iPS 建系经验

细胞海洋

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: v: G7 @, t. a% O本文系ｘｙｚｅｎｇｈ版主原创 非常感谢
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IPSC Generation by Lentiviral System Protocol
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+ ^' t. b/ z& j4 M# L. W( d$ bLentiviral Packaging ; k' T3 O/ w7 Z0 Y! a; {0 P

# ]% t5 h" A! \* G8 M4 j+ eMEDIUM
4 r( i# D5 G( t( k6 y" a& x293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
  X: z( |& y7 T' J8 V( s$ f, j% l) q4 D293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
. V4 x0 R5 Y6 _% {0 o5 M* X+ x6 w
. x- ?% ~1 ?6 [4 z9 Q6 D293FT CELL CULTURE
% g% x7 Q& [7 H% f( y* w) KMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. " c# B* `9 b, K5 K5 x' L( d

: V3 t  H% b; CREPROGRAMMING FACTORS
" p8 H8 K, _9 q; |$ H  G3 fOct4，Sox2, Nanog, Lin28, c-Myc, Klf4* h: u, ?' W" l/ k
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)- S6 u2 y  z8 L& \3 \4 p$ y2 j% m0 i3 S

) x) {; ^$ ^1 Z7 l" p7 jMaterials Each T75 flask
7 J$ A! ~0 {' t+ ^. f293FT cells    10-15x1062 H6 ^/ w" T1 k2 w* m" L  H
MD.G (VSVG)   5 µg
8 e9 \2 T8 ]9 `# p7 t& Q4 ?& @. R" JPsPAX2   10 µg- O7 L. Q# Z( l7 P  ~3 Z) ^
PSIN vector (~10kb)   5 µg6 M; e2 p1 H9 j+ D
Superfect (Qiagen)   40 µl
! q6 J* u6 U5 N3 Y2 O) zIMDM   400 µl# G( A' h' k6 O8 a' e3 v0 G2 |
293FT medium    10ml2 U% i5 [! Z/ U" z" O3 ]1 E: n, H
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
! g- `; v5 E! `! `2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin)./ v9 J7 k$ `( Y6 o- @( r% E$ r
3.  Add 10 ml of 293FT cell suspension to each T75 flask.
; Z* Y$ n( m; |5 D4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
: g, {. M7 R; @, |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.) @. e9 {" d( @) p; W% a4 M
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
4 _' R, v5 C* ~2 G7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.9 J  E4 U" `. C5 ?
8.  Incubate the cells at 37ºC O/N.7 J) o0 u1 j7 U: ?
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.; }* F1 L" |4 c3 O2 l8 ?
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
2 N1 u6 Q+ h9 P; P" e+ o11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
; b) X0 k! E2 b7 Q' k12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). ! s: r2 f$ L- B( V5 j
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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* T, v" s5 G" P' \) F8 GPreparation of human fibroblast cells (IMR-90)2 d* ], D) [3 J+ f. y) b; m
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.
) J/ U% w9 m2 K: n2 k+ P2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
/ a  W/ w' \9 n" c. K7 E  h3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
2 D4 R/ U2 ^  O6 t( ?; H! g# ?% [4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.. J4 X/ U; A% K3 Q5 J+ C1 E
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
# Y) n% f$ H" w" @6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.  \  ]# p+ I* E3 N' X
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection; Y$ b. t( I+ t9 f/ u4 v, q
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
1 a" G! c9 q( B4 M* ^2 ~5 L" C2. 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.- E+ K* \; P4 j% }; o! `* i
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.. |+ P! p  q) h& H6 O
4. Repeat transduction (including virus harvest) as described above.
% _# t+ I/ E) l0 S( `5. A 3rd transduction may be necessary.( _# r+ l5 Y' p( V. q
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).
7 y2 R1 H* i/ a3 |" o2 ]7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
* c6 P2 f9 s5 e8. 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   u8 S! i5 V! M4 i4 U

' N- r* |; [* j5 X7 f4 P; r1 D) ?% V5 hMEDIUM0 W+ P" z' j4 X3 p' x
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.$ i$ b% F* k  ^+ y& _
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.0 h5 h5 u* U0 c
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293FT CELL CULTURE
; ]" ]0 D  M" r; \* g# G! ?9 qMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. % G4 @% Z6 v( N) P+ w2 G
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REPROGRAMMING FACTORS
3 M# s8 {, P$ {pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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6 T  ^% H$ d  sTransfection of 293 FT Cell with Lipofectamine 20003 M) {1 R7 N/ _4 l* \

5 h- y) p5 t' \( `8 xFor T-75 flask
/ ?! T! j8 S/ |7 k' ?% ~( |Prepare 293 FT cell:
  C5 p4 L; k6 C6 G1 wPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
, \: C/ {% d: t3 U% ZObserve 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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+ i: t! g) p% G6 w# H# t+ `1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
/ {8 u. Q: c9 r5 w2.  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.  9 {1 a" s( N" B! F3 {, T
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. 0 I2 P' b2 K0 ~6 k
4. Mix and incubate at room temperature for 15 min.
1 N( E: x3 {# \, L5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.' A4 ^" K8 W1 O% C+ K0 g3 ~
6.  Incubate at 37 ºC, 5% CO2 for 48 h.0 I& o! t! \4 z" l) [
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
( T6 s( `+ _: q) M8 y! P  T8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
; T6 |6 y4 a3 f/ z9 g  n3 s9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). % E4 g7 h  X: ~5 l( C/ e
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.$ L0 ~+ }9 y. p0 a0 C: M( m- H
' z  K% u. V  M
Preparation of human fibroblast cells (IMR-90)" \0 l/ [0 \; `% N/ y
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 i$ ^; ^8 g% e  m, m6 G' z2 d
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.( K% h) I. K5 Z; w" n. Y
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.$ f0 }3 l& c% E) b+ u1 T
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.9 }3 S1 a% u4 W$ y) Y# t
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
# T" A% w5 R: H, }' e6 c6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.# m2 m$ A  W9 D. s# b2 z4 i* z
7.  Incubate at 37 ºC, 5% CO2, for 6 h.  ~  H, H) m% T  {+ `
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Retroviral infection1 z( K0 V; @3 h
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.# r4 L  _7 y8 {: d& l- i
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.$ l+ q' p2 i+ h8 Z# `' O, r
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.7 ^4 c" M3 M# M+ e# b' g8 n
4.  Repeat transduction (including virus harvest) as described above.
6 R! {$ ^- [. A! O( g7 o8 ?5.  A 3rd transduction may be necessary.. I1 @0 i$ o% K2 F  m# I# P1 Z0 x
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).
$ h; y6 H  `, |1 b7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.2 C8 B2 g( u7 Z+ m. _- }6 z; @, ?/ P) ?
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) @5 `8 x5 |. r" ]% s1 i; i
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问：unconditional human ES medium 和conditional ES medium的差别？ & N3 O$ g) F- ~* x' W  E+ k
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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。

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！