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

近年来，在人工组织的工程化以修复或替换受损组织方面已经有了巨大的发展。科学家们现在可以生长软骨，生产人造皮肤，甚至打印3D气管，并正在朝着从患者自身细胞中生长替代器官的理想目标前进。虽然目前的进步已经有一些患者的好处，这是有限的，仍然需要更好地了解如何控制和确定人工组织的生长，如果我们要实现广泛的改善健康。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支架上的细胞生长。然而，这些技术中的大多数要么需要用特异性标记物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，从而结束该过程并仅提供有限的信息。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记的方法，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;这种效应称为拉曼散射。从分子得到的拉曼散射将取决于化学结构，并且对于生物分子，其还将取决于生物物理结构。拉曼光谱仪的最新进展使得能够快速收集活细胞的拉曼图谱，从该图谱可以产生细胞的图像，从而识别随着细胞生长发生的生物化学和生物物理变化。因此，我们将开发拉曼光谱作为一种新的方法，用于直接在原位分析生长在不同形状的三维支架上的活细胞。存在各种方法来生产由各种聚合物制成的人造3D支架。最成功的方法之一是直接激光写入，它可以使用一系列形状，尺寸和材料在微观尺度上打印支架。通过了解不同的支架拓扑结构如何影响在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.