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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# q7 t9 i4 E" U7 eIPSC Generation by Lentiviral System Protocol
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Lentiviral Packaging ' X+ o, g( c" Z- S+ J
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MEDIUM2 n6 x$ R' c! O4 x3 T% I! F
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
: y4 P) P5 g! |& F293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.+ o- b; o$ b' S# \- z+ k

1 k0 d" m8 I/ P9 ^293FT CELL CULTURE) |7 {7 s, I' S( b- I8 _5 Z
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS6 ]7 E9 r6 D) v$ w% T* @0 m
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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: |9 I: F- d4 h% m' a+ [LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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Materials Each T75 flask' l4 V% m5 h" ~2 _/ ^  I
293FT cells    10-15x106) I' C' A8 [' d! d
MD.G (VSVG)   5 µg! b" b0 P! Q* ?
PsPAX2   10 µg+ E# X- h3 H6 P7 R
PSIN vector (~10kb)   5 µg9 N' v- ~+ Y) P+ x1 }( N- @+ W' E
Superfect (Qiagen)   40 µl " [* J( F" x+ S6 p+ S, _( L
IMDM   400 µl
3 R/ d  e' e6 ~; {6 o, k293FT medium    10ml
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# Z  t6 {+ x4 s' W/ A. D: T6 @1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
4 S8 b* Q3 T" j, v- u) V% ]8 G2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
/ J" O- V' [8 }3 M3.  Add 10 ml of 293FT cell suspension to each T75 flask.9 {* q+ f0 o" D9 b6 M
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
" l% ^4 E9 ~$ d, r5.  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 ?( C! B$ ?# G% o1 l6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
4 F: _6 X" \# e7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.( B, T, b! `+ m0 y# w
8.  Incubate the cells at 37ºC O/N.
- j& J9 D9 j* U& b) {2 h9.  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.
- q; W: b3 [/ [. O4 T; n  T10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
. P9 R+ {# z0 _1 r* a$ ]11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris., B# L+ @2 \2 K
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
: l. e! ^! k  z8 C, H! A, [  [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.0 U3 t5 x, u7 z6 H+ s+ Q
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Preparation of human fibroblast cells (IMR-90)
$ a+ I+ @" r7 Y) V6 b+ |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.+ L. p! A( J7 e% @
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
  Y/ M7 }6 l2 {5 k0 J2 ~( j, \6 l3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
* U6 e! P# c8 W" f# U1 Z- Z  z: ?4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
1 Q/ N' M/ {1 F5 _, O5 }) M8 E- z5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
/ `/ X( J1 L8 K& \6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.0 V. w, x! t* Y( K5 A% N4 i7 f) ]
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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0 J$ u4 n5 |, s4 J% iLentiviral infection* O9 U2 K3 T, Q
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
' l/ [4 Z: C, u$ m/ H$ ~; F2. 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 B" p! F3 W8 C; k( |; ~* a3. 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.
* o7 y; C% D3 D. W) u: h4. Repeat transduction (including virus harvest) as described above.' \& _( z1 J: P; I- [
5. A 3rd transduction may be necessary.& S  V9 r( o* y+ r
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).! s& T" k2 b* W% [: R
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
; U4 N2 u5 w" A( V; Q8. 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 8 C/ B  X5 M/ M: `' o3 t( ~
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MEDIUM0 j  g5 j) h& L4 P1 l4 y) q: N
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
% {6 }# }( a( I* e8 E293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.* d9 y0 r' O6 W& k9 K

7 w( i0 v6 y# V6 f* @293FT CELL CULTURE
' q& r, G  R: }+ T  oMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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$ L, @, U% B! ]9 H9 s8 t1 v5 X  uREPROGRAMMING FACTORS% N5 Y1 k, A; j: Q, m
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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! X( M4 `8 a; j8 X, P' sTransfection of 293 FT Cell with Lipofectamine 2000! a* }* L+ M6 M: I2 P! U
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For T-75 flask" H# @/ q: s- j8 o$ T& T
Prepare 293 FT cell:
' h; i( ^% k8 g) }Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
9 T2 Y  p: C% t; d9 h" `4 ]2 K% mObserve 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. $ ~9 l- `. A" T' M3 I
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.  * C# D5 r6 W1 M
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
: c/ U+ J2 S. @, }4 B% i5 N( e4. Mix and incubate at room temperature for 15 min.! l1 f! c9 ^* m  |) T. i
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 k5 f8 }! ~" ]* n6.  Incubate at 37 ºC, 5% CO2 for 48 h.
" T- z5 o6 q6 e( c" o( n7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.. e% q% j# V5 |! R8 G# n' q% k/ F
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris./ ~4 \6 G) Q$ y& Z
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
- Q  |5 b2 G3 ]6 u10.  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.. s5 d) f2 n! K+ b% B
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Preparation of human fibroblast cells (IMR-90)7 F2 l  M. d3 s3 T" D7 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.
/ l; e) F+ b6 _. r' a6 K3 Z* n2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
; I# ]' ?( ~6 S' |! y3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.& [1 z0 k" a! s! ?: y1 s
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.& \) z; u6 E/ s9 a; x; Y0 k5 ]
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
" u* ]! ^! q4 `) d& ]+ d6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.  A& b6 _0 N3 ~2 A0 b. k
7.  Incubate at 37 ºC, 5% CO2, for 6 h.7 v! i7 C5 K# c5 r" y  C  k( L

1 v1 L, s0 e% I' S. dRetroviral infection. C6 V! J6 h  k2 E! d5 c
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
4 R' C) y' x1 Z6 d2 ^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.
, V  a4 ?" `; f" R3.  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.
: g2 ]# p5 S. E* e7 b$ l/ F4 y4.  Repeat transduction (including virus harvest) as described above.1 A! _, [% w  I0 }% ~
5.  A 3rd transduction may be necessary.8 i  O+ z8 Z# G1 K
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).+ H3 L# q! z  D. t+ t  C* C. ~! Y/ k
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
8 d! p  y7 l1 }+ u8.  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$ S' P5 U1 @4 y5 d8 z: i+ \
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问：unconditional human ES medium 和conditional ES medium的差别？ ) n) ?: H; P: N/ ]8 X9 f

1 @) D# N1 T+ U0 A( f) I# B: J! Oｘｙｚｅｎｇｈ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！