iPS 建系经验

细胞海洋

IPSC Generation by Retroviral System Protocol
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2 D" y, L$ m* V5 z2 i) FRetroviral Packaging 2 y; f% V5 l9 u
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MEDIUM
; E4 _" x3 ~9 u2 g: F- S293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids./ S" W: B7 P( c; d( j. L9 Q
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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0 d* {2 {4 w9 K7 W" B, C" v/ w4 u" [293FT CELL CULTURE0 v" g0 S( q1 g( q
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. ! P# c/ @* s  A+ F" a6 D% r
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REPROGRAMMING FACTORS1 G/ e, T1 u* }8 G$ U6 \+ [* r
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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- t* P& x" D* X( S' HTransfection of 293 FT Cell with Lipofectamine 2000
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For T-75 flask9 b4 u& l' V* l" Q
Prepare 293 FT cell:% q* n4 C) u9 Q7 D7 z* r
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.+ Q+ d8 W. L) u* t- v3 X3 h6 m
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. 8 u% k) U3 A: S- Q! ?
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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium. $ A( a9 n- p; A. ^9 r0 m( u
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.  8 H4 l' S+ ^: V+ r
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. 6 [+ A+ |2 a; u& e. H- ^& F$ J
4. Mix and incubate at room temperature for 15 min.. V3 B; F2 ~$ V( X% ], q) 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.
: N; b# ?) h3 {+ f7 u8 a& p6.  Incubate at 37 ºC, 5% CO2 for 48 h.
$ G. Q. R2 w4 d  c0 J7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.4 q' t+ k- b; \! k1 {) v# [. L
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.2 u* ~3 o5 X' o. [
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). 8 Q5 n1 l/ \2 R% v" C  ?
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.
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Preparation of human fibroblast cells (IMR-90)/ t* o  i5 L0 i4 \, x% M) _
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.
! S5 k/ G, y- m7 y& F2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.7 t9 u. l8 Z3 B  o
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
' v  e3 r! z5 P. i/ _* L4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
' n' B+ i: D  B  e1 m. y5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
" B  O! P9 d, V* n1 d6 r6 Y" S3 C. V6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
% ?6 E$ H5 H) ?" \7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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  E; j: L& P) }( }' |) [! j' P5 ^, G; bRetroviral infection/ C4 h2 t; C1 U& T
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
, p5 G# @7 B2 Z" 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., ^3 ?6 N9 n" m
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.
9 Z0 o6 n7 l/ ]- t- f/ d4.  Repeat transduction (including virus harvest) as described above.- \0 B8 t7 Z2 S2 f  c
5.  A 3rd transduction may be necessary.
' ]2 ]+ z$ y) `! @' j% @6 u. r6.  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).
: e/ }, D' M) ]7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.3 }! F' E4 ^* x  _( n+ z0 w
8.  Day 17 (9~10am) Change to conditional ES medium. Observe any colony formed and pick them up when they grow up.