iPS 建系经验

细胞海洋

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6 L6 D" A* M1 J. ^/ D5 L  t本文系ｘｙｚｅｎｇｈ版主原创 非常感谢; D" G8 }6 Q; R/ O0 U2 H, M6 a
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IPSC Generation by Lentiviral System Protocol
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( V+ e, {+ m' l+ F/ MLentiviral Packaging
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" t8 c& _+ j3 j% U: MMEDIUM
$ x% P$ @6 M9 }293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
- g$ ]* O! E% h& [1 g293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE& M& K  l! b) o6 g) k
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. * x% ?' {2 P4 s

' R* D- ^6 U3 _0 f1 |2 Z9 PREPROGRAMMING FACTORS+ a9 s- U, z0 I: M
Oct4，Sox2, Nanog, Lin28, c-Myc, Klf4
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* a9 m/ @; p5 yLENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)! `4 u- m5 Z- f

* d0 I# Z: K+ f7 S7 QMaterials Each T75 flask3 r! V2 U1 _4 W5 k
293FT cells    10-15x1060 X% v0 K6 {4 P( x" L* B
MD.G (VSVG)   5 µg
3 n  }* r; H$ c9 T& jPsPAX2   10 µg
" F1 J9 q' [: Y, c3 o8 pPSIN vector (~10kb)   5 µg
1 V- ~3 O, h0 K7 o1 ISuperfect (Qiagen)   40 µl
' T  Z, L$ g* V# ]IMDM   400 µl
/ U1 D4 h1 y3 ~* Q  m0 k293FT medium    10ml$ d5 z+ q' |, E! I
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization., E, D2 |3 e. o2 G& t& [
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).
6 [) y/ L+ V# ?6 k  L+ W3.  Add 10 ml of 293FT cell suspension to each T75 flask.
  |# _9 \; z/ Y! ~" U' Q. P8 I( K0 I4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).2 ]+ ]& X" ?7 `' V* q% O6 I
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.8 L: @- k7 j3 P5 ]
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
, G% f) {" l& C% V7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
4 l2 Z7 V8 R) R4 l8.  Incubate the cells at 37ºC O/N.
8 T: K4 w5 n; u# d, O7 N" b* a% }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.2 Y. p) u) C8 ]* a
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
2 g: x) b* z% F# ~5 q$ _11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.9 i- e  d" ]( g  A4 w
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). " B# h4 u) q2 u# V8 [
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.% @% w0 j* [8 g  P# }

7 h6 _  s. b8 gPreparation of human fibroblast cells (IMR-90)
+ K; x- M  [; Z( v1 f1. 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.: o# c. n$ M  `/ p2 x" V
2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
2 Y+ g" |! P: G% T" x5 T3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
. p# ]: w/ l+ r! N/ m- G1 [4 ~, Z4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
$ C) z: w' m5 M: k' R, C- a5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
0 V& ^$ ^$ j1 {9 Z4 H2 n' n) f6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.- C: i6 ]) D1 F- }5 g, B' b
7. Incubate at 37 ºC, 5% CO2, for 6 h.
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Lentiviral infection# M& C5 C* }9 i; q. o* N
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.3 I% b; |8 ~" y- {5 i, `( X
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.- f% J8 y$ u) A- ]8 p/ N
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.
6 [1 X5 R& W0 W2 X+ w, f4. Repeat transduction (including virus harvest) as described above.
. J" B2 d& A5 L5 C6 p5. A 3rd transduction may be necessary.9 f* k7 Q9 b  ]
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)." O5 |9 a. Z2 C' O4 \; {
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.6 x' P/ p+ F2 k) B2 R5 B! {
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/ L, n9 \5 {# M: f7 j7 G3 y. u. i  P
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Retroviral Packaging
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MEDIUM* c) V  J* s9 [( @, e; |* Z9 H1 r
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
* ]% E; e+ C: l293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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5 _; w5 A& P/ _- y6 x) C293FT CELL CULTURE
! [0 B3 r, f/ D; ]Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 9 I( D: R8 p  x( h/ T
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REPROGRAMMING FACTORS7 K/ M6 l/ E# ~% }; z7 c
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4
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Transfection of 293 FT Cell with Lipofectamine 20001 S' |  W5 C% a1 ]+ w$ E

. _3 c0 g) |# s9 Z: {5 iFor T-75 flask1 ?/ x- e  J. y
Prepare 293 FT cell:
% @7 i3 q/ k0 ^$ X: APassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.0 R" w# m8 w7 L: h1 H  l
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. 5 Y7 x2 _  P% z" h
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1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
. v) M" T  V, w3 C5 Q; |% 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.  
  g6 M0 Z0 v7 q8 B1 O3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
+ v* n. b7 u+ r4. Mix and incubate at room temperature for 15 min.
( X0 }2 L/ F8 \  ~2 ~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.- H& ?8 {, U/ [" g) G7 |3 G, z
6.  Incubate at 37 ºC, 5% CO2 for 48 h.8 X* b0 j4 o; ~( D/ b1 o+ O6 A6 g
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
3 E, S3 g8 A1 V8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
6 ~7 r! Y* U  h2 B) u' V7 i" r9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). $ T' {, {( q8 F
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 A+ s8 h, Q0 C# ~" |: O& Y: [* Y& M9 |! M7 O" j; X$ b
Preparation of human fibroblast cells (IMR-90)
- I" Q5 _5 `; L% J* V0 R1.  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., U0 o% p: _) Q: Q
2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.( H3 T, X9 m2 }
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
/ W3 Y  E" V7 I4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.8 J. l# b. R" F9 P
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.- `+ K6 Z8 u8 V  g0 x9 a
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
( t- u, {! c9 e* j7.  Incubate at 37 ºC, 5% CO2, for 6 h.
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* q# q( ?# W* i. ^Retroviral infection7 F/ L9 a  ^0 n7 s
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
/ E4 G5 z" l; |; f8 P: I9 u0 @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.
. O6 ^3 f) z; b+ V" C8 r# U" X3.  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 e, l: r/ L% K& W% ]* l9 J
4.  Repeat transduction (including virus harvest) as described above.
, t! {& W: e  u; M" r) |5.  A 3rd transduction may be necessary.
* T4 f, }" F+ J, [: i6.  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)." A5 H/ {5 I6 [
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday." `* Y9 I8 |, p" f  h) T
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
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问：unconditional human ES medium 和conditional ES medium的差别？ 7 k( l- c) k; p1 _8 U: A& \

: e( J2 t- N3 ?- |$ \5 Y0 iｘｙｚｅｎｇｈ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！