请问神经干细胞可以冻存吗？

安毓菡

我看到的nature protocols上介绍的，希望有帮助~
0 ], X1 f' r# q7 `! |Cryopreservation and re-establishment of neurospheres:
* z2 I- X/ e) T1. Pool the culture medium with the neurospheres from a 24 multi-well plate into a 15 ml conical tube.( y1 n; A- b& C; E7 S+ u$ b  v
2. Centrifuge for 5 minutes at 200g.4 n; N4 r8 S# O& N0 i5 P
3. Remove the supernatant and re-suspend the cells in 10.0 ml SFM (without  EGF and FGF) containing 15.0%
( R. P" W  p4 I; T; Zdimethyl-sulfoxide (DMSO).
7 \7 ]2 C3 X# N9 M0 Y* b# |4. Gently mix the neurospheres in the SFM with a Pasteur pipette. Aliquot  1.5 ml of the mixture into polypropylene
7 D3 V; Q2 ]( P. W4 n6 F% Z0 bcryovials (Nunc Cryo Tubes cat. no. 357418).
) f+ G3 e+ a# M3 z- G5. Transfer the tubes to a -80°C freezer overnight.
- K- [& {4 ?& f/ S6. The following morning, transfer the vials to a liquid nitrogen cryofree zer.$ C* a( D4 P! V9 H% {- v* h
TIP:
% }  A: b' w* e1 c& K0 O9 mIf you do not have access to a liquid nitrogen freezer, the neurospheres c an be kept for several weeks at -80°C. The number
+ l9 R# I; {, u8 Wof viable neurospheres obtained in culture will decrease with prolonged st orage at -80°C.
2 e% D9 n  y  e1 Y8 n7. To re-establish the neurospheres in tissue culture, remove the cryovial  from the freezer and let the vial warm up to room
/ @( f) M2 W. m) ltemperature on the bench.
! v& ~7 k) B0 c4 ]8. Add the contents of the cryovial slowly to a 15 ml conical tube contain ing 10 ml of SFM.
/ F1 D! G+ H) n- ?1 w9. Centrifuge for 5 minutes at 200g and remove the supernatant.
2 ]! X7 k; S9 q2 N7 a& _# I6 d10. Gently mix the neurospheres with 4.0 ml of the SFM with a Pasteur pipe tte.3 _. U" a4 I1 |, G+ z& O( a  q. G
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.
# m: B$ l$ P0 s3 i4 k) i* y" e4 lTIP:# \, U- `6 N9 ^$ F; O  ?
Smaller neurospheres will survive the freezing process better than larger neurospheres (in excess of 100µm in diameter).* c7 S3 x/ \9 D) x  k9 b
The viability of neurospheres following the freezing process in this tissu e culture medium is low (< 20%). It is# ^! g4 X/ ]3 p' M( K
recommended that the individual wells contain at least 50-100 neurospheres . You want to freeze a high concentration of
: L& s0 q0 C# y; B- T1 Y( Wneurospheres to enhance neurosphere re-establishment after freezing. Neurosphere viability can also be enhanced by the3 c- O  O4 x; b; X+ `. O
addition of 20% fetal bovine serum (FBS) to the freezing medium. However, keep in mind that FBS will induce3 a8 o% v3 P% D  x2 C% `2 {2 R
differentiation.