iPS 建系经验

细胞海洋

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  q9 {) s/ {6 h. R本文系ｘｙｚｅｎｇｈ版主原创 非常感谢$ E# [( l* C3 D6 |9 T# A: f
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IPSC Generation by Lentiviral System Protocol8 ^2 l8 R: {# u7 }
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Lentiviral Packaging 3 O. w" D  i' @( B  Q* F8 ~5 a
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MEDIUM
1 e0 Q6 z' L4 b; n2 a) `$ D293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.& H8 h# u/ K5 r
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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( J7 l5 P. h) D5 u% b" A293FT CELL CULTURE  u# ~' ?; Y9 k7 J6 o
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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1 c5 F0 J! V3 m! C, o8 B) VREPROGRAMMING FACTORS
& ]6 {, m- \& e& NOct4，Sox2, Nanog, Lin28, c-Myc, Klf4% }8 E* [) C, x& N/ b

. W. D0 e( c/ |2 N4 @3 yLENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer): s8 M  d; A! g# \9 k4 }
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Materials Each T75 flask) r! y* c, E& t# q* ?7 M0 ^4 }
293FT cells    10-15x106
) a& [- P6 m# W2 MMD.G (VSVG)   5 µg( m( h! G5 X; a6 H5 X
PsPAX2   10 µg1 A1 @5 I9 e- o3 I: }( S
PSIN vector (~10kb)   5 µg
5 U* l3 y1 T7 ySuperfect (Qiagen)   40 µl % p7 x. G4 x/ \9 L& }. i- n
IMDM   400 µl0 C& Q) O8 v; Y4 i
293FT medium    10ml  v: l5 `! {) `* Z6 j: S

* `7 e- d  B4 V1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
: k( A& q5 }( v% `  y3 p2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
' o6 ~  P' V8 v3 L4 y8 q* |3.  Add 10 ml of 293FT cell suspension to each T75 flask.1 S! B0 ~  p. P% d2 D
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).4 u( Z3 D- G' O( @( O; P! B( {
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.
. `/ L; ^2 }+ v' L+ J6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
# \" l0 e! D( j7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
  k  Z  M4 e5 |3 t6 e& S0 T; N8.  Incubate the cells at 37ºC O/N.# R4 n8 l# H# |: U" c( f3 }
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.
& ]; P- o) j# |/ Q7 M, T; }% t10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.# M* G4 {# y5 Z6 f% L' y% ]
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.) n3 m: v! n1 F# @5 a1 ^: J
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
! }& k4 n+ ]! T13.  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.. Z# }' H" K* t% f9 E

$ R, [# d% O4 r( @Preparation of human fibroblast cells (IMR-90)6 G& N5 ]2 y8 }2 |- I" G: N& v
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.
$ s) \6 E7 S0 E* U4 }9 Y2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
5 m7 O. P0 P9 }5 G  b3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.3 L& |3 n8 U- P( g* K8 \
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.8 C! Y- ~, ^' w6 E
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
+ e) L. \% ^/ S5 h! V6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.5 i, y- K2 f0 Z  ]/ c3 q
7. Incubate at 37 ºC, 5% CO2, for 6 h.8 m# g% i8 y  B! V8 n. }  i% G

  }% w+ I8 V3 YLentiviral infection
: |' Y8 h( [$ E  v; H  m: \7 o1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
7 w* |' W0 s/ Y$ g* |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 L5 E, A0 i3 f' x$ G, s" p3. 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., V! s% V$ x# v/ Z
4. Repeat transduction (including virus harvest) as described above.
9 Q6 }9 V  S0 \- B8 U5. A 3rd transduction may be necessary.- k& P/ `/ c0 F, \' V3 H$ T% W- L
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).; w9 }# b# X% _( u$ a( E- y
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.  ~8 p7 p) e1 h) S2 @0 C
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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& I" \6 O2 Q) u; I  J, vRetroviral Packaging 7 E# y6 s2 [* l; n  p
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MEDIUM, Q2 U9 I0 a+ U! p, |5 M
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.4 y2 h5 R5 C; W
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE+ V( B1 H# e) {& L
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 9 k7 ~1 _  `* |' B5 m( K9 N
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REPROGRAMMING FACTORS
. }( {# T9 [, G% _, J7 ]7 v/ @6 NpMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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0 z# J& U5 Z1 V: mTransfection of 293 FT Cell with Lipofectamine 20006 K3 Z0 [+ ]# ?+ J" {
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For T-75 flask
, P( U/ l4 m; h! gPrepare 293 FT cell:2 S/ Q8 [+ p: @. k& R
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.8 P$ K  N$ e6 h; l/ 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. + `6 W! n/ K  Q# R( ~5 ^

! }- F  O6 R: G  o7 Y% h1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
4 a3 f) E# Q& c* U1 m2 \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.  
* L5 e  s1 p# A3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. 8 u4 m+ ^/ c7 \7 N
4. Mix and incubate at room temperature for 15 min.
) i4 l: ?0 J- s9 U5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.
" N! o: E% @8 \! K6.  Incubate at 37 ºC, 5% CO2 for 48 h.; ^  f* p- x" b" j" c
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.5 W9 ?' G  f" r) [
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.: V* o. K  L$ ~* |3 g' K4 ^/ g
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). ' K$ k$ a  ]& L
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.( X" ?% z# ^8 X% R% H
% f" L; N  \5 [  v! E% F5 \  {5 t
Preparation of human fibroblast cells (IMR-90)4 n% N3 r' P" ]! K' M( R/ V9 ?2 G
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.$ A' w. R; p3 ~, t5 z6 x' j" \" @% u2 l0 O
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.7 |# L+ R4 [( h5 E9 o5 n4 Z" k
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.. G& S8 n, y; _- r( J0 d$ W
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
! L+ T+ L: l1 _4 D  e5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.+ p8 X( S: t& `6 n# V
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.1 s/ i  v0 l( I: |
7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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0 d" s# H" @1 J7 v5 A9 D' l0 `Retroviral infection
+ L& L6 w3 K- y: E, T3 J# o. n: ^1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
* y: ]$ R: Y% X% m* h2 n2.  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.
1 C1 S0 o7 ?! v7 Z: x, {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.
! X3 D  ?0 F5 t( v3 [4.  Repeat transduction (including virus harvest) as described above.5 g- o, j' x4 r, d. M
5.  A 3rd transduction may be necessary.
. ?& M# r& ]5 b! M9 H# `5 j6.  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).( d7 D$ z! I. |8 L( l: O% ?
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
  Q$ L7 n6 ?$ S% e+ 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
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3 l: c: ]# L+ ]问：unconditional human ES medium 和conditional ES medium的差别？ 6 [  E' T  K2 Q. e% J, k/ A& c* B

2 i; i1 w$ B& Y% x+ k! U% iｘｙｚｅｎｇｈ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！