iPS 建系经验

细胞海洋

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+ T% Q4 w' v. t0 V1 r本文系ｘｙｚｅｎｇｈ版主原创 非常感谢- f1 J0 `! E; S  r. P, x1 S
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IPSC Generation by Lentiviral System Protocol
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Lentiviral Packaging ( `/ W+ }, R$ b7 A* ?3 Q
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MEDIUM
4 P7 M% _& X1 Y% E4 f293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
+ ?: e: ^) D/ b8 x' y/ D293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.* o- O3 d. P4 Q- f4 R
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293FT CELL CULTURE
9 H, r3 S: e% @5 Z4 ?% UMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 2 C* ?: F9 F5 \) s( [* A
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REPROGRAMMING FACTORS' z/ {5 l* o# D& c* g' V
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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Materials Each T75 flask5 s& q0 u" R' u( g) |  D8 _
293FT cells    10-15x106' Y7 y/ m/ m3 R1 I
MD.G (VSVG)   5 µg
+ {$ J- }: ^+ Q6 M9 TPsPAX2   10 µg( e5 u6 L8 u; @- R
PSIN vector (~10kb)   5 µg
6 s% g9 r) O3 K, RSuperfect (Qiagen)   40 µl 0 ~" P" s; P$ B( M+ c; d) J/ ~
IMDM   400 µl
" t4 Q( x+ x- F293FT medium    10ml
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) Y$ [  G! e4 M; l4 {) q1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
; [- ^; H, `% w$ u+ C2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
- q3 a% G' L! Y. }2 B% c. X3.  Add 10 ml of 293FT cell suspension to each T75 flask.
  T0 T2 z; N3 `4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).$ y% L+ ]8 Q2 j/ t
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.
, y$ l5 M2 V3 x- m8 G6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
* |4 A/ l; B- H5 p, e7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
! E3 c# x6 J2 a  o7 a8.  Incubate the cells at 37ºC O/N.
# @8 z0 K* t* v* [& M+ r3 S9.  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.* w& `/ c0 e: ~+ s; h; ~; N
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.. Z2 o, U2 ^: A
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.6 E. u: y. X; J1 e
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).   E- `  \7 R7 g  j# V+ C
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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Preparation of human fibroblast cells (IMR-90)
9 ?; A1 t" U& y0 R! l' B1. 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.
8 m5 \/ }0 o* K5 O4 v1 |2 k% @2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
; @/ K8 e- L$ Q0 K/ \- Z: l3 G, E3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
  x- G1 ]9 Z8 z( D: X9 x: B& K7 E4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.: Y9 Q% _3 @  A2 E, v
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
4 N4 u' g* @" {, }1 W+ b* Y! Y8 k6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
+ {6 o/ t# i' e: _/ T. o7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection
% ]! U  v# f4 w: N1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
* c: @( r+ f) t% r' P) a2. 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.! o0 v$ v0 u& y  q0 G7 _* `; Q2 q! Q
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.
; P7 ?% Q% d) \; y. L4. Repeat transduction (including virus harvest) as described above.
* W7 |9 s0 W+ z7 D- {5. A 3rd transduction may be necessary.
* Y* }! U7 p& A& ?2 w6. 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).' n4 _5 o% Y, O2 r
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.2 R( P- `/ p, W$ h* n; |6 V
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
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  w5 k4 J+ x/ G3 _; q, c' gMEDIUM
  M2 ^  ~, X! z7 S1 \  H293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
7 h6 @0 l. d+ I& i4 d; C293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.. I5 |7 G3 g5 W
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293FT CELL CULTURE% s% b. d( m% E. @
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. " U- M# r0 }) n$ G5 F

1 R2 v3 M9 g) b. jREPROGRAMMING FACTORS7 q; \# e  z, T
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4& b7 ?' v9 t+ M; h7 E2 V

5 |" L. s4 B2 [Transfection of 293 FT Cell with Lipofectamine 20004 ?4 s  w6 j: X5 @; F5 K& U

, `3 ?' h$ B$ ]- U. gFor T-75 flask5 }% F3 t0 C( l0 ~4 ?3 p3 V; E, m
Prepare 293 FT cell:8 A& J: \, p& e* j$ }
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.* Y: E! h6 I! `7 m) N  Y' o9 H& s
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. 6 n' i' s% m, c" x! h! d

) H+ U+ i1 ]) G2 E3 f1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
1 C4 ]. K& E9 X- ]* l2.  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.  - h. Q' _- V7 ~7 `) P8 [6 P
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
3 M; ~; g; M/ _  ?1 O, U& I9 F4. Mix and incubate at room temperature for 15 min.5 Q: Z& r  N8 C  R7 S& j) 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.
, q1 I; _0 n  }. K6.  Incubate at 37 ºC, 5% CO2 for 48 h.( |0 Y. r. t1 T+ Z) p0 U/ e! x: Q1 L
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
; }/ O; L9 a1 y; C$ m0 ?8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.. L' @6 K, x+ b
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
1 s- _4 o& r8 u0 ?! a$ m10.  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.+ b- {7 s4 v: |/ T' x* C2 o* e
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Preparation of human fibroblast cells (IMR-90)+ F( I6 ^0 g9 o! H8 L& w
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.
0 Z2 i! B) Z1 p6 \3 k2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.. f2 W5 P  f; e2 d
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
; V# m5 h- [1 i2 C. @4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.2 `# Z$ G& d! c7 ~
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.3 `. K$ i( y$ ?! i' f: ?
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.% \% W+ v% s5 {( S
7.  Incubate at 37 ºC, 5% CO2, for 6 h.7 x2 [* Z+ w- ~9 S% e; D* P

) e6 F0 K, C9 A5 L% uRetroviral infection" u" F$ a& s8 C8 w3 ]1 v0 e
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.7 R1 a$ Y/ j( n! j
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.
) K/ x) w# w( O8 _  e3.  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 X) f! d) f, t, n9 `# `. j. p+ n
4.  Repeat transduction (including virus harvest) as described above.
3 a% q1 S! y! @2 d' s5.  A 3rd transduction may be necessary.
% S2 x7 d  `4 f' R# {) E6 o6.  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).
5 p; i% B* G  F  E$ o: P/ ]7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
' _5 D3 h1 M$ n5 Y3 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
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问：unconditional human ES medium 和conditional ES medium的差别？ ) [8 ?2 S# {+ a

+ A3 w5 D- ~/ T- ?7 @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！