iPS 建系经验

细胞海洋

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- I4 @( r6 W) t# T8 C( c本文系ｘｙｚｅｎｇｈ版主原创 非常感谢
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IPSC Generation by Lentiviral System Protocol' x# [$ l* C# C0 r
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Lentiviral Packaging ! ]! S; q1 @* l* M0 G

4 }1 E3 m( x8 f. b) HMEDIUM
  h* ]% i: v, b1 V. Y5 p293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.$ D3 P1 ~3 d$ h8 [
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate./ n7 u. d3 A/ u# K  M, `" p, ]
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293FT CELL CULTURE/ h' e2 f1 L6 z( ^
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS2 ]. O1 G; E' }9 g# @
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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1 ^: p' h! l6 k/ ~6 B  B4 k! E0 `LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)* }  K, a- X' h  z5 a
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Materials Each T75 flask) u6 c* I5 F* W: d
293FT cells    10-15x106% [/ ~# k% J" E
MD.G (VSVG)   5 µg
2 r( [4 L3 I. l: `) [PsPAX2   10 µg
! G/ g8 w) a. M( }; m6 HPSIN vector (~10kb)   5 µg% E& J1 }/ ?. ]5 V# x3 O- Y& W* K( v" B
Superfect (Qiagen)   40 µl " _3 I3 x* w- M, _1 x) `
IMDM   400 µl% m' p. z+ C. S" l$ L
293FT medium    10ml
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
2 H+ U' o. x2 e& [2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
! e( C  C5 ?2 [1 G8 E3 m& Z3.  Add 10 ml of 293FT cell suspension to each T75 flask.' j6 f) ?+ _- l1 I" m; Q# j
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
4 e! _+ G* C% E: d* x/ i5.  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.0 Q! ?# v( D2 g, T1 H
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
( p9 q6 b' C. u/ H6 ]7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.. K0 V' \9 B' e$ U
8.  Incubate the cells at 37ºC O/N.9 l5 Q, W/ z8 G* L+ B6 W
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.# x% _! `0 R5 u
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
9 i  }# G: J  v! z6 n! \4 ?3 C+ p11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.' r0 j4 P& y! A# a% R2 P
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). ( I% y. s2 S0 ?) I2 }3 f
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)) L6 {5 Z3 X) `- ]8 \
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.( {+ N3 R: x' v& g) S8 [$ L/ `
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
& ?  Q! c" o3 ~6 t- i# W9 h3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant." e, N+ j, ?- Y- G. d
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
. n5 S7 |- S" V: o4 P% I5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.0 S' N3 r1 C; D) 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.8 B/ [8 e- Z" s8 o5 Y
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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4 Q7 z7 r0 X! {Lentiviral infection
* F1 g( I, {7 m  C6 ^1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
* N) j+ ~/ J+ Q/ E& ]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.
2 a( ]2 J% \0 L- o! @1 I  h3. 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.
7 d9 T. n  ~) i4. Repeat transduction (including virus harvest) as described above.
* ]+ f3 p0 n* H& L# k# [5. A 3rd transduction may be necessary.9 W4 |' N: U7 Y8 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).& d' H# Z' {  A2 k7 i% i, E
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
  s* s* y0 h$ t5 _5 K2 Z' G8. 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, y' j0 f" b0 t* t0 I/ ]

+ Q) W  O" j2 S$ jRetroviral Packaging . P1 `0 M+ \, G3 q+ M% m8 ~# ?

8 W* W- k: Q8 G, uMEDIUM/ w  S& ]$ u4 j$ W9 L! U
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.  I4 L% \3 F+ I! Z
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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, b- \( e: [# U2 r3 `/ d! m8 M6 s293FT CELL CULTURE
& F. S% u9 D9 E1 C) VMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS
: O& c# ?+ T8 u5 X3 l: P& W( dpMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4* v. I: C3 O  p$ K3 H& [" I  E

3 o4 L4 U' s/ }# nTransfection of 293 FT Cell with Lipofectamine 20001 b" D( j6 w' i, F" v6 c
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For T-75 flask5 j4 B3 s! f; N- ]  B& I* {6 q
Prepare 293 FT cell:
1 E4 t& z; P* [Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
0 A. e+ \" r7 I- H9 X. v7 K4 p3 ^0 n+ QObserve 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.
5 @" J8 w9 J2 w% p6 B! }3 D" e2.  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.  
8 n* D. ~5 T0 F7 Q3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
- y8 G2 `1 g3 o1 |' l4. Mix and incubate at room temperature for 15 min.6 i4 W* U3 k* K3 a! N
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.
$ U/ o. ]4 Y/ ]0 a7 C6.  Incubate at 37 ºC, 5% CO2 for 48 h.9 U7 A2 s3 N6 Y8 k+ U) k1 w
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
) G3 p( z3 J" ?. n; D! n/ p. o( a  P8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
( q  J; Y3 Q: A9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
$ A: b; A! L- L3 }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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+ ?6 {9 a( M! k7 Z; PPreparation of human fibroblast cells (IMR-90)- t% m; w" L& O( h3 f, _6 X
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.
* Z+ s9 U+ o9 w- B6 Z/ `8 R: G2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
6 X1 E/ U" l$ t& @3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.5 |/ U7 u8 j/ o8 m: c
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.$ D& P* ?4 ]3 d6 q! w( y
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.1 q* Z3 u& D8 m
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
2 c, b3 z0 c' [" C( ~0 [7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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2 N6 e3 i+ p5 X+ WRetroviral infection
6 w3 {* i2 l5 v1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.! N4 _  }+ Y: u
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.
) g8 `. x5 |9 z: X; z1 T: V3.  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.
; a5 W# Y$ z; K7 M& A6 |4.  Repeat transduction (including virus harvest) as described above.
8 z2 \. I+ s" a  f5 k5 Y5.  A 3rd transduction may be necessary.
0 w+ o) `$ y3 |# H8 f- i; A9 S6 v8 V6.  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).* S7 i! u( |* h7 S- M9 o3 `
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.- ^+ Z  e! {+ T4 R% @
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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  c6 I; c" a9 h问：unconditional human ES medium 和conditional ES medium的差别？
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0 }9 D1 G: j  D5 ^, J6 N6 Pｘｙｚｅｎｇｈ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！