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
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4 W6 H( o" d4 s( w/ Y8 j8 H' b! B6 E5 yIPSC Generation by Lentiviral System Protocol% B) U0 D  t; s  T: D2 a! I
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Lentiviral Packaging
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MEDIUM# F/ V! S" W+ h% [) d
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.5 z. E; v9 C# p* b& \+ i  }% w
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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4 Q- U# A$ D! G# R4 y293FT CELL CULTURE1 _! e4 g- K* U! u, b
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 5 g: u+ W- E9 n$ m
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REPROGRAMMING FACTORS& b. }4 [3 G# c, z3 s* b' I  {
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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2 L( j6 O2 t: B# Q6 a& ^; y  ?LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)' e" L/ a1 U) ]; E

- B: t/ Z" G# \( y- O# `Materials Each T75 flask! b2 V* @& Q4 A# a1 U
293FT cells    10-15x106
: Q0 v* G0 C1 @MD.G (VSVG)   5 µg
8 W  N: y& C- x* v/ M+ vPsPAX2   10 µg
* Y( m! h, ^1 m+ s+ f+ |6 y/ ZPSIN vector (~10kb)   5 µg$ b7 z+ H1 T) \: X4 Q- Y8 F
Superfect (Qiagen)   40 µl / ^, [# T/ I4 D* t) _) H% t0 _
IMDM   400 µl
) k3 j. I6 l( m293FT medium    10ml7 @- N) k7 Q, f4 ], l
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
, F! ^, {9 }  Q2 P0 O! L1 N2 x8 D2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
6 S' m* A7 M5 D: R3.  Add 10 ml of 293FT cell suspension to each T75 flask.0 [5 u$ k  W% j
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
9 P. _8 b5 v, F& T. r1 b5.  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/ i$ _) i; x" p5 B6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
7 @! v- z) i0 c$ q2 Z7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
0 X3 H" m' [, J3 G8 l/ E, X8.  Incubate the cells at 37ºC O/N.
+ |- Z8 L: g/ [, W2 p+ x9.  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.. _7 X- B, ]2 x. n6 z& r! \
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
% @- J" ^% q8 Q" F( O11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
2 f" s. X1 V* {7 {12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). ' p- d( X' g# e7 J6 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.
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, q% t0 w: a2 F1 _! CPreparation of human fibroblast cells (IMR-90)- ]' k0 l& \4 J% P* M
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./ r. l* r  h) V( C( Q: n
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
( s9 N/ Y% o6 Y: x5 H1 L3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
5 f& T: {) ]2 G1 i4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
1 j! q/ W* E# i5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.. [6 i' `( {6 i& j/ @7 Y: F& d) L
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.4 Q$ Y9 e) Z% I/ `% K9 e
7. Incubate at 37 ºC, 5% CO2, for 6 h.! D+ t9 o' d: W
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Lentiviral infection
; z+ E: V' ~8 X( |' v1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
+ K# e: H4 n  ~* ~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.& Y" ]5 i3 U6 ]# n
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.9 s4 k0 C) ^5 {8 u3 P: d
4. Repeat transduction (including virus harvest) as described above.
5 |1 j. K. Z! R! z5. A 3rd transduction may be necessary.
( z! [/ L: \$ F+ R( A6. 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).( e# N3 Z# E2 o% ]7 G
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.* B' f/ T. C% g/ w7 L, y
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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Retroviral Packaging 9 j' o- }0 _9 v
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MEDIUM* V. c* \( n7 i. ^& U$ e$ v
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids./ a6 ^4 s0 \$ X
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.1 w6 N1 b! S- D1 w
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293FT CELL CULTURE" F" h( l: l, i9 s# G- q
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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% Q0 R% f( I+ h. B! h' J1 OREPROGRAMMING FACTORS
  S9 P8 Q) z( epMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4% ?. Y# V9 {( x& S

; p* a- F4 p' y$ w9 a4 ^0 XTransfection of 293 FT Cell with Lipofectamine 2000
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; ?! \% _7 O/ Z& d$ w& YFor T-75 flask
! K( D/ v; @: u* y& P  yPrepare 293 FT cell:1 L$ ~4 b* k3 M/ ]; Z% k
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.1 w, V+ f5 x$ s- Q. f
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. - q$ o: t- H, I4 f
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.  9 u% E* J4 D; g! q, O+ J& d
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. ! t6 f8 J, f; x3 r! O
4. Mix and incubate at room temperature for 15 min.
  F8 {1 |$ e( ^, 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.& X: f& K  n, D( d5 x
6.  Incubate at 37 ºC, 5% CO2 for 48 h.4 y% V2 }' l; c0 D$ j
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.+ o- L# j- {, }8 n" s" f+ X
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.* Z: ^2 [8 C) b0 v) f: n
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). ' @- `* P  f$ l; {  }; j  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.
- m4 T2 V% R) e! s; M' @3 U% N! u1 o9 Z* [4 t2 x2 j
Preparation of human fibroblast cells (IMR-90)
( Q3 X, @4 n7 z% P/ @. ^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.) Z$ ^* S- [  U3 H. L9 L' ~
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
% _. O1 K% d. W3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
1 o2 z6 e' r9 _- {7 X' A' V3 s4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.. [3 i; `! L. W" p% c/ U
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.( J3 v. ~6 ~$ p
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
/ q' z/ r  U! Q7 v* X7.  Incubate at 37 ºC, 5% CO2, for 6 h.; H( x& @/ l3 u' l2 i4 z, ^9 K
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Retroviral infection6 m/ Q* `4 S/ N1 F
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.# y0 ?# i' W/ {
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.# F: J; @" Y! e. 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.
; J! V, N8 q5 w7 b4.  Repeat transduction (including virus harvest) as described above.% W1 y6 Z2 y* Y' j) ]9 M6 ]
5.  A 3rd transduction may be necessary.
' c0 u& U2 s% ^0 v* D6.  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).
& S" w$ d2 R# P3 K6 R7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.: X5 W: C+ R. W
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3 @6 \0 d. r4 y; p5 f5 \0 ^

+ ^8 z9 f% Q( }9 F2 x( v9 V问：unconditional human ES medium 和conditional ES medium的差别？ 0 @" N# ^  L# ]
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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！