Phenotype and behaviour of dental pulp cells during expansion culture
Phenotype and behaviour of dental pulp cells during expansion culture
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DOI：
10.1016/j.archoralbio.2009.06.008
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发表时间：
2009-10-01
影响因子：
3
通讯作者：
Cooper, Paul R.
中科院分区：
文献类型：
--
作者：
Patel, Minal;Smith, Anthony J.;Cooper, Paul R.
目的：牙髓细胞原代培养常用于研究细胞反应、成牙潜能和干细胞反应。他们通过一系列技术方法孤立和扩张的情况得到了广泛报道。设计：采用胶原酶、胰酶、透明质酸酶和无花果蛋白对啮齿动物切牙牙髓进行酶解离，以确定活细胞的分离情况。将释放的细胞在DMEM和α-MEM培养液中进行扩增培养。结果：摘除的和机械切碎的啮齿动物牙髓组织在0.25%胰酶：EDTA中孵育，然后在α-MEM培养液中培养，为最大限度地生长和扩增提供了最佳条件。在这些条件下，延长培养时间会降低细胞的增殖能力，直到第7代，而传代较高的细胞则表现出恢复的生长速度。总的来说，成骨和成牙本质相关标记物的基因表达分析随着传代次数的增加而减少。值得注意的是，转化生长因子βS-1、-2和-3的表达在第10代时增加，干细胞和周细胞/肌成纤维细胞标志物CD74、神经丝蛋白和α-SMA的表达也增加。分子表型分析表明，传代前后牙髓细胞的分化能力差异不大。结论：本研究确定了原代牙髓细胞分离和扩增的条件，表明传代前和传代后期均能维持分化能力。然而，继续传代可能导致选择具有周细胞/肌成纤维细胞表型的细胞。(C)2009爱思唯尔有限公司。保留所有权利。
Objective: Primary pulp cell cultures are frequently used to study cellular responses, odontogenic potential and stem cell responses. Their isolation and expansion via a range of technical approaches are widely reported. The purpose of this study was to investigate the influence of isolation approach and extended expansion on cell phenotype and behaviour.Design: To determine viable cell isolation, enzymatic dissociation was performed on rodent incisor pulps using collagenase, trypsin, hyaluronidase and ficin. Extended expansion culture of released cells was performed in DMEM and alpha-MEM media. Cultures were subsequently analysed for gene expression, cell proliferation, cell morphology and differentiation capacity up to passage 20.Results: Data indicated that incubation of extirpated and mechanically minced rodent pulpal tissue with 0.25% Trypsin:EDTA and subsequent culture in alpha-MEM medium provided optimal conditions for maximal cell growth and expansion. Under these conditions, extended culture decreased cellular proliferative capacity up to passage 7, whilst higher passages demonstrated recovered growth rates. In general gene expression analysis of osteogenic and dentinogenic associated markers decreased with increasing passage number. Notably expression of TGF beta s-1, -2 and -3 increased up to passage 10 as did the stem cell and pericyte/myofibroblast markers, CD74, Neuroserpin and alpha-SMA. Analysis of molecular phenotypes indicated little difference in lineage differentiation capacity between earlier and later passages.Conclusions: The present study characterizes conditions for primary pulp cell isolation and expansion and indicates that both earlier and later passages maintain differentiation capacity. Continued passage however may result in selection for cells with a pericyte/myofibroblast phenotype. (C) 2009 Elsevier Ltd. All rights reserved.