Optimal design of metabolic flux analysis experiments for anchorage-dependent mammalian cells using a cellular automaton model.
Optimal design of metabolic flux analysis experiments for anchorage-dependent mammalian cells using a cellular automaton model.
复制标题
使用细胞自动机模型对贴壁依赖性哺乳动物细胞的代谢流分析实验进行优化设计。
DOI：
10.1002/bit.21414
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发表时间：
2007
影响因子：
3.8
通讯作者：
Blanch,HarveyW
中科院分区：
文献类型：
--
作者：
Meadows,AdamL;Roy,Siddhartha;Clark,DouglasS;Blanch,HarveyW
代谢通量分析（MFA）被广泛用于量化代谢途径活性。典型的应用涉及同位素标记的底物，其需要代谢和同位素稳态以简化数据分析。对于细菌系统，这些稳态在恒化器培养物中容易实现。然而，哺乳动物细胞通常具有锚定依赖性，并且实验通常在分批或补料分批系统中进行，例如组织培养皿或含微载体的生物反应器。表面粘附可能导致偏离指数生长，导致代谢异质群体和可能影响观察到的代谢的不同数量的细胞“最近邻”。在这里，我们讨论了适合于去卷积这些效应的不同生长模型及其在采用表面贴壁哺乳动物细胞的MFA实验的设计和优化中的应用。我们描述了一个随机的二维（2D）细胞自动机模型，与细胞数量和非生长细胞分数的经验描述，适合于易于应用于大多数锚定依赖性哺乳动物细胞培养。通过研究接触抑制对MCF 7细胞（一种通常研究的乳腺癌细胞系）的生长速率、比细胞外通量速率和乳酸盐中的同位素标记的影响来验证模型效用。该模型成功定义了可以假设指数生长和代谢同质生长细胞群的时间。元胞自动机模型的开发被证明是一个有用的工具，在设计最佳的MFA实验。Biotechnol. Bioeng. 2007; 98：221-229.© 2007威利期刊公司
Metabolic flux analysis (MFA) is widely used to quantify metabolic pathway activity. Typical applications involve isotopically labeled substrates, which require both metabolic and isotopic steady states for simplified data analysis. For bacterial systems, these steady states are readily achieved in chemostat cultures. However, mammalian cells are often anchorage dependent and experiments are typically conducted in batch or fed‐batch systems, such as tissue culture dishes or microcarrier‐containing bioreactors. Surface adherence may cause deviations from exponential growth, resulting in metabolically heterogeneous populations and a varying number of cellular “nearest neighbors” that may affect the observed metabolism. Here, we discuss different growth models suitable for deconvoluting these effects and their application to the design and optimization of MFA experiments employing surface‐adherent mammalian cells. We describe a stochastic two‐dimensional (2D) cellular automaton model, with empirical descriptions of cell number and non‐growing cell fraction, suitable for easy application to most anchorage‐dependent mammalian cell cultures. Model utility was verified by studying the impact of contact inhibition on the growth rate, specific extracellular flux rates, and isotopic labeling in lactate for MCF7 cells, a commonly studied breast cancer cell line. The model successfully defined the time over which exponential growth and a metabolically homogeneous growing cell population could be assumed. The cellular automaton model developed is shown to be a useful tool in designing optimal MFA experiments. Biotechnol. Bioeng. 2007; 98: 221–229. © 2007 Wiley Periodicals, Inc.