Orgovan Norbert, Ungai-Salánki Rita, Lukácsi Szilvia, Sándor Noémi, Bajtay Zsuzsa, Erdei Anna, Szabó Bálint, Horvath Robert
Department of Biological Physics, Eötvös University, Pázmány P. stny. 1/A, H-1117 Budapest, Hungary and Nanobiosensorics Group, Hungarian Academy of Sciences, Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege út 29-33, H-1120 Budapest, Hungary.
Nanobiosensorics Group, Hungarian Academy of Sciences, Centre for Energy Research, Institute for Technical Physics and Materials Science, Konkoly-Thege út 29-33, H-1120 Budapest, Hungary and Doctoral School of Molecular- and Nanotechnologies, University of Pannonia, H-8200 Veszprém, Hungary.
Biointerphases. 2016 Sep 1;11(3):031001. doi: 10.1116/1.4954789.
Monocytes, dendritic cells (DCs), and macrophages (MFs) are closely related immune cells that differ in their main functions. These specific functions are, to a considerable degree, determined by the differences in the adhesion behavior of the cells. To study the inherently and essentially dynamic aspects of the adhesion of monocytes, DCs, and MFs, dynamic cell adhesion assays were performed with a high-throughput label-free optical biosensor [Epic BenchTop (BT)] on surfaces coated with either fibrinogen (Fgn) or the biomimetic copolymer PLL-g-PEG-RGD. Cell adhesion profiles typically reached their maximum at ∼60 min after cell seeding, which was followed by a monotonic signal decrease, indicating gradually weakening cell adhesion. According to the biosensor response, cell types could be ordered by increasing adherence as monocytes, MFs, and DCs. Notably, all three cell types induced a larger biosensor signal on Fgn than on PLL-g-PEG-RGD. To interpret this result, the molecular layers were characterized by further exploiting the potentials of the biosensor: by measuring the adsorption signal induced during the surface coating procedure, the authors could estimate the surface density of adsorbed molecules and, thus, the number of binding sites potentially presented for the adhesion receptors. Surfaces coated with PLL-g-PEG-RGD presented less RGD sites, but was less efficient in promoting cell spreading than those coated with Fgn; hence, other binding sites in Fgn played a more decisive role in determining cell adherence. To support the cell adhesion data obtained with the biosensor, cell adherence on Fgn-coated surfaces 30-60 min after cell seeding was measured with three complementary techniques, i.e., with (1) a fluorescence-based classical adherence assay, (2) a shear flow chamber applying hydrodynamic shear stress to wash cells away, and (3) an automated micropipette using vacuum-generated fluid flow to lift cells up. These techniques confirmed the results obtained with the high-temporal-resolution Epic BT, but could only provide end-point data. In contrast, complex, nonmonotonic cell adhesion kinetics measured by the high-throughput optical biosensor is expected to open a window on the hidden background of the immune cell-extracellular matrix interactions.
单核细胞、树突状细胞（DCs）和巨噬细胞（MFs）是密切相关的免疫细胞，它们的主要功能有所不同。这些特定功能在很大程度上由细胞黏附行为的差异所决定。为了研究单核细胞、DCs和MFs黏附的内在和本质动态方面，使用高通量无标记光学生物传感器[Epic BenchTop（BT）]在涂有纤维蛋白原（Fgn）或仿生共聚物PLL-g-PEG-RGD的表面上进行了动态细胞黏附测定。细胞黏附曲线通常在细胞接种后约60分钟达到最大值，随后信号单调下降，表明细胞黏附逐渐减弱。根据生物传感器的响应，细胞类型按黏附增加的顺序排列为单核细胞、MFs和DCs。值得注意的是，所有三种细胞类型在Fgn上诱导的生物传感器信号都比在PLL-g-PEG-RGD上大。为了解释这一结果，通过进一步利用生物传感器的潜力对分子层进行了表征：通过测量表面涂层过程中诱导的吸附信号，作者可以估计吸附分子的表面密度，从而估计潜在地为黏附受体呈现的结合位点数量。涂有PLL-g-PEG-RGD的表面呈现的RGD位点较少，但在促进细胞铺展方面比涂有Fgn的表面效率低；因此，Fgn中的其他结合位点在决定细胞黏附中起了更决定性的作用。为了支持用生物传感器获得的细胞黏附数据，在细胞接种后30 - 60分钟，用三种互补技术测量了细胞在Fgn包被表面上的黏附情况，即：（1）基于荧光的经典黏附测定法，（2）施加流体动力剪切应力以冲走细胞的剪切流室，以及（3）使用真空产生的流体流提起细胞的自动微量移液器。这些技术证实了用具有高时间分辨率的Epic BT获得的结果，但只能提供终点数据。相比之下，高通量光学生物传感器测量的复杂、非单调细胞黏附动力学有望为免疫细胞 - 细胞外基质相互作用的隐藏背景打开一扇窗口。
