iPS 建系经验

细胞海洋

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! H: S- q, Q, r) R本文系ｘｙｚｅｎｇｈ版主原创 非常感谢7 B7 r+ x9 n1 W4 o
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IPSC Generation by Lentiviral System Protocol
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Lentiviral Packaging
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MEDIUM1 z- u/ T0 h( t5 G5 w, y
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
2 S0 R/ e! j: H1 p9 w1 n# Q" w293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE, C: A* M, j' C& a" ]
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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Materials Each T75 flask# e* Q% g2 t: q
293FT cells    10-15x106
: K1 V+ V. z4 q( oMD.G (VSVG)   5 µg8 K8 ]6 Q$ ]3 f$ L; v1 |
PsPAX2   10 µg6 R  m) G2 u( H" l
PSIN vector (~10kb)   5 µg$ ]% G! X& ~. M  d
Superfect (Qiagen)   40 µl
) a8 M1 q1 B& N. }% x) TIMDM   400 µl# m7 w' {0 e6 J) q
293FT medium    10ml
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' U+ s4 b7 ^1 y" H! Y$ h1 [1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
7 }% H1 [! r) k0 c6 o) e9 U. p4 ]/ G2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).! h% y  F3 F, ~
3.  Add 10 ml of 293FT cell suspension to each T75 flask.9 L% F3 O. P' d2 U# e7 q$ {
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).& J. m! Q7 Q- Q" c$ |! T
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.
7 T8 A- b$ H3 g9 X# d( Q( G- g: f6.  Incubate the DNA/superfect mixture at R.T. for 10 min.1 L& h5 r) h' z! e! |! `4 X% J0 ~
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.0 p1 W- M$ V$ D4 g
8.  Incubate the cells at 37ºC O/N.
" e3 `4 ^( P$ N. i# O- ^- g9.  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.
- O# ]7 I3 ^* v1 G10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
" z* K: O! F+ I9 M" A, N$ @6 O11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
1 y4 z4 ^$ a8 L$ v: y6 ]+ w12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). $ ^$ s3 x5 ^( |; K
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.
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Preparation of human fibroblast cells (IMR-90)) b1 \1 u7 o+ u3 l2 l9 ?
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." {! Z$ M: q0 h2 N8 S
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
2 t7 m7 N4 }5 h" w! w# Q( Q3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
+ V/ U3 \2 }$ G* N4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.( L6 \, F7 t7 `& D0 u' t/ y& `
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.  ~6 L. L/ M' I
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.1 |* _- t/ e5 l5 a5 }& O" Y
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection* X! M% w- u( P- }9 h" p+ X
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.  L* T, D. u* Y; }
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.  e) \' B$ a% r) S
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.
" R) w, E, `; D1 K3 h- ^0 a4. Repeat transduction (including virus harvest) as described above.
0 V- C6 D; L7 e4 s- H5. A 3rd transduction may be necessary.. W! l7 a  i. C  j( d- `) a
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).
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8. Day 18 (9~ 10am) Change to conditional ES medium. Observe any colony formed and pick them up when they grow up.

第一心音

牛人啊

zgyexh

好东西

grassroot

病毒感染之后再把细胞转到大皿里，那不直接在大皿里感染病毒来得简便些？

jameng

怎么都是293FT？这个比293T包装效果好吗？

zhangtcm

回复 Jonathan 的帖子
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6 S+ c! y2 ]0 p& T; [谢谢

Jonathan

回复 zhangtcm 的帖子  w; d2 P1 u& U+ S; b6 Z: F
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polybrene是在病毒感染细胞的时候用的，提高病毒感染的效率。Y27632是做完感染后换ES media过程中加的，用于提高ips的效率的

zhangtcm

回复 细胞海洋 的帖子
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这两篇文章有很大的参考价值，但具体有一部分细节上的操作，还要结合别的文献；另外，别的文献资料中提到的polybrene 以及 Y27632 是否可以 不用，对诱导有无影响？

zhangtcm

回复 细胞海洋 的帖子
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Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. 我看了很多文献资料，好象都要是Ug，而不是mg，是不是这儿错了。

周馨宁

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