CN101348767A - 一种无边缘效应的96孔细胞培养板 - Google Patents

一种无边缘效应的96孔细胞培养板 Download PDF

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CN101348767A
CN101348767A CNA2007101303478A CN200710130347A CN101348767A CN 101348767 A CN101348767 A CN 101348767A CN A2007101303478 A CNA2007101303478 A CN A2007101303478A CN 200710130347 A CN200710130347 A CN 200710130347A CN 101348767 A CN101348767 A CN 101348767A
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CN101348767B (zh
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张�杰
陆军
沈时扬
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Suzhou Prantel Skin Cell Technology Co ltd
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Abstract

本发明涉及细胞生物学领域，细胞培养过程中，在96孔细胞培养板上边缘孔与内部孔之间存在着边缘效应，这种效应致使基于细胞培养的各种检测及观察均存在系统误差，影响试验结果的可靠性。本发明在96孔细胞培养板的外周增加了新的结构，这种结构可以在细胞培养时减少内、外孔之间的热动力不平衡问题，使细胞沉降均一，从而消除边缘效应。

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一种无边缘效应的96孔细胞培养板
技术领域
本发明为细胞生物学技术领域，具体地说为一种可以避免细胞培养边缘效应产生的96孔细胞培养板。
背景技术
细胞培养中的边缘效应是指在使用多孔板的细胞培养中，在周边孔中的细胞会集中在孔的外侧，这种现象在实验中常用的96孔板中十分明显。边缘孔的数量占到总数的1/3，这就意味着因为此误差所导致的影响相当巨大。
由于大多细胞的生长会受周围接触细胞的抑制，所以边缘效应导致边缘孔内细胞平均生长速度要比其他孔来得慢，从而在实验过程中引入系统误差。在很多大规模药物筛选实验中，人们甚至弃去周围孔的实验结果，这就意味着每三块96孔培养板只能被当作两块板使用。而且由此我们可以得出结论，如果边缘孔的外侧加以结构变换便可以减少边缘效应的产生。
边缘效应是由于在室温下的培养板转移到37℃培养箱后，培养板上产生的温度差影响细胞沉降引起的。我们的另一个实验证据是细胞培养板在37℃条件下放置一段时间以后取出，加入细胞而不放入37℃环境，发现细胞呈相反的沉降趋势。这两点足以证明产生细胞边缘效应的决定性因素就是细胞沉降过程中内、外孔之间热动力学失衡。
目前克服该现象的方法是在培养板内分入细胞后，在室温下放置1-2个小时，让细胞在没有温度差的情况下沉降，然后再转移到37℃培养箱内。该方法至少适合CHO等在室温下能贴壁的细胞。该方法的缺陷是对于脆弱的细胞外界环境温度降低会导致细胞应激反应产生，从而改变细胞的性状，尤其是原代培养的细胞更加不能忍受如此苛刻的生存条件，因此会发生细胞生长状态不佳、细胞凋亡甚至死亡。
发明内容
通过改变原有96孔板的结构，设计和研制不同宽度及厚度的新结构，达到最大程度消除边缘效应的目的，同时又保证了细胞培养的最佳状态。
实施例1  无边缘效应96孔板的培养效果试验
优先选用CHO细胞作为宿主细胞，宿主细胞购自美国菌种保藏中心(ATCC)，为二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞(CHO dhfr-)。将此细胞培养于100mm培养皿中，37℃培养，待细胞长至70～80％满时，吸出培养基，以胰酶消化细胞，按100个/ml稀释细胞，按200ul分别加入10块96孔细胞培养板，继续培养72小时。进行如下指标观察：
1)光学显微镜下观察：观察整个培养板96个孔的细胞生长状况，包括细胞在孔内的分布，细胞生长状态差异。
2)细胞克隆数形成实验：给每个孔接入一定的细胞数，定期观察细胞的克隆形成情况，并观察每个克隆的细胞数量，以统计学方式记录。
3)细胞染色观察：用经典的台盼兰染色法观察细胞的分布，不同于光学显微镜观察的是它可以观察到整个96孔的总体水平。
4)细胞活力观察：细胞用经典MTT法染色，细胞活力强的MTT活力高，显色深。酶联分析仪读取数据，以数字方式记录。
6、统计学分析：将上述数据进行统计学分析，得出结论。
实施例2  实验结果
1)光学显微镜下观察：各孔细胞生长均一性较好，外孔与内孔无大差别。
2)细胞克隆数形成实验：各孔细胞克隆数平均值如下表：
Figure A20071013034700041
统计学分析表明，外侧孔的克隆形成数与内侧孔相比无统计学差异。
3)细胞染色观察：染色结果表明内、外侧孔无统计学差异。
4)细胞活力观察：10块板的各孔平均值如下表：
Figure A20071013034700051
统计学分析表明，各孔的吸光值无统计学差异。

Claims (10)

1.一种细胞培养板，其特征在于为96孔有上盖培养板，为8×12分布，每个孔为大小、形状均相等的正圆形，底端封闭，上端敞开便于培养液加入，周边有侧壁围绕孔的周围。培养板孔的高度稍高于侧壁形成的平面，形成一圈突出的槽。上盖可以平整的覆盖在培养板上，保证与每个孔形成的平面平行。上盖上有凸起的圆圈，正好覆盖在孔的上方并保持0.2-0.5cm的间距。
2.权力要求1的细胞培养板，其上盖平坦，与培养板上面孔形成的平面平行，有裙边，与培养板之间存在缝隙，间距为0.1-1mm。
3.权力要求1的细胞培养板，其孔的分布为矩阵形，每个孔的中心距相等，距离为0.64-1mm。
4.权力要求1的细胞培养板，其制作材料为透明的塑料。
5.权力要求1的细胞培养板，其特征为在最外一圈孔与培养板侧围之间存在间隔，距离为：12孔长边侧1-4mm，8孔短边侧2-6mm。
6.权力要求5的细胞培养板，其特征为在最外一圈孔与培养板侧围之间存在一个槽，该槽为四周连通的方形或者四边各有一条或多条的槽，槽的方向平行于侧围的方向。
7.权力要求6的细胞培养板，其特征为槽的宽度为：12孔长边侧0.5-3.5mm，8孔短边侧0.5-5.5mm，槽的内径为：12孔长边侧0-3.5mm，8孔短边侧0-5.5mm，可以是实心槽。
其长度覆盖周边的孔。
8.权力要求6的细胞培养板，其特征为槽的材质可以是培养板的材质，也可以是任何其他材质。
9.权力要求6的细胞培养板，其特征是槽可拆卸，或者与培养板作成一体，槽内可以加入任何气体、液体或固体。
10.权力要求6的细胞培养板，其特征是槽可以是上端敞开的，或者上端封闭，在一处或多处有孔与外界连通。
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Publication number Priority date Publication date Assignee Title
CN101962613A (zh) * 2009-07-24 2011-02-02 无锡耐思生物科技有限公司 一种无边缘效应的96孔细胞培养板

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101962613A (zh) * 2009-07-24 2011-02-02 无锡耐思生物科技有限公司 一种无边缘效应的96孔细胞培养板

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