iPS 建系经验

细胞海洋

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, n  [* J/ ~7 \" j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 - G7 M. r$ _. v* |" K% `

, a: C9 N+ k0 _7 f3 {5 _MEDIUM
/ r4 w. R( f- D6 X5 e4 }  c293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.0 ~5 ^# v' H, f) H" F6 n2 ?9 ]8 G
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.. h! x1 D  A$ K6 S% D
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293FT CELL CULTURE
. W1 [  _0 K% V3 MMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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: ]- T/ H  \0 `" }REPROGRAMMING FACTORS
$ }/ K1 r; l  r# v# m, I0 {3 pOct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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' g' G" b& j$ V3 J' Q$ S5 @LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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7 j7 h: [/ ^- D8 HMaterials Each T75 flask
( c( e7 T2 q0 s293FT cells    10-15x106
! K* Q. u9 K9 u8 v6 iMD.G (VSVG)   5 µg
3 P. k* O- M  uPsPAX2   10 µg
" C+ B" K# t( g1 S7 ZPSIN vector (~10kb)   5 µg9 X7 h8 L# f# i0 M4 F
Superfect (Qiagen)   40 µl - Q' r. W- B) V* ?4 J6 T9 ^1 D: @
IMDM   400 µl5 p: e* u. f2 X0 O( _7 W& |$ W2 J
293FT medium    10ml3 E5 P' W+ D( M
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
- J1 s' n+ z$ H. C# M2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
' P) U" [$ I/ V1 K' t/ T& ?3.  Add 10 ml of 293FT cell suspension to each T75 flask.
! E3 z/ n% h1 g; b) |2 T4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).* U+ k$ r1 J5 m! a; x
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.
: S6 I: y' {. i8 ^  Q3 }6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
* V3 ], N2 o# d( f/ Y1 r8 `7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix./ M  m! B, W( o' E! T( F
8.  Incubate the cells at 37ºC O/N.
" `" E$ q3 K8 B, D4 w9.  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.
( l  n3 V6 K2 [10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
. c1 L  N# k7 e6 K2 a- H( Y2 y# B11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris." G' `' e8 v2 ~! A9 s$ u/ o# V
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
1 C2 ]9 t" I1 y" G+ {$ Y13.  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.& t6 Z) U9 a7 W$ O3 h7 z

+ C! t: O; w7 l/ ?8 nPreparation of human fibroblast cells (IMR-90)$ L0 v  G. z# y( X- J  N" G  A
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.
/ K% V+ ~( u2 C, Q) u2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
0 W7 y' m7 c) `* r3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.. G) @8 s: s; @* V" T
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.5 O: o9 |) A3 [' Q
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.# P6 j1 Z' g5 W' o2 r5 a( q
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.1 T5 I- d% b! }/ _
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection
$ W, \& T5 G# `& ]3 V2 S1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
7 a! o3 `! }8 E; V2. 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.
6 ?6 n& N' p) s3. 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.
7 r9 o! q$ @# K7 f' v7 X% I" e4. Repeat transduction (including virus harvest) as described above.% ]9 u( i* }( U  x# A6 ]$ V
5. A 3rd transduction may be necessary.
0 s. b0 b5 k# a$ I6. 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).
8 [" y7 M! P9 U9 C7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
' T3 C$ M7 X! R6 Y, p8. 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( R! f  {8 C2 N. {/ F

& {& _. O. U+ j: ~7 c; BRetroviral Packaging & x- }: p: ?8 k! Q

5 }3 R0 c3 Z/ n1 }9 z/ YMEDIUM
1 M* |- l- \9 k4 O) u293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
! x; S$ p/ W. g4 B9 D  w9 Y6 x  }' \293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.* f1 |  j/ N) l

- B! r8 q$ F! Z. ]8 ~. s( p: G: R293FT CELL CULTURE
0 v- u# F8 h. q  Z% yMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS
& b" D, D) g  X& qpMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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Transfection of 293 FT Cell with Lipofectamine 2000% A. e6 b1 c8 l& h

5 S2 E! I6 b- q* @For T-75 flask
5 i+ m% g3 }1 e+ ]Prepare 293 FT cell:9 W0 v8 X& j- a5 L: a4 \
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
" Q$ ~6 D1 Y' E) d7 b6 vObserve 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 p% @) ~3 G& y+ E7 h2.  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.    S9 M# `# _/ [  g5 [: d6 Y" |' L! m
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
% ], T, }7 b8 M& Z4 K4. Mix and incubate at room temperature for 15 min.
6 }) g: e& f3 E/ b  Z5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.6 A* t% L1 T8 ?  I+ e
6.  Incubate at 37 ºC, 5% CO2 for 48 h.
2 V+ g" i5 `. x$ ?+ l( P- \; {7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
8 q7 P$ K8 V3 c' R4 _; g8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
; f- Y, G/ L' w9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
% X" `) q( N1 V- Y2 ]1 e10.  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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Preparation of human fibroblast cells (IMR-90)$ Z& v  I2 l, H% v; W
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.8 R; f6 s  s& O6 N
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.$ y# d( u' C( g* {: Y2 }
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
1 J0 F+ Z7 Z5 U( S6 V4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.8 x, B$ H3 R) E& e" X; D
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.7 Z: b2 @, h  }3 m1 `
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
" B7 M! O: S* o: a8 E5 ~; C) E7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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/ s: W# z' W# j+ lRetroviral infection
3 e2 d2 v$ E5 c, S. j1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
! y4 ~+ V: l" H5 G; m4 C2.  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( Q5 v  v' ?. p! H9 U( ]
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.
$ H! M' D$ [* i# m/ J2 N4.  Repeat transduction (including virus harvest) as described above.
0 N8 G8 b, Y) Y& V* y5.  A 3rd transduction may be necessary.7 A, V6 w+ }* o. Y# h6 [
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. z4 P6 C8 ], o1 L, h7 b8 }) }7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
; \9 Q! Q! |; r/ c  F0 H* `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 D# F! S, W2 v- _

5 B$ k5 t- Z& g/ p- i问：unconditional human ES medium 和conditional ES medium的差别？ % K( K5 j1 d4 T. B9 k( y, e7 ?- J2 }! \8 N

! p2 A1 j2 r9 ]0 d* wｘｙｚｅｎｇｈ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！