iPS 建系经验

细胞海洋

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/ J, P" x1 i$ T本文系ｘｙｚｅｎｇｈ版主原创 非常感谢
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IPSC Generation by Lentiviral System Protocol  t+ X3 N9 a, [9 y. C- f* ^( f- T
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- y* g' m- D/ H6 G, e# PLentiviral Packaging
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" V6 o/ Q1 X  E: F' UMEDIUM
# M9 g, ]; T; G: q1 E293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.( Y- D4 p7 N8 P; o
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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5 ]5 R. H: c0 K$ s293FT CELL CULTURE5 c' c( ~2 u# S( j/ @, `
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. + Z! J; z- t: E1 k2 M

# [+ A3 }1 g4 j5 k: f) ^REPROGRAMMING FACTORS
& a( r. r: s7 v" h2 g4 N5 vOct4，Sox2, Nanog, Lin28, c-Myc, Klf4! Y, G6 q) _& i, w1 O! k8 w. g3 |3 i

0 V' a6 r5 G, r7 W2 m+ kLENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer): ], P' g  g. ]% W
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Materials Each T75 flask
; q! Z  n# ~2 Y" P( y* _293FT cells    10-15x106
& L- r2 N) @9 U; ^2 s( aMD.G (VSVG)   5 µg( n( J4 E2 Z1 }: A
PsPAX2   10 µg) D7 a- j9 z+ j
PSIN vector (~10kb)   5 µg
' R& U  y0 A9 Y' }9 A& I; mSuperfect (Qiagen)   40 µl - Y& d9 u* V% j' {/ a
IMDM   400 µl
) z$ ?8 Q9 {  t0 F6 P! P293FT medium    10ml
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1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization.
& ~; k# z+ p4 B' y& l) @2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin).7 \3 `. J' I% u- u
3.  Add 10 ml of 293FT cell suspension to each T75 flask.% y& Z! Y" g  ~0 ^7 |
4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).  S, K" u; Q- P2 I! l: 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.. H/ I# L( Z! f: \0 Q/ K% W. t
6.  Incubate the DNA/superfect mixture at R.T. for 10 min.$ x; h& T; W/ W  a9 l
7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.
9 ?  H3 A6 E) T% b4 N, L! i8.  Incubate the cells at 37ºC O/N.
3 D! o. ~/ ^9 y* `- S9.  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.
: j9 K& j% d- k4 a$ R# b* [10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
% z  f! z$ ]: @" |9 ]11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
& @$ o) J: ^" H" Z# @( Y0 ]' n% X12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
" [! e6 z  a5 _8 |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.' I" m1 z. T. _# C* h2 Z1 b/ N

! m4 O  h" C# Q& o6 gPreparation of human fibroblast cells (IMR-90); k" N4 j8 W0 ~2 S- g# q
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.
% z4 o+ I% c: l; A/ q$ b2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
4 K$ f. X) H7 U" h) o3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.$ \; H) _0 U$ \, ]  S( Z8 x: @
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.
( r. H* o1 D$ ~, c6 P( v; T5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
) T# C6 w6 M" F6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
. E/ T) n3 |: v) {7 }8 n7. Incubate at 37 ºC, 5% CO2, for 6 h.
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! s& N7 |2 T- h$ v; s7 P6 sLentiviral infection( O6 @! ^9 B3 ~5 q: j+ M) a4 g& s. P
1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.3 n" t  R  p( g7 c" x* [) L
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.
- X5 a  F# n' z- w9 I3. 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.3 o7 {4 E- K) k8 c
4. Repeat transduction (including virus harvest) as described above.
. b0 f$ ~' w# q& v5. A 3rd transduction may be necessary.' ^2 |# y7 y6 t; |6 i; i
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).
7 q) L% ]2 v) [. v- E; F7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.
1 t, [" r" W$ m$ f' 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 Protocol+ [( O# t" B  b& ?  Z3 ^% c
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Retroviral Packaging
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MEDIUM! d' J- R. ?% O9 j
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
" T0 y8 u" g4 N( a" u  u  i293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.
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293FT CELL CULTURE' [" w& s  J7 j
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin.
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  O4 Z9 T1 b; M& O0 xREPROGRAMMING FACTORS* k5 ?+ `2 ~; @8 g4 x. z) ], j
pMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4' o$ R5 Z- Z  A. w: c! K6 ]

+ L( a; S* P5 T. v4 Y! O4 jTransfection of 293 FT Cell with Lipofectamine 2000
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For T-75 flask
# K* x. |7 M& V2 Q! J( DPrepare 293 FT cell:
& O/ O  h. @( p9 ^& UPassage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
$ p) e# ]# U' n! V6 bObserve 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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* k8 j6 W1 Z# X2 a1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
* Z5 N: J8 H% d, J* a2.  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.  9 g6 Q. N: M8 h" X
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
; {( Q# w. ~# v; n- X- _4. Mix and incubate at room temperature for 15 min.6 C, X* ~1 S# r% J+ \
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.$ G! J0 W  G1 h: b# r" x: J
6.  Incubate at 37 ºC, 5% CO2 for 48 h.$ _, k7 ?' R4 [7 L
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.
* b! w7 s$ `% a3 J( ~+ m8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.
8 q3 I5 M" p3 l9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS). ) U+ W2 o7 M$ d' w8 h
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.4 f8 E, r, f/ Y4 `  h
: K/ g& }& T$ L
Preparation of human fibroblast cells (IMR-90), v6 l3 R; t# @
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.
/ ]* u; [6 X9 B- J2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
/ v8 T6 G# U, N) P3 c' r3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
; l9 X# Z5 C# r# s4 G- |0 K4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.& P6 t; L' h; @8 F6 a5 W9 J
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.
; Z5 e/ Z6 g  h2 ]: i+ y6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium., f  I) u# l: P8 O
7.  Incubate at 37 ºC, 5% CO2, for 6 h.6 @& ]; \# c) L

9 v2 }  h) C1 j9 nRetroviral infection+ k. }0 [4 ~6 q# Y% @8 d
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.9 ^) s: H4 G% j9 D) p' P4 U
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.$ d; ?/ h' D' _- [
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.
( d/ Q1 R) f$ H$ f4.  Repeat transduction (including virus harvest) as described above.
7 B' r8 d; k, N6 A5.  A 3rd transduction may be necessary.: F, r4 N+ |' k% W. }
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).4 g, d" Y; U& n5 Z
7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
/ g% w, |9 g1 W* i7 @2 ?) m8.  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# ~  V- H  ]# ?3 g) Q$ r8 c! m
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问：unconditional human ES medium 和conditional ES medium的差别？ ) o& b- Q( |) ~

! b% F7 d4 z1 o. Y9 m# f8 Z: ~1 Eｘｙｚｅｎｇｈ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！