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
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' s" f" e& [! T* u9 MIPSC Generation by Lentiviral System Protocol
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Lentiviral Packaging # F6 `" W8 w. J% m9 F

; `) a, j# N4 l* ~, m2 pMEDIUM
6 z; e' [+ R1 j0 y# E& x% v293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
1 O* b. |- h2 ~* ?- o2 J293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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9 p5 y8 t, W% o" \! a, d& e( _293FT CELL CULTURE
9 X9 J7 l) ^1 v& f; b5 PMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. ( H) M! I' u# @& B: Q- I# I% f: Y

  @' m- B5 C; u6 e& R2 T/ aREPROGRAMMING FACTORS
8 g4 v4 y. {* D9 dOct4，Sox2, Nanog, Lin28, c-Myc, Klf4( ?! W3 J+ [7 I# P/ [

0 z5 u- f7 X$ H6 \; pLENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)
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) k6 X! b. L& G" r8 ?; ?* @9 jMaterials Each T75 flask
- I) W; n& U/ b  ]; o2 j+ b6 i! K293FT cells    10-15x106
0 e; Q$ N/ o' Q3 V1 LMD.G (VSVG)   5 µg# q: j# p! G# v# _
PsPAX2   10 µg, ^; J% `% r) z7 s/ K2 r
PSIN vector (~10kb)   5 µg
1 o% ]" g* `' e/ U% [6 VSuperfect (Qiagen)   40 µl
+ Q8 Z& @! h; b4 g2 R* f" sIMDM   400 µl
  J5 ?) T9 B% F293FT medium    10ml
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
9 K1 d2 A2 C  e; Q4 Q7 r2 O2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
4 c: A) }. l1 p" p8 @3.  Add 10 ml of 293FT cell suspension to each T75 flask.  b2 Y' M9 V4 m" S
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).6 V: D6 ]( p8 x2 J
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.
* Y6 `4 v0 R0 T7 g9 B6.  Incubate the DNA/superfect mixture at R.T. for 10 min.- B/ z) `. M7 b' ?+ ^/ |3 C
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
( w& c% ~: y- T8 m/ @( g8 h$ F8.  Incubate the cells at 37ºC O/N.
9 O! s; u7 x' u. {7 O9.  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.
7 N, z8 U% ?2 s, C3 k+ i& Q10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.( s8 R! z0 s' U3 j2 T# c# S( Q, M
11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
  d4 z  [; ~9 k) v2 z, X+ j, x$ Y12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). : I8 G, Z" I/ N! ]6 R! }) x
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.& K6 u. \$ z) S5 g9 }8 t

. o: y7 U3 m! k3 ?4 MPreparation of human fibroblast cells (IMR-90)! K( U5 `% u. C6 c) T2 j: H
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.5 e' n: \* v- H+ ^9 K! Y: y7 S2 a2 {/ q( _+ Y
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
& A" e2 q; U9 v3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant." |6 c- P. E$ q9 `4 W9 f) i
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer., a; `  |" U& p3 O7 `5 |( E
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.9 ^* U) e8 ~' d4 m" E
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.+ j7 }8 C3 }9 V: ?, c, b* w& `
7. Incubate at 37 ºC, 5% CO2, for 6 h.2 f# T) ?, A3 o  q' W: r
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Lentiviral infection
2 x7 [1 Y/ D8 Y* Y& M0 O& ]- @/ h1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.' ?6 i& E$ R$ g3 ~: r  {" w
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.
- q9 X! o5 ?4 Z8 }+ c  ?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.+ [* e  s9 a! O4 B4 v" D! r' V/ b
4. Repeat transduction (including virus harvest) as described above.
& `& `. \3 B2 Y0 X+ X, |6 N0 {# H* }5. A 3rd transduction may be necessary.
) w, o# \; y9 d. 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).
# R+ ?  J/ U6 u; m" U7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.' H' {. o* Q% R% ~6 s% Q4 D( B& Z) u
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
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0 m; s) @" \0 ~1 ?; J4 ~Retroviral Packaging
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MEDIUM! A2 @5 B, D6 y/ a
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
, y& N; w; ?+ R293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.3 }" x4 O  U" j( \- m' R7 x
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293FT CELL CULTURE5 A8 c/ R0 j) p2 {1 W- V$ F
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 3 p3 E- ]1 t. ?: U+ ?: R0 b
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REPROGRAMMING FACTORS6 N, g4 ~7 R* A* ?: E' d' G
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4  ?* x" }  k9 O: x% w+ `) P( h
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Transfection of 293 FT Cell with Lipofectamine 2000% t- J) l( }% X' j

# ~6 D' d! ]; |For T-75 flask
: n/ X# l% X+ c+ a4 Z; J! @Prepare 293 FT cell:, T# o; H0 S3 m$ E
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.2 A; E, b3 {5 I% \  Z3 T
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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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
& u4 g9 y1 i0 o: _' u; s) O2.  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.  & B* T7 L6 U/ ]9 D
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol. & `' a; }$ Q, S9 B, K3 P
4. Mix and incubate at room temperature for 15 min.$ o2 t8 ]4 _, B, o# ?
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.
5 \( {& q2 d: I4 Z/ `6.  Incubate at 37 ºC, 5% CO2 for 48 h.9 i& H+ q2 l( _, c3 b1 c
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.) k8 `% {$ n" @! [" o
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
/ G& s  v- U* W1 q: d2 A! {9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
2 ?# N! ?6 O" B+ |10.  Use filtered virus directly or store in 600 ul aliquots in cryovials at -80°C until use. Retrovirus can be stored at -80°C for several months without loss lot of infectivity.- k6 a# U$ T- _( W
& W* ?& Q( d% |5 h9 F9 B
Preparation of human fibroblast cells (IMR-90)
9 k7 ^  j+ w, v+ z* ]8 q1.  Day 3 (~ 10am): when fibroblast cells have reached 80% confluence, aspirate medium, wash once with PBS, cover cells with 0.05% trypsin, incubate for 5 min at 37 ºC.
- H3 l+ \) z& Y# {' @1 J+ C2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
% d2 Z$ \1 k) g3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.0 B6 V* u& x+ t" ]8 e1 O9 j
4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
8 A- A' g, Q1 f- _5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
2 V2 c, v0 b& A+ \6 Y. r5 ~; o# V6 n6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.& q) W1 A5 D3 |/ h5 _9 K( {. [  E% y
7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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( ~: l2 F/ d! ^+ t! cRetroviral infection
9 [7 }$ J, ^- p# @. V1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.* S/ @0 _) @+ u# d9 u7 {* _
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.4 \- }* {2 t& 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.
! |# ?, ?" A4 \# `. ?2 s4.  Repeat transduction (including virus harvest) as described above.
  ?9 Q6 K( S9 j  b! ^8 t5.  A 3rd transduction may be necessary.* d# O9 H% m9 J- x" z& @0 u
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).- v3 ^6 n; l( v) l
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
9 w) r9 \  D+ I8.  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' Z, P, b! u2 f1 {
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问：unconditional human ES medium 和conditional ES medium的差别？ 8 J& O* U% ~& K/ e/ ?/ W6 k

/ K1 d6 p+ d& P  p  \% C# rｘｙｚｅｎｇｈ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！