Evaluation of in vitro cytotoxicity and paracellular permeability of intact monolayers with mouse embryonic stem cells
Evaluation of in vitro cytotoxicity and paracellular permeability of intact monolayers with mouse embryonic stem cells
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DOI：
10.1016/j.tiv.2008.02.023
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发表时间：
2008-08-01
影响因子：
3.2
通讯作者：
Barile, Frank A.
中科院分区：
文献类型：
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作者：
Calabro, Anthony R.;Konsoula, Roula;Barile, Frank A.
使用细胞外基质（ECM）组分和生长因子（GF）的组合，在细胞培养插入物中诱导小鼠胚胎干（mES）细胞形成完整的单层。使用跨上皮电阻（TEER）和细胞旁通透性（PP）标记物监测完整单层的逐渐形成。mES细胞最初接种在灭活的小鼠胚胎成纤维细胞（MEF）加白血病抑制因子（LIF）上。在75%汇合时，在不存在MEF和LIF的情况下传代细胞以刺激圆形多细胞聚集体（MA）的形成。4天后，将含有MA的培养物转移到仅用ECM组分包被的培养插入物中，并在所选单个GF存在下生长。再过10-14天，发现单层细胞融合，TEER值为500-700 ohms cm（2）（Omega cm（2））。与无GF或ECM生长的培养物相比，在培养基中存在表皮生长因子或角质形成细胞生长因子的情况下，在涂覆有ECM组分（如纤连蛋白或胶原-IV）的插入物上生长的单层产生最高的TEER测量值。在96孔板中对融合单层mES细胞进行的急性细胞毒性（AC）研究表明，在24小时暴露时间内，细胞活力与TEER之间存在高度相关性（R-2 = 0.91）。此外，TEER的降低与标志物PP的增加成反比。与标准化细胞毒性登记（RC）数据和TEER测量值相比，mES细胞的MTT IC 50值较低。因此，在相同化学品的等效浓度下，细胞活力在单层完整性受损之前降低。该系统代表了一种新的方法，用于操作mES细胞向特定的完整单层，作为生物单层形成的体外模型，最重要的是，用于细胞毒性试验。(C)2008爱思唯尔有限公司保留所有权利。
Mouse embryonic stem (mES) cells were induced to form intact monolayers in cell culture inserts, using combinations of extracellular matrix (ECM) components and growth factors (GFs). Progressive formation of intact monolayers was monitored using transepithelial electrical resistance (TEER) and passage of paracellular permeability (PP) markers. The mES cells were initially inoculated on inactivated mouse embryonic fibroblasts (MEFs) plus leukemia inhibitory factor (LIF). At 75% confluence, cells were passaged in the absence of MEF and LIF to stimulate formation of rounded multicellular aggregates (MA). After 4 days, cultures containing MA were transferred to culture inserts coated with ECM components only, and grown in the presence of selected individual GFs. An additional 10-14 days revealed confluent monolayers with TEER values of 500-700 ohms cm(2) (Omega cm(2)). Monolayers grown on inserts coated with ECM components, such as fibronectin or collagen-IV, in the presence of epidermal growth factor or keratinocyte growth factor in the medium, yielded the highest TEER measurements when compared to cultures grown without GFs or ECM. Acute cytotoxicity (AC) studies with confluent monolayers of mES cells in 96-well plates indicated that there is a high correlation (R-2 = 0.91) between cell viability and TEER for 24-h exposure time. Also, decrease in TEER is inversely proportional with increase in PP of markers. In comparison to standardized Registry of Cytotoxicity (RC) data and TEER measurements, MTT IC50 values for mES cells are lower. Thus, at equivalent concentrations for the same chemicals, cell viability decreases before the integrity of the monolayer is compromised. This system represents a novel approach for the manipulation of mES cells toward specific intact monolayers, as an in vitro model for biological monolayer formation, and most importantly, for applications to cytotoxicity testing. (C) 2008 Elsevier Ltd. All rights reserved.