293FT CELL CULTURE
( t4 p, K  U/ X) N  nMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 9 s" ~; R8 ~$ |! |1 G& Q2 n, u: ?

) F/ _$ |2 r0 l  }8 |REPROGRAMMING FACTORS
4 \3 z( |" R) p4 l, a0 ]) x$ T) W) lpMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4; p+ G. S: D. }2 Q

. d5 N$ h2 [. z1 N0 o( KTransfection of 293 FT Cell with Lipofectamine 2000) A9 F2 @% N; d5 Q# W) a5 B

% J; a- H6 u2 o/ B/ w: E. |For T-75 flask
( s0 u- j( M: W. U- G- mPrepare 293 FT cell:
  U' W- Q, J5 k; ~0 ~7 H1 U; i' fPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
3 _& D) a$ Y0 v0 DObserve cell dish before transfection. The density of the cell should be 80-90% confluent, and the cells should be evenly distributed and attached on the dish.
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$ J2 Y3 M0 C9 t1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
) s. A4 @' Z. N7 r2.  For each T-75 flask, add 40 ul Lipofectamine 2000 transfection reagent, 600 ul of IMDM, and mix; incubate at room temperature for 5 min.  
, J! P6 i6 ^' z8 A; C$ N8 T2 N- f( K3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
% d- Z; j  s1 \  |4. Mix and incubate at room temperature for 15 min.
! g% O' }: ~6 H8 S' |; o9 E5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.
0 M, G( x) G8 V/ T6.  Incubate at 37 ºC, 5% CO2 for 48 h.
' h* B7 Q# h% ~4 }) v7 D) }7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
. ?' d- d9 f6 p+ J. ^6 i2 }' `* X$ Y8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.- X; @: {: E* H; j1 B6 t. u3 j
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
$ _  u6 ]0 j' f8 E% |10.  Use filtered virus directly or store in 600 ul aliquots in cryovials at -80°C until use. Retrovirus can be stored at -80°C for several months without loss lot of infectivity.+ r" S  F+ a! }" S: Y
# S$ Y  s! E2 N4 z" _
Preparation of human fibroblast cells (IMR-90)5 F8 A" ]3 F3 S( v4 d( F
1.  Day 3 (~ 10am): when fibroblast cells have reached 80% confluence, aspirate medium, wash once with PBS, cover cells with 0.05% trypsin, incubate for 5 min at 37 ºC.
! m6 J" L; X5 Q# I( z2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
9 ~! {# s% L4 O8 @1 F1 ~3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
0 N; F9 e. \1 p0 Q4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
2 n/ v: @, L! Z1 N2 y6 B5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
1 K  F& I. P, E" R7 l6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
' Q% p; S% c4 G% i7.  Incubate at 37 ºC, 5% CO2, for 6 h.% v6 I# f5 e, V( ^; Q

) O% S/ k1 j. \" ^, U  e* Q" \4 t/ PRetroviral infection5 J# v- ^7 R: |, c3 Y1 U
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
; O! X( j: s' D2 Q5 i2 w2.  Day 3 (~ 6pm) Add equal volume (600ul) supernatant of each factor ( OCT3/4, SOX2, c-MYC, KLF4) to fibroblast dish in the presence of 10ug/ml polybrene, incubate at 37 ºC, 5% CO2, for 2h.$ Q3 a% I  ]2 b4 X
3.  Day 3 (~ 8pm) Add another 2ml fresh fibroblast medium and incubate at 37 ºC, 5% CO2 for overnight, then aspirate the virus medium and replace with 2ml fresh fibroblast medium.% e# z& N! p( @7 o$ F( u3 y
4.  Repeat transduction (including virus harvest) as described above.1 D  M$ r! X2 b- ?
5.  A 3rd transduction may be necessary.
, m4 x1 j( X6 w% J8 C6.  Day 7 (9~10am) Trypsinized fibroblast cells completely (4 days after 1st transduction), transfer to two 100-mm MEF dishes (3 x 10^6 MEF cells /dish).
/ H, X9 W; M# t+ y, d7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
0 ?) V4 q) z4 G4 A4 Z8.  Day 17 (9~10am) Change to conditional ES medium. Observe any colony formed and pick them up when they grow up.

细胞海洋

转帖一个问题
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+ N; T6 \6 T6 \9 H1 F问：unconditional human ES medium 和conditional ES medium的差别？ ; K% G) C1 Z1 C5 \& B- n: m4 \
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ｘｙｚｅｎｇｈ版主答:unconditional human ES medium 在feeder cell(如MEF)上培养20-24小时后收集起来过滤便成为了conditional ES medium，加入bFGF后便可用于直接培养hES细胞了。

xyzengh

感谢超版费心转贴，愿于园中各位高手相互学习交流。

stemcell8

向你学习

stemcell8

plus 500 µg/ml Geneticin. 什么意思

zlx0814

我理解，加G418，终浓度达500 µg/ml

lorey

好东东！支持斑竹！

zjkenan

我也是学习的

双刀

有逆转录病毒诱导ips建系的经验吗？谢谢！