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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IPSC Generation by Lentiviral System Protocol
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Lentiviral Packaging
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' s& H' D9 G1 }* H: a- W. IMEDIUM3 J& S- f$ B9 ]- J
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
# q- {5 `" r3 Z/ U) e; Y293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.6 R) ?1 H; z8 W( R5 P
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293FT CELL CULTURE) M, u4 k! O. G! _% L5 D3 t% I# ?
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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6 A: K* O- B2 H' D9 o  E6 w* vREPROGRAMMING FACTORS% g( t2 w" L# E3 W9 c1 L% d& t' }
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4& y: R7 t% c, j: `* ?! |7 z

9 s" u, D+ O9 o9 YLENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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Materials Each T75 flask) f! Q* U) O7 [/ O
293FT cells    10-15x1063 h5 M) E% C/ [8 P
MD.G (VSVG)   5 µg
" \4 [4 v( X" u/ E+ U6 W- l/ iPsPAX2   10 µg
/ _3 k2 t  \5 _" M" k: U& `; ePSIN vector (~10kb)   5 µg
2 N3 ?7 h1 p/ R6 Q9 ?  i/ @' _Superfect (Qiagen)   40 µl
1 U" Z6 l. j; c6 hIMDM   400 µl( ~, L  }4 f1 M0 ~
293FT medium    10ml5 q3 A$ c( G& \! s

, Q+ t2 ?+ u+ m. }6 E) ]1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.7 e1 e. O4 I; ]. d
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
! f$ P5 R" G. k/ Z3.  Add 10 ml of 293FT cell suspension to each T75 flask.
/ p& _4 @( b# ^2 B: |4 Y1 j6 B4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).) K  F/ o2 q! b  P' M7 k% z& `) _
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.- O, `2 `! C& H' B- ~
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
8 h: u" a% A2 t4 P8 A! a7 n( Z7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.& e! E5 u8 w6 h6 V7 ]2 j% Q
8.  Incubate the cells at 37ºC O/N.
  b$ Q3 e" A6 y( D* k) K- x9.  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.
5 X* }' W& d3 l6 |5 c  b' _10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.+ e8 }& C6 A4 u5 s8 W
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.4 O& `& ~% D4 N4 L* U# C% h5 ^( G
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). 9 c6 N; r- S# T3 Q
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.
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2 }% s2 h: b3 H( v* x! o' hPreparation of human fibroblast cells (IMR-90); K* X9 _4 {& t2 M% P4 s$ {! E
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.
7 B, C2 J/ y+ @2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.9 e& i5 v0 o; t8 `  S* N
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
' l- h; O5 ?) I: j. F4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
$ E" q7 n$ c# c5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
# |5 ~; R& c( }. P* r+ I* Y( ]6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.( R3 G( _* I0 ~" a1 K5 H
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection
& x; m  ]" j! y2 @1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
* R; V" q, m+ [2. 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.- d7 o, b0 P8 N$ T
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.
& u5 f0 o" {( q3 Y( D4. Repeat transduction (including virus harvest) as described above.
% X" o7 Z1 K, _5. A 3rd transduction may be necessary.
9 ?6 t5 E# T- j' o& D5 v- l6. 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).
" T( G  l$ V# I7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
/ J5 O' E% x/ U7 ]: [& @1 |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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Retroviral Packaging
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MEDIUM
2 V3 D) t" R7 D' P5 k8 K293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.8 [( V, h2 Y  s  k
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.- b% p6 W; Y. @- G5 T: M
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293FT CELL CULTURE+ C( q# C5 _4 B
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 6 h% m( J/ s* _* |; U2 P7 @
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REPROGRAMMING FACTORS
& F. n+ J! t7 r  r' EpMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF49 \+ q7 O1 D3 s' m  l5 Q

9 ^+ T' o# l: \/ Y( w* C1 @Transfection of 293 FT Cell with Lipofectamine 2000
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, I( Y  n2 Q- O; w6 o/ eFor T-75 flask
  T( W: S9 p" \+ t! W$ A5 G0 aPrepare 293 FT cell:1 b+ S: d3 }7 X6 @
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.' V/ v  A. Z3 Z
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.
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, _) ]5 z0 f' _1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. ) e- ~( ?6 O0 k9 M
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.  
) c( e, c% ^# V3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. ) Y9 C1 W5 u( r* f* O
4. Mix and incubate at room temperature for 15 min.
6 V/ U! D/ S! f! l+ t- J2 ^$ f* O) j) H5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.
7 ^) m* W. i  T  ]9 M: A6.  Incubate at 37 ºC, 5% CO2 for 48 h.
; |4 Y: g! q% J7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.( ^* }& u" b5 N0 h: Q: A
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
3 f1 }; J. ^' x' N5 ^+ k9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). : H5 d  i/ T8 }! M
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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2 L/ X% e2 f) [5 gPreparation of human fibroblast cells (IMR-90)  F  E5 r/ @, j* X3 Z! l
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.
& C. u: {! V6 `% m% \6 R2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
+ d% R7 c9 t7 `5 L3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.  T4 ^9 i0 L6 K: R+ U" x  G
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
$ ~- s3 y9 w% l+ l4 r5 U5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.; v# ~' ]- V0 Y# {
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
  x$ |+ D6 E. k" ~4 l9 e. f! P7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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& w5 P% a6 }5 t' KRetroviral infection  A1 E, s  Y' u4 \3 h
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
+ j: L" E6 M) r5 C5 U2.  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.
) l% f* s$ p" Z; U" t3 z" J- ^* a7 C3.  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.
& \& q7 w$ @6 l4.  Repeat transduction (including virus harvest) as described above.
, m- K2 o: s7 g7 _1 P$ s& i5.  A 3rd transduction may be necessary.
% O/ K: c6 j' P. H& I- \( R- B- p9 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).
6 {3 m5 O: M0 U5 l6 v$ i) G7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.! {+ g$ K! {6 l5 H) 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1 \/ E- d* W- Q1 t& b+ d
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问：unconditional human ES medium 和conditional ES medium的差别？ 6 n+ W! n! R5 \& h  y  G- P- u+ d

2 x( \0 X9 U' `0 h% s7 A6 j; s) jｘｙｚｅｎｇｈ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！