本帖最后由 细胞海洋 于 2010-5-5 23:12 编辑 ' J0 B! Q& u$ O0 K1 z6 v+ x0 y$ H( m6 m' P7 e2 [- x+ ]
本文系ｘｙｚｅｎｇｈ版主原创 非常感谢 8 y% k E+ v5 Z: a. t0 @2 M" v6 u& u2 j1 U- K- ^# I+ r5 x
IPSC Generation by Lentiviral System Protocol & f8 L6 @* Y3 R2 F4 X4 f O5 ^) P" Z& n$ h/ p
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Lentiviral Packaging ' j0 ^( G; Q/ S/ F. Z) y
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MEDIUM : u& L; A1 W, C* c293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids. " Q/ s4 Z4 D f6 `% n, D293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.# s/ ?6 \# h9 X4 l' {& h
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293FT CELL CULTURE 3 |6 z6 J k: LMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. $ p; L8 Q- a" E5 z7 [( b2 j" r; Y0 d- k" C5 ^
REPROGRAMMING FACTORS " w. u* m5 c f; D3 QOct4，Sox2, Nanog, Lin28, c-Myc, Klf4) e! a5 @- M$ S- s8 [/ a
6 o+ }/ ~- e* o ?LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)8 j, D5 w8 X1 t0 R
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Materials Each T75 flask 3 u( F& ]3 h1 ]( B6 D293FT cells 10-15x106 2 C3 J- `4 v5 ?% i& wMD.G (VSVG) 5 µg 3 ?- U( e! }- m) m0 r' bPsPAX2 10 µg 8 a& h6 z! T* APSIN vector (~10kb) 5 µg6 m+ O& K& _2 ^
Superfect (Qiagen) 40 µl j: x) i1 ?. }0 ~+ XIMDM 400 µl! r& U5 B2 v/ U ]! l" |3 s+ _4 b
293FT medium 10ml @4 Z1 y$ R" @4 l( }5 ~9 A9 ]
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1. Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization. , m2 r5 ^$ n# d0 I7 Z) H6 Q2. Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).3 _. X4 a% `1 U- Q: u
3. Add 10 ml of 293FT cell suspension to each T75 flask.& _( t, S* X) R
4. Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).3 s S8 i! M9 |1 E. Y& N+ h
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) d' C9 R2 y, S9 |6. Incubate the DNA/superfect mixture at R.T. for 10 min. , Y, A2 L& Y7 S" s; P3 @ u7. Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix. & H. V z( j" b6 `- ?. i8. Incubate the cells at 37ºC O/N.% Y7 Y' m1 }+ P0 N& i7 w
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. * t2 E2 @0 P) c9 t) I. J) L! [% q* a7 _* `10. Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.' S2 l9 Y2 G3 o
11. Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris., {) ?. F! k* U4 T; }! @' D. o) ^
12. Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). + e! N0 |- K" w' q# r/ A# U. S* f
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.; M" h. |( i5 Q8 ^7 b8 O6 {: J6 h
V, C: y+ R) V0 N( m+ ?Preparation of human fibroblast cells (IMR-90)3 @. p0 u" k$ U9 U& t+ j
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.1 d1 p! D: a1 S: L/ Q
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube. & b0 ]$ \0 w3 r; c; B( t+ k' l3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant. ) E, n* {! ?# `: h! m5 F4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.) {4 i- t1 l/ M. x# f n9 a
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.' @# ?) T9 M ^% n3 q- |7 D
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium. 0 Q' L! H1 r3 i6 ?9 i8 a7. Incubate at 37 ºC, 5% CO2, for 6 h. - i7 \$ I$ r5 P% T; t# ^+ C7 e( v* S; _
Lentiviral infection! c0 S" Q0 c$ A2 w9 O( {
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction., p! G: m: G" H/ _+ q
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. - @2 N' F1 Q2 Y/ B) S3. 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.8 W, V" r- y2 ?5 K s6 ]
4. Repeat transduction (including virus harvest) as described above. / h1 B/ D. E0 E8 b1 ~( |5. A 3rd transduction may be necessary. 4 X- i+ W" q! W4 k3 T6. 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). " t6 k! @7 h: J8 I: Y- V7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.* }6 r/ B) ?! ^0 t
8. Day 18 (9~ 10am) Change to conditional ES medium. Observe any colony formed and pick them up when they grow up.作者: 细胞海洋 时间: 2010-5-5 23:10
IPSC Generation by Retroviral System Protocol5 _2 g# z t( p$ i! w0 z& n
6 r% P& b( m0 ~3 ?Retroviral Packaging ) l4 O' X) G. o) g
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MEDIUM , N5 M9 q1 z7 ]+ y293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids. {- n! k7 R" p7 r$ @) x2 o
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.' Q. k- h+ h* A3 }
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293FT CELL CULTURE $ w8 p; O3 _) U/ f) l- vMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. $ I: c- h( z( `, Q9 c. m 0 g2 Y" N' c0 lREPROGRAMMING FACTORS 5 m3 ~- J7 G7 `pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4 % e2 z( O4 F7 p1 m3 { ! d2 M$ T% B+ G) b# @Transfection of 293 FT Cell with Lipofectamine 2000 _; s. k% U. O) d' m3 R" B
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For T-75 flask 2 h/ M6 j; W1 a: MPrepare 293 FT cell:6 y8 K6 \1 _) e Z
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.1 ?& f4 M# R& e! m8 O, ?$ T/ C* W
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. 8 D, ]& i) }6 V0 f; J! F8 A
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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. + r ?; |9 G1 [- D& p# g" g/ ]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. $ d' j' A" y' A5 [+ A) o+ e3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. ! H2 S" i9 _& R% Z( B
4. Mix and incubate at room temperature for 15 min.; J O1 Z6 s- u L4 n; o/ e5 K. A! Y
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. 4 T- P5 T! U" f1 F7 b6. Incubate at 37 ºC, 5% CO2 for 48 h. 1 { v3 L& j+ |7. Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.6 `: k5 Q7 T& `" d
8. Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris. 1 O: R( R! x3 _# x$ _/ x, v9. Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). % C: M. m7 g7 @/ `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. , ]& C; X* F9 o3 w4 j3 t ( G- g! N& \# e& ^5 ^& f& c9 wPreparation of human fibroblast cells (IMR-90) . T: C4 Q2 s3 T& ?6 m6 m# T1. 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 z$ [$ z0 p/ q& _2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.' N$ e0 y% a; d6 T
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant. ) X- D+ ]* z# ~* s4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.0 j( C0 o1 r4 t1 f6 w7 M$ @6 f! z3 Y
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml. / ?7 G4 z" `2 c; c5 @0 Z6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium. + |5 |) z9 V& c$ F1 o& e+ l' J! ^7. Incubate at 37 ºC, 5% CO2, for 6 h.$ g& S/ X/ r% u" R/ J5 v
: J7 e) [7 c1 B( ~# x( B1 e+ ~& BRetroviral infection ; h( t0 m3 u) i7 q1. Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction. 1 v) Z6 d' c: z8 r K2. 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. $ r* E; u9 G! s3. 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. 1 N2 W; b$ w& x. ` C6 Y* V4. Repeat transduction (including virus harvest) as described above.* d" d, Y7 ^# {$ ~
5. A 3rd transduction may be necessary. N( V( E3 r4 E4 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). ( {% S7 ~0 @! j# Q7. Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.1 e: ? i" {# _& ?: c
8. Day 17 (9~10am) Change to conditional ES medium. Observe any colony formed and pick them up when they grow up.作者: 细胞海洋 时间: 2010-5-5 23:12
转帖一个问题) J A: c. s& l
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问：unconditional human ES medium 和conditional ES medium的差别？ 7 W9 b- @: z, ?& ^
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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。作者: xyzengh 时间: 2010-5-5 23:34