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
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6 I3 s/ P* S! d/ oIPSC Generation by Lentiviral System Protocol
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: C% d/ i6 |0 aLentiviral Packaging
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MEDIUM
% Q' _* X2 H; Y) x, Z/ N7 B! ^7 G1 H293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.% \: F# [1 v2 S/ J3 `9 H6 W9 }
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.; s7 s: s2 W( x# Z: @' e# ~) e

! u* w. R5 C$ y2 [- w293FT CELL CULTURE% ]% w* E6 W& K$ @4 {' o
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. . O0 {3 N- c' W& b
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REPROGRAMMING FACTORS
9 \& H3 [# g4 o  `Oct4，Sox2, Nanog, Lin28, c-Myc, Klf46 K& V6 V3 O& ]. E) V% \. Q7 q
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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Materials Each T75 flask
7 Q9 y$ m3 j# [  _" K8 V$ R! R, f7 q293FT cells    10-15x106
6 M6 @9 D1 o( M% F: HMD.G (VSVG)   5 µg
3 j" F. y* y7 y. ]2 }3 rPsPAX2   10 µg/ ?! |8 w; T( }: c6 S6 s# p# W1 U( z
PSIN vector (~10kb)   5 µg
0 ?6 k% _4 M( }$ p: D  l( HSuperfect (Qiagen)   40 µl
7 r" C% d9 ^: x# IIMDM   400 µl  `6 F" J7 @1 S! r+ F  Z
293FT medium    10ml  t8 ^& J, Q4 A8 \- S

/ a9 w& t; a. j" s" Z: J: n7 o( f1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
3 ?+ ^: L0 B( r" m2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).. o% T: Q" m, X6 p
3.  Add 10 ml of 293FT cell suspension to each T75 flask.
" V: r. ~* l2 t4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
9 k, k$ d7 u5 t$ ~% D5.  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.+ W1 _6 T0 J6 R$ M/ |( X
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.0 s6 k9 x3 `5 e
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.! U: H- {7 e8 J8 V9 l9 H$ i6 Z
8.  Incubate the cells at 37ºC O/N.
+ G* K! s! r* r2 h9.  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.
) d% a1 i; B- x, p7 ]10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.6 Y  \% r6 J7 H( @% T+ A2 Y
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
* q; c) D( I( A$ c4 a12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). 4 ?, \7 V3 O" q5 W" w/ @0 @2 y
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.' P/ A% M# w# }: `- [7 |1 J( P9 x

: I* Z: g) \  BPreparation of human fibroblast cells (IMR-90)) ]$ H# a; y0 f# w' F5 x
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.
7 Z( w. {" P) v0 f8 k) ~2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.' [" ?- A5 O1 n& q# o
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
5 b2 z3 ^+ g( f; U# @4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.9 A( u+ x, i: w& V0 G6 J
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
8 W( J( m8 e3 V! h/ C6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.' I. B' y* H$ `+ u
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection' y' b2 b( V3 _
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.' d% }6 h: d0 Q3 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.& ]3 \8 n6 W2 M* s4 {- M) ~1 X6 t6 W
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.- Y+ R1 F6 q- ?
4. Repeat transduction (including virus harvest) as described above.% V( |3 N2 X9 v' K* g
5. A 3rd transduction may be necessary.
& ?: t7 ]$ Q+ Z2 p6. 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).
& j9 S+ Z' L- W7 L7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
6 ?+ ]# |* `% L' ^2 u% X; s6 u- ]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; K) Y. N4 L7 J8 c+ z/ ?# Z1 c

, W, v2 o$ y: O; `9 N+ \3 J6 G5 M3 v& mRetroviral Packaging 7 K' L1 W$ m# u8 ~
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MEDIUM! ~; {& f" h) J
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.. W3 R$ j1 i, X8 [* Z: b
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.$ a+ S8 p5 h* L; c9 A
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293FT CELL CULTURE' y8 ^/ P: ~, n6 k
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS
5 J$ ~/ v1 m; h; j& L1 j7 npMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4/ W3 \3 l: \! p1 x+ A

+ }  Y4 l7 z2 }Transfection of 293 FT Cell with Lipofectamine 2000
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9 y0 \7 ]$ g$ O, ^- x2 u& j3 a8 w4 HFor T-75 flask
1 N6 s; K: K) G( ~Prepare 293 FT cell:
7 F/ l  Z" |" |6 U- b* x! aPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.( F6 ]5 h- ~/ t! L7 j/ e
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. " o* M4 U( R4 v7 H6 @' \# m
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.  , }0 F) ]- c4 [7 T# T
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. 4 X0 o4 B6 ^  V- f/ O9 }0 W% \
4. Mix and incubate at room temperature for 15 min.
! J6 r, o/ w. K; J4 P" r$ T" r5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.
: q0 u* V  v% `: g: O) I6 |  m6.  Incubate at 37 ºC, 5% CO2 for 48 h./ p4 K9 E- f# b) V9 x
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.# ~1 g6 z$ e3 [( |5 W) @9 \
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
0 t& J. t" p. {8 ^9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
6 O) e4 f% I' B+ u6 m( c1 K5 L10.  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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' r0 f: {# S. aPreparation of human fibroblast cells (IMR-90)
/ G' |( k& `  d2 N1.  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.. u. r4 D/ Z8 ]4 N0 h; j
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
/ o3 x) p' e/ O) B' z3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant." \8 t% z; t; ~' j1 U% r/ S2 T4 i5 f  @7 S
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
* t/ |. d$ j! p5 o0 m: l: X; W5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
) z' W3 S4 E) v" I. P" D6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.. H9 x' u" v4 p6 [) r3 b
7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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Retroviral infection
4 i, a3 v5 S) U3 x1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
, Y- U. I; Y0 u: R! c2.  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.
+ F0 F* I% @  B: c" {% m5 a3.  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.
- ]( _  a/ L- q1 O' M4.  Repeat transduction (including virus harvest) as described above.
. x3 G; b9 V- x# `( m5.  A 3rd transduction may be necessary.
8 s0 C0 {, S' D; L9 j6.  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).# Y5 D3 x! V3 J) m
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
/ G! Z1 {# Z" H0 }" N8.  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6 o! B5 A" [$ \" D) Y
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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！