iPS 建系经验

细胞海洋

. e8 u# L& q+ y/ k; v' d
( s- {: F; V# n" K本文系ｘｙｚｅｎｇｈ版主原创 非常感谢
4 }! i$ H" D0 h* B: c6 t- Y& ~5 C9 {3 i2 q3 [% m! l
IPSC Generation by Lentiviral System Protocol; n  n' @9 d& F- h7 C+ H4 l5 w

9 Z1 s; B/ s6 Q# ]6 U. X- Q  / l2 t4 k% ]1 d6 l' u
Lentiviral Packaging 8 c8 m* `, X1 q5 U" z- ^' R% @2 R

( y- I/ E1 j! |5 L5 _0 N3 B3 E; uMEDIUM
' I- c" D- L6 M4 @: H' g293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.' K9 D8 H9 L2 N& A) @7 L) \
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.. B' L$ l& [1 D+ D) b2 ~

, y: C3 i  J- e* j293FT CELL CULTURE
4 W9 g: ~- A* ^0 r# d4 @( NMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 7 H. q) Z! u( g8 |1 Z0 f/ }; z
: h" M, k1 `% T! @( k' s2 s$ j3 G
REPROGRAMMING FACTORS; D: b2 s' o# O
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4
- j# V5 K+ {2 a, h6 ]* ]* d
% H$ n6 g; Y# D$ U5 E* g7 ~LENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)6 x  V+ ]$ c% L2 D- b' O7 R

8 O' B8 `$ W5 ^% L8 W+ k4 IMaterials Each T75 flask
4 b6 S% a7 h- Q$ X293FT cells    10-15x1065 W! j: V' A! Z/ F5 ~0 C
MD.G (VSVG)   5 µg
8 X6 ~$ A' h! {# pPsPAX2   10 µg
3 z6 {/ |& V+ K: qPSIN vector (~10kb)   5 µg7 K7 R) m; |" R& @& J+ S* J0 R
Superfect (Qiagen)   40 µl * v' w, b* m9 N8 H% N' d  n) u
IMDM   400 µl
& {. u1 u) k8 B9 p2 E293FT medium    10ml$ X( k, s/ u& L5 |

9 g* R7 U4 U/ A! [5 D6 L* N1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
4 C4 y" Y5 o+ ]* o0 b: U2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).- x7 c5 `$ ^" N
3.  Add 10 ml of 293FT cell suspension to each T75 flask.
8 a0 E/ E7 u/ {, d) Q4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).& w- o& F6 B5 N1 q4 r2 n  {
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." x. S* s" w8 X8 D8 f
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
5 m9 \$ P; M3 q' C! W. X- }7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.# m  D: o4 W' L  F7 u& W5 O; S8 Q" n
8.  Incubate the cells at 37ºC O/N.
. A5 ~# ?' F$ e0 ]9 ]( g! E+ a2 c9.  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.- u9 v7 \' h* b& l
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
0 k0 ~5 |1 T8 w8 N) `11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
9 Z4 G  F) o: Y( p. |, A3 ?+ E/ t9 h12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). 0 F, z' r1 s. H  m
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.* h& r+ s) N8 d+ l. y5 ^+ v3 i- [
, ]0 k4 m0 q/ i7 O, L( N  P
Preparation of human fibroblast cells (IMR-90)
+ f) C9 K' z' ~! [9 r1. 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.+ n+ H8 g3 P0 U! d
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.& C; D# m1 u8 `7 _4 C! C& s
3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
$ P% X, }+ S1 H- K4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.2 O: d* y% |: G5 t- b8 L" l* W5 Q
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
  ?9 G% b, d8 r/ u9 ]5 L5 q6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
8 Z: W, r1 ]. h1 V6 C) W7. Incubate at 37 ºC, 5% CO2, for 6 h.
* w; T: M& g  p+ Q% _
& k2 F; @: o  ?) a+ |Lentiviral infection
* ?3 n1 H; m" U5 {# z0 [1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.6 w& [1 b" e( S2 f3 ]. a
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.
4 \7 \+ k, H- L1 r0 h' F2 R) o3. 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.# }, J) U. a1 g; X6 A) u5 v4 H9 G. ?
4. Repeat transduction (including virus harvest) as described above.& M, J! z' R: T. q( @* I
5. A 3rd transduction may be necessary.
. J. R( P8 y$ B; h2 D! y1 m6. 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).( \" S; v% _$ Y5 \" ~# w0 M
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
, D! P3 C0 J# R9 O# B; E8. 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 Protocol6 B# q6 M) y: x( c$ V6 u) L

: _9 g$ T* c, p2 [4 \  H  `Retroviral Packaging
. b, G# l% ]! _! ^
& l  F4 M7 E5 GMEDIUM
' C/ j5 X* t- P0 w8 G1 w293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
4 A* n: E  U- w& t293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.6 Z3 b" E& j& P& g9 J

7 r4 c, t. e+ }3 U! t! v6 `293FT CELL CULTURE3 e5 R$ @4 D7 i. m
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
" G& j+ L- ?+ L" V$ q! B2 o! L* A. r  d
REPROGRAMMING FACTORS5 p- |/ b- e; j
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
, m/ `) k: n* v6 s
5 F, E" P2 f% `9 M4 x5 d/ s9 OTransfection of 293 FT Cell with Lipofectamine 2000* N7 C/ Q# i0 v
0 E1 f- K* |9 G# I( \! s- \2 |* Z5 u
For T-75 flask" Z7 i3 o4 n" W) F! K9 c  A
Prepare 293 FT cell:
1 V, O# S+ }7 p% S$ F6 z: C+ o( ?5 n% zPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.! d5 Y4 @5 P8 W$ S9 c" P5 w- S& o
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.
9 z5 h8 `/ r) I
# U. t. v* j! I4 j7 O" X4 n. n/ n1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
. _- U0 o- \. B3 C8 D2.  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.  
. \) r# e. E/ m. S3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. : x/ s: R! v4 L. X( X
4. Mix and incubate at room temperature for 15 min.+ A3 [; H; W' b6 \0 v
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.2 K; g4 |9 z9 z; A/ N/ \1 p
6.  Incubate at 37 ºC, 5% CO2 for 48 h.
8 n' F, O4 L* ~& h7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.) S7 T) k- F( s0 V+ w. S
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.% G9 o! m" T$ l
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). 7 W7 J- E, j# o2 b
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.
3 {  x6 y! C( D( H5 O3 b# D% ]+ P+ J% r! _% Z$ ?% i
Preparation of human fibroblast cells (IMR-90), y6 ?1 c# i# q) k( X2 b
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.3 b- t+ J, x% d8 G" {3 ]5 r9 y
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.7 I8 T6 a/ P1 S5 k; r. n- ?& Z
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
4 O. t/ i1 y* v; Y6 U  Y+ P4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.0 C9 [" t1 l/ _; F! y# y: n
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml., X( a% K6 w1 ~$ t
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.! i8 \& I8 b8 \% _& z
7.  Incubate at 37 ºC, 5% CO2, for 6 h.- N" y& Z" [1 e8 E

$ b  G  V7 H6 Y3 p% MRetroviral infection0 z( g# H2 k* K( L; o2 o/ [% x
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
- g# _" o  g1 }$ y! ?7 X2.  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 P; l4 G' J; h7 B
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.. Q" Q  v4 k7 m" Z" g
4.  Repeat transduction (including virus harvest) as described above.
/ r  G4 U  `% N) [5.  A 3rd transduction may be necessary.
, e4 S8 w0 W4 i, Q* |/ @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).7 U7 K$ b3 ]1 C) u: i  q; d6 M
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
/ I. g; s& c" _- h: i0 c$ E6 H8.  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
* V3 {* U6 @# j# l' I
+ l& c* g4 b% ?' p8 I- X问：unconditional human ES medium 和conditional ES medium的差别？
# l& p! b; H- N/ }' B3 r  K3 I  l+ P1 s# m$ s& U6 f
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。

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！