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$ f  V3 ^0 `/ _8 l9 B' g
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IPSC Generation by Lentiviral System Protocol
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Lentiviral Packaging
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! a0 j' U6 A; O$ y1 c; IMEDIUM; Q9 i, `) J# Z  V1 P% m  `
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.1 f0 @$ b2 m: P' r  P' w$ d/ u; H1 ~
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.( x( G4 u* u. V# a& n1 G

2 z( `+ S% `# Y' J; \2 {9 G" z: _293FT CELL CULTURE
# Y# M7 ?0 ?& a$ XMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. % e. c+ Y/ z* Y

) Y4 N$ W- z' n' ^REPROGRAMMING FACTORS
, X" ^! |2 Z: q  t6 n6 uOct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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Materials Each T75 flask
2 `' p5 p! ]( V' p293FT cells    10-15x106
7 f3 _, V' A% {$ }MD.G (VSVG)   5 µg( s) X, X  R4 m
PsPAX2   10 µg
6 n3 p3 F  @* G  H' n3 M1 i1 EPSIN vector (~10kb)   5 µg
, t+ a0 q+ Y' y" n, O0 G! ]8 h5 ESuperfect (Qiagen)   40 µl
+ R* P7 H4 G5 w" E3 ^' `9 \$ [! ?9 }IMDM   400 µl
; {2 R1 i6 G' ]+ J293FT medium    10ml
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1 l/ ~2 q9 `: B$ L: q1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.: f& j0 B6 q# T  Q. O
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
7 Q( e) o1 X/ E. R4 y0 K3.  Add 10 ml of 293FT cell suspension to each T75 flask.  i- M) K2 X0 t5 G6 a( u+ ~
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
1 M6 ^, X9 o7 t: I5 b2 R5.  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.
# ]) ~+ `4 H8 \3 r! i: w6.  Incubate the DNA/superfect mixture at R.T. for 10 min.( a5 @0 P  @/ E3 Q
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.2 l/ G  t6 t4 g1 _  [& g5 l
8.  Incubate the cells at 37ºC O/N.
9 S8 c* E1 g+ U9.  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.
) c- Z; F6 `. ]# \; m10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.5 }$ P( m$ M+ K; o
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
3 @! ^! }4 ^# H  ]; r12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). 8 r/ ^, k  B, E+ z: r) g2 P/ `. }
13.  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.8 w6 l! v& M3 M

% U& t4 _! k2 B7 X4 R7 EPreparation of human fibroblast cells (IMR-90)* Q/ O: g7 Q. h- b( f
1. 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.1 [: T, `6 c" j& w
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
" Z/ T$ `5 w9 k3 u- b7 P3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant., f- B6 ~! B( F1 C; j/ V$ @
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.* X+ n# S' |  h. I- |0 u
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
, h" v' Y: w* o2 g$ ?6 y6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.6 `- C6 F' B% _6 v
7. Incubate at 37 ºC, 5% CO2, for 6 h.: A3 u! k1 K8 R% o9 B' ^' i% l
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Lentiviral infection
8 o6 O. ]7 k+ r& I; y7 q1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
6 J; x* O/ C+ E5 q3 X; w& h, U2. 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.
5 U& K* Q8 m/ q3. 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.; I. Q* E& Y3 q2 Z5 ]% G: J* x
4. Repeat transduction (including virus harvest) as described above.
. h/ P% S2 A! a5 e. z- X# c5. A 3rd transduction may be necessary.: w' K. _& d+ ~  U
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).
9 \; |& k. m3 o# P7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday., n$ @. `* S$ X& L% F
8. 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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9 b1 E. B9 V' N3 j; }+ M  E, Y; VMEDIUM' ~# [) H* H0 a" e/ ^
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
+ ?% r4 U* L' Y4 r' c, v. c293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE9 ]: h+ Z0 e# c8 Z2 M( x$ t8 h
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 3 E5 X- A3 w1 r1 ~
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REPROGRAMMING FACTORS) O. }7 W2 h9 L1 Y
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4; J9 b% m6 O! C' l
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Transfection of 293 FT Cell with Lipofectamine 2000
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1 \* V% V0 z& S& \For T-75 flask
! c5 t* A$ ~! C& SPrepare 293 FT cell:6 \# f7 W- ^- X: X
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
! _4 M; U! N/ r; v# P" gObserve 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. ( n6 n6 A# l' t
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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. # M0 l; P% J, d6 O7 |. r$ A# v
2.  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.  
2 X6 b/ c. a4 O: k6 `3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. " Q; X" g, S) D) F6 j; G. T) n
4. Mix and incubate at room temperature for 15 min.
! t0 t/ p% Y- B/ u  t5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.- k0 f7 Y; i9 i4 v* f! D. w/ C" X
6.  Incubate at 37 ºC, 5% CO2 for 48 h.
2 I  F1 E0 K% w3 q7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
' J0 ?; h. k% K4 }8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.) R, _1 T6 D$ v6 ]* e
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). + N+ n/ C% s( o* |5 Z; d
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.
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, ^: R6 O" Y) C% [9 wPreparation of human fibroblast cells (IMR-90)
( J8 z' I( G8 P) V3 i3 Z5 U1.  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.( d$ K+ }) W! x5 A
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.( r& t6 H* ^/ J; [  f' V
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant., E. e+ Z+ d" e5 v5 t/ [
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.' L* X) }: o- ~& a
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
9 S+ k* M8 T5 I  y6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.2 G+ i7 _) c# h+ _' k. I0 ]. Y
7.  Incubate at 37 ºC, 5% CO2, for 6 h.- G! o! }7 N1 [2 H. j. P' y

! G+ w4 H" V0 E; ~# }9 }Retroviral infection
- m9 _+ K* `' \5 S8 L! R1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
' y; ?$ N! g9 v& p4 B2.  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.
. e, @9 p+ K+ r7 j! J- p3 M3.  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.  \2 ~* x' f9 Q, L% M, M
4.  Repeat transduction (including virus harvest) as described above.
+ E$ O, d# ^* O$ A: v2 v" n  J5.  A 3rd transduction may be necessary.5 n/ y* p8 L1 {7 N! L! u
6.  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).
. ~" U1 ?9 S4 [$ [7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.+ d5 I1 C! J0 d. b  [
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  H3 v% b* Z3 U0 o5 {! v0 ]8 C+ f

& @' r: \* q+ q2 A: ]+ [# }问：unconditional human ES medium 和conditional ES medium的差别？ 4 c. V8 Y$ K! d1 z, j8 f

$ ^9 y5 v/ R. _% `" Sｘｙｚｅｎｇｈ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！