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Label free cell tracking in 3-D tissue engineering constructs with high resolution imaging

机译:具有高分辨率成像的3-D组织工程构造中的无标记细胞跟踪

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Within the field of tissue engineering there is an emphasis on studying 3-D live tissue structures. Consequently, to investigate and identify cellular activities and phenotypes in a 3-D environment for all in vitro experiments, including shape, migration/proliferation and axon projection, it is necessary to adopt an optical imaging system that enables monitoring 3-D cellular activities and morphology through the thickness of the construct for an extended culture period without cell labeling. This paper describes a new 3-D tracking algorithm developed for Cell-IQ®, an automated cell imaging platform, which has been equipped with an environmental chamber optimized to enable capturing time-lapse sequences of live cell images over a long-term period without cell labeling. As an integral part of the algorithm, a novel auto-focusing procedure was developed for phase contrast microscopy equipped with 20x and 40x objectives, to provide a more accurate estimation of cell growth/trajectories by allowing 3-D voxels to be computed at high spatiotemporal resolution and cell density. A pilot study was carried out in a phantom system consisting of horizontally aligned nanofiber layers (with precise spacing between them), to mimic features well exemplified in cellular activities of neuronal growth in a 3-D environment. This was followed by detailed investigations concerning axonal projections and dendritic circuitry formation in a 3-D tissue engineering construct. Preliminary work on primary animal neuronal cells in response to chemoattractant and topographic cue within the scaffolds has produced encouraging results.
机译:在组织工程领域中,重点是研究3-D活组织结构。因此,要针对所有体外实验(包括形状,迁移/增殖和轴突投影)调查和识别3-D环境中的细胞活性和表型,有必要采用能够监视3-D细胞活性和构筑物在整个培养期的厚度范围内的形态学,无需细胞标记。本文介绍了为Cell-IQ®(一种自动细胞成像平台)开发的新3-D跟踪算法,该算法配备了经过优化的环境室,可以长时间捕获活细胞图像的延时序列而无需细胞标记。作为算法不可分割的一部分,针对配备20x和40x物镜的相衬显微镜开发了一种新颖的自动聚焦程序,通过允许在高时空条件下计算3-D体素来提供更准确的细胞生长/轨迹估计。分辨率和细胞密度。在由水平排列的纳米纤维层(它们之间具有精确的间距)组成的幻象系统中进行了一项初步研究,以模仿在3-D环境中神经元生长的细胞活动中充分体现的特征。接下来是有关3D组织工程构造中轴突投影和树突状电路形成的详细研究。响应支架内趋化因子和地形线索的原代动物神经元细胞的初步研究已产生令人鼓舞的结果。

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