如何将胚胎干细胞和支持他生长的饲养层分离开来？

blue11sea

MEF贴壁速度和明胶关系不大吧

blue11sea

应该使用空板来爬，因为有明胶ES也容易贴

冰罗布

谢谢，学习了

mominglily

回复 5# czzhaozhu : D2 D8 n! n* X9 F( R% I" _+ g
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    谢谢您！我自己也打算用这种方法来做，但是是否必须选用和原板面积一样大的板来爬呢？用较小一点的面积的板可以吗？！~还有就是爬的时候需要加凝胶铺板还是空板？因为有人说使用空板来爬，谢谢！~

czzhaozhu

对于mES细胞和MEF feeder如何分开来，我们实验室一般采用差速贴壁的方法，MEF feeder1h左右在明胶包被的皿上有70-80%贴壁，而ES细胞基本不贴。

mominglily

万分感谢，我先下载来学习下，但是目前我们在做的是含有饲养层的mES细胞，就想寻找针对有饲养层细胞的方法。

待鹤

这儿还有Nature上的一篇protocol。
1 D7 u0 J* r3 `0 h) Q5 D% iFeeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.pdf+ k. B( [: P2 i1 Z+ Y

待鹤

使用MEF来源的条件培养基代替feeder。
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! V* Z; ]7 a( M0 kPreparation of MEF- Conditioned Medium (MEF-CM)
# ~) F" C3 ?8 s7 `1. Plate 4 x 106 mitomycin C treated MEFs in a T75 Flask coated with 0.5% gelatin, in complete MEF medium.
! l" F7 Z3 G* Z0 j  p2. The following day, replace the MEF medium with 37.5 ml 20% KSR hESC medium containing 4 ng/ml bFGF, and incubate for 24 hrs at 37°C, 5% CO2.# z6 e8 `% h$ F
3. Collect MEF-CM from the flasks after 24 hrs and 0.22 μM filter sterilize. MEF-CM can be used fresh or can be frozen." X4 G  P+ q% k8 a0 r/ p
4. Add fresh 20% KSR hESC medium containing 4 ng/ml bFGF to the flasks.
) g* b1 H4 F3 S- \5. Collect MEF-CM for up to seven days using this procedure.7 @  X( a( {; j. ~  `
6. Depletion of L-Glutamine and bFGF from the MEF-CM is assumed, and L-Glutamine (to 2 mM final), and bFGF (to 4 ng/ml final) are therefore added back prior to using MEF-CM with hESCs. Freshly thawed ß-Mercaptoethanol (ß-Me) is added to 0.1 mM final fresh each day of use.