iPS 建系经验

细胞海洋

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本文系ｘｙｚｅｎｇｈ版主原创 非常感谢
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9 |' W" h# Q& g" }IPSC Generation by Lentiviral System Protocol
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* G$ P- a5 \$ j( hLentiviral Packaging
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; U9 n7 G6 k* `% RMEDIUM
! {$ X7 k% c, e: r% k293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
# t) H2 p9 ]) n3 q/ \) n/ e293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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3 e  d+ I3 \* S/ B3 o! I$ X& {293FT CELL CULTURE) B8 x' r! }; X" S- A  \# y* V
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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1 ^* e5 X8 R, E' s' A6 O! _" s- MREPROGRAMMING FACTORS
. J) i8 w% g- h# C5 G- P8 ?Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4/ H2 Y2 V- n2 @/ m  Z+ d

  C4 z9 q/ W5 ?9 YLENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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Materials Each T75 flask
) B0 C; W% `, N3 ?293FT cells    10-15x106
& E4 g, V, b, N3 |& y% iMD.G (VSVG)   5 µg
% r. G* N3 [" F! K8 PPsPAX2   10 µg' }: T, f' r7 M) W
PSIN vector (~10kb)   5 µg
7 e, D# G3 S1 m" y* kSuperfect (Qiagen)   40 µl
# W" x! K. x$ o3 \! z3 s- nIMDM   400 µl( \/ @* S; O: F7 |2 a% h
293FT medium    10ml
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.0 B6 v& [( ^! Y9 l$ l8 d
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
' ^$ f4 E# q- V* T5 l* n3.  Add 10 ml of 293FT cell suspension to each T75 flask.
7 S' k) o* W  u" e4 }4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
* t+ s% B4 j, g8 t" o5.  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.
3 g; P, G+ P4 d& G5 p* [6.  Incubate the DNA/superfect mixture at R.T. for 10 min.$ Z" A7 g# U# X- _& J0 o3 P
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
% b' k% _+ P8 L2 P: U8.  Incubate the cells at 37ºC O/N.
, C! I4 P% ~1 X: j8 j5 ?; F9.  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.
# U# z$ L4 Y! e+ t- u0 B10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.# D) A9 y$ @/ D5 |
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
! n5 e5 d- F; G1 d& M/ r! l12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
  T& O* y2 \* O3 Y* m& v7 j6 t13.  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.5 \& W( c6 c2 X; y
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Preparation of human fibroblast cells (IMR-90)
6 `- j  r9 U: |: S1. 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.
+ [4 `3 ^" U# V; a7 u' u2 v, m2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.% C/ p: L0 {, R
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
0 W3 e3 p* G- z2 s2 x4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.9 e1 _. y: ?5 a% f6 d
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
8 a& |( h% T5 a+ Q3 k6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
* k/ I1 N( c0 D, {2 Q4 ?7. Incubate at 37 ºC, 5% CO2, for 6 h., P* x# H- s9 t1 o9 k
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Lentiviral infection+ l0 l9 p$ i' y* C" I) b$ l
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
# t$ s  j9 R- w! M) J0 q: i2. 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.9 @" i$ E1 d6 F& m& q
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.
. V, h9 q" w) A) E# g7 G4. Repeat transduction (including virus harvest) as described above.& }; J  D5 j, W# \3 V" ]$ t8 u% U6 l
5. A 3rd transduction may be necessary.- p% M" }% {& f* h0 Z2 X
6. 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).5 C7 l1 j! v8 H! f! |: p
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
5 D$ R3 }/ w4 z: [! j8. 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% ~) v8 A9 z' }$ G4 l1 C. ~

+ e/ F% y( L, e9 j: o4 e" I% x) SRetroviral Packaging " u2 v2 W2 I* S5 |; ^# j
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MEDIUM5 W4 A- e4 t/ }5 N7 G6 b
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
" u# h7 r  g; `/ \) n& s  }293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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3 z4 L4 D* G' J+ a2 @293FT CELL CULTURE
6 Q1 V+ }+ C. v9 xMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS. [5 e1 j$ o* W
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF47 j7 @; Y8 B2 ^% I7 c  {

+ w3 ^: {& [4 Q. V0 NTransfection of 293 FT Cell with Lipofectamine 2000
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For T-75 flask& n4 t2 f+ w  H9 Z
Prepare 293 FT cell:
) I, y5 Q1 q: l8 q5 FPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.# M5 P4 C  T+ v. U
Observe 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. $ I$ B# x* k$ v5 d- X- p

# j/ V6 R1 z* J$ t. p9 i" I1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
3 p, O& W  v! m- `6 p2.  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.  
! z) c3 ?& _2 V3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
  g( ?5 U6 \1 v& p6 d5 G0 P* `4. Mix and incubate at room temperature for 15 min.
- B' K8 h; V) P5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.
( y6 G$ C2 Q, ^% y6.  Incubate at 37 ºC, 5% CO2 for 48 h., z' f# K3 E7 L8 V( k! m8 g+ [
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
6 f) q4 S4 n1 e: d! ?- o) \8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.  p9 ~- ?, n! O8 r0 X; [
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
* k* S+ g, J5 i# }5 w/ j* |6 s10.  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.& w& I  q( e; S7 I
; m8 k' p% a# _% ~7 ?( m  s
Preparation of human fibroblast cells (IMR-90)( y& q% g1 S7 e! D% F$ e
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.7 t7 F' p4 J+ ~. K5 M% h3 M' ?: q& A
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.* M3 ~+ r  ^1 W5 S, a
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
/ c/ M$ r. ?% \  j4 c' H! T) [4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.7 h( n) E9 B9 B# R
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.* m* F# d% c* u& B) y+ w8 h; @, T6 q
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
5 o  e; U! y9 i! }1 S  _+ I# d+ Q7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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) c$ b$ z" |& i$ v4 j5 C+ JRetroviral infection
' f# ~* A$ w9 W; r6 O  X. j, L1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.- ~5 H2 P/ M+ @3 i$ H2 _0 N) j5 W
2.  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.
, k+ S1 U; _/ j1 b& C( G/ Z: t3.  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.
+ `8 d6 Y& w& V, Y4.  Repeat transduction (including virus harvest) as described above.
: n+ m% @( V4 `" Z9 [5.  A 3rd transduction may be necessary.
0 M  ^9 L, Y4 R! \$ E0 v6.  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).
9 Z- Z* A; C% a1 s7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.% x! [, P7 t! d; X) T
8.  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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- O7 ^% Y' S; W+ j  F$ g问：unconditional human ES medium 和conditional ES medium的差别？
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: E9 S" u4 l" i: Q( C4 D* Iｘｙｚｅｎｇｈ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！