Raman Spectroscopy of Live Cell Invasion of 3D nano-fabricated scaffolds

近年来，在修复或替换受损组织的人工组织工程方面取得了巨大进展。科学家们现在可以培育软骨，制造人造皮肤，甚至打印3D气管，并且正在朝着用病人自己的细胞培育替代器官的理想目标前进。虽然目前的进步已经给病人带来了一些好处，但这是有限的，如果我们要实现对健康的广泛改善，仍然需要对如何控制和确定人工组织的生长有更深入的了解。困难在于细胞不能单独形成组织，还需要细胞基质或支架。目前最有前途的组织工程分析技术集中在人工打印的3D支架上细胞的生长。然而，这些技术中的大多数要么需要用特定标记标记细胞以检测它们的存在，要么需要从细胞培养条件中去除细胞和支架，从而结束该过程，仅提供有限的信息。我们的目标是开发使用拉曼光谱，一种替代的非破坏性和无标签的方法，来研究不同拓扑结构的3D支架上的原位细胞生长。当光照射在物质上时，会发生散射，有时光会以不同的波长散射；这种效应被称为拉曼散射。分子产生的拉曼散射取决于化学结构，对于生物分子，它也取决于生物物理结构。拉曼光谱仪的最新进展使活细胞的拉曼图能够快速收集，从中可以产生细胞图像，识别细胞生长过程中发生的生化和生物物理变化。因此，我们将开发拉曼光谱作为一种新方法，用于直接原位分析生长在不同形状的3D支架上的活细胞。有多种方法可以生产由各种聚合物制成的人工3D支架。其中最成功的是直接激光书写，它可以在微尺度上使用一系列形状、大小和材料打印支架。通过了解不同支架地形如何影响在3D细胞培养环境中生长的细胞，我们将更接近于控制和决定细胞和最终组织生长，从而进一步推进组织工程领域。

In recent years there has been a vast development in the engineering of artificial tissues to repair or replace damaged tissue. Scientists can now grow cartilage, produce artificial skin and even print 3D tracheas and are advancing towards the ideal goal of growing replacement organs from a patient's own cells. Whilst present advancements have had some patient benefit this has been limited and there is still a need for a greater understanding of how to control and determine the growth of artificial tissues if we are to achieve extensive improvements to health. The difficulty is that cells do not form tissue in isolation but also require a cell matrix or scaffold. The most promising analytical techniques for tissue engineering at present focus on the growth of cells on artificially printed 3D scaffolds. However, the majority of these techniques either require the labelling of cells with specific markers to detect their presence or the removal of the cells and scaffolds from cell culture conditions thus ending the process and only providing limited information. Our aim is to develop the use of Raman spectroscopy, an alternative non-destructive and label free approach, to investigate in situ cell growth on 3D scaffolds of differing topology.One process that happens when light is shone at a substance is that it is scattered and sometimes the light scatters at a different wavelength; an effect named Raman scattering. The resultant Raman scattering from a molecule will depend on the chemical structure and for biomolecules it will also depend on the biophysical structure. Recent advancements in Raman spectrometers has enabled Raman maps of live cells to be rapidly collected from which images of cells can be produced identifying biochemical and biophysical changes that occur as cells grow. We will therefore develop Raman spectroscopy as a novel method for the direct in situ analysis of live cells growing on 3D scaffolds of different shapes. Various methods exist to produce artificial 3D scaffolds made from a variety of polymers. One of the most successful is direct laser writing which enables the printing of scaffolds on a microscale using a range of shapes, sizes and materials. By understanding how different scaffold topography affect cells grown in a 3D cell culture environment we will be a step nearer to controlling and determining cell and ultimately tissue growth necessary to advance further the field of tissue engineering.