Development of nanopatterned substrates for the delivery of high quality stem cells

Lead Research Organisation: University of Glasgow
Department Name: School of Engineering

Abstract

The use of stem cells in regenerative medicine holds great potential and with an increasingly aging population, we need to look for new opportunities. Their potential use span from orthopaedic applications such as arthritis and osteoporosis to neurodegenerative disorders such as Parkinson's and Alzheimer's, to name a few.

The body has a constant source of stem cells located in niches within the body. From a scientific and clinical point of view, one of the most exploited sources for adult stem cells is the bone marrow. The bone marrow is relatively easy to access and stem cells can be easily isolated from the extracted cell population.

However, until recently, a major hurdle is that the stem cells cannot be cultured for extended periods in culture and maintain their regenerative potential (multipotent). The very potential of stem cells, that they can change into many different cell types and so help repair damage on demand, means that their profile (phenotype) is unstable in culture. Hence, as we culture stem cells they soon lose the very potential and potency we want to exploit. We have recently (2011) demonstrated that by culturing the cells on a uniquely nanopatterned surface (nanopits, 100 nm in diameter and 100 nm deep, arranged in a square lattice) it is indeed possible to keep the cells in the multipotent state in prolonged culture as well as expand the number of cells.

These nanopatterned surfaces resemble the tracks on a Blu-Ray disc and indeed our technology is very similar to the production of optical media where nanopatterns can be injection moulded into polymer discs in high volumes and at a very low cost. We can change the arrangement of the nanopatterns, thereby tuning the stem cell response to growth without profile (phenotype) drift and to target desired changes to tissues we want (known as differentiation). This is has important implications on the design of implants (like a hip replacement implants) where a specific cell changes (differentiation) is desirable. An example of this is again, orthopaedic implants where differentiation of cells to bone is desirable, or an area with perhaps even more potential is the growth of large numbers of stem cells. Thus, a key research goal is to take a patient's stem cells, grow them in the laboratory to useful numbers, and then place them back into the patient to spark regeneration. Scale up of our technology will allow this.

The technology we have used so far has only allowed us to explore the stem cell interaction to a very limited number of different geometries (<10). In this proposal we will develop a new platform where a single sample will contain 1000 different patterns thereby allowing us to investigate a much larger library of nanopatterns and their ability to influence the fate of the stem cells. From these libraries new patterns will be identified and we will investigate them in more detail using mass spectrometry to identify small molecules influencing the cell fate.

Importantly, to see real benefit of these discoveries, it is vital that we are able to scale the materials used to large areas to sustain the growth and expansion of stem cells used for regenerative medicine or pharma. As described above, our technology is very similar to the production of DVDs and Blu-Rays, which means that it lends itself to a cost effective mass production of the nanopatterned surfaces. To demonstrate this potential, we will expand extracted bone marrow stem cells to 5 million cells, the number of cells used for the fully tissue engineered trachea demonstrated in 2008.

Technical Summary

Our preliminary work on a few set of topographies (<10) has demonstrated the potential of using nanotopography to control the fate of mesenchymal stem cells. Using these, we have made two fundamental discoveries. The first is that our nanotopographies can be used to either drive differentiation of the stem cells towards an osteogenic linage or to retain their multipotent properties in prolonged culture - depending on the nanopattern. The second is, that only small variations in the nanopatterns, the addition of as little as 20 nm of positional error, illicit very different responses by the stem. Thus is it very likely that other nanopatterns will have a strong influence on the cell fate.

Here, we proposed to develop a high-content nanotopography platform, similar to a gene array chip, where a single sample/substrate will contain 1000 different patterns to which cell response can readily be screened. With the use of fluorescent staining for a range of phenotypes (osteoblasts, chondrocytes, adipocytes, neuronal, glial and phenotype retention) and automated fluorescence microscopy, it will be possible to rapidly identify hit patterns for different phenotypes. Hit patterns will be explored in more detail using mass spectrometry to identify small molecules influencing the phenotypic changes induced by the nanotopography.

Our established links with clinic and industry will help us to exploit and translate key discoveries. And to demonstrate the industrial potential, we will produce nanopatterned samples large enough for expansion of mesenchymal stem cells with a target of generating 5.000.000 cells from a bone marrow aspirate.

Planned Impact

The proposed research is at the forefront of stem cells research globally. Through a number of "World's firsts" we have published results based on our nanotopography strategy in leading journals. We have excellent experience in identifying potential IP, protecting it and developing the innovations for industrial uptake. Our stem cell work has been supported by BBSRC follow-on funding (2x). We have carried out market research and have a good idea of the commercial landscape and the penetration. We are willing to share these resources with interested industrial partners in the club.

As we generate further proof for our surfaces and awareness of their potential, the impact will be wide-ranging. We will engage with interested BRIC partners to maximise the exploitation of the differentiating surfaces.

The proposed technology is generic and will be able to accelerate the development of existing and new products. As the technology can be scaled to industrial production, it has robust manufacturability and the full potential to be cheap and reliable, a prerequisite to yield cell based products at lowest possible cost.

We believe that it would be possible to produce 100.000 parts with an additional cost per part of less than 40 pence. If the technology is to be incorporated in more products it is likely that the costs can be further reduced.

The team is well-placed to bring the technology towards market. The in-house injection moulding facility can be run as a pilot line and in collaboration with a global cell culture plastic manufacturer, prototypes of cell culture flasks have been made based on nanopatterned polymer microscope slides. Moreover, the University is a corporate member of the Scottish Plastic and Rubber Association and Gadegaard has excellent connections to Engel UK through which OEM partners for full-scale production can be identified.

With the injection moulding facility in Glasgow, we are able to produce samples (1000-2000) for trialling with interested BRIC partner. We have previous experience working with multinational companies on producing prototypes and test them subsequently.

Furthermore, understanding how stem cells are controlled by small molecules (metabolites) and biochemical pathways will help generate new pharmaceuticals to target stem cells in vitro and in vivo.

Non-academic beneficiaries will include:
Clinic - most clinicians now accept that tissue engineering, stem cells and nanotechnology will be important in the medium-term future. We believe it is pivotal for clinicians to engage with early-stage research to inform a realistic compromise between material function and clinical application and we will do this through our established orthopaedic and regenerative connections.

Patients - the public are interested in stem cell therapies and our surfaces hold the potential to help deliver these therapies.

This proposal will augment previously funded BRIC projects such as bioreactor design, large-scale production of multipotent cells, scalable selection methods for therapeutic cells, and charged and topography based cell separation.
 
Description This is a highly interdisciplinary project where engineers and biologist work closely together at the interface. From the engineering side we have developed a number of methods for more efficient lithography and patterning. This is important when the technology needs to be scaled to an industrial setting/product. It is also important as it will enable us to explore larger parameter spaces for new substrate designs to control cellular function. We have also developed new tooling technologies which have enabled us to manufacture a standard multiwell plate but with the ability to explore combinations to nanotopographies.

From the biological side of the project, we have demonstrated the use of the multiwell plates, but the analysis is still underway. More importantly, using mass spectrometry it has been possible to identify small molecules which are relevant in regulating the stem cell functions. We are now verifying the impact of the molecules on the different pathways in the cells.
Exploitation Route we are now in discussions with several companies to take the technology forward
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description Development of scaffolds for hepatocyte regeneration 
Organisation Oslo University Hospital
Country Norway, Kingdom of 
Sector Academic/University 
PI Contribution We have developed materials for the initial proof of concept data. This has been followed up by a student exchange where a Glasgow student spend 1 month in the stem cell centre in Oslo.
Collaborator Contribution They have provided the cells and expertise for this project
Impact We have had student exchange and have submitted one grant to the Norwegian research council.
Start Year 2013
 
Description The use of micro and nanopatterning for cartilage regeneration 
Organisation Aarhus University
Country Denmark, Kingdom of 
Sector Academic/University 
PI Contribution We have expertise in injection moulding of patterned samples for cell culture experiments
Collaborator Contribution They are carrying out the biological experiments.
Impact We are still developing the materials for the experiments. In the process we have also submitted on grant application to a Danish charity.
Start Year 2013