iPS 建系经验

细胞海洋

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% F. v, C7 H3 |/ t( \& e本文系ｘｙｚｅｎｇｈ版主原创 非常感谢+ W2 x( y; n  g: |& T8 T) D

$ w+ Y) L; |" Y6 [" q1 jIPSC Generation by Lentiviral System Protocol
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, p. ?, X( S. |* g# LLentiviral Packaging
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( b# g% g( n- |% |' a- XMEDIUM5 F5 R+ V. b" _# G5 `# l
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
" F9 M, ?3 B) o, U) a, Z2 [8 G8 ~293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE& i! E# M! V$ x) y3 R5 @
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.   a( Q# w5 T" V* @4 b! p' T) |2 L' Y

( N0 @8 ?, s! C7 o; l# }REPROGRAMMING FACTORS
5 I# D. i" d7 W2 [* `" S% y" FOct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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1 T& s* [3 U' W- D6 o7 K7 s( sMaterials Each T75 flask
2 L( y7 |) _. b# \293FT cells    10-15x106
9 x: H0 [0 ?1 W! `3 W  RMD.G (VSVG)   5 µg' S# ~. @, Z! E) s; J
PsPAX2   10 µg3 E! A, ~2 X/ a  j2 G- m0 q3 U
PSIN vector (~10kb)   5 µg' s' I* `& K" Y% @1 _
Superfect (Qiagen)   40 µl
1 D* L* N( J* x, x7 i/ x9 @- ^IMDM   400 µl
2 U( i  u! [3 s6 L9 r: |' \- D293FT medium    10ml, T' G0 R% d0 l- ~

6 N, x9 L6 G2 \3 c1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.5 F6 X! F8 Q/ Y5 L( B. d3 Y! p
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).( z9 P* L& y+ X  U$ W5 o- X
3.  Add 10 ml of 293FT cell suspension to each T75 flask.
: M2 ]. b( s" M( Q, |) L( K4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).3 f# I# R& V! w; D1 u7 \% \' P0 p
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.) _, l5 t- `9 y
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.9 W6 n% P; J3 ^! _- h$ e! i
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix., w$ x9 M( U) S: T( \
8.  Incubate the cells at 37ºC O/N.* z( a6 P6 i) V: M% f+ p5 h
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.
% u" n; p( N0 m' K- r2 H6 B+ M10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
/ r2 B! W8 `+ ]" b' `11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.$ j" l% @' u+ E" H' W/ p2 u" Z( Q
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
3 b9 k% z/ i4 f' ~0 q13.  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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, c% s% U! F& n, z+ p- g% ~3 K- @% UPreparation of human fibroblast cells (IMR-90)- X0 ^" J& S0 p3 B! z
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.
2 \2 r6 l  W) D5 @' l2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
  |0 D3 w* F- U/ S  t3 ~3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.$ b3 O, X+ g( j) Z" x2 X1 S
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
6 b9 G3 n1 Z) v% K0 W5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
3 n, c3 L" C+ ^2 R/ `: [2 N* h, c8 C6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
9 j* l/ N' |' @7 J! s$ j5 W0 I7. Incubate at 37 ºC, 5% CO2, for 6 h.* a- ?/ {7 _$ c
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Lentiviral infection7 U% F! ]6 D' e: U$ ?5 d3 _
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
: p# K; `" Q3 {0 i% @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 R8 u0 |( W; w8 S. v- B3. 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.
# o1 V5 q# ?/ A! H4. Repeat transduction (including virus harvest) as described above.
, ?) ?" w4 a& o# ~/ O7 w% f5. A 3rd transduction may be necessary.3 e9 H/ _( T- ~/ y/ n
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).
% r% x: V! ?0 [; S. G: ?7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
  c8 t& Y% e7 U2 L& e4 k4 b7 ?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# x, P2 a& c! o( m2 Q3 D: I. M- t4 h

0 e( z0 q' V2 j3 o$ @Retroviral Packaging
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# A5 \. c. q  T, T/ c0 uMEDIUM4 |% T: D2 h0 f# j' v
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.6 k+ j+ J' s7 }! L. z; g- A
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.' v; A6 N- z, ~+ J/ w
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293FT CELL CULTURE* X& F+ G3 `/ k' G$ U% f
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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" U6 r/ H5 A8 K7 G  p2 E7 E6 O  g. cREPROGRAMMING FACTORS2 ~( t7 Y* n+ ?. n0 q4 \
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF45 Z0 `) e$ A: E6 E, a/ o: e8 \

# q6 Z/ r4 L* q8 u& v1 QTransfection of 293 FT Cell with Lipofectamine 2000
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For T-75 flask* I/ u3 {9 }0 u4 Y
Prepare 293 FT cell:
( @" _) @, J. K; p" zPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.+ _9 z' c& i7 X  Y1 J3 Y
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.
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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
6 `) x0 N$ z+ k2.  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.  1 r3 @" E7 }3 a& A2 z0 d7 @
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. . H. p5 o  J9 k4 j0 a5 ^5 A
4. Mix and incubate at room temperature for 15 min.! N5 ?* R* f. q7 Z
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.( m3 o! o+ {/ i, y" P
6.  Incubate at 37 ºC, 5% CO2 for 48 h.  v5 @( y: x3 v7 V: x
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
) Z9 C+ ~3 V4 W& N7 {2 r) \8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
) p8 ^$ n- V. w7 O7 G, W* Y' C6 L9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
7 y6 z3 C$ f! t2 ]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.
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+ A% `$ }3 c$ P* q& y; G  vPreparation of human fibroblast cells (IMR-90)
* @  h- @! {6 L$ w' C1.  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./ M# @: i' ]7 d& Y# ]9 U
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
- ]9 W  d2 X0 W7 [" Y2 c3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant., L4 R+ `3 s4 |7 \/ w3 i( \/ ]
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
* F' Q- H$ r6 V8 N7 N8 m" m5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
- B3 v# E" r7 F2 d, r/ Q" K% M; R6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.9 u4 E9 B2 ?) `% X
7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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Retroviral infection
8 p4 M- c& _7 v% V, b+ W6 @: P1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.7 [; e6 ^, o; _9 |+ C
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.4 t2 O; @( \. |* u- @
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.2 _) j$ P8 Z6 R+ P# ]4 p; w
4.  Repeat transduction (including virus harvest) as described above.5 z- E5 m3 {" [: j, t
5.  A 3rd transduction may be necessary.
' y1 X$ p# o% x, p# F% q/ W6.  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)., R3 L; \# |6 D5 r. ~
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.7 X! V* Y) {* H% N1 k
8.  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？
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9 z0 n& Y7 {6 _) n6 T5 l2 [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！