A novel characterisation and separation technique for pluripotent human embryonic and hematopoeitic stem cells
Lead Research Organisation:
Heriot-Watt University
Department Name: Sch of Engineering and Physical Science
Abstract
Realising the promised benefits of stem cells and their derivatives in regenerative medical therapies or in high throughput screening platforms for drug development necessitates the development of tools to purify cells to homogeneity. Ideally, such tools should be non-invasive with a capacity for separation several orders of magnitude beyond current methods. Industrial systems for the purification of therapeutic proteins provide precedents for large scale bioprocessing. To date, the vast majority of these have focused on column chromatography to achieve desired levels of purity. However, the size, sensitivity and complexity of cells present particular challenges to the downstream engineer which are unlikely to be solved by any further evolution or modification of traditional column chromatographic techniques. Consequently, novel techniques are needed. This proposal intends to develop a novel, simple, scalable and commercially useful technique for the separation/purification of human stem cells irrespective of their tissue of origin. This will be achieved by using Atomic Force Microscopy to define the topography and electrical charge distribution on the surfaces of human embryonic and adult haematopoietic stem cells followed by the use of this information in computational models to design complimentary surfaces. Surface prototypes will then be used in small-scale experimental work with living cells to demonstrate their ability to reversibly bind and separate cells and their subsequent viability. By focusing on both charge and topography of stem cell-surfaces the project will produce an adsorption-based separation technology more specifically suited for cell purification than current chromatographic techniques. The use of both adult and embryonic stem cell populations will exemplify the utility of this technology to both current and future clinical and research applications.
Technical Summary
This proposal intends to develop a scalable separation/purification technique for human stem cells capable of non-invasive and reversible processing of cells that will supercede current methods based on flow cytometry, centrifugation or magnetic separation. By using Atomic Force Microscopy with charged tips to generate charge maps the research will identify surface electrical charge differences associated with specific human stem cell populations, namely embryonic and adult haematopoietic (CD34+) stem cells. This information will be used to computationally model complimentary interactive substrates providing reversible affinity. Small-scale experimental work will be used to evaluate substrate prototypes for their capacity to enrich for stem cell populations without altering their viability or pluripotency, assessed using a range of standardised in vitro assays for cell molecular marker expression, and differentiation potential. By focusing on both charge and topography of surfaces the work will produce an adsorption-based separation technology more specifically suited for cell purification than current chromatographic techniques. This novel technology should be scalable to processing larger quantities of cells (10 E9-10) than current methods, thus making it a commercially useful solid-phase separation technique.
Publications
Choudhury D
(2011)
A femtosecond laser inscribed biochip for stem cell therapeutic applications
Choudhury D
(2011)
Ultrafast laser inscription: science today, technology tomorrow
Choudhury D
(2010)
Sorting stem cells using a femtosecond laser inscribed biochip
Choudhury D
(2012)
A 3D mammalian cell separator biochip.
in Lab on a chip
Corradetti B
(2012)
Paracrine signalling events in embryonic stem cell renewal mediated by affinity targeted nanoparticles.
in Biomaterials
Ermakov A
(2012)
A role for intracellular calcium downstream of G-protein signaling in undifferentiated human embryonic stem cell culture.
in Stem cell research
Kiss R
(2011)
Elasticity of human embryonic stem cells as determined by atomic force microscopy.
in Journal of biomechanical engineering
Nicholas Willoughby (Author)
(2010)
Identification of variable physical properties of cell types suitable for stem cell purification processes
Nicholas Willoughby (Author)
(2011)
Too Big to Bind? Novel Techniques for Separation of Human Embryonic Stem Cells Based on Physical Properties
Willoughby N
(2016)
A scalable label-free approach to separate human pluripotent cells from differentiated derivatives
in Biomicrofluidics
Description | We have developed a novel platform for separation of pluripotent human embryonic stem (hES) cells from differentiating derivatives. In the first instance this is based on the greater elasticity of the former. This was discovered following mapping of cell surfaces by atomic force microscopy (AFM). This also revealed that hES cells have a more positive surface charge, providing a second modality for separation. We have created transparent femtosecond laser-etched multichannel devices for proof-of-principle demonstration. This method offers extremely high levels of control over channel size and flow characteristics and clear imaging of cell separation behaviour. Specific channel sizes permit deformable (pluripotent hES) cells to pass through while more stiff (differentiated) cells are excluded. We have developed scalable membrane separation devices from this proof of principle and demonstrated their capability to process 10^7 cells at a rate in excess of 10^6 cells/min by separating pluripotent hES cells from differentiated cell types (osteogenic progenitors and fibroblasts) |
Exploitation Route | Identification of key physical property variation in cell types during differentiation and development will allow other researchers to develop scalable separation processes based on cell physical property. Results will also be critical in understanding how physical properties of cells vary with biological changes. |
Sectors | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Description | The emergence of therapies derived from stem cells requires a new direction in separation techniques to assist scale-up and fully realise societal impact. This research demonstrates scalable non-invasive non-tag-based cell separation methodology and drives a collaborative BBSRC FLIP commercialisation project (FLIP2012/08, Heriot Watt and Edinburgh; Roslin Cells Ltd) as well as key sections of the translational collaborative SFC Horizon "New Blood" project (227208649, £3M, Glasgow, Heriot-Watt, Edinburgh and Dundee Universities; Scottish National Blood Transfusion Service, Roslin Cells Ltd) leading to potential commercialisation of new separation methodologies. Whilst the realisation of cell therapies produced using technologies developed in this project is of course some way off in terms of timescale, the outcomes of this research have been instrumental in progressing the development of a viable large scale cellular therapy (red blood cells) |
First Year Of Impact | 2011 |
Sector | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Impact Types | Societal,Economic |
Description | Acquisition of experience in industrial project management and commercial product development in support of non-invasive stem cell separation technology |
Amount | £125,157 (GBP) |
Funding ID | FLIP2012/08 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2013 |
End | 03/2015 |
Description | Industrially generated red blood cells for transfusion |
Amount | £2,500,000 (GBP) |
Organisation | Government of Scotland |
Department | Scottish Funding Council |
Sector | Public |
Country | United Kingdom |
Start | 09/2011 |
End | 08/2016 |
Description | Manufacturing with Light |
Amount | £100,247 (GBP) |
Funding ID | EP/L022060/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2014 |
End | 10/2015 |
Description | De Sousa Group |
Organisation | University of Edinburgh |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Development of separation tools for cell therapies Development of techniques and methodologies for analysing physical properties of cells |
Collaborator Contribution | Biological analysis, understanding and development Development of reporter cell lines |
Impact | 10.1115/1.4005286 |
Start Year | 2008 |
Company Name | Decima Biomed |
Description | Decima Biomed is a company resulting from the collaborations developed within BBSRC BRIC project BB/G010374/1 to facilitate commercialisation of novel cell separation devices. The results and outcomes of the above project have been used in the design of Decima's first cell separator, which is being prototype-tested in collaboration with several partners |
Year Established | 2015 |
Impact | prototype elasticity-based cell separation and purification device |