Department of Chemical and Biomolecular Engineering, University of California-Berkeley, Berkeley, California 94720, USA.
Soft Matter. 2014 Apr 14;10(14):2372-80. doi: 10.1039/c3sm52265b.
The ability to independently assemble multiple cell types within a three-dimensional matrix would be a powerful enabling tool for modeling and engineering complex tissues. Here we introduce a strategy to dynamically pattern distinct subpopulations of cells through genetic regulation of cell motility. We first describe glioma cell lines that were genetically engineered to stably express constitutively active or dominant negative Rac1 GTPase mutants under the control of either a doxycycline-inducible or cumate-inducible promoter. We culture each population as multicellular spheroids and show that by adding or withdrawing the appropriate inducer at specific times, we can control the timing and extent of Rac1-dependent cell migration into three-dimensional collagen matrices. We then report results with mixed spheroids in which one subpopulation of cells expresses dominant negative Rac1 under a doxycycline-inducible promoter and the other expresses dominant negative Rac1 under a cumate-inducible promoter. Using this system, we demonstrate that doxycycline and cumate addition suppress Rac1-dependent motility in a subpopulation-specific and temporally-controlled manner. This allows us to orthogonally control the motility of each subpopulation and spatially assemble the cells into radially symmetric three-dimensional patterns through the synchronized addition and removal of doxycycline and cumate. This synthetic biology-inspired strategy offers a novel means of spatially organizing multiple cell populations in conventional matrix scaffolds and complements the emerging suite of technologies that seek to pattern cells by engineering extracellular matrix properties.
能够在三维基质中独立组装多种细胞类型将是建模和工程复杂组织的有力工具。在这里，我们介绍了一种通过细胞运动的遗传调控来动态模式化不同亚群细胞的策略。我们首先描述了经基因工程改造的神经胶质瘤细胞系，这些细胞系在四环素诱导或 cumate 诱导启动子的控制下稳定表达组成型激活或显性负 Rac1 GTPase 突变体。我们将每个群体培养成多细胞球体，并表明通过在特定时间添加或去除适当的诱导剂，我们可以控制 Rac1 依赖性细胞迁移到三维胶原基质中的时间和程度。然后，我们报告了混合球体的结果，其中一个亚群的细胞在四环素诱导的启动子下表达显性负 Rac1，另一个亚群的细胞在 cumate 诱导的启动子下表达显性负 Rac1。使用该系统，我们证明了四环素和 cumate 的添加以亚群特异性和时间控制的方式抑制 Rac1 依赖性迁移。这使我们能够通过四环素和 cumate 的同步添加和去除，以正交方式控制每个亚群的迁移，并通过同步添加和去除四环素和 cumate 将细胞空间组装成径向对称的三维模式。这种受合成生物学启发的策略为在传统基质支架中空间组织多个细胞群体提供了一种新方法，并补充了新兴的一系列通过工程细胞外基质特性来模式化细胞的技术。
