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, V6 a6 e5 O, Q2 V' A% f' u
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IPSC Generation by Lentiviral System Protocol
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Lentiviral Packaging
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% k( z7 ~: a. v9 q! O- }, iMEDIUM/ Q: I2 R( |0 S# ~
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
  b0 q6 k+ _+ k293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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7 F1 {' V- y& T293FT CELL CULTURE
8 E+ ]5 `) W" I& VMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. & G$ l3 f, Q( L, B+ T* t. N
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REPROGRAMMING FACTORS
% T6 m  T  W% A# S5 h  r0 A4 BOct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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Materials Each T75 flask; d  v5 `. K- P) z3 U- [/ w
293FT cells    10-15x106( p. h/ @' L& D3 ?
MD.G (VSVG)   5 µg3 b/ T  A3 K, r5 Z
PsPAX2   10 µg) S: I9 g1 y, v- T# s
PSIN vector (~10kb)   5 µg+ l6 Z# N; n- U7 S" e2 F. C" c8 L
Superfect (Qiagen)   40 µl
0 W: m. s6 J, r/ a2 x: GIMDM   400 µl
% J% f2 _) p0 @% H" ?5 R% e293FT medium    10ml
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3 `3 |% b- h; O  U4 ^* K, s& d1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.. J8 C" D" W7 [9 r* S
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
. o/ h/ J& P7 x. [/ C3.  Add 10 ml of 293FT cell suspension to each T75 flask.# x! k: |# u( T6 V$ D$ U' L: \
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
4 w% t3 Z0 s; n& p* Q: {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.
3 g4 F8 n" R3 j* \" q8 s& d! N. {6.  Incubate the DNA/superfect mixture at R.T. for 10 min.0 d9 a  Y2 z5 b/ {) Q) \
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.8 p6 E$ v. b- h
8.  Incubate the cells at 37ºC O/N.
3 i$ i* G3 d; B2 v1 s) \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.8 j$ C9 A- N  Y* z' [8 I* L  J" Z
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
7 i* ?; ^& Q, |' x/ v11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
3 [9 j  U- j6 {- t6 ^7 l12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). 3 n  x8 E: |& p9 V5 f7 n+ w/ U; r
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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( S' b! w# ~3 ]Preparation of human fibroblast cells (IMR-90): N! G' `% l, X- s* W" G
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.
$ j# g# o' U" |9 L/ t* T' f( \2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.( q" k3 ?/ |( v2 m0 g# r) G+ ^3 f
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
* o6 J3 `) Q# B7 D' [4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
4 P: z" m% h, F- U5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml." o$ {1 w$ j8 r" G8 x
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.0 j% {6 A# Z7 B- b1 v4 F
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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, t) C. F4 e8 F# JLentiviral infection
2 |5 ?- r' w6 e8 D* [1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
, E. H3 f( q7 R4 p" N9 b! g! E2. 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.7 ?1 [: W: l8 |# G# H# b
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.
. L; Q* s- M$ F# O  L8 x( E: D4. Repeat transduction (including virus harvest) as described above.
' @! a+ b) D  m5 g9 v1 x; t5. A 3rd transduction may be necessary.1 o1 |. [" y: Y3 k
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).
3 L) Y& e6 ^0 C" c% l. D7 h! f7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
5 U, w, U4 ]. W  u  K( {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 Protocol3 ], o% n1 H( a4 p! W0 c7 ?

8 Z7 [- A+ ^7 L2 h' P  WRetroviral Packaging
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( ^3 v/ V7 X% K9 lMEDIUM% z  {+ c2 r6 u) n5 H& e6 s' L) G; a
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.7 W' c; V8 o2 l  l. w
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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3 R; H9 T$ j5 {293FT CELL CULTURE
0 J: t1 L( `# @Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS1 y1 K8 ~- [. w; q3 i! r) I
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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Transfection of 293 FT Cell with Lipofectamine 2000
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For T-75 flask
% F' U+ ^) ^3 CPrepare 293 FT cell:3 R$ j8 D. d3 F
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.: O* C& b) J( G$ G$ a
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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8 \6 D9 o: u9 C" I/ v1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
$ o# A. y, J4 h" c) w0 A; x2.  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.  1 C  ]: Q8 l# N: A! v! P
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. 6 Z  {! q4 S3 \% |" i7 c: I3 K
4. Mix and incubate at room temperature for 15 min.
9 m1 x- n$ M: h# _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./ m+ X+ Z" S  b1 C
6.  Incubate at 37 ºC, 5% CO2 for 48 h., \2 D& T2 `8 M! X/ l& u
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.: G1 k; |$ p0 c5 D
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.  x  F9 [  t* C: f+ I
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). # }) V* g2 r5 }/ K" l$ f2 Y
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.. h* b2 o# {" N& K$ B. h/ M
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Preparation of human fibroblast cells (IMR-90)4 @- s9 I# Y. N, P$ ^1 t; o
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.
, C: p: L6 k) p0 t4 s' v* h2 L$ q2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
- L" {5 }* M- S* x4 k4 X& b3 e3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.0 {+ q- O& h1 o2 ^- D% I& ?
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.- k: d! Z0 t' b) T. U
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.2 z- H  u0 v5 c6 |7 L1 ^: }& y
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
. i) b0 N: D* }/ a" c2 Q, I7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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Retroviral infection
) G; P  E, ?3 p7 P* A5 J/ G1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
2 z) [! x/ O4 n3 W' 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.0 i1 L# D# m9 z4 i
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.2 a  `7 E3 ~! O7 P: k6 f
4.  Repeat transduction (including virus harvest) as described above.7 b) c; l5 g4 ^( |8 s# z
5.  A 3rd transduction may be necessary.
- q& t; x: a- [5 F4 u3 B' g; a4 U6.  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).
5 B% w3 Y7 u! D5 W7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
; S. }7 [4 @/ 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! O8 I* ?* z" L( ]. Y7 h

7 P7 f% p5 |; N) ]问：unconditional human ES medium 和conditional ES medium的差别？ 6 t, t" l0 J/ e* T. I

/ R( Q/ v" w5 _3 I% yｘｙｚｅｎｇｈ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！