干细胞传代时的数量为多少合适？

sarah19860420

回复 fengxuechunyang 的帖子
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还是不是很理解，状态好，传代的比例反而小？胰酶消化的过程能不能也详细阐述一下呢？我的是消化1min后去胰酶，加培养液终止消化，事实上也丢失了一些细胞。而这边的老师告诉我1-2min后直接加培养液终止消化，然后转移至15ml离心管离心后，再分瓶。哪种操作比较好？

松鼠

回复 sarah19860420 的帖子7 C( G, q( t1 S6 ~5 ?
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抱歉，我这儿也没有全文 把摘要贴给你看一下~ 希望你权限大一些能下来全文 呵呵
5 }' @1 a3 m9 s- f, EGenomic alterations in cultured human embryonic stem cells.
: j: b/ k5 }( _* m8 q2 _" ]# OMaitra A, Arking DE, Shivapurkar N, Ikeda M, Stastny V, Kassauei K, Sui G, Cutler DJ, Liu Y, Brimble SN, Noaksson K, Hyllner J, Schulz TC, Zeng X, Freed WJ, Crook J, Abraham S, Colman A, Sartipy P, Matsui S, Carpenter M, Gazdar AF, Rao M, Chakravarti A.
4 ^  Q8 d. M, D/ b8 ASourceMcKusick-Nathans Institute of Genetic Medicine, Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.' H7 ^6 L! l' [( S9 h

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Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10(-9) per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.
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sarah19860420

回复 松鼠 的帖子8 c( R2 [# e" O, o- |; y/ A

2 n7 r  O' D3 g$ g) i6 n; dO(∩_∩)O哈哈~，有全文了，大家共享吧。

流浪的波斯猫

回复 sarah19860420 的帖子; l. S$ q5 p: E, K7 @! V4 v
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细胞数量还算正常 不同个体来源的间充质干细胞表观形态会可能差别比较大 但是经过流式检测可能不会有什么差别的6 n* o/ V. o  s8 J7 Y: O
我们在实验过程中也遇到过这种情况 5 K  d9 h8 a; t0 a
我说的分化只是一种可能 再说分化到什么阶段也没办法确定的
" {) P4 m* ~: O  T5 t8 A# j% {5 D4 }分化是个过程 呵呵 检测蛋白的表达有个范围 8 d0 D& [" O9 ~  I  H: I9 c2 Z0 o: F
所以可以检测 不用担心的

song58

回复 流浪的波斯猫 的帖子
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谢谢啊

azzyou

回复 sarah19860420 的帖子
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( I" V  v' g+ \4 U6 B. B5 }; p& J1 M我们这是用的皿，基本铺满整个皿的时候细胞量比较多了，而且随着代数的上升，细胞形态会越来越大，越来越长，可想而知铺满整个皿的细胞量也减少了，所以3-4代以后，我们就采用了1：3这样的传代比例了。还有培养的时候我们一般不去换液哦，如果你觉得细胞状态不行，还是全换液的好。

sarah19860420

回复 流浪的波斯猫 的帖子
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好的，谢谢

sarah19860420

回复 azzyou 的帖子
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6 p9 ^; R6 t2 M2 V) ^  Q) v, Q# I你是说培养的整个过程都不换液吗，培养周期是多久，2,3天还是更久。我是想细胞长得慢，是不是要经常换液才长得更好一点呢。我的细胞传代太稀了，估计要下次传代应该是一周之后了。

sarah19860420

回复 流浪的波斯猫 的帖子3 z4 f; _( F- u

% A5 F- w! n* v% b! e! G$ q1 y. Z* @谢谢。
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shashou_com

细胞基本长满瓶底就可以传代。传代的密度在（2-6）*10的6次方每平方厘米