An Automated System for Delivery of an Unstable Transcription Factor to Hematopoietic Stem Cell Cultures
An Automated System for Delivery of an Unstable Transcription Factor to Hematopoietic Stem Cell Cultures
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DOI：
10.1002/bit.22297
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发表时间：
2009-06-01
影响因子：
3.8
通讯作者：
Zandstra, Peter W.
中科院分区：
文献类型：
--
作者：
Csaszar, Elizabeth;Gavigan, Genevieve;Zandstra, Peter W.
用于细胞培养应用的自动化递送系统将允许研究更复杂的培养策略，并简化将细胞暴露于不稳定分子的措施。我们有兴趣了解细胞内TAT-HOXB 4蛋白浓度如何影响造血干细胞（HSC）的命运;然而，目前这种不稳定蛋白的手动给药策略是劳动密集型的，并且产生的浓度范围很宽，可能无法促进最佳生长。在这项研究中，我们描述了一种可编程的自动化输送系统，该系统旨在整合到一个临床相关的，一次性使用的，封闭系统的生物过程，并促进转录因子输送研究。报告细胞试验的开发允许动力学研究以确定TAT-HOXB 4活性的细胞内（1.4 +/- 0.2 h）和细胞外（37 ℃和4 ℃下分别为3.7 +/- 1.8 h和78 +/- 27 h）半衰期。将这些动力学参数纳入数学模型，该模型用于预测TAT-HOXB 4的动态细胞内浓度并优化蛋白的递送。使用人外周血患者样本对自动化系统进行原代细胞培养验证。在添加TAT-HOXB 4后，在没有使用者干预的情况下获得人原始祖细胞的显著扩增。因此，该递送系统能够用作用于探索和优化时间敏感的干细胞培养系统的临床相关工具。Biotechnol. Bioeng. 2009;103：402-412. (C)2009 Wiley Periodicals，Inc.
An automated delivery system for cell culture applications would permit studying more complex culture strategies and simplify measures taken to expose cells to unstable molecules. We are interested in understanding how intracellular TAT-HOXB4 protein concentration affects hematopoietic stem cell (HSC) fate; however, Current manual dosing strategies of this unstable protein are labor intensive and produce wide concentration ranges which may not promote optimal growth. In this study we describe a programmable automated delivery system that was designed to integrate into a clinically relevant, single-use, closed-system bioprocess and facilitate transcription factor delivery studies. The development of a reporter cell assay allowed for kinetic studies to determine the intracellular (1.4 +/- 0.2 h) and extracellular (3.7 +/- 1.8 h and 78 +/- 27 h at 37 degrees C and 4 degrees C, respectively) half-lives of TAT-HOXB4 activity. These kinetic parameters were incorporated into a mathematical model, which Was used to predict the dynamic intracellular concentration of TAT-HOXB4 and optimize the delivery of the protein. The automated system was validated for primary cell culture using human peripheral blood patient samples. Significant expansion of human primitive progenitor cells was obtained upon addition of TAT-HOXB4 without user intervention. The delivery system is thus capable of being used as a clinically relevant tool for the exploration and optimization of temporally sensitive stem Cell Culture systems. Biotechnol. Bioeng. 2009;103: 402-412. (C) 2009 Wiley Periodicals, Inc.