如何将胚胎干细胞和支持他生长的饲养层分离开来？

待鹤

使用MEF来源的条件培养基代替feeder。" Q% x3 \7 w# d
条件培养基制作方法如下：+ ]2 j, x! X+ v# j$ y4 C8 I4 Z
Preparation of MEF- Conditioned Medium (MEF-CM); |$ Q2 B2 x/ y" E& C7 V7 Q  G- Y9 T
1. Plate 4 x 106 mitomycin C treated MEFs in a T75 Flask coated with 0.5% gelatin, in complete MEF medium.) C7 O7 g, B8 j
2. 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.* {) p! ^) u; ]( `
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.
0 }# V) V) _7 ?4. Add fresh 20% KSR hESC medium containing 4 ng/ml bFGF to the flasks.
* w  u0 x" y9 S% i* q) V6 x# q5. Collect MEF-CM for up to seven days using this procedure.9 R: ^1 C! c+ N
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.

待鹤

这儿还有Nature上的一篇protocol。) H! b! I  a: c  @
Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.pdf/ N/ m% Y+ |  J