Defining conditions for the co-culture of Caco-2 and HT29-MTX cells using Taguchi design
Defining conditions for the co-culture of Caco-2 and HT29-MTX cells using Taguchi design
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DOI：
10.1016/j.vascn.2010.02.004
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发表时间：
2010-05-01
影响因子：
1.9
通讯作者：
Kitts, David D.
中科院分区：
文献类型：
--
作者：
Chen, Xiu-Min;Elisia, Ingrid;Kitts, David D.
简介：用于测试肠道药物和营养转运和代谢的Caco-2和HT 29细胞的共培养提供了吸收细胞和杯状细胞两者的存在，这两者对于最佳生长和功能具有不同的培养要求。关于Caco-2和HT 29细胞共培养的研究在改善减少实验室内和实验室间差异的特定条件方面非常有限。在本研究中，我们报告的条件，使可重现的结果，以获得药物渗透性使用Caco-2和HT 29-MTX的体外共培养的基础上田口实验设计。研究方法：确定了培养基、接种时间、接种密度和Caco-2：HT 29-MTX比例4个因素对普萘洛尔、酮洛芬和呋塞米的TEER值和个体渗透系数的影响。基于所选择的用于共培养的条件，我们还使用一氧化氮作为肠道炎症的指标来确认最终选择的培养条件的功能。结果如下：细胞培养时间和培养基的选择是影响TEER值和模型药物渗透系数的两个最重要的因素。另一方面，接种密度和Caco-2：HT 29-MTX比例对TEER值和药物渗透系数没有显著影响。对于所有药物，无法获得绝对最佳的细胞培养条件;然而，随后的确认实验得出结论，使用以下条件组合，即初始接种密度为1 x 10（5）Caco-2和HT 29-MTX细胞/cm（2）（比例为9：1），随后在MEM培养基中培养21天，由两名操作员获得了TEER值和药物渗透系数的极佳精密度。最后，使用上述选择的体外条件的共培养模型系统的功能导致与Caco-2细胞单层相当的一氧化氮合成。讨论内容：田口实验设计使我们能够定义一种体外培养条件的组合，该条件导致在Caco-2和HT 29-MTX共培养系统中测定药物渗透系数的良好操作重现性。此外，所选择的条件下使用的吸收和杯状肠细胞的共培养没有妥协的一氧化氮的合成，炎症的指标，在Caco-2单层测量。(C)2010年爱思唯尔公司All rights reserved.
Introduction: The co-culture of Caco-2 and HT29 cells for testing intestinal drug and nutrient transport and metabolism provides the presence of both absorptive and goblet cells, both of which have different culture requirements for optimal growth and function. The research on the co-culture of Caco-2 and HT29 cells is very limited in respect to refining specific conditions that reduce intra- and inter-laboratory variations. In the present study we reported conditions that enable reproducible results to be obtained for drug permeability using in vitro co-culture of Caco-2 and HT29-MTX based on Taguchi experimental design. Methods: The selection of four factors that specified cell culture conditions, namely culture medium, seeding time, seeding density, and Caco-2:HT29-MTX ratio on TEER value and individual permeability coefficients of propranolol, ketoprofen and furosemide was established. Based on the selected conditions for co-culture, we also confirmed the functionality of the final chosen culture condition using nitric oxide as an indicator of intestinal inflammation. Results: Choice of cell culture time and culture medium represented two of the most important factors that affected TEER values and the permeability coefficients of the model drugs. On the other hand, the seeding density and the Caco-2:HT29-MTX ratio exerted no significant influence on TEER values and the drug permeability coefficients. No absolute optimal cell culture condition could be obtained for all drugs; however subsequent confirmation experiments concluded that excellent precision for TEER values and drug permeability coefficients was obtained from the two operators using the following combination of conditions, namely an initial seeding density of 1 x 10(5) Caco-2 and HT29-MTX cells/cm(2) at a ratio of 9:1, followed by a 21 day culture time in MEM medium. Finally, functionality of the co-culture model system using the above selected in vitro conditions resulted in comparable nitric oxide synthesis to that of a Caco-2 cell monolayer. Discussion: Taguchi experimental design enabled us to define a combination of in vitro culture conditions that resulted in excellent operator reproducibility for determining drug permeability coefficients in a Caco-2 and HT29-MTX co-culture system. Moreover, the selected conditions used in co-culture of absorptive and goblet intestinal cells did not compromise the synthesis of nitric oxide, an indicator of inflammation, measured in Caco-2 monolayers. (C) 2010 Elsevier Inc. All rights reserved.