小鼠胚胎成纤维细胞的制备及处理(饲养层)

qgjin

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0 R6 H9 u% S' X+ ]. w3 q: hPREPARATION OF MEF CULTURES. j" ^  B! l4 R# e$ E6 t
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
* x1 D6 U: [% G7 T, w3 |6 |2 _* qplacenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.! A2 V& W9 E# X6 J
2. 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 on3 Q# e9 ~* Z) D; J
magnetic 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,4 a9 `2 a5 @, G( F  V# N$ T. {2 a
the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).: _' a# J+ D# A2 t' X8 O
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 of9 n: J' [% |  ~0 Q
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.) U8 Q. U, \' l! Q7 Z; T
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%9 a* w: J& J; l% f( {- a/ G
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.
- }' k! N. y- F4 n; f% I0 l4. 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& H: a7 ^# N* Z6 c: j9 P! o' [
MEF growth medium at 5% CO2 and 37℃ for 24 h.2 I5 H* a4 I! G' p, X, f! G
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
6 N) n( y5 J: ]" M4 E) Z6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.5 I3 Q) r# h: v3 X/ ^2 b: t
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.
" W- O6 `, ^: Y0 o4 {8. Add trypsin solution to the culture plates and incubate for 1–2 min.4 k4 l3 c# e" B+ ^' r9 d1 R
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).2 h# G# [: W4 w" @( G7 a
10. Freeze any MEFs not needed immediately.  F, [% W8 m$ y/ O& _
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year. 4 ?4 n$ [, F0 m  s: ^& _+ W; Y
11. 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 of6 Y9 Q  M# |* c) v
undifferentiated maGSCs; prepare as follows.
# |+ P" o' g" p12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.
+ ]" u/ Z6 T; Y+ r13. Aspirate the mitomycin C solution and wash three times with PBS.
0 Y' M+ j) h* Q: \. e! p14. 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 B( [* @# U; A8 k$ cat a density of 50,000–60,000 cells/ cm2.7 k1 N1 g% s9 T
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
6 ^, V% g) @5 ^, \0 V1 p3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical
: i( t. v( _8 m& gpassaging 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 Z5 l1 O: n% Tremove and discard placenta and fe ...
% |; L; ^& p% s6 ]ppcl2 发表于 2010-5-7 09:50

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. P5 ~: i, m+ q) ~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。