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

近年来，人工组织工程学在修复或替换受损组织方面取得了巨大的发展。科学家们现在可以种植软骨，生产人造皮肤，甚至打印3D气管，并正在朝着从患者自己的细胞培养替代器官的理想目标迈进。虽然目前的进展已经给患者带来了一些好处，但这是有限的，如果我们要实现广泛的健康改善，仍然需要更多地了解如何控制和确定人造组织的生长。困难的是，细胞不是孤立地形成组织，还需要细胞基质或支架。目前最有前景的组织工程分析技术主要集中在细胞在人工打印的3D支架上的生长。然而，这些技术中的大多数要么需要用特定的标记标记细胞来检测它们的存在，要么需要从细胞培养条件中移除细胞和支架，从而结束这一过程，并且只提供有限的信息。我们的目标是发展使用拉曼光谱，一种替代的非破坏性和无标记的方法，来研究细胞在不同拓扑结构的3D支架上的原位生长。当光线照射到物质上时，发生的一个过程是它被散射，有时光以不同的波长散射；这种效应被称为拉曼散射。分子产生的拉曼散射将取决于化学结构，而对于生物分子，它也将取决于生物物理结构。拉曼光谱仪的最新进展使人们能够快速收集活细胞的拉曼图，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，我们将发展拉曼光谱作为一种新的方法，用于直接原位分析生长在不同形状的3D支架上的活细胞。存在各种方法来生产由各种聚合物制成的人造3D支架。其中最成功的一种是直接激光写入，它能够使用一系列形状、大小和材料在微尺度上打印支架。通过了解不同的支架结构如何影响在3D细胞培养环境中生长的细胞，我们将更接近于控制和确定进一步推进组织工程领域所需的细胞和最终组织生长。

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.