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标题: 小鼠胚胎成纤维细胞的制备及处理(饲养层) [打印本页]

作者: qgjin 时间: 2010-5-7 04:20 标题: 小鼠胚胎成纤维细胞的制备及处理(饲养层)

本帖最后由 qgjin 于 2010-5-7 04:30 编辑 9 A* w& p( j9 L7 N; j
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PREPARATION OF MEF CULTURES) y/ y4 x9 a) Y$ ]7 t
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard+ x! g5 i9 C# a! s$ v1 g: U
placenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS.
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 on- `) \$ F3 ?$ `& H5 `0 \
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,
the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).
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 of7 @. ?+ m7 e% c7 \$ M
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.' y3 L( H1 G8 X) c# G) l+ @! {' T8 w
4. 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
MEF growth medium at 5% CO2 and 37℃ for 24 h.! F% P5 ^- ^. [
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.& A: R1 W% b* {8 A5 n2 {% y& F
8. Add trypsin solution to the culture plates and incubate for 1–2 min.
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).
10. Freeze any MEFs not needed immediately.
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
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 of- K, Y: s  m- b
undifferentiated maGSCs; prepare as follows.
12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.: A4 x. r% E# ]( J  @5 J
13. Aspirate the mitomycin C solution and wash three times with PBS.3 V# [7 H# k/ z. q, J
14. 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 dishes0 L  e8 b$ v3 f9 `
at a density of 50,000–60,000 cells/ cm2.
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical# L! u! h6 ]& O4 J5 Q  H* F
passaging and thawing).

作者: ppcl2 时间: 2010-5-7 09:50

俺感觉这个protocol不咋地哈，有几个人用来自E16胚的MEF来做feeder？
remove and discard placenta and fetal membranes, head, liver and heart.这个也粗放了点哈！
E16那么多细胞，3ml培养基，不是开玩笑吗？

作者: w1986725 时间: 2010-5-7 10:25

都没有人用胶原酶处理的吗？我想知道胶原酶处理的时间啊！~

作者: ppcl2 时间: 2010-5-7 12:31

回复 3# w1986725 4 C- W. ^5 [" |) P) _8 Y4 x( I
差不多，也是15到20分钟左右。

作者: w1986725 时间: 2010-5-7 13:07

请教一下，是用胶原酶Ⅱ还是Ⅳ？

作者: qgjin 时间: 2010-5-7 16:05

本帖最后由 qgjin 于 2010-5-7 16:21 编辑 ; y5 Y6 {4 C3 q' C0 Y% n- W

俺感觉这个protocol不咋地哈，有几个人用来自E16胚的MEF来做feeder？5 S" W$ O4 y" V: Y ~1 m+ }- P
remove and discard placenta and fe ...
' d" v% D. e: Q3 p2 b# |5 bppcl2 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。

作者: ppcl2 时间: 2010-5-8 01:07

回复 6# qgjin 4 s) ?1 `: p0 E$ ^5 r
Feeder一般用E12.5或E13.5，最迟不超过E14.5，俺取的MEF都是去头，掐尾，去四肢，内脏全去掉，剩下的躯干剪碎，再0.25%的含EDTA胰酶消化20分左右，加含FBS培养基吹打均匀并停止反应，离心，用吸管小心去除大多上情，再用10多毫升培养基重悬，培养，第二天换液，然后一直养到差不多满了。

作者: majing217 时间: 2011-7-6 16:17

顶楼上，我基本上用13.5天的做饲养层

作者: llecstem 时间: 2011-7-26 14:30

回复 ppcl2 的帖子6 U6 Y$ x9 t: [4 K' Y

想问一下，为什么去掉头内脏四肢？如果说头和内脏的细胞类型太多，所以要去掉，那四肢呢？谢谢！

作者: hcluo 时间: 2011-7-26 19:21

我个人觉得有一个时间范围，基本都可以使用，但不能太超过。

作者: majing217 时间: 2011-7-28 09:26

我通常取13.5天的，也是去头去尾去内脏去四肢，也是用胰酶消化15分钟左右，然后用含FBS的培养液终止，一般酶与培养液的比例为1:2.之后一只胎鼠接种到一个大皿中。

作者: 褚cell 时间: 2011-8-4 23:11

不知道大哥验证过没啊！建议谁能把做的MEF细胞照片展示一下，这样大家交流一下怎么才能做得好看并纯化MEF细胞。

作者: dongxin 时间: 2011-9-1 15:30

本帖最后由 dongxin 于 2011-9-1 15:31 编辑 , I4 x9 Z4 c; l3 x9 ^

取12.5d—13.5d的小鼠胚胎，将孕鼠断颈处死；取出子宫，将含有胚胎的子宫置于干净的100-mm培养皿中，将子宫用PBS清洗三次，去除血迹；分离胚胎，去掉小鼠的头部、肝部、四肢；剪碎，0.05%胰酶消化；用 MEF培液终止并吹打组织块；用70um滤器过滤；离心，一般1个胚可培养一个35-mm培养皿。供参考，祝好运！5 p, Z6 E I% t, g& K

作者: irise1006 时间: 2012-3-9 17:10

ppcl2 发表于 2010-5-8 01:07 ; ?# w( N1 r9 I/ E0 W4 C/ i. \
回复 6# qgjin
$ u" E% _# u4 X P5 `Feeder一般用E12.5或E13.5，最迟不超过E14.5，俺取的MEF都是去头，掐尾，去四肢，内脏全去 ...

是这样，但胰酶消化时间要根据你买的厂家，这个掌握好，别消化过！

作者: irise1006 时间: 2012-3-9 17:10

13.5较好！

作者: qwch1988 时间: 2012-3-29 22:35

回复 w1986725 的帖子
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好像一般都用胶原酶Ⅳ

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