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小木虫论坛-学术科研互动平台 » 材料区 » 生物材料 » 生物效应评价 » 材料表面细胞活性怎么衡量
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bbhgchina

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[求助] 材料表面细胞活性怎么衡量

各位兄弟姐妹们， （之前发信问了几个牛人同学，都没有回，无意冒犯，但是这个问题还比较急，现在卡着我，所以又发帖来咨询一下）请教一下关于生物细胞试验的一个问题，(非生物专业，只在网上搜索过MTT相关内容）望不吝赐教。

我的目标是观测一种材料表面形态对细胞的影响。 初步设计的试验方案是在实验组和对照组材料上面分别培养细胞，一定时间后（24h,48h,72h）对比两边细胞的差异。
现在的问题是 由于不是生物专业，不知道用什么方法来 对比两组细胞，甚至不知道培养何种细胞比较好。   我目前的想法是 1. 用电镜直接观看细胞形态变化 /粘附 2. 测量活细胞密度（不知道怎么测） 3. MTT 分析细胞活性
否麻烦大家给点建议
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1楼 2013-01-24 00:38:21
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aircraftvip

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红舟流星: 金币+5, BM-EPI+1, http://emuch.net/bbs/viewthread.php?tid=5464228投票通过~ 2013-01-26 17:33:55
引用回帖:
10楼: Originally posted by bbhgchina at 2013-01-24 19:36:19
Actually I am planning to bring down the surface topography to nano meter scale, to explore the details how surface structure affect cells.

I have read couple dozens of articles, they observed th ...

sounds interesting! wish u good luck.
you'd better have more discussion with biologists, especially with a developmental biology background or cell biology, to design a suitable but valuable model to study (cell type, influencing factor...)

Here is one about nanotopographic influences:

1.        McMurray, R. J.; Gadegaard, N.; Tsimbouri, P. M.; Burgess, K. V.; McNamara, L. E.; Tare, R.; Murawski, K.; Kingham, E.; Oreffo, R. O. C.; Dalby, M. J., Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency. Nat Mater 2011, 10, (8), 637-644.
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11楼2013-01-24 21:01:26
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bbhgchina

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好像问题太模糊了。再增加点描述

想对比 经过处理的材料表面形态，比如说 表面沟槽， 洞，突起 等等 对细胞的增值，粘附，活性的影响； 对照组就是一个 ‘平板’
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2楼2013-01-24 00:45:39
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aircraftvip

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红舟流星: 金币+2, 专家考核, 欢迎来到生物功能材料板块积极应助交流 2013-01-24 04:34:02
ATP analysis could be used to measured the loss in viability because a decrease in ATP production can be linked
to a decrease in cell number.

Specifically, ATP could be quantified by measuring the light produced through ATP's reaction with a naturally occurring firefly enzyme-Luciferase using a luminometer. The amount of light produced corresponds proportionally to the amount of live cells present in the sample.
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3楼2013-01-24 01:03:24
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aircraftvip

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markar: 金币+1, 应助指数+1, 感谢回帖交流！ 2013-01-25 12:23:20
Are u using multipotent cells like hMSCs?
if yes, you could try high-content imaging (see the proliferation) and immunofluorescence Staining (see the expression of alkaline phosphatase (ALP), a marker for early osteogenic differentiation)
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4楼2013-01-24 01:55:37
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bbhgchina

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引用回帖:
3楼: Originally posted by aircraftvip at 2013-01-24 01:03:24
ATP analysis could be used to measured the loss in viability because a decrease in ATP production can be linked
to a decrease in cell number.

Specifically, ATP could be quantified by measuring t ...

Thank you sir.   So the name of this sort of analysis is ' the ATP analysis‘?
Are these reactions you mentioned?
luciferin + ATP → luciferyl adenylate + PPi
luciferyl adenylate + O2 → oxyluciferin + AMP + light
But, are these reaction happening in every type of cell like fibroblasts and epithelials, which are likely to be used in my experiments according to articles in this field.
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5楼2013-01-24 02:18:57
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bbhgchina

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引用回帖:
4楼: Originally posted by aircraftvip at 2013-01-24 01:55:37
Are u using multipotent cells like hMSCs?
if yes, you could try high-content imaging (see the proliferation) and immunofluorescence Staining (see the expression of alkaline phosphatase (ALP), a mark ...

Thank you sir, this makes more sense to me now.

To be honest,at this stage,  I still have not decided yet which type of cell to use.   I will probably go for fibroblasts or epithelials.
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6楼2013-01-24 02:25:28
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aircraftvip

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6楼: Originally posted by bbhgchina at 2013-01-24 02:25:28
Thank you sir, this makes more sense to me now.

To be honest,at this stage,  I still have not decided yet which type of cell to use.   I will probably go for fibroblasts or epithelials....

Why fibroblasts?

For your study, the influence of geometry on cell fate, only multipotent stem cells are applicable. Otherwise, the influence might not be so significant.
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7楼2013-01-24 02:52:11
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bbhgchina

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引用回帖:
7楼: Originally posted by aircraftvip at 2013-01-24 02:52:11
Why fibroblasts?

For your study, the influence of geometry on cell fate, only multipotent stem cells are applicable. Otherwise, the influence might not be so significant....

May I ask why ' only multipotent stem cells are applicable' ?
Sorry if this is too simple, I'm not a biology majored.
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8楼2013-01-24 03:41:11
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aircraftvip

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红舟流星: 金币+1, 应助指数+1, 专家考核, 欢迎来到生物功能材料板块积极应助交流 2013-01-24 12:43:09
bbhgchina: 金币+10, ★★★很有帮助 2013-01-24 19:19:01
引用回帖:
8楼: Originally posted by bbhgchina at 2013-01-24 03:41:11
May I ask why ' only multipotent stem cells are applicable' ?
Sorry if this is too simple, I'm not a biology majored....

during differentiation, multipotent stem cells could read their microenvironmental signals, integrate them intracellularly, and make varying decisions accordingly. E.g., mesenchymal stem cells, could differentiate into both fibroblasts and osteoblasts according to the matrix stiffness. These points help to explain the development of different tissues.

Question:
why do u perform these experiments?
what is your novelty of your work?
have u read some relevant literatures on this topic?

As I know, it is generally not new. See the former work of Christopher Chen and Dennis Discher:

Geometric Control of Cell Life and Death
Science 30 May 1997:
Vol. 276 no. 5317 pp. 1425-1428

D.E. Discher, P. Janmey, Y-L. Wang. Tissue cells feel and respond to the stiffness of their substrate. Science 310:1139-1143 (2005).
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9楼2013-01-24 08:21:32
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bbhgchina

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引用回帖:
9楼: Originally posted by aircraftvip at 2013-01-24 08:21:32
during differentiation, multipotent stem cells could read their microenvironmental signals, integrate them intracellularly, and make varying decisions accordingly. E.g., mesenchymal stem cells, coul ...

Actually I am planning to bring down the surface topography to nano meter scale, to explore the details how surface structure affect cells.

I have read couple dozens of articles, they observed the cells' attachment and proliferation on certain nano-patterns, but I have not looked into how surface structure affect stem cell differentiation.
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10楼2013-01-24 19:36:19
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