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细胞海洋

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本帖最后由细胞海洋于 2010-5-5 23:12 编辑 4 i; r/ `4 S5 a# @# ]8 ~

本文系ｘｙｚｅｎｇｈ版主原创非常感谢
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IPSC Generation by Lentiviral System Protocol) M2 W) ?1 T. M: t( |, o! z


Lentiviral Packaging * [6 G3 w0 \! {6 t9 w# q. ?! a
5 J2 \  `$ e2 ]+ `
MEDIUM
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.1 O, b5 A1 {7 x  i" v
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.$ m% q' W6 O# ^1 Z

293FT CELL CULTURE
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4

LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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Materials Each T75 flask3 S/ R( J0 y5 X0 b; r7 q
293FT cells 10-15x106
MD.G (VSVG) 5 µg3 s. V7 `9 @. H& d+ _$ m# ]
PsPAX2 10 µg( O9 M" a8 _8 H9 ^( q3 d
PSIN vector (~10kb) 5 µg( N  v# U* f) S% k: j8 F( C; j
Superfect (Qiagen) 40 µl
IMDM 400 µl2 u! K$ ~; K1 n; I0 F
293FT medium 10ml( U3 s8 s, o" B1 t4 L. `
9 [+ z3 T9 G5 q  b% n  A: p: }
1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).9 ^. q* Y9 y( ]' D: D
3.  Add 10 ml of 293FT cell suspension to each T75 flask.
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).% K% D' \8 m) D! _
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.
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix., g- G  q0 `5 K5 [# d, f4 D& k
8.  Incubate the cells at 37ºC O/N.
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.
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
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.$ `2 e- R7 T' F- z9 q0 U7 w) ~3 D& i
; `0 F& G& q- Y
Preparation of human fibroblast cells (IMR-90)0 h) c' G+ c* p' X( z% D0 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.. m0 c0 m6 j: \( K. D$ f# @4 {$ n
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.- @4 T+ L4 e  ~( c
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.# A* P0 b0 N7 D, w+ O
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.! N/ }, s: `( }% e. \" l, m' Z
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
7. Incubate at 37 ºC, 5% CO2, for 6 h.( A" t. ^3 n; ~( E1 d

Lentiviral infection! u$ L; |. f! s$ \. t# A# Z2 }
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction./ h% G( J' ?4 x6 D
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.
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.. C& ]6 L% N- K- S, T3 c
4. Repeat transduction (including virus harvest) as described above.- u( i  D% s: [) @1 ?0 p0 f: v% h, R9 ^
5. A 3rd transduction may be necessary.
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).
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.! \! ~: U) T9 p  Q4 A- O; l1 l6 C
8. Day 18 (9~ 10am) Change to conditional ES medium. Observe any colony formed and pick them up when they grow up.

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发表于 2010-5-5 23:10 |只看该作者

IPSC Generation by Retroviral System Protocol4 ^! h9 d, t7 B

Retroviral Packaging 7 B2 X+ Z+ ~- ^& V7 g, o8 [

MEDIUM
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.

293FT CELL CULTURE
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. : I3 a; Z( j% Q( S; F- ?6 o9 F) }9 m, [
# B, {% s0 e: i8 J
REPROGRAMMING FACTORS
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF49 B! N& c0 B! t+ T% Z
1 d3 d* }; z  z0 @* G) O+ {
Transfection of 293 FT Cell with Lipofectamine 2000
. \9 `2 f& X6 `: U! j" j
For T-75 flask0 f% ]' f! m3 z# f( V' T
Prepare 293 FT cell:4 H+ x: ~9 k3 X1 B0 J3 T5 k
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.# A; C& [2 [) A9 H9 X- c3 w8 o% d
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. " y/ o: I6 H; E2 c! s
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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
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.
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. 1 {0 I& G4 l. D1 g. a4 x
4. Mix and incubate at room temperature for 15 min.* i# U) r4 R& h8 n
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.% g3 M. e/ d% |7 a8 m  |! H
6.  Incubate at 37 ºC, 5% CO2 for 48 h.* L4 B7 i0 I4 P. O
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). ; G0 A8 T) d% x, p9 J8 C4 u
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.$ F6 [% p' w8 ?! J' `
- b- G$ |% Q4 I1 ~) A
Preparation of human fibroblast cells (IMR-90)
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.
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube./ l& H. l9 n' G6 W, [
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.) r, W: J+ ^: \8 u+ X" b
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.- n1 }6 T" I1 k" d, a' c
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.  ^# w; F3 h6 @- A4 P1 {# ~
7.  Incubate at 37 ºC, 5% CO2, for 6 h.+ e3 e- x8 z  S1 ~& W* a( H% d9 y& r
7 x* K0 x6 K: V. U6 ~
Retroviral infection3 m2 p8 f8 G5 C' e  F9 ]2 ~7 q
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
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.
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.
4.  Repeat transduction (including virus harvest) as described above.4 L: T4 ?3 j# N, V  j
5.  A 3rd transduction may be necessary.! y" |, E0 V5 b4 }! a
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).
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
8.  Day 17 (9~10am) Change to conditional ES medium. Observe any colony formed and pick them up when they grow up.

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细胞海洋

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藤椅

发表于 2010-5-5 23:12 |只看该作者

转帖一个问题

问：unconditional human ES medium 和conditional ES medium的差别？
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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。

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