iPS 建系经验

细胞海洋

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  E4 i- f. E6 q! r本文系ｘｙｚｅｎｇｈ版主原创 非常感谢) x  n# i; I1 M" r% |- F7 }

5 Q2 Q  L3 g* P: ]$ oIPSC Generation by Lentiviral System Protocol% ]& j% N2 _: o( B8 a3 x$ O8 e

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Lentiviral Packaging
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MEDIUM
  o! M$ E. G4 d' [293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
6 G7 k7 i7 @6 Y1 n7 E* ?* c293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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# P* N0 n  X* l5 m% U293FT CELL CULTURE
6 {/ P; B- k' V  T' zMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 3 f# {5 K, ?1 Q- j
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REPROGRAMMING FACTORS4 Z! ~( [. g' S& B$ M# h
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4( q9 L2 l( k- g2 N+ v4 H6 ]

+ B/ l: `  a+ {LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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2 T2 B6 t$ \+ T  pMaterials Each T75 flask
, k6 @( w- [) ]; k  i293FT cells    10-15x106
2 T3 G. c* B! LMD.G (VSVG)   5 µg; H; e: K2 I1 R! @1 Y: f7 V& \
PsPAX2   10 µg' {' C, A$ y1 S& l5 A
PSIN vector (~10kb)   5 µg5 e+ n6 Q- o* k$ S+ F- d- t) b6 u* g
Superfect (Qiagen)   40 µl
0 q4 d, Q% @  y2 g5 t% H2 AIMDM   400 µl
2 }. I8 m5 f. Q6 n- D& o293FT medium    10ml
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
& a; A; j+ m* T1 J8 u2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).3 W- a; L3 g0 h6 {' u* z
3.  Add 10 ml of 293FT cell suspension to each T75 flask.# R# q. v1 z, N
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).# {& x9 _$ o$ h. r/ F0 s
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.
  M  N% E/ J7 D: g  a7 O. E# s" x6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
9 d" Y" D3 f+ d1 S7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.: a8 h* ]9 f7 K$ A1 ^
8.  Incubate the cells at 37ºC O/N.
8 L& V. ]1 M: k# }' M# c9.  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.
: x5 P. m2 i0 c6 s' h10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.; Q4 {* k+ V( F" n& D$ e* J. l
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.2 R3 o7 ~! d% G; g$ x& h
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). 7 i" x" h; a% h  r
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.  o8 ~; m1 n8 |+ h' ~7 x
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Preparation of human fibroblast cells (IMR-90)
4 ?* T5 e! {1 s; \2 I7 b" E1. 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.
/ D- C- H- N3 N! Y5 f  z# Z2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
4 |/ F1 |1 i" k, A8 @0 p/ `8 t! a3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.8 _. a4 J8 x+ Q7 t
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.: Y0 n7 o3 y2 A3 a) h6 q3 b( v2 w
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.. t: s: z( G- E
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
0 ^7 E  _# p2 b: Y) [7. Incubate at 37 ºC, 5% CO2, for 6 h.$ I  [4 n5 c3 K( X+ j2 Q/ C
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Lentiviral infection5 T$ k5 f7 _( \) h# U
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.' O4 u% I# S% C. K
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.
" p3 z  z; v: Z  W) m3. 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.4 A; Y9 _% ]+ l: j" ~5 T
4. Repeat transduction (including virus harvest) as described above.
. {9 z& S  l0 Q$ \; z, z$ F8 k, X5. A 3rd transduction may be necessary.5 V8 Q3 P* f3 _5 }
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).
+ G2 X& j2 j( g) G" }% M/ Z7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
4 B7 z5 m& G3 Q8. 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* p& L, C$ {' o% g0 Z  V

/ b0 |  u+ M; L% B9 TRetroviral Packaging 9 O& y; V1 t$ x$ g8 V

2 |8 x) G3 h1 P" u% S2 T. O& `MEDIUM  {& {( Z3 C4 m. x8 r- F* T8 m
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.) ^0 \& i0 q) @# N+ Y3 J& z
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.7 X/ W8 J% [1 d7 @5 X

9 @9 C1 }. R/ {" K" m293FT CELL CULTURE
% g% E* |8 p& F0 F! hMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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) {& Z& v! z0 q0 m* f: w9 Y3 f& AREPROGRAMMING FACTORS: y. j/ Q( F* O
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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Transfection of 293 FT Cell with Lipofectamine 2000
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% i# p0 k3 o* Z- N; R# S- Z' vFor T-75 flask0 {3 f% ^: L5 h: C/ w0 }
Prepare 293 FT cell:, F4 v$ V% R% S+ I/ ?
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
0 W) B) L* B& j5 _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. 8 x6 V+ k) a/ _( \
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/ X3 r3 G  i: t( h6 k+ n
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
+ @- l5 ?2 e, k. W4. Mix and incubate at room temperature for 15 min.
# U7 F1 H' E8 C& ^5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.- \0 ]& R$ ~7 I. X8 Z( M
6.  Incubate at 37 ºC, 5% CO2 for 48 h.
2 U4 u) Z1 A( X2 R6 h- c7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection." @( r3 ~) m; B% ]: P+ u1 w
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
. K) `7 x  S/ k# P0 I9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
  a$ C, s. i; _5 O10.  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.* y! r- A( F; M! \, z/ c! b# V- e
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Preparation of human fibroblast cells (IMR-90)$ v3 `5 E( c/ ]0 [" z9 _' ?
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.1 N( u& O4 d! B$ n+ [
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
1 A2 I. i1 Q! B8 |0 b3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
9 S- Z5 H( A0 E* x9 H( o' g( _2 m/ S4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
* u8 P% V6 b0 S$ E6 x5 _5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
) i4 o, o- J& f/ X, e6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium." S4 b; H8 g8 u% o1 T" A
7.  Incubate at 37 ºC, 5% CO2, for 6 h.- O* u7 i$ j) f& x% n$ {
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Retroviral infection1 s( h  j: q( }6 [- h$ g# `
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction." V- Z6 e9 J6 L. n! J3 e
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.1 w) Q, u% U5 b7 p
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.$ P8 K  h/ X( x# n/ M4 L6 V
4.  Repeat transduction (including virus harvest) as described above.
' j! z6 Z$ Z% B, ?: ^3 ^5.  A 3rd transduction may be necessary.3 O; j' g$ ?' b7 G. m2 E' e
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).
# M5 M( _' E) e7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.7 b; r  I& M) f! y
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: e$ ^  V5 w; ~6 K- k6 ~( R

- x% f9 h1 l  W6 P7 G+ r问：unconditional human ES medium 和conditional ES medium的差别？
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. {( I1 c. ?( l, F5 `% l5 _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！