Tissue Microfabrication Lab, Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Lab Chip. 2013 Aug 21;13(16):3246-52. doi: 10.1039/c3lc50493j. Epub 2013 Jun 20.
Gradients of diffusive molecules within 3D extracellular matrix (ECM) are essential in guiding many processes such as development, angiogenesis, and cancer. The spatial distribution of factors that guide these processes is complex, dictated by the distribution and architecture of vasculature and presence of surrounding cells, which can serve as sources or sinks of factors. To generate temporally and spatially defined soluble gradients within a 3D cell culture environment, we developed an approach to patterning microfluidically ported microchannels that pass through a 3D ECM. Micromolded networks of sacrificial conduits ensconced within an ECM gel precursor solution are dissolved following ECM gelation to yield functional microfluidic channels. The dimensions and spatial layout of channels are readily dictated using photolithographic methods, and channels are connected to external flow via a gasket that also serves to house the 3D ECM. We demonstrated sustained spatial patterning of diffusive gradients dependent on the architecture of the microfluidic network, as well as the ability to independently populate cells in either the channels or surrounding ECM, enabling the study of 3D morphogenetic processes. To highlight the utility of this approach, we generated model vascular networks by lining the channels with endothelial cells and examined how channel architecture, through its effects on diffusion patterns, can guide the location and morphology of endothelial sprouting from the channels. We show that locations of strongest gradients define positions of angiogenic sprouting, suggesting a mechanism by which angiogenesis is regulated in vivo and a potential means to spatially defining vasculature in tissue engineering applications. This flexible 3D microfluidic approach should have utility in modeling simple tissues and will aid in the screening and identification of soluble factor conditions that drive morphogenetic events such as angiogenesis.
在 3D 细胞外基质（ECM）中，扩散分子的梯度对于指导许多过程（如发育、血管生成和癌症）至关重要。指导这些过程的因素的空间分布很复杂，由血管的分布和结构以及周围细胞的存在决定，周围细胞可以作为因素的来源或汇。为了在 3D 细胞培养环境中生成具有时间和空间定义的可溶性梯度，我们开发了一种在 3D ECM 中对微通道进行图案化的方法。在 ECM 凝胶前体溶液中包埋的牺牲导管的微模网络在 ECM 凝胶化后溶解，从而产生功能性微流控通道。通道的尺寸和空间布局可以使用光刻方法轻松控制，并且通道通过密封件与外部流动连接，密封件还用于容纳 3D ECM。我们证明了扩散梯度的持续空间图案化取决于微流控网络的结构，以及独立地在通道或周围 ECM 中填充细胞的能力，从而能够研究 3D 形态发生过程。为了突出这种方法的实用性，我们通过在内皮细胞衬里通道来生成模型血管网络，并研究了通道结构如何通过影响扩散模式来引导内皮细胞从通道中发芽的位置和形态。我们表明，最强梯度的位置定义了血管生成发芽的位置，这表明了血管生成在体内受到调节的机制，以及在组织工程应用中空间定义脉管系统的潜在手段。这种灵活的 3D 微流控方法应该在模拟简单组织方面具有实用性，并将有助于筛选和确定驱动形态发生事件（如血管生成）的可溶性因子条件。
