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5 f8 {5 b/ W* ~' @% g; v7 }! J
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IPSC Generation by Lentiviral System Protocol% W9 V( s% |; b9 v
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. h7 H# K1 n% }- h" _Lentiviral Packaging 9 _) \* @4 K- K7 W
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MEDIUM
1 B+ Q# `1 r) v3 z& g7 R+ |! x293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
- [( L0 b1 L) S4 x" W; q8 @293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.4 l4 X$ }( R8 g- Y

' G$ f' p1 Z% |" A1 A; l5 B293FT CELL CULTURE" l( @( M: E9 `0 H6 w9 i  j) a! q
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS
* {/ S* C+ @. Q+ ~' aOct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)1 M+ g* J6 q9 x0 N

9 H  o5 d( W& j- X* @0 hMaterials Each T75 flask' I2 w6 U4 t  ?
293FT cells    10-15x106. p) c. t# D# d* e: m3 M* v0 G
MD.G (VSVG)   5 µg
  G# k' H2 m! P! \3 _+ Q' E- yPsPAX2   10 µg# Z- _" Y) W9 B5 K9 T( c
PSIN vector (~10kb)   5 µg7 L+ U! Y* M5 A- N
Superfect (Qiagen)   40 µl ; F. m9 y9 a& K
IMDM   400 µl
$ m) _) }* B" L4 @293FT medium    10ml
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.7 F: i+ s6 z1 a2 e0 s% N3 n
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
0 W6 B* E( W* ^3 t3.  Add 10 ml of 293FT cell suspension to each T75 flask.% o/ T1 ?  H' Q8 u( @# s) ^
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).9 c% H6 ~5 A. l# {: @
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.) e. y; O" K3 f  K6 z( p
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
/ I& V0 I1 J$ e/ Y7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix./ g% V8 a- g8 `. g6 H. D  [' y
8.  Incubate the cells at 37ºC O/N.6 d2 A2 ~! [9 f) f, h1 |/ N. W8 I
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.
/ D$ t2 K: v! O& g10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.2 g! Z( m$ t- G' M! v9 t& T% m
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
" K4 K5 [( `( q3 ^- C: q) ~; b12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
) k. Q: C' _, B7 d, `0 M5 {8 ?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.; D9 E- E# D3 Y5 ]) \' F
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Preparation of human fibroblast cells (IMR-90)- h  I/ `1 Q7 J. I& l: U
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.6 i) S5 Z0 i9 E# p" Q: E9 u, O; G
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.+ n+ C. r: u/ ^. m
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.3 o" f% [* e% c. H( @
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.6 |1 C  D  S1 F6 T
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.1 \6 }4 h) }$ M' `# s, K
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
8 ~4 Q. ]1 w; e  t3 C+ U% v# [7. Incubate at 37 ºC, 5% CO2, for 6 h.) j: w( U" O% h! F1 J% M

7 a; {  @; E* J" {9 y7 }, xLentiviral infection* |' E; f1 H* p
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.; s: B, H8 l6 W* ~4 W5 _
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.
0 _# r* v  Q+ d8 w. b$ O3. 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.
1 g! v+ j$ y: D( W! t( v! y4. Repeat transduction (including virus harvest) as described above.# b3 E: c& \: X( C( m
5. A 3rd transduction may be necessary.
! u2 ~: `4 ]% F, T0 S6. 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).% ^" O: Z, b' E
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
" B% H1 R0 Z7 d9 c, }, d0 ~0 V8. 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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+ _) S; M9 p9 T7 B3 ^Retroviral Packaging
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. s; o. D. a; o1 v! w( r1 SMEDIUM2 p" J; P6 i$ m1 H
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
1 x+ M5 j: M- F" }293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.& y9 K, ^+ C% F; a2 \
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293FT CELL CULTURE
% L7 ]% @# k9 e  E: N( eMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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0 J6 _; h! V5 P' H6 l7 _REPROGRAMMING FACTORS
3 _/ G( C; w1 S1 E1 CpMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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+ J; I# f& Y- [3 N' h, OTransfection of 293 FT Cell with Lipofectamine 2000
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( ?) T/ {! u% [. u7 N: [) j' ]! LFor T-75 flask
* ~+ T* p  o2 d3 d9 t! I8 p$ uPrepare 293 FT cell:
8 Q0 k: ?! i: k9 ?4 EPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.2 y" L2 _* j) f: W! ?. \, r
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. ) O" C1 t& q. A, v% G( B7 K
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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. . I  h3 k& h) S" @1 J$ 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.  $ n% v: C8 q+ }7 A
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. # K$ U, }: q/ C
4. Mix and incubate at room temperature for 15 min.
# B( S0 ]8 G1 s: H$ f2 v1 k; U4 x! X5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.3 p) `. b6 N; {" Y
6.  Incubate at 37 ºC, 5% CO2 for 48 h.3 K7 r& r5 V: b" f" ~: ?) ]$ B. I- U
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.6 g, E: @1 i" ]! }0 z/ J' ^
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
" d6 R" ^2 j* k) Q; B9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). ( D4 a1 K1 m: q3 v# L7 O0 E8 N- 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.
! G5 b3 G3 e! }3 f+ `7 q# S4 m) E" B9 c
Preparation of human fibroblast cells (IMR-90); n1 @! d! F; I; ?+ ]8 Y
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.3 M: S$ a$ Y; D! R3 W5 z
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.5 R2 {( Q, Q1 y: [& w' T! z: Q
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.& k3 ~7 n/ v9 w3 K+ Q# C" k! F
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
! V, F$ r, I; w5 O9 U5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.- T1 B: k. l( T: k. E
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.- z5 z" m3 P. h7 c/ h2 i$ P/ }; c8 e
7.  Incubate at 37 ºC, 5% CO2, for 6 h.: \# o% }) Z) R2 X2 j
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Retroviral infection6 [. _; @# \: l4 E
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
- n7 [( @' V0 `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.
- D) y% Z* @4 m) B$ f3.  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.
) f; _9 }6 I/ x+ G4.  Repeat transduction (including virus harvest) as described above.7 H/ |& c: F) U# P
5.  A 3rd transduction may be necessary.
/ S4 H0 ]2 g# n$ _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).$ b, [- B/ Y1 f4 H
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
- [; A7 f1 P" S8 ?$ @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: \5 K$ Z8 W5 O: F

' v9 i( M1 P9 i问：unconditional human ES medium 和conditional ES medium的差别？ 1 K; R# a  B4 H' @  M

1 |% z' c& i; G" r. _0 |( Uｘｙｚｅｎｇｈ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！