小鼠骨髓间充质干细胞

viv2005

我做rat的，小鼠的没做过，不知道我的protocol有参考价值没，感觉MSC是最好培养的了，比什么osteoblast和osteoclast好养多了+ }* Q( t9 _8 Q0 q0 s5 S

/ g8 [$ e( \/ I- dMSC  Isolation from Rat Femur and Tibia
, G. R* J3 N4 e1 W' X% f) f1.        Euthanize the rats with CO2 for 5 mins.) K6 B0 {( n, o7 H0 i- b& o: w) V
2.        Isolate femur and tibia by disconnecting them at the joints.$ @, ~  K$ H6 }3 O
3.        Clean the bones of soft tissues.
5 }( b" _  M3 X1 z. z$ F9 n4.        Wash both femur and tibia in PBS containing 1% penicillin/streptomycin twice.
  t3 _! {8 H/ q3 D5.        Under the cell culture hood, clip-off the epiphyseal plates to expose the bone marrow.* o- D+ e6 Z/ W# e7 P/ z# g) ~
6.        Using a syringe, flush the bone marrow with 10 ml of media into a dish.
/ w# b2 ~# J* Y/ I. T7.        Break up the clumps by aspirating the cell solution multiple times.  `% u& `3 x( ^. X# g
8.        Using a 70μm cell strainer, filter the cell solution into 15ml tube. This will remove any unwanted debris and bone fragments.
& O) N6 b( s" V# j' o% F9.        Centrifuge the filtered cell solution at 1000rpm for 10 min.) Z/ j3 V4 P2 m+ `$ j% r; A
10.        Resuspend the pellet and pipet this solution into a 100mm dish.8 u/ s- q) `% e" k" ~" l9 n
11.        Incubate for the next 48h.% Q# Z8 m, o. R2 N/ h
12.        Change media to remove non-adhering cells., E* v% H' F4 [4 y- L  n
13.        Change media every 2-3 days, even the media doesn’t change the color.. M7 B5 n( {. i1 l. l
14.        Usually the cell will be confluence at 7-10 days, and then are ready for passaging.
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