小鼠胚胎成纤维细胞的制备及处理(饲养层)

qgjin

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PREPARATION OF MEF CULTURES
# E% u4 p4 n3 m& `6 l$ d1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard, ~' U/ e  m% \8 D: E/ Z
placenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.
5 i8 b: R7 D) |% G% f7 G* p- q2. Mince the embryonic tissue in 5 ml of trypsin solution, transfer to an Erlenmeyer flask containing a stir bar and a few 5mm glass beads, and stir on
" W5 ?! o* ]( Q6 t- xmagnetic stirrer for 15–25 min (use shorter incubation times if the embryos are E13 and use longer incubation times if the embryos are E16). Ideally,
6 C3 \# K0 S& E& |the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).
, n; {6 y5 r, b4 ^3. Pipette the cells with a 2-ml glass pipette to achieve cell suspension. Filter the suspension through a sieve or a screen, add 10 ml of
' t7 e: u) O8 |MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.9 \7 R" N0 K; A3 z
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%0 }& n: B1 [) N2 Y6 f
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
1 D4 c/ _! [, d4. Plate the cell suspension onto cell culture plates at a density of about 2*106 cells per 100 mm plate (P0; i.e., passage 0) and incubate in
1 U0 [9 S  Q. |  V3 H$ iMEF growth medium at 5% CO2 and 37℃ for 24 h.$ D5 y* S; z# F3 ]
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
  _- j1 J/ _5 Y8 P8 q$ I6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.) k( [4 _- g- [. T. c
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.- q, A+ j6 U+ {' H5 {' d
8. Add trypsin solution to the culture plates and incubate for 1–2 min.
) z) h# k4 a  U9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).+ [4 G& L# j4 d+ ?# Q
10. Freeze any MEFs not needed immediately.4 x; A: M$ h, ?1 T
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
6 N% L, n+ Z0 e( K$ v" q" _( w11. Continue to passage cells as described in Steps 6–9 of this Box. MEFs at passages 2–4 are most suitable as feeder layer for cultivation of$ ^+ C/ v; @2 k& l9 W
undifferentiated maGSCs; prepare as follows.
, }/ ~0 K7 Y2 \" M5 y# v" [2 T12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.2 a9 k2 G9 g/ \9 J4 ~4 N& V
13. Aspirate the mitomycin C solution and wash three times with PBS.
5 R0 B! U( o/ @/ D14. Trypsinize MEFs as described in Steps 7–9 of this Box and replate them on new gelatin (0.1%)-treated microwell plates or to Petri dishes
2 L7 n3 u, w: x4 F$ ]# e$ v' G2 Zat a density of 50,000–60,000 cells/ cm2.8 j  z& f- \- s0 V& m% [+ K
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
  G* w, {) v. {+ J8 I- W% ]3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical: ^+ A% ]' l! ~7 a
passaging and thawing).

qgjin

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俺感觉这个protocol不咋地哈，有几个人用来自E16胚的MEF来做feeder？* Y2 A+ m9 D: U7 H
remove and discard placenta and fe ...
8 D7 t/ _8 F" tppcl2 发表于 2010-5-7 09:50

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只是在论文中搜到的，请讲细一些好吗？（譬如：E？胚最合适？； 取哪个部位的细胞效果更好，怎么取？；终止消化要添加多少培养液？为什么？） 我是新手不太懂 请教一下。