iPS 建系经验

细胞海洋

5 |% L5 ^. A0 ?7 s' y
( n+ s  [: j/ k, B0 |3 i2 r* Z, I本文系ｘｙｚｅｎｇｈ版主原创 非常感谢
2 d+ M5 W, R* r) Z) y, c
3 a6 M- U0 h( q8 I6 l9 ZIPSC Generation by Lentiviral System Protocol
& I: z7 l$ F3 Q+ g: P- O( K$ J5 [! A% \* q8 t  q* u* K+ s. I4 q
  
6 l5 R- t. x' N1 T9 qLentiviral Packaging + w! v2 h8 \2 k" w  ?) j
, P# q9 j: N* A% @4 |( V4 }# w$ R+ h! y
MEDIUM4 V$ v8 l& v$ |. }, Z3 D
293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.
$ f8 F( d9 v8 U293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.$ l" z% l1 L; x. [7 X

' p" F, l) p/ \) Y$ g8 w293FT CELL CULTURE" ~# V& Q# d# {. y. k
Maintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. 5 `, P+ L( X9 }. H" m% p7 s5 Q

1 x) s! @, B& A/ W) W. \* l8 \1 d8 QREPROGRAMMING FACTORS
  [4 k% v' Z' U7 y/ l. gOct4，Sox2, Nanog, Lin28, c-Myc, Klf4
+ Y) i; J; D; i( j" R' e
0 z2 e; s. t2 d5 j5 uLENTIVIRAL PACKAGING (~10^5-10^7 particles/ml titer)* i' @+ t! `8 g; W7 u+ X6 V
5 G+ v1 ^, {) y- P9 R6 [" B
Materials Each T75 flask; h, a/ S, v* H* _" p" ?0 \2 }
293FT cells    10-15x106
6 C0 a: C# ?! y  J0 I- _0 sMD.G (VSVG)   5 µg
* z, u: g7 ?/ k1 |" }* @: B9 LPsPAX2   10 µg0 U$ Z. d+ D( {2 |& c7 k- I
PSIN vector (~10kb)   5 µg  R+ U% x' w! X3 J5 F( C
Superfect (Qiagen)   40 µl 4 F1 r$ p1 `/ H' ?2 `* i8 c7 _
IMDM   400 µl6 t1 V: o9 F5 X  k' d0 H
293FT medium    10ml
4 c( {, s& G# D$ k) o; y
* g0 d* a7 B6 v/ U( Z; u. V( C1.  Day1 (~ 5-6:00 pm): Collect 293FT cells by trypsinization." [# Q7 n6 U! p# y5 ^, C
2.  Count cells, and resuspend to 1.5 x 106cells/ml with fresh 293FT medium (without Geneticin)./ @1 K8 H& @" A7 g; k
3.  Add 10 ml of 293FT cell suspension to each T75 flask.
9 U+ `, z" [8 E  {' h4.  Mix corresponding amount of each plasmid in R.T. serum-free medium (IMDM).
$ N% l4 ]/ T5 a! I2 D7 d, t8 k5.  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.
3 g/ Q" R. o6 ]9 n4 M7 i4 Q$ w6.  Incubate the DNA/superfect mixture at R.T. for 10 min.
: ?1 J9 I0 K- g- O" n+ T! O7.  Add equal volume of fresh 293 FT medium to the DNA mixture, mix and add dropwise to the cell suspension, and mix.: x8 D; d5 B8 E
8.  Incubate the cells at 37ºC O/N.6 y# ^. Z5 O8 r' p
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.- H4 g( h& n( P
10.  Day 4 (~ 6pm): Collect virus at ~ 68 to 72 hrs post-transfection.
9 D5 y) `- o: ?# @* X: @; z% K; q11.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.0 |- s' r2 g2 b) H
12.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
+ u; b) V9 h  U: V13.  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.0 R3 \7 Q7 a% N& K: o6 ^$ m

3 z* Q3 b' \) W" Z. r, N( ~Preparation of human fibroblast cells (IMR-90)+ T& W. p8 k, I; n: n  }  \
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.
6 c) t. Z6 p6 `2 `0 K, R! n2. Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.
, o/ j; ^! T4 @; ]! i2 f0 Z/ e9 u$ a( d3. Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.6 b0 v7 j0 i( a- X
4. Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.% b1 l! a9 b7 R8 A4 c
5. Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.7 u) J! b. f' u% p& c
6. Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.
, E. v: j) W9 E2 f$ l+ I7. Incubate at 37 ºC, 5% CO2, for 6 h.1 n* C+ {' X) {3 `
9 N, Q% L, L/ ^) C8 j
Lentiviral infection
$ [+ a' T' x% e+ z# @' D1. Day 4 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.
1 x1 ]; C7 O) T. ^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.
0 j6 `7 Y# O! Q, d9 n( s3. 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.
- K1 G- m& a' e6 i' y5 R4. Repeat transduction (including virus harvest) as described above.+ t8 E  @! H' Q7 g
5. A 3rd transduction may be necessary.) r2 V* Z8 a  c. ?
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).' C$ X$ P3 W3 _: `# F- r
7. Day 9 (9~ 10am) Replace with fresh unconditional human ES medium everyday.6 @$ i' Q: N8 O7 v: ~
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 Protocol1 w% T' L3 B1 {' N: U
) Y. K- S- \, H" \: N: q. w5 C
Retroviral Packaging ( ?; A) u, l4 m2 ~7 f+ ~# Y* b
9 H. N/ l# P0 V+ c& l- _
MEDIUM
! [3 G5 k$ Z5 c( d* D7 Y8 Z' O293FT medium: D-MEM/10%FBS, 2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids.* y, M& d% M( L4 d2 r, H
293FT complete medium: 293FT medium plus 1 mM Sodium Pyruvate.6 S7 @9 `% R7 ]) o/ `" G

2 T! I2 i1 o% }( F5 q  e2 }293FT CELL CULTURE
/ U% {3 E& S) BMaintain 293FT cells in T75 flask with 293FT medium plus 500 µg/ml Geneticin. % M6 |8 M) f& ^% l* v' J! Q+ p

, o7 ~  @1 v+ C! H+ LREPROGRAMMING FACTORS
+ b- e, t2 i2 _4 `5 a1 npMXs-hOCT3/4, pMXs-hSOX2, pMXs-hc-MYC, pMXs-hKLF4& }) p/ v: S! S- b3 L

# r3 H! j' U' ?/ BTransfection of 293 FT Cell with Lipofectamine 2000% u! S3 U0 \0 x' c- A! k
6 Q5 E1 l  g! V/ }* i
For T-75 flask) B# G  F+ J6 w# a9 D" U9 e; m
Prepare 293 FT cell:* C- a7 N3 ]6 J
Passage 293FT cells (4-6 x106cells) one day before transfection to T-75 flask.
( j( ?/ p+ H* Y& HObserve 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.
! A9 p  Y- ?3 f% t* m& n/ c: u+ }4 T* \5 J- q6 B6 |2 ?; }
1. Day 1(~6pm): aspirate medium from 293FT cells and replace with 10 ml fresh 293FT medium.
( y5 M' u" ^7 h+ N2.  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.  . K, s6 D/ G4 |& v9 N1 _6 z9 n% [
3. Add 5 mg retroviral vector, 0.5 mg VSV-G and 4.5 mg Gag-Pol.
1 o5 |' x( q1 D& c4. Mix and incubate at room temperature for 15 min.
! ?/ A) H" K2 D8 h# ^. m5. Add the transfection reagent/DNA complex to the cells in a dropwise manner. Swirl the dish to ensure distribution over the entire plate surface.
9 H" W* ^! S3 F/ Y# L6.  Incubate at 37 ºC, 5% CO2 for 48 h.& L7 D) w/ d- i1 |! N
7.  Day 3 (~ 6pm): Collect virus at ~ 48 hrs post-transfection.1 C: b: l2 E6 W) X& d1 F1 p( V
8.  Centrifuge at 3000rpm for 5 min at 4ºC to remove cell debris.* o7 w' n) u0 H0 j. I
9.  Filter the supernatant through Millipore Millex-HV 0.45µM PVDF, Cat# SLHVR25LS).
9 \+ }! O: N0 g8 i10.  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.% {" V" @$ }0 ?5 j

; t/ g. k0 h! b4 ]% {/ i* aPreparation of human fibroblast cells (IMR-90)
0 U( R0 C& i$ {+ Z; R. ?# p1 y1.  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.
+ }. W* J1 C8 v2.  Inactivate trypsin with fibroblast medium, collect cells in a 15-ml conical tube.2 G: k0 d7 ~" `% H% a  X2 M; H
3.  Centrifuge the cells at 1200rpm at room temperature for 3 min and discard the supernatant.
& F1 \! Z  H* e4.  Resuspend the cells in 1 ml fibroblast medium and determine cell number using hemacytometer.- |3 e. w: A% o* L$ m/ x
5.  Dilute cell suspension with fibroblast medium to 2 x 10^5 cells/ml.6 X9 b0 r# v: x! P/ c' j
6.  Transfer 1 ml fibroblast cells suspension to 35-mm dish (coated with gelatin 30min in advance) and add 1ml fresh fibroblast medium.% f9 u4 b9 v9 ^% U* j" e
7.  Incubate at 37 ºC, 5% CO2, for 6 h.
7 _, U- S3 t4 \1 R3 y( r2 L
/ {( w; R8 |1 l3 h" i5 R" `( SRetroviral infection, q+ `' K+ t6 J5 }5 u* ^2 D
1.  Day 3 (~ 4pm): For fibroblast cells, replace with 2ml fresh medium 2h in advance of transduction.1 j8 k/ B. R" B% J
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.7 y7 o  k4 K5 S
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.! m$ _0 [* T/ Z0 b3 ]$ i! f
4.  Repeat transduction (including virus harvest) as described above.
6 R' P: I* |# W$ {* o% L% G% }! T5.  A 3rd transduction may be necessary.
. {4 P2 P9 B, J4 \1 l) X: b6.  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).
/ g' P2 ]" p- Z; @$ h  H7.  Day 8 (9~10am) Replace with fresh unconditional human ES medium everyday.
  \1 [, v; {" p3 f! }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
8 h% B# X; |0 d' m7 M
$ P# Z1 l# j) a. A) U6 u$ L* |2 `问：unconditional human ES medium 和conditional ES medium的差别？
. G0 i- G0 n: V% A
& P2 U, v+ V3 ~! ^8 e* Xｘｙｚｅｎｇｈ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！