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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IPSC Generation by Lentiviral System Protocol( J5 e  ^3 V6 v: ?

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Lentiviral Packaging % Z- }% g6 [! [( \
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MEDIUM5 Y+ S) X- i+ ?$ ^2 ~' X
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
. H+ l3 {% _" ~# P293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.& T0 n" {( g7 }& Q: e7 @; \

8 V% F  q, \0 z$ y1 ?293FT CELL CULTURE
6 X; W! c6 [+ L2 J( ^/ P# t. @) @Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. : g/ l2 p0 ]2 j3 x4 ?5 D0 |0 }

- N' O0 s4 d; _8 wREPROGRAMMING FACTORS- O4 d& n/ Y" A
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf47 g( h% E# H) V7 p# _& B" _
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)6 C* i8 c% G- c  ]/ F$ l! [

0 j+ R4 ?5 ]8 ^: j4 m5 jMaterials Each T75 flask/ M$ @) j4 W/ w; X" w5 T6 m! m* c& c
293FT cells    10-15x106
' ^, s* q/ N+ z+ fMD.G (VSVG)   5 µg" e8 ~) _5 j7 k3 X  [; O
PsPAX2   10 µg4 A  M' d* c9 C+ h3 p* N0 r
PSIN vector (~10kb)   5 µg# B+ b0 U: H" n
Superfect (Qiagen)   40 µl - Q. t: o& v- s' H9 e% m8 p5 s/ g$ S+ X+ s
IMDM   400 µl
. t! N0 V- v! g; h, _, Y293FT medium    10ml
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7 u0 D; _: d8 u2 q; _* w# ]1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.! Y' e8 F8 B. y0 ^
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).9 F6 ?! w1 Q" C4 b# C
3.  Add 10 ml of 293FT cell suspension to each T75 flask.
5 S# ?5 g6 N$ K2 l7 s8 u5 G4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).# O6 b& q4 ?1 x0 t
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.
( h$ C! ?5 n" D( {" Z- a; q6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
- O* }; {5 B& I- [# A7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.' U% Y: p! j" J- p! A5 }
8.  Incubate the cells at 37ºC O/N.. C1 C" i6 U& G+ Y9 D
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.
7 g- C8 h5 B+ `- q8 L9 V! V10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
" o9 a( A- L0 g/ p" a. ~11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
2 s1 D, F" b( h( H  _12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
+ z( y3 f$ _* R2 ]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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/ c# S/ Y' u. H' A- MPreparation of human fibroblast cells (IMR-90)3 C; K$ J1 b: Q6 E- S. ~, N
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.6 \/ P) L- W: t  B( y. r0 ]
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.. K/ G; }% l, E' {* H7 z! e( H
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.+ ^* S' J$ O! x( ^3 M
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.2 \8 E/ w$ g0 K' m- c8 h0 d3 X9 n6 x
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.8 [2 x* b( ]% G: D, O
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.7 w2 H! g1 P* ?8 F0 A; T8 p
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection
0 d% X& v1 v, H% K/ p1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.. d# D4 z# p. C  h1 n
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.- r6 P. L0 i8 h. r0 ]
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.7 x1 k6 O' R  f6 W1 @, o: q# r+ _
4. Repeat transduction (including virus harvest) as described above.
/ S, _& z# G; C) ^" G0 ?5. A 3rd transduction may be necessary.
/ v$ _* {: v  I* o6. 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).  J) i9 a3 e1 \7 s" t+ G  Y$ a
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
1 C! E! ~9 z1 \6 n8. 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 Protocol0 M6 w3 P  g0 ~6 m7 f" j
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Retroviral Packaging 4 a" k/ `! L1 J( B1 _7 Z

" g* [& v. f, M: g- Q7 T; FMEDIUM
, [: Y9 _* K6 _$ o293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.  |$ N# z7 S2 C( A
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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; l! J7 Y# S/ i- P293FT CELL CULTURE
5 O! `. h% f2 N4 _, U4 H" ]2 ~Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS
' o+ P# h& {4 D3 i4 v# bpMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4( y% R# P# u: \

4 O4 f1 S; e8 q5 r. UTransfection of 293 FT Cell with Lipofectamine 2000% [& ~9 |+ Y3 n0 ?, r. F  [! X

% _  i, S5 d: a% zFor T-75 flask
# O3 D4 c# r% n% E% ~Prepare 293 FT cell:
+ L/ L" s7 `3 ?% n7 oPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
4 [2 Z+ K* V5 P. u6 @' k( _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. 3 [% F9 f, J( f1 g; Z7 D

5 U; I) S# W3 P1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. - Y. C4 P+ i, N; c0 |5 _
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 i8 Z& V$ R% ^
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. * ?2 |! S" _8 [: Y4 m) {3 a, j
4. Mix and incubate at room temperature for 15 min.4 |( J1 m( i1 F6 U5 z" G
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.1 Q* \3 M' O/ c5 F. l. \
6.  Incubate at 37 ºC, 5% CO2 for 48 h.
$ M; G6 k3 o5 i. U1 D3 C( h: u7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.% I* z! E: t- n+ a  K
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
1 r2 c' }, ~9 ^9 u& I9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). 6 z4 J" P3 p3 U( r
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.+ [: m- @( W2 ?; o+ R! [3 d1 S# o0 G
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Preparation of human fibroblast cells (IMR-90)
# j2 H; S7 k+ m( J" H9 G1.  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' Z1 P5 g* {# e7 ~5 H
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
1 w$ c$ [- x# d- y3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.$ N9 {- _% Z  }) u/ H7 _
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.& y) \! n1 w$ K4 Q" {
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
& M$ v, k2 c) m6 b) K6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
! e4 E- F3 p, y; R. a7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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* m" F4 I# ~2 o0 ^, u, gRetroviral infection$ G9 ]2 z! C8 q7 U. o& o
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.: G8 M( n3 ?2 A% L1 O% a1 b% b8 v- a" p
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.
$ c5 E/ U, a- J3.  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.. L: V- g; _  _  B0 A& y
4.  Repeat transduction (including virus harvest) as described above.( \6 p, G( p6 Q
5.  A 3rd transduction may be necessary.
. x) }* K* z# e! }5 q2 o6.  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).8 C9 O+ d1 x/ C9 g& ^
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.& h& |& G; ]8 h  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？ * Q- r+ x3 l, M6 P' R' S, B

: ~( U0 b9 E9 w. n2 q" L2 B+ Cｘｙｚｅｎｇｈ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！