Shamloo Amir, Ma Ning, Poo Mu-Ming, Sohn Lydia L, Heilshorn Sarah C
Department of Mechanical Engineering, Stanford University, Stanford, CA, USA.
Lab Chip. 2008 Aug;8(8):1292-9. doi: 10.1039/b719788h. Epub 2008 May 30.
The directed migration of endothelial cells is an early and critical step in angiogenesis, or new blood vessel formation. In this study, the polarization and chemotaxis of human umbilical vein endothelial cells (HUVEC) in response to quantified gradients of vascular endothelial growth factor (VEGF) were examined. To accomplish this, a microfluidic device was designed and fabricated to generate stable concentration gradients of biomolecules in a cell culture chamber while minimizing the fluid shear stress experienced by the cells. Finite element simulation of the device geometry produced excellent agreement with the observed VEGF concentration distribution, which was found to be stable across multiple hours. This device is expected to have wide applicability in the study of shear-sensitive cells such as HUVEC and non-adherent cell types as well as in the study of migration through three-dimensional matrices. HUVEC were observed to chemotax towards higher VEGF concentrations across the entire range of concentrations studied (18-32 ng mL(-1)) when the concentration gradient was 14 ng mL(-1) mm(-1). In contrast, shallow gradients (2 ng mL(-1) mm(-1)) across the same concentration range were unable to induce HUVEC chemotaxis. Furthermore, while all HUVEC exposed to elevated VEGF levels (both in steep and shallow gradients) displayed an increased number of filopodia, only chemotaxing HUVEC displayed an asymmetric distribution of filopodia, with enhanced numbers of protrusions present along the leading edge. These results suggest a two-part requirement to induce VEGF chemotaxis: the VEGF absolute concentration enhances the total number of filopodia extended while the VEGF gradient steepness induces filopodia localization, cell polarization, and subsequent directed migration.
内皮细胞的定向迁移是血管生成（即新血管形成）过程中早期且关键的一步。在本研究中，检测了人脐静脉内皮细胞（HUVEC）对血管内皮生长因子（VEGF）定量梯度的极化和趋化作用。为此，设计并制造了一种微流控装置，以在细胞培养室中产生生物分子的稳定浓度梯度，同时将细胞所经历的流体剪切应力降至最低。对该装置几何结构的有限元模拟结果与观察到的VEGF浓度分布高度吻合，且发现该浓度分布在数小时内保持稳定。预计该装置在诸如HUVEC等对剪切敏感的细胞以及非贴壁细胞类型的研究中，以及在通过三维基质的迁移研究中具有广泛的适用性。当浓度梯度为14 ng mL⁻¹ mm⁻¹时，在所研究的整个浓度范围（18 - 32 ng mL⁻¹）内，观察到HUVEC会向更高的VEGF浓度进行趋化运动。相比之下，在相同浓度范围内的浅梯度（2 ng mL⁻¹ mm⁻¹）无法诱导HUVEC趋化。此外，虽然所有暴露于升高的VEGF水平（无论是陡梯度还是浅梯度）的HUVEC丝状伪足数量都增加了，但只有发生趋化的HUVEC丝状伪足呈现不对称分布，在前缘有更多的突起。这些结果表明，诱导VEGF趋化需要两方面的条件：VEGF绝对浓度增加了伸出的丝状伪足总数，而VEGF梯度陡度诱导丝状伪足定位、细胞极化以及随后的定向迁移。
