iPS 建系经验

细胞海洋

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# f6 N+ l6 ?' e- [# K/ N本文系ｘｙｚｅｎｇｈ版主原创 非常感谢
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0 ]3 C- D$ r7 L6 N0 PIPSC Generation by Lentiviral System Protocol9 [  K" u1 D8 K7 W# b: \

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+ y. t+ a0 E' e7 b9 A, O6 ^Lentiviral Packaging 4 V; I8 G8 Z3 F# d3 T$ _5 N5 Z
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MEDIUM6 [. k& u$ a/ x
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.. J/ \- W3 ~0 l4 R0 j, ^
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.5 J8 \4 |3 j; \

2 t3 d) y0 }+ P# r# J7 T293FT CELL CULTURE4 l, [! [/ `5 z# l3 S
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 3 \( g8 o7 U5 E$ l' i/ T

# R+ r; e3 B0 l9 }REPROGRAMMING FACTORS
& q7 x5 D8 ]! y# @- E# h6 Z( \/ AOct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)) b0 j* `2 d9 A* I% Q" v
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Materials Each T75 flask
  E3 w: H5 H2 a293FT cells    10-15x106: M3 w5 U$ n# M0 k# V6 W4 K9 k3 }/ D
MD.G (VSVG)   5 µg
& i9 D; m+ {' J7 a0 O. OPsPAX2   10 µg
8 Q  R  b6 N* c* c1 ~PSIN vector (~10kb)   5 µg5 [( }( f8 o2 W9 Y0 `+ |8 h
Superfect (Qiagen)   40 µl
8 m8 o5 A, Y9 X; a8 o" I0 i$ D; n& cIMDM   400 µl
/ J+ P: r* o2 T! W% N7 |! s1 g! ~$ a293FT medium    10ml
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.; i; S2 O0 S5 A/ x- W
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
6 U2 `" L0 a4 V, R$ S3.  Add 10 ml of 293FT cell suspension to each T75 flask.  x5 X5 S( U$ s& ?# |
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
: b6 y1 G. |1 x& c; A5.  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.
( J9 H. c, W$ L1 S8 K4 @, |6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
' u: m2 p  c6 g  O7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.0 V: @# ?* q( q
8.  Incubate the cells at 37ºC O/N.
- x% }* ~  t1 [' k: I0 E' f9.  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.: }6 O7 H* d$ ?$ R* U5 R# ]5 n
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.2 D; @* \' \8 C0 d% b% X) y
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
& e7 i. i) R( X' ^12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
7 T" E- W& P: x4 i13.  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)
, k3 N  L/ P) w0 Q+ U1. 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." i+ ~- l6 w7 {" s1 v/ A0 A4 {
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
6 z5 Z8 ?6 V, Y: V# T3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
; s  i' Y7 d6 O. |1 O& v4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
: C. H0 i: b( a; @$ F0 l5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.. ]0 J& x5 i% h
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.5 N- ]+ D2 t6 ~
7. Incubate at 37 ºC, 5% CO2, for 6 h.9 ?7 `5 I3 c; w$ G. z0 G7 a0 U

5 d) B7 \! r+ vLentiviral infection  y/ G# U2 }& v3 @/ l( E! Q4 D
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
# i/ f$ E+ [) h" F/ M2. 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.1 D$ n: D" t: ^
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.( H( r/ W! e" z+ l* b
4. Repeat transduction (including virus harvest) as described above.& x* x$ P! B2 N9 m5 T# f# C
5. A 3rd transduction may be necessary.
0 Y, |1 H: b3 W' [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).: e" d. y( h+ R1 A, H) P8 d) v
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
' B7 L* w! ^) e  Q4 s4 A' R( M2 h5 Q8. 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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) [" s$ X# M  G4 kRetroviral Packaging 2 [1 [3 C% w4 w7 F( u. _

6 S0 I- J/ W3 @( T  B' B; qMEDIUM! A& ^) N9 J: r3 d
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids." m2 S# _& T9 y' i2 f1 t2 J5 b( e
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE2 C7 s6 V' H6 a6 Z! p; I
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. & k% }+ x4 @( s' L6 M3 x8 L
% s4 w( r' Q( o) I+ A% Q5 D4 F
REPROGRAMMING FACTORS# R$ C) N$ z( u6 [+ n6 M
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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: Y0 E7 w, Y; C3 qTransfection of 293 FT Cell with Lipofectamine 20006 C) A$ }9 W- R9 [
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For T-75 flask, F* t0 L' Z0 M5 }! n. \' F
Prepare 293 FT cell:
' D8 f( W2 y, T# c: JPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
+ m8 o7 F; w# z' B5 _1 MObserve 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. ; l4 @1 ]! ^' I9 f. x
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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. ' B6 q% q% A/ G3 C1 M7 {
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.  
* s" \! Z0 w: B. C/ l+ m$ [6 T0 v/ S3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
! Q3 o9 w7 E" o" |  @" p+ a4. Mix and incubate at room temperature for 15 min.
) e$ c! g6 \( j9 F7 F, K4 Z5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.
3 ^/ l4 M4 h; u9 m9 ^6.  Incubate at 37 ºC, 5% CO2 for 48 h.
& v; L, C6 L: A4 b7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
) V) I$ Y$ I2 H, I6 o5 l- s* o% z8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris." b! v7 E# A. h4 ?- i0 }: ]
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
# h; ?& @8 r2 j  d" E8 k10.  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.
4 Z4 ~6 [8 X! m( v  m/ @+ K
1 p% t7 L/ p# S, S- x, PPreparation of human fibroblast cells (IMR-90)
+ R' y+ P' X8 Z3 J4 x9 k1.  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.
* r( x; P  }5 g, u: J6 ]2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
' [4 }! ^' e9 a3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
- H3 i# ~+ R  V! \3 q0 K$ R4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.% \2 P& Y1 g( k7 j8 ]; b/ o
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.( Y( X! t) b6 P* [; l/ D6 {
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.( [: V% F3 Q6 i4 e; [
7.  Incubate at 37 ºC, 5% CO2, for 6 h.0 n* e: n) j$ N. g3 D9 ~6 c% Y& P: R; Q  w

" P; e( d; Q% i8 aRetroviral infection
% p' `1 n8 u5 y9 a+ K$ T1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
6 U$ U9 m# o7 R6 v( A2 B2.  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.8 u: K( D& s9 H, u6 K( g( I) e
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.5 L8 U9 V" Z/ P$ i
4.  Repeat transduction (including virus harvest) as described above.
1 V- U) U2 O% h; T8 ]5.  A 3rd transduction may be necessary.
3 F4 q! ?0 Z& I( z6.  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).
' n  l' u1 l9 Z5 X7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
0 n. D$ i  b; z/ a0 D* w8.  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) p0 W' e- S. Q) L' N9 C3 Q
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问：unconditional human ES medium 和conditional ES medium的差别？ ) z) x. S$ K/ U; n7 C

- x% P$ l# U* U) ?9 Qｘｙｚｅｎｇｈ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！