Effect of different mitogens and serum concentration on HUVEC morphology and characteristics: Implication on use of higher passage cells
Effect of different mitogens and serum concentration on HUVEC morphology and characteristics: Implication on use of higher passage cells
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DOI：
10.1016/j.tice.2011.03.004
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发表时间：
2011-08-01
期刊：
影响因子：
2.6
通讯作者：
Gohil, Nivedita Karmakar
中科院分区：
文献类型：
--
作者：
Bala, Kanchan;Ambwani, Kiran;Gohil, Nivedita Karmakar
人脐静脉内皮细胞（HUVEC）在两种不同的培养基中培养，即含有20%胎牛血清（FBS）和内皮细胞生长因子的常用M199培养基和含有2%胎牛血清（FBS）的确定培养基EGM-2以及已知浓度的生长补充剂。本研究的目的是确定不同培养基对随后传代的生长势和细胞形态的影响。我们已经确定明胶和人纤维连接蛋白细胞外基质的双重涂层提供了最佳的细胞附着。在确定的培养基中，原代培养的生长速度几乎翻了一番。对于二次培养，在确定的EGM-2培养基中观察到两倍高的增殖率。利用相衬、共聚焦和扫描电镜进行组织学研究，发现在M199中培养的细胞从第3代开始失去其形态特征，第6代后出现衰老阶段，而在有定义的培养基中，细胞到第10代没有变化。在不同培养基培养的细胞中，可溶细胞内粘附分子-1 （sICAM-1）的表达有显著差异。此外，我们还观察到细胞脱离过程中胰蛋白酶- edta的暴露时间在维持细胞形态特征方面也起着重要作用。这些结果表明，如果细胞在常规培养基中长时间生长，高阶传代会出现明显的形态学变化，因此可能不适合细胞信号传导实验。(C) 2011 Elsevier Ltd.版权所有。
Human umbilical vein endothelial cells (HUVEC) were cultured in two different media, viz. the commonly used M199 containing 20% fetal bovine serum (FBS) and endothelial cell growth factor and a defined media EGM-2 containing 2% FBS along with growth supplements in known concentrations. The purpose of this study was to determine the effect of different media on the growth potential and cell morphology in subsequent passages.We have established that a dual coating of gelatin and human fibronectin extracellular matrix provides optimal cell attachment. Growth rate for primary culture was almost double in defined media. For secondary culture a two fold higher proliferation rate was observed in defined EGM-2 media. Histological studies were done using phase contrast, confocal and scanning electron microscopy which showed that cells cultured in M199 started losing their morphological characteristic from 3rd passage and after 6th passage appeared to come in senescent stage, while in case of defined media there was no change observed in the cells up to 10th passage. A significant difference was found in the expression of soluble intracellular adhesion molecule-1 (sICAM-1) which is an endothelial cell marker on cells cultured in different media. Additionally it was observed that exposure duration to trypsin-EDTA during cell detachment also plays an important role in maintaining cell morphological characteristics.These results show that significant morphological changes appear in higher order passages if cells are grown in routine medium for a long time and therefore may not be suitable for cell signaling experiments. (C) 2011 Elsevier Ltd. All rights reserved.