iPS 建系经验

细胞海洋

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4 m/ ?6 P% G0 k, M本文系ｘｙｚｅｎｇｈ版主原创 非常感谢
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IPSC Generation by Lentiviral System Protocol9 ^" ~. w- ]% y. }0 C+ ^
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Lentiviral Packaging ' S0 ?; Q3 i7 H9 ~) W4 f  w
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MEDIUM% X. J. G: {9 p' u8 x! {
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.% W9 V! S" k- |+ n$ G
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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+ J5 H, p5 B9 |% Q5 G" j- x! i8 s293FT CELL CULTURE% N/ L! @4 \$ G5 u1 z
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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/ z! W, t5 [  V0 y2 pREPROGRAMMING FACTORS
# v( p7 ^4 O" Q; X  YOct4，Sox2, Nanog, Lin28, c-Myc, Klf44 C! [. g* N/ \: h- E$ s9 x
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)  F& a9 j; w" ?$ @- ^4 R% k
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Materials Each T75 flask' ~7 G3 w; n* M' ]4 j/ j
293FT cells    10-15x106
  @+ X3 ^- W1 [' ^# x$ z+ V' JMD.G (VSVG)   5 µg; E4 K2 C6 q# K9 X0 F- Z# B+ Y
PsPAX2   10 µg
% p" W' p+ ~( g* n& |, QPSIN vector (~10kb)   5 µg0 J( z$ `* N  {7 l! w6 X: T! F/ r) I
Superfect (Qiagen)   40 µl ' T, k1 x6 R/ u: O9 x" z
IMDM   400 µl
1 v, S: u( ?1 v, ^; w293FT medium    10ml
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
8 U" ?$ l3 \6 b1 A2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
1 J% a/ L: C3 f8 I% o3 _5 J3 Q3.  Add 10 ml of 293FT cell suspension to each T75 flask.
  R7 f# Q7 W2 ]6 L  d: h8 f4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
1 Q1 R) v* G2 l, M0 _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.
8 X9 j& f% }3 _+ E5 N  Y6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
0 ?. R, r) W; a4 J" }  W- G7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.5 W/ H2 Y3 @" r4 \& O0 p9 W
8.  Incubate the cells at 37ºC O/N.$ y/ F1 M/ V5 x. n- [1 k1 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.
  z  m% g3 S/ C- P7 [% }2 c10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
  c( u8 H  q% O- Z: C1 W5 ]0 H11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.$ s, Z" {( ?6 W, M; s- A" T
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
, M, n5 T% `2 x; i4 ]; Z' ]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.; k8 c% s# p& T2 D
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Preparation of human fibroblast cells (IMR-90)
- X! n8 x6 w! ?4 W9 i. }9 I1. 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.
" [- n# O1 B$ F8 P: C# f( w2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.4 V1 k. B# Q7 c4 z5 D* |! C
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.3 J- ~* L0 L- b! `( R* [
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.- N5 E8 }) M. _/ q- _& f$ T- Z1 V+ a
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
. F2 f. t6 A5 J: a" H7 J; v) T6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
8 x9 i7 ~& [! j7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection3 C+ }- h) k8 h3 z6 G
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.1 x6 p8 W+ ~( o# ~  u1 g8 u* l. I0 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.4 z6 L$ y+ h. G" Z+ ?  H
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.  o$ A0 R. |! O/ D
4. Repeat transduction (including virus harvest) as described above.
" ~& T% D/ _( A2 l, e: k, Y5. A 3rd transduction may be necessary./ q* d) H$ o9 ~" T# ~1 [+ p
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).
! M* w) H+ Z0 L1 E+ f( h7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
2 X0 E6 T: x0 h- C8. 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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/ \4 f, y: }. A* U% J0 r' l% tRetroviral Packaging
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- P+ c2 K6 V" u0 g3 L" mMEDIUM* I" E7 E7 B+ c* m) s
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.5 V. o! F; ~8 B/ U& ~. C
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE
' @2 |( N* ]' c! u8 HMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 6 I$ x5 ]+ [3 l7 t8 _1 F

1 J% T8 Z  H1 F( A/ rREPROGRAMMING FACTORS- q- v4 a7 K3 [9 n1 `
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4! E( o: `1 X% Q) v7 r% |: z) e
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Transfection of 293 FT Cell with Lipofectamine 2000# M% r" S0 J) F3 x: V
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For T-75 flask3 O; Y( U% H3 K* ]
Prepare 293 FT cell:
  _+ A9 g4 S0 dPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask." O  O0 V& r# p! K
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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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. 4 u4 P* A; |, ^4 G
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.  
3 S- V$ v; a! K+ d3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
2 M4 {5 D' i. J3 k4. Mix and incubate at room temperature for 15 min.
) q: y: r- f, [! x5 ^' I6 f! l5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.
1 [" x, O4 d4 ?$ u. d6.  Incubate at 37 ºC, 5% CO2 for 48 h.9 b& m0 C8 G. W4 ?" n8 |  {
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
6 F1 s5 v) ]4 d* C6 J% N: P8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.. c( a) Z- {/ I- t
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
* K& e; h) g& W6 j1 ?9 ~* ^9 b10.  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.8 s  Q2 f9 a# V

+ i% O3 _6 L) [5 MPreparation of human fibroblast cells (IMR-90)
( \: K2 Z8 h9 L8 W5 P% |/ V$ O1.  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.
; T% i1 ?2 ]( r6 |* N# \2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
, W7 n; Z; `* Y3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
9 Z% y$ Z8 T9 h- L4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
, @9 g+ L! g3 r! {% P5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.$ P  }: F4 O0 M2 Q7 `
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.' a. E4 `9 \  y) `! r  S
7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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) {+ X. I% ^7 l( IRetroviral infection# |% }- g+ h5 R; Q
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.. P' q5 V6 X- h" d1 v4 G
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., h# z/ {0 O; h2 F; }
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.
3 x0 V1 D5 I( s  o$ u" S" K: P3 R4.  Repeat transduction (including virus harvest) as described above.
' O4 F8 P; U/ P2 A2 G( O5.  A 3rd transduction may be necessary.5 v. E! {5 ?4 c- k0 C
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).
4 k5 Q- h2 W0 o! \8 u# X+ y, m7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
8 w+ `* B% v' i2 Y8.  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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问：unconditional human ES medium 和conditional ES medium的差别？
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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！