Raman Spectroscopy of Live Cell Invasion of 3D nano-fabricated scaffolds
Lead Research Organisation:
Lancaster University
Department Name: Chemistry
Abstract
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.
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.
Planned Impact
Beneficiaries
Due to the rapidly increasing interest and exploitation of 3D cell culture the proposed project has a wide range of potential beneficiaries. Four important areas of interest are cell culture engineering, cell biophysics, mammalian cell technology and stem cell research and therefore we will target beneficiaries with investments in bioprocessing applications. In particular we will work with industrialists, academics and other specialist groups within the UK bioprocessing community through BioProNET. However, as outlined in the Pathways to Impact we will also work closely with trained professionals to identify impacts and other potential beneficiaries in research and business throughout the project.
Manufacturers of Raman spectrometers and commercially available 3D cell culture scaffolds will also benefit from the proposed research. In particular Renishaw PLC (Raman spectrometer manufacturer) will benefit from developing the use of Raman spectroscopy with live cell imaging and Reinnervate (provider of 3D cell culture products) may benefit from this research as it will give a greater insight into the fabrication of a large variety of scaffolds.
Benefits
Advances in mammalian cell culture are essential for the development of new biopharmaceuticals and 3D cell culture models allow the characterisation of cellular processes that are closer to the in vivo conditions than 2D cell culture. Furthermore, 3D cell culture environments provide flexibility for experimental variation in cell culture methods and media supplements. Bioprocessing research and industry will benefit from the proposed research as it will provide fundamental knowledge of the effects of scaffold topography on cell invasion in 3D culture and will enable new protocols to be developed to increase cell growth and enable 3D scaffolds to be designed specifically to increase cell proliferation and to control and direct cell adhesion and migration. This will not only benefit bioprocessing and research by improving the efficiency of 3D cell culture models but will also be of benefit to companies producing 3D cell culture products, such as Reinnervate.
Bioprocessing research and development will also benefit from the development of Raman microscopy for live cell-scaffold complexes. At present there is a lack of suitable bioanalytical techniques to monitor cell behaviour on 3D scaffolds without either having to label the cells and/or stopping the cell culture process, thus disturbing cell culture. Raman microscopy offers an alternative label free, none destructive approach that can be used to characterise biomolecular changes in cells in 3D culture in response to changes in cell culture methods and media supplements. The development of Raman microscopy as an alternative bioanalytical tool for 3D cell culture will also have much longer term benefits for all areas of 3D cell culture based research.
This project will provide training for a PDRA who will become skilled in micro-fabrication of 3D scaffolds, Raman spectroscopy and mammalian cell culture, skills not only required in bioprocessing, numerous areas of 3D cell culture research and instrumentation development. Successful funding of this proposal will provide the PI the opportunity to build their research group and begin research into 3D cell culture with the potential for long term impact and career development.
Due to the rapidly increasing interest and exploitation of 3D cell culture the proposed project has a wide range of potential beneficiaries. Four important areas of interest are cell culture engineering, cell biophysics, mammalian cell technology and stem cell research and therefore we will target beneficiaries with investments in bioprocessing applications. In particular we will work with industrialists, academics and other specialist groups within the UK bioprocessing community through BioProNET. However, as outlined in the Pathways to Impact we will also work closely with trained professionals to identify impacts and other potential beneficiaries in research and business throughout the project.
Manufacturers of Raman spectrometers and commercially available 3D cell culture scaffolds will also benefit from the proposed research. In particular Renishaw PLC (Raman spectrometer manufacturer) will benefit from developing the use of Raman spectroscopy with live cell imaging and Reinnervate (provider of 3D cell culture products) may benefit from this research as it will give a greater insight into the fabrication of a large variety of scaffolds.
Benefits
Advances in mammalian cell culture are essential for the development of new biopharmaceuticals and 3D cell culture models allow the characterisation of cellular processes that are closer to the in vivo conditions than 2D cell culture. Furthermore, 3D cell culture environments provide flexibility for experimental variation in cell culture methods and media supplements. Bioprocessing research and industry will benefit from the proposed research as it will provide fundamental knowledge of the effects of scaffold topography on cell invasion in 3D culture and will enable new protocols to be developed to increase cell growth and enable 3D scaffolds to be designed specifically to increase cell proliferation and to control and direct cell adhesion and migration. This will not only benefit bioprocessing and research by improving the efficiency of 3D cell culture models but will also be of benefit to companies producing 3D cell culture products, such as Reinnervate.
Bioprocessing research and development will also benefit from the development of Raman microscopy for live cell-scaffold complexes. At present there is a lack of suitable bioanalytical techniques to monitor cell behaviour on 3D scaffolds without either having to label the cells and/or stopping the cell culture process, thus disturbing cell culture. Raman microscopy offers an alternative label free, none destructive approach that can be used to characterise biomolecular changes in cells in 3D culture in response to changes in cell culture methods and media supplements. The development of Raman microscopy as an alternative bioanalytical tool for 3D cell culture will also have much longer term benefits for all areas of 3D cell culture based research.
This project will provide training for a PDRA who will become skilled in micro-fabrication of 3D scaffolds, Raman spectroscopy and mammalian cell culture, skills not only required in bioprocessing, numerous areas of 3D cell culture research and instrumentation development. Successful funding of this proposal will provide the PI the opportunity to build their research group and begin research into 3D cell culture with the potential for long term impact and career development.
Organisations
Publications
Baldock S
(2018)
Single-cell Raman microscopy of microengineered cell scaffolds
in Journal of Raman Spectroscopy
| Description | We have for the first time successfully acquired Raman images of single CaCo-2 cells adhered to biocompatible 3D scaffolds made from the polymer Ormocomp and fabricated in a range of sizes (10-100 µm in width and height) using direct-laser writing. In order to reliably achieve cell attachment to the scaffolds we have developed a scaffold preparation protocol which includes coating scaffolds with an extracellular matrix glycoprotein fibronectin. Whilst cells did adhere to the scaffold without the presence of fibronectin the process was difficult to control; however, once coated with the glycoprotein cells reliably adhered to the scaffold surface. Raman mapping of the cell-scaffold complexes has been successfully carried out on both fixed and live cells. Cell culture conditions for cell growth on scaffolds has been effectively maintained within the Raman microscope chamber and individual cells have been repeatedly mapped as they continued to grow on the scaffold surface. Raman maps have been acquired in a single plane across the cell and in 3D achieved by collecting spectra across several planes at different depths of the cell-scaffold complex. We are currently in the process of optimising the data processing and false-colour shading approaches needed to analysis the Raman maps. Results to date indicate that a protocol of noise-filtering to enhance the quality of the spectra combined with shading to peak area intensity of cellular features observed at 775-790 cm-1 (nucleic acid content) and 1330-1350 cm-1 (protein content) as well as an Ormocomp assigned peak at 1705-1757 cm-1 enables clear images of the cell to be generated from the spectral data. Using this procedure we can identify spatial distribution of the cell nucleus on and off the scaffold as well as detailed structural features of the scaffold. We are now in the final stages of the project which includes designing and fabricating scaffolds with more complex 3D architecture and evaluating the spatial distribution and chemistry of the cell nuclei in response to scaffold topology. |
| Exploitation Route | Our research so far has focused on one cell line (CaCo-2 cells) to take this research forward we now need to test further cell lines. In a new collaboration with Prof Ihtesham Rehman (Department of Engineering, Lancaster University) the investigation of further cell lines and scaffolds will be carried out by a PhD student and will lead to further new collaborations in 3D cell culture. One paper of our results has already been published in the Journal of Raman Spectroscopy, |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |