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6 E- r4 G( v/ {1 t7 t/ M' e
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IPSC Generation by Lentiviral System Protocol6 P! F0 T0 x. o+ [$ _# H

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5 K" m: h" p7 J3 ZLentiviral Packaging
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; L% Q* _- Q& M( l  TMEDIUM! C& B0 w' w! @. O$ s" z
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.0 T) [* I: R$ W
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE
$ @! _4 G% S9 h# F/ j+ {Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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/ i% W& t# }6 F" Q- Z2 B$ SREPROGRAMMING FACTORS; j' t+ j8 A+ L% I8 b
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf49 K6 d8 F* n0 j+ z5 v" m2 b: u5 j

: P2 L* z/ n, k, c7 F4 p' N: a( C3 dLENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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Materials Each T75 flask# |7 J5 X2 h6 y, g& q
293FT cells    10-15x106/ o& o. P- X# X6 H
MD.G (VSVG)   5 µg5 K1 f: C( |% M1 E$ x6 @' U
PsPAX2   10 µg* A) ?2 J. z- }$ u) S2 V
PSIN vector (~10kb)   5 µg. G# s) g/ R9 e3 B) c5 u! N* v8 I5 q  s
Superfect (Qiagen)   40 µl
. s7 w) \9 c1 q" q9 OIMDM   400 µl' s6 N8 n4 T, u& k3 c4 M- o
293FT medium    10ml
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
( X- C* p6 u% y' c2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
1 x) {' s) h& K( p4 H* F! F+ |6 T" H3.  Add 10 ml of 293FT cell suspension to each T75 flask.1 f- v' f( E! i! j  h) ]" j
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM)./ j' _- l; v3 \3 R( t$ C% I' E
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.- x6 i: O: o) x( U# ?9 ?/ O+ `
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.0 W5 ?" J* Q; Y
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
( h- P9 u0 _& |7 Z8.  Incubate the cells at 37ºC O/N.2 @1 \$ i" B( v) s' ~2 w" I
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.& O  y3 N3 z5 }. j' M2 t
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
  K" |& \; \" \7 x: o: o11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
9 J1 N  w' H% r% e12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). 1 Y8 N* w+ `  Z1 Y8 e
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)
# |  o0 G8 B2 K1 e1. 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.: _7 |' s' l* o
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.! K4 h' _. E) l# I
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.# Q+ L9 @6 a0 l, A# n
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
3 q# r6 b/ I* `, |5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
, e! [, w5 Z" ~8 }# r0 G0 N6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.1 C+ `: p* S% \& @
7. Incubate at 37 ºC, 5% CO2, for 6 h.0 i0 g( }; z4 m4 h7 P& s. C
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Lentiviral infection1 p+ ]3 w1 w- {; O, a9 t
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
: {; k1 w" m" Q5 }% B2. 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.6 z4 u9 V& q, A$ b% Q+ P, l# ]
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.
' a3 M! P/ ?: j& X0 B4. Repeat transduction (including virus harvest) as described above.) p% y" N+ a# x, Z3 c' ~0 m; l
5. A 3rd transduction may be necessary.% i/ Y% x2 R1 S' u) f
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).
6 n& V: [/ b, n( Q. ^7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday./ m& V& q$ ?: i# m9 q2 N& a4 ]9 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 / L2 d7 a: p) _1 K( W1 |. z( L8 f/ l

7 H# K  ^% d, d6 x5 E' [4 F0 `- PMEDIUM' c5 r2 O2 a. x0 e
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.* n; l3 X0 {& W8 B9 i8 m: o
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate., T% r2 M, q+ K- v! Z* [
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293FT CELL CULTURE
1 @: c  |% G7 _; _0 aMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. ; s! k3 v& S  {5 F: C! j$ [% m6 w
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REPROGRAMMING FACTORS
8 K) J* p+ {  L# c0 GpMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4! u; H4 e; O" {8 x0 q" T1 }$ p
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Transfection of 293 FT Cell with Lipofectamine 2000
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+ t% P6 `) K6 d% j) J9 a, YFor T-75 flask
# f" G  Q4 N7 {  TPrepare 293 FT cell:; k  m$ c4 S+ ]0 L$ ^6 o1 \! R
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
! v( O0 r& f4 g, q4 R- V6 Y5 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. ; _- M! r) t1 K$ x% u
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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. * }$ f( Y# [' u# _
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.  
! X8 V; \8 K, x! y: p& V3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
: Z6 Y4 q- \5 A+ U5 @' o4. Mix and incubate at room temperature for 15 min., i3 p  r; P3 ^* K: e" D9 b0 ^
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.( I% d+ [8 ]1 v' n; {' P
6.  Incubate at 37 ºC, 5% CO2 for 48 h.
+ R9 ~8 I* k/ N) k/ m7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.$ B7 T6 l  T+ k" t
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
  L5 f7 q/ b1 {9 b; z8 U9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
' ]% l9 q, Q" M' ~6 L, g( {, ^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.
, C9 p# T$ u1 p' B. [6 V2 F! z  _/ }/ v6 W. e! S( |" L; F; S" x$ P
Preparation of human fibroblast cells (IMR-90); {" k. ~- ^- E* A# g6 m" ~& }
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./ \. ]3 r2 G- D, c
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube., g' S5 C( S, F, n( G1 E
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
  k5 q9 C% U5 F5 Z4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
, M& s! V) G8 o4 D, U  y: d5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.' `( S0 n- \, ]7 J7 Z
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
, W9 ]/ ?! {* R: a( l7 Y! L7.  Incubate at 37 ºC, 5% CO2, for 6 h.$ f2 d) `! Q: c/ C
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Retroviral infection
' `/ h! Y# W1 a! s/ I& v8 }1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
/ d+ a8 ?9 B1 T  K  {5 Y& d5 M2.  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.. e2 o" H: U) v! I) H3 @6 k3 x
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.6 V# H" }; K0 z
4.  Repeat transduction (including virus harvest) as described above.
3 @( P/ r/ u* x' g! a: j) l5.  A 3rd transduction may be necessary.8 G: n2 y4 @3 Y! p$ D, h0 Q: 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).; |$ X: E# a( k. a1 R& U
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
9 n; v+ s# c5 I$ U- N5 }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& x! W' Q$ j' s% c6 X. d
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问：unconditional human ES medium 和conditional ES medium的差别？
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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！