Department of Industrial Engineering, University of Padova, Padova, Italy.
Biophys J. 2013 Feb 19;104(4):934-42. doi: 10.1016/j.bpj.2012.12.049.
Adenoviruses are commonly used in vitro as gene transfer vectors in multiple applications. Nevertheless, issues such as low infection efficiency and toxicity effects on host cells have not been resolved yet. This work aims at developing a new versatile tool to enhance the expression of transduced genes while working at low viral doses in a sequential manner. We developed a microfluidic platform with automatically controlled sequential perfusion stages, which includes 10 independent channels. In addition, we built a stochastic mathematical model, accounting for the discrete nature of cells and viruses, to predict not only the percentage of infected cells, but also the associated infecting-virus distribution in the cell population. Microfluidic system and mathematical model were coupled to define an efficient experimental strategy. We used human foreskin fibroblasts, infected by replication-incompetent adenoviruses carrying EGFP gene, as the testing system. Cell characterization was performed through fluorescence microscopy, followed by image analysis. We explored the effect of different aspects: perfusion, multiplicity of infection, and temporal patterns of infection. We demonstrated feasibility of performing efficient viral transduction at low doses, by repeated pulses of cell-virus contact. This procedure also enhanced the exogenous gene expression in the sequential microfluidic infection system compared to a single infection at a higher, nontoxic, viral dose.
腺病毒通常在体外作为基因转移载体用于多种应用。然而，低感染效率和对宿主细胞的毒性影响等问题尚未得到解决。本工作旨在开发一种新的多功能工具，以在低病毒剂量下以顺序方式工作时增强转导基因的表达。我们开发了一种带有自动控制顺序灌注阶段的微流控平台，其中包括 10 个独立的通道。此外，我们构建了一个随机数学模型，考虑到细胞和病毒的离散性质，不仅可以预测感染细胞的百分比，还可以预测细胞群体中相关感染病毒的分布。微流控系统和数学模型被耦合起来以定义一个有效的实验策略。我们用人包皮成纤维细胞作为测试系统，用携带 EGFP 基因的复制缺陷型腺病毒感染。通过荧光显微镜对细胞进行特征描述，然后进行图像分析。我们探讨了不同方面的影响：灌注、感染倍数和感染的时间模式。我们证明了通过细胞-病毒接触的重复脉冲，可以在低剂量下进行有效的病毒转导，这一过程也增强了顺序微流控感染系统中外源基因的表达，与更高、非毒性的病毒剂量下的单次感染相比。
