Establishment of a triple co-culture in vitro cell models to study intestinal absorption of peptide drugs
Establishment of a triple co-culture in vitro cell models to study intestinal absorption of peptide drugs
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DOI：
10.1016/j.ejpb.2012.10.003
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发表时间：
2013-04-01
影响因子：
4.9
通讯作者：
Sarmento, Bruno
中科院分区：
文献类型：
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作者：
Antunes, Filipa;Andrade, Fernanda;Sarmento, Bruno
体外细胞培养模型用于研究药物开发早期阶段的口服药物吸收，在物质吞吐量和重复性方面已成为药物发现和开发的有用工具。本研究的目的是建立一种基于人结肠癌Caco-2、产生粘液的HT29和Rail B细胞的体外细胞模型，以设计一种更准确地模拟小肠上皮层的模型。将Caco-2和HT29细胞共培养于Transwell滤器中，建立正常定向模型，并在相同条件下加入Raji B至基底侧室。建立倒置模型，将Caco-2和HT29细胞接种于基底侧室，然后将Raji B添加到根尖室后，将上皮细胞移入Transwell装置，使上皮细胞面向基底侧室。用荧光显微镜观察到细胞膜的大小和厚度在形态上的差异。在三重共培养模型中，从Caco-2到Caco-2/HT29和Caco-2/Raji B，细胞膜的大小和厚度都在增加，而且细胞核似乎比其他模型大。与Caco-2/HT29共培养模型相比，三重共培养模型的胰岛素渗透率更高。此外，在正常取向的Caco-2/HT29/Raji B模型上，纳米颗粒介导的胰岛素渗透和胰岛素溶液渗透比反向模型更高。总之，我们的结果表明，Caco-2/HT29/Raji B三重共培养面向正常的细胞模型可能是可靠的，可以获得更具生理性、功能性和重复性的体外肠道屏障模型，以研究蛋白质在溶液中和通过纳米载体输送时的吸收。(C)2012爱思唯尔B.V.保留所有权利。
In vitro cell culture models for studying oral drug absorption during early stages of drug development have become a useful tool in drug discovery and development, with respect to substance throughput and reproducibility. The aim of this study was to establish an in vitro cellular model based on human colon carcinoma Caco-2, mucus-producing HT29, and Rail B cells in order to design a model that more accurately mimics the small intestinal epithelial layer. Normal oriented model was set up by seeding co-cultures of Caco-2 and HT29 cells into Transwell filters and maintained under identical conditions following addition of Raji B to the basolateral chamber. Inverted model was set up seeding Caco-2 and HT29 cells on the basolateral chamber and then transferred in the Transwell device with the epithelial cells facing the basolateral chamber following Raji B addition to the apical compartment. Morphological differences on size and thickness of cell membranes were detected between the models studied by using fluorescence microscopy. On the triple co-culture models, cell membranes were increasing in size and thickness from the Caco-2 to Caco-2/HT29 and Caco-2/Raji B. Also, the nuclei seem to be larger than in the other studied models. Insulin permeation was higher on the triple co-culture model when compared to the Caco-2/HT29 co-culture model. Also, insulin permeation as mediated by nanoparticles and insulin solution permeation was higher on the normal oriented Caco-2/HT29/Raji B model as compared to the inverted model. Overall, our results suggest that Caco-2/HT29/Raji B triple co-culture normal oriented cellular model may be reliable to obtain a more physiological, functional, and reproducible in vitro model of the intestinal barrier to study protein absorption, both in solution and when delivered by nanocarriers. (C) 2012 Elsevier B.V. All rights reserved.