iPS 建系经验

细胞海洋

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6 ?) }/ @! j" I- z本文系ｘｙｚｅｎｇｈ版主原创 非常感谢
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IPSC Generation by Lentiviral System Protocol
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. f  X8 ?/ D: ]: @( B+ [Lentiviral Packaging
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% p; h! b8 O0 D1 v, N4 qMEDIUM4 `7 C( k. J5 z$ Y+ E* t( p) {/ M
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids., L7 l% ^! H5 F* q, \9 ]
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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- w8 l4 _$ O5 ^) f( n, t3 M293FT CELL CULTURE
: F+ Z- A- M5 {- c6 _9 cMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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/ M+ W0 Y, ^! \% F; rREPROGRAMMING FACTORS
3 _+ U# ~, y, _Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4" K3 C1 k$ S+ B0 d+ N
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LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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0 e( z2 R! T5 S1 i" OMaterials Each T75 flask- n$ q1 f# O' Z" B+ |6 j3 W" E
293FT cells    10-15x1067 s) Y" l) B& A& {$ D
MD.G (VSVG)   5 µg" j2 f1 h) u) V! o* ?" \  G
PsPAX2   10 µg5 ]  o! G4 }/ r4 \" w! @7 W
PSIN vector (~10kb)   5 µg' `: L; ?$ R3 R7 ?( X
Superfect (Qiagen)   40 µl
$ N- q. L5 T' sIMDM   400 µl) C/ X. Z: d# p
293FT medium    10ml$ ^0 A5 N% s; s4 v. \5 p# t  ^

1 m4 }$ z. P: m1 }; ~1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.$ W3 g) C! D# c
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
8 p& Q/ s+ Z5 o" ~; T2 G0 O4 H3.  Add 10 ml of 293FT cell suspension to each T75 flask.2 J: f5 X2 {6 J# d( ^
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).- @  ?$ w' u, U3 O
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.
& D2 p. G$ N4 c: W1 T( r5 Z6.  Incubate the DNA/superfect mixture at R.T. for 10 min.8 L' }& b1 b- f3 }8 g7 V- Y
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
9 p( r  M) }6 K: D- t, o8.  Incubate the cells at 37ºC O/N.  v" N2 t. h! o6 I; d7 z
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.% j$ v% o$ k$ I) e& D
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
' w  d8 T8 ~5 h3 Q4 M$ o! K- w( G11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
( V: L0 o; h" N8 T12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
% f% z0 B. l4 l" `' U% p13.  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./ Z% Y0 M# p3 V6 S, R

+ X6 I$ t) K' `! |6 Z( i' xPreparation of human fibroblast cells (IMR-90)
5 [6 i' u$ C. T1. 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.2 U7 S/ W4 d: W. w* x7 z
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
  {1 P0 ?' l: R. D4 o8 n9 O3 h, C3 z3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
, D6 O$ ~# n7 ?" ^! U) b4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
$ F6 {& f0 t2 b5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.' Z7 c0 M2 E3 Z- X
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.. {/ t5 |3 W8 h* u3 S
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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' \' n+ M# g  O$ `Lentiviral infection
* O& q2 V; p% @1 [* {4 u+ Y4 M1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
9 a' Y' A: ~% v# `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.& M6 S# c9 M/ {$ M6 R7 i! \8 e1 r  p
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.
5 Y/ f6 F7 E7 g& h1 _, q6 S5 E4. Repeat transduction (including virus harvest) as described above.
( r5 _# i2 [) H+ k8 F5. A 3rd transduction may be necessary.1 A" f% b& \2 J: y& T! P5 H! J/ }
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).
& J5 F& b( X* S7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.* d% b2 @* h1 k, h1 x' }8 x
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  R+ H; R9 R5 Y, j7 t" J; e7 U. H. v& Z
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Retroviral Packaging
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4 k3 {& M* G7 P; g2 @. OMEDIUM
# b, D9 f5 U& F: k! w. j293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.' P2 Y, {7 x  p
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE+ P, ?3 d7 _  M$ E, b# v" w5 {
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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REPROGRAMMING FACTORS
% S; M& T+ b: e. `$ epMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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Transfection of 293 FT Cell with Lipofectamine 20004 Q  h7 P" W: g5 d/ [+ v
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For T-75 flask
) d3 B. @# U& \Prepare 293 FT cell:
% @% u& ~+ u( l& j' A! S9 FPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
& a- H8 `6 G  h& r  _, TObserve 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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, h1 ~$ F- L. ~- n. Y3 e. E1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. ' C8 Z/ O; W' [& T. }( |0 [! ?* x
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.  
% T" h/ F$ b( \  S) _% Y3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. 4 Z, F( w( m2 ~1 |
4. Mix and incubate at room temperature for 15 min.
3 K: e! a" ]1 K: k2 q7 C  E' _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.
( L, J- e$ V* j! F6.  Incubate at 37 ºC, 5% CO2 for 48 h.. n% z( u2 `+ |$ Y3 w
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.3 M  Q: Y5 K2 y$ x
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.3 ?9 k* p% n6 A: V
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). 5 n6 f, F! X: B3 G- j0 K' |
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.- w+ `% s6 n$ T$ c6 ~. {; i- p

( |# ^8 |0 h, A9 c* f/ u; sPreparation of human fibroblast cells (IMR-90)9 S. O/ _# S5 Q7 Z( `* k7 [
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.% V4 v+ ~8 S( ^. V' x
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
1 r# B# w- v2 z) |" E& ]3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
" L  a0 y$ N8 F1 |+ h3 `5 H4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
6 I4 t9 U) s8 p3 X. `- ]# ?5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
* ?5 E! {; e/ U6 \( r1 A, S6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.( F( L$ w5 [5 n: t7 ?9 A* @
7.  Incubate at 37 ºC, 5% CO2, for 6 h.0 ^7 [: q' v! g! @* Y  ^
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Retroviral infection
" _& v% Q& l9 n# Q- q: J6 j1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
6 t; l1 r  }( F5 [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.. D3 C/ q) Q: J5 R( x" p
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.
, R* h$ s" m2 v4.  Repeat transduction (including virus harvest) as described above.% K) j3 c* e; v& y& x8 g) S
5.  A 3rd transduction may be necessary.! H3 r, x) J- B
6.  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).. i  D: \+ C+ l) U% q) d
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday., E, p' w/ d2 E1 s
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9 V+ ?+ d, U& N+ D/ H
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问：unconditional human ES medium 和conditional ES medium的差别？ ) b, u% L# r/ E0 ]
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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。