iPS 建系经验

细胞海洋

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: S$ I# ?; y& |$ s/ X, l! s本文系ｘｙｚｅｎｇｈ版主原创 非常感谢! m' k4 t% K$ t% e+ p* |1 e6 U

* ]/ k( ~4 b. ?/ z6 m6 L- ^& xIPSC Generation by Lentiviral System Protocol
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- y4 o$ x# Q% Z( P: YLentiviral Packaging
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7 _; \- a$ X' P8 DMEDIUM' g2 K2 u" x6 E& T
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
" [0 c! `/ V0 Z/ S- K$ y. k" Y293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.& r7 `- G  H3 F5 D3 Z; {5 j
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293FT CELL CULTURE& o' n; Z' r3 i: C# S" [+ ]
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 9 s8 t- n0 h+ t+ E$ q4 W# P
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REPROGRAMMING FACTORS
3 z% T2 Z4 D- T7 U1 k5 hOct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)5 r, E4 w" S7 {8 o
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Materials Each T75 flask) }$ d+ W7 `# I5 B5 X5 c' w
293FT cells    10-15x106
. }$ L7 Y0 m+ Y. D' Y. W3 b+ jMD.G (VSVG)   5 µg
2 Z. w  y( @- VPsPAX2   10 µg% J* V! P" U0 O
PSIN vector (~10kb)   5 µg
$ U" L6 t5 N3 q/ ~Superfect (Qiagen)   40 µl
+ F! ~, {8 j6 |2 S+ YIMDM   400 µl! \7 o4 n/ m9 Q& \9 z
293FT medium    10ml
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/ N& ?/ Y: V8 y1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
, L, O; {) @: Q2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
8 }! ?# G- f4 R+ U* t3.  Add 10 ml of 293FT cell suspension to each T75 flask.
& O3 m- P4 u2 l( m: k4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
$ j. A' o8 U  _, g! x% i5.  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.
5 ~5 }1 l( J9 D  h4 _6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
/ [# D; x3 [$ a3 f# {, Y; T7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
, ]1 r$ V6 p2 @3 l" f- B8 \8.  Incubate the cells at 37ºC O/N.- u1 ]% Z' O8 e* [2 M& ?* j
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.! @1 q% `$ F: K' r: w) `% S/ k. x
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.4 E! R2 A7 g" N$ n1 g* Y+ A
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
+ V- |  ?( r$ m) g8 s; o7 c12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
  @3 C9 }1 v9 O13.  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.- ^6 Q* ~) l+ n4 k6 j' w0 W7 {
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Preparation of human fibroblast cells (IMR-90)3 _/ s9 z  y+ |, }
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.
/ g% t' U% k+ q3 [  J7 A2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
6 d* F9 U) X2 j0 @3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.  ~# X1 A; b* _- t
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.; S0 N. t1 {, m( {) v! M6 U
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.$ s( T. {+ u' i9 o& b9 C
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
7 }6 U% [" D+ `4 Z; E& ?7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection0 g' s$ F4 ?3 q6 Y1 ?) M
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.: j9 Y0 X: [) a9 H. j
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.8 |6 d( R" w- |5 Q2 i2 C: x& D* c
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.
1 d6 ~1 [) T+ }. a: g4. Repeat transduction (including virus harvest) as described above.* @6 ^" s( c' \/ T- c
5. A 3rd transduction may be necessary.
4 p0 I7 Y, Z: m# i9 X! e/ M6. 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).
, Y( R- J; h8 F$ j$ t4 B7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
. {, ^5 _9 ~+ R2 {- M# e& }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 1 ?  p9 ^2 R0 R' X0 n: \4 _
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MEDIUM
3 S7 Y: v4 ^0 O, ?( t/ ~293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
9 e( X9 D2 y' U( w1 m9 x293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE5 g2 A9 m1 X: K- Q: B3 c1 m
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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8 q& x4 B; A/ O. vREPROGRAMMING FACTORS
" z5 H) D8 {9 n' [2 R4 o) K( `pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF48 e- z6 b* J5 c6 b7 B2 d  c2 V

% N, h# ]. ^7 k( {. bTransfection of 293 FT Cell with Lipofectamine 2000" z* R6 l4 o( u: O' r* |0 c

/ a& ^: l6 j& _9 f( jFor T-75 flask
( ?( n: O' l- s6 kPrepare 293 FT cell:7 Y% ~* C5 o: I+ h. E$ g
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
$ e# I) e  `4 f: a1 xObserve 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. , e7 u2 {1 I' r8 K" |# z

$ \; D( c8 A) D6 x1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
7 p( e9 t, @  b9 ^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.    `) m! m8 P( Z' x5 J# L2 X
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. 2 I% z+ v( U$ h8 p9 P$ j3 r! x7 I. W
4. Mix and incubate at room temperature for 15 min.# Y. p3 U0 \- k
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.
+ ^- ?3 J/ p& K6 U6.  Incubate at 37 ºC, 5% CO2 for 48 h.
7 ]9 I! P; j' _3 |2 s4 O7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.& f' i) u7 R" I8 d+ p  e( L% \
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
; z* _# V7 P2 ~3 W" W9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). . H& K! f* x/ b  C: N* X
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.5 ]( X0 V9 Z* ], I$ g! Q

; k4 n" s2 ]. Q% a% I2 L3 j  @Preparation of human fibroblast cells (IMR-90)
1 y8 g* V7 i) n9 S( l8 y1.  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.
5 |3 r' t& K- T# k* }2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
6 y! z9 Y6 F$ W! v% a0 f3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.9 P8 D9 y1 v3 r6 N1 O; }& e
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer." L; E! L  B( N5 {" o3 h
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
- G9 W% D9 v) t2 `! i6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.6 O1 a) v# e8 O: ]# f
7.  Incubate at 37 ºC, 5% CO2, for 6 h.% L2 ^1 n$ d* W' O0 f

0 R. t% F+ @  zRetroviral infection
$ Y6 X  f! G7 n- z4 C$ o  O1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
+ o$ Y; T0 {# o/ X2 P  g2.  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.
- g0 ?0 K: q7 s* L' l" d) D3.  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.. K0 F+ c4 g( l+ x
4.  Repeat transduction (including virus harvest) as described above.
3 y: y! t7 P, ]" I+ R7 w) I5.  A 3rd transduction may be necessary.  k  g1 ~, E2 |; K% I
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).: n; |  L* L* I: t
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
- z$ D4 Y" l+ `# F/ u  B8.  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
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3 H7 ^( i, Y: S( B. s$ G, ?) Y问：unconditional human ES medium 和conditional ES medium的差别？ 8 t* G/ q% y; \5 ~$ Q& a8 ~

# B* D% }5 c/ h+ {7 e3 Xｘｙｚｅｎｇｈ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！