请问神经干细胞可以冻存吗？

aifei001

做实验有时候对代数有要求，每次都提细胞，费时费力费经费。哪位前辈有冻存的方法？还有，冻存后对细胞的性质有影响吗？谢谢！

ljx

回复 aifei001 的帖子: r/ ]+ H# p: n# _8 Q7 N
9 ~  B& S6 |" N( B8 R, L8 N
http://www.cqvip.com/qk/91237A/200802/27139868.html

aifei001

回复 ljx 的帖子, b3 ?9 @0 v* S
5 t/ h5 L1 I1 s( m' E
谢谢。这类文献是看过些，但实际做的话并不能重复出来。最好有英文文献，或者哪位前辈亲自做过。

黄毛

有文献报道用9份NSC培养基加1份DMSO，我试过，是可以，另外用9份的DFBS加一份的DMSO也OK，但这些都要保证有足够多的细胞才行，神经干细胞的生长状态有很强的细胞密度依赖性。

安毓菡

我看到的nature protocols上介绍的，希望有帮助~3 r$ Y# t( W: x! a
Cryopreservation and re-establishment of neurospheres:
, j0 u+ J; _7 H  e2 P; e, {1. Pool the culture medium with the neurospheres from a 24 multi-well plate into a 15 ml conical tube.
1 v4 `1 F7 Y- }* a8 i2. Centrifuge for 5 minutes at 200g.5 J1 Y' B- P; f2 R
3. Remove the supernatant and re-suspend the cells in 10.0 ml SFM (without  EGF and FGF) containing 15.0%
, `; Q3 B/ f- H: J( bdimethyl-sulfoxide (DMSO).
) [" v, T  j7 H& A4 _4. Gently mix the neurospheres in the SFM with a Pasteur pipette. Aliquot  1.5 ml of the mixture into polypropylene% E/ h* E& `! c9 N% a! e5 t" W" w
cryovials (Nunc Cryo Tubes cat. no. 357418).
; n3 ]) M, @: Q5. Transfer the tubes to a -80°C freezer overnight.. c3 I% m' j9 {$ Y' e7 @- E
6. The following morning, transfer the vials to a liquid nitrogen cryofree zer.
& y; ^, ]1 _* KTIP:
2 T  C# v) L& ]+ N* m! J; ^If you do not have access to a liquid nitrogen freezer, the neurospheres c an be kept for several weeks at -80°C. The number
# g0 ~2 [5 k8 U9 E( ~of viable neurospheres obtained in culture will decrease with prolonged st orage at -80°C.9 p. X# k+ ?9 f* @. i' d% c
7. To re-establish the neurospheres in tissue culture, remove the cryovial  from the freezer and let the vial warm up to room5 x- z$ Y' k, S6 E, U
temperature on the bench.* \8 S" D9 ^# ^! |5 c
8. Add the contents of the cryovial slowly to a 15 ml conical tube contain ing 10 ml of SFM.( B! ?# @3 j6 W3 [# M/ p6 Z7 P0 h9 Z% m
9. Centrifuge for 5 minutes at 200g and remove the supernatant.
& ^; n0 j8 X; F! j8 k! M; }; D10. Gently mix the neurospheres with 4.0 ml of the SFM with a Pasteur pipe tte.
8 N/ k' D) R* l) {11. Fill 8 wells of a 24 multi-well plate with 0.5 ml of the mixture and incubate at 37°C with 95% air and 5% CO2.
- ]& C9 Z" Y$ X7 [TIP:. W8 U; h; I8 n3 v
Smaller neurospheres will survive the freezing process better than larger neurospheres (in excess of 100µm in diameter).
" U: Z3 G; |* w6 i' FThe viability of neurospheres following the freezing process in this tissu e culture medium is low (< 20%). It is
  u: }# y- o. _$ G4 d& O2 Yrecommended that the individual wells contain at least 50-100 neurospheres . You want to freeze a high concentration of
5 j1 e. B1 H& }; g  e4 h9 O% Mneurospheres to enhance neurosphere re-establishment after freezing. Neurosphere viability can also be enhanced by the, n2 j  h' e) Q% i$ `# R
addition of 20% fetal bovine serum (FBS) to the freezing medium. However, keep in mind that FBS will induce4 K& c+ h% V. ^& p- m; N
differentiation.

yzhang_pumc

神经干细胞冻存液：20%BSA+神经干细胞培养基+5%DMSO，我冻过几次，复苏出来的细胞挺好的，希望对你有用。。。。

chengyun

5%DMSO+50%FBS+45%神经干细胞完全培养基也能很好的冻存