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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% [. m8 i9 b( h& M
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MSC Isolation from Rat Femur and Tibia
) z" F5 q! p8 S! F% x1. Euthanize the rats with CO2 for 5 mins.+ A& M8 o9 D5 }
2. Isolate femur and tibia by disconnecting them at the joints.0 u" R6 [6 x6 a9 Q& {5 }% m( R& ^
3. Clean the bones of soft tissues.9 ]9 K! d9 S6 @2 a2 ^% w. p
4. Wash both femur and tibia in PBS containing 1% penicillin/streptomycin twice.
: F# L4 Y, U) ]5. Under the cell culture hood, clip-off the epiphyseal plates to expose the bone marrow.0 F! E# Y L# Q# u! `7 e, ^
6. Using a syringe, flush the bone marrow with 10 ml of media into a dish.
1 r. g0 ]9 U) d" Y/ }6 V9 N6 b7. Break up the clumps by aspirating the cell solution multiple times.
' l! W# y3 W: \1 s4 c8. Using a 70μm cell strainer, filter the cell solution into 15ml tube. This will remove any unwanted debris and bone fragments.; @; h3 g9 {2 y. C
9. Centrifuge the filtered cell solution at 1000rpm for 10 min.8 E% h0 b6 q2 D9 L7 a8 L
10. Resuspend the pellet and pipet this solution into a 100mm dish.
3 |; ]) Z Z2 v" M% n11. Incubate for the next 48h.& V$ n1 _ ^8 Z* F3 l: P' d7 D b
12. Change media to remove non-adhering cells.
, ` l" Q7 L/ t. }; Z13. Change media every 2-3 days, even the media doesn’t change the color./ L: E, E3 A3 B% J) R, O( z3 g9 T
14. Usually the cell will be confluence at 7-10 days, and then are ready for passaging.
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发表于 2011-11-15 20:30
发表于 2011-8-16 21:43
