Regenerative medicine: instrumentation for flow cytometry and cell printing

Regenerative medicine (RM) aims to develop stem cell and biomaterial therapies to restore function to tissues burdened with injury or disease (A Strategy for UK Regenerative Medicine, 2012). The need is huge, with chronic wounds, traumatic injury and degenerative diseases costing the NHS >£1bn per annum. RM is a major MRC strategic priority and a focus of the EPSRC challenge theme 'Healthcare Technologies'. Its strategic importance is highlighted by the UK Regenerative Medicine Platform (UKRMP) that addresses key challenges in clinical translation. The University of Manchester (UoM) is a partner on all three of the newly-funded national UKRMP hubs: Engineering and exploiting the stem cell niche (Kimber, Kielty, Merry); Safety and efficacy (Kimber); Acellular technologies (Downes).
This proposal is for capital equipment that is fundamental to our ability to deliver cutting-edge stem cell research and translation in RM. We request flow cytometry and Fluorescence-Activated Cell Sorting (FACS) instruments to enable us to analyse and sort different stem cell populations and cells generated from them, at the highest possible level of reproducibility and accuracy, and cell/gel printing facilities to produce live cell arrays and to enable 'printing' of stem cells precisely within gels containing biological molecules and other support materials for tissue repair. Our users will be the UKRMP hub teams, within the University of Manchester (UoM) and nationally, in order to ensure standardised stem cell analysis and selection. Within the 'niche' hub, we will analyse and select human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and mesenchymal stromal/stem cells (MSCs), and characterise surface markers in these cells and the specialised cells generated from them. Within the Safety hub, we will analyse the effects of introducing tracer constructs and surface labelling on stem cell phenotype. The instruments will be accessible to other stem cell researchers in UoM and UKRMP hubs.
At UoM, we are building on interdisciplinary research platforms and state-of-the-art biomedical campus to establish an international centre for RM. We have an excellent research and training record in the biology of tissue formation and repair, the design, engineering and fabrication of biomaterials, and clinical translation. We focus on: characterisation of stem/progenitor cells; understanding how cell microenvironment (niche; extracellular matrix and soluble mediators) directs cell fate; designing biocompatible materials that mimic the niche and interact with cells to control their fate; regulating inflammation and responses to implantable constructs. Our research is integrated with the Manchester Collaborative Centre for Inflammation Research (UoM, GlaxoSmithKline, AstraZeneca), with biomaterials, tissue engineering, cellular and in vivo imaging, nanotechnology and Good Manufacturing Practice (clinical-grade stem cells). Clinical translation is through NIHR/Wellcome Trust Clinical Research Facilities within the Manchester Academic Health Science Centre (MAHSC). Translational themes include:
- Repairing/regenerating musculoskeletal tissues
- Enhancing chronic wound healing
- Stimulating neovascularisation to treat ischaemic tissue injury
- Developing therapies for stroke and peripheral neural repair
- Gene therapies for inherited diseases.
Requested: (i) Flow cytometry analyser and fluorescence-activated cell sorting instrument to enable efficient characterisation and selection of cell subsets. We urgently need this equipment to replace current 7-9 years old equipment that is no longer fit-for-purpose. Preferred options are: BD FACSAria Fusion with sterile hood, and BD LSRFortessa X-20 Research Flow Cytometer System. (ii) Cell/gel printer to enable us to print self-assembling viscous hydrogels encapsulating stem cells, for live cell arrays and tissue engineering. Available option is: CJ100004-2 CellJet, 4-channel (Digilab).

Cell analysis and sorting
Fundamental to stem cell research is high-quality instrumentation for analysis and selection of cell populations for research and translation. Flow cytometry and FACS instruments (with sterile hood) are essential to the regenerative medicine hub research in all 3 UKRMP hubs to which UoM is contributing. The requested machines would replace current obsolete 7 and 9 year old machines. Most suitable options are:
- BD FACSAria Fusion with sterile hood (4 laser, 16 colour) which has fluidics allowing aseptic sorting, exceptional optical detection and fluorescence sensitivity, high numerical aperture light collection, precise coordination of optics and fluidics.
- BD LSR Fortessa X-20 Research Flow Cytometer System delivers high performance multicolor analysis with up to 5 lasers, enabling detection of up to 20 parameters simultaneously.
Cell/gel printer: a high-capacity cell/gel printer will transform our ability to test multiple cell/gel niche combinations, enabling evaluation of the complexity of stem cell niches through 'printing' stem cells within injectable self-assembling hydrogel solutions (fully-defined, animal product-free and relatively cheap to manufacture to GMP). Short peptides will be tested that self-assemble into beta-sheet type structures to create hydrogels suitable for encapsulation of stem cells (Merry CLR et al, PCT Submission GB2013/050295). These can be modified to display functional motifs (e.g. for binding cells) and for mechanical properties, and can support stem cell transplantation.. The only suitable instrument is:
- CJ100004-2 CellJet, 4-channel (Digilab) (c£100k) can take up cells suspended in a pre-gel from a multiwell plate and decant them onto slides (or plates) in 10-100 nanolitre drop/spot sizes, using non-contact printing to maintain cell viability. Up to 4 channels/nozzles offers the opportunity to print in less restricted 3D patterns to better replicate tissue-specific environments.

This proposal is for capital equipment that is fundamental to cutting-edge stem cell research and translational regenerative medicine (flow cytometry and Fluorescence-Activated Cell Sorting (FACS) to analyse and sort stem cell populations at the highest level of stringency, and cell/gel printing facilities to produce live cell arrays and to 'print' stem cells (within niche extracellular matrix-containing hydrogels) onto biomaterial scaffolds. Our delivery focus is UK Regenerative Medicine Platform (UKRMP) hub partnership teams within the University of Manchester (UoM) and nationally (we are partners on three UKRMP hubs: Engineering and exploiting the stem cell niche; Safety science; Acellular technologies), to standardise stem cell experiments. Within the 'niche' hub, we will analyse stem cells in (de)differentiated states. Within the Safety hub, we will analyse the effects of cell transfection and labelling on stem cells. We will compare datasets with UKRMP colleagues. The instruments will be accessible to all stem cell researchers in UoM, including members of a prospective MRC Centre for Regenerative Medicine, and UKRMP colleagues nationally. Thus we will address the MRC Priority 'Resilience, Repair and Replacement'.
Impact of requested instrumentation on:
(1) Biomedical scientists and UK regenerative medicine community
It will enable us to deliver essential new insights into how stem cells interact with their extracellular matrix-rich microenvironment (niche), and to bioengineer niche mimetics to regulate stem cell fate.
Realising the benefits: Added value will be achieved by stringent stem cell analysis and selection, stem cell collaborations and data and reagent sharing (UoM, UKRMP hubs, international). Research will be translated through UKRMP hubs and the Manchester Academic Health Science Centre.
(2) Biopharma
Our expertise on stem cells and their niche is sought after by cell therapy/regenerative medicine/tissue engineering companies wishing to develop regenerative therapeutic products, and to Biopharma to inform the design of niche-based biologics and stem cell-based therapies.
Realising the benefits: University of Manchester Intellectual Property (UMIP) will commercialise our research, e.g. selling/licensing of reagents, provision of research expertise, in-house assays/techniques, co-development of technologies or licensing of IP.
(3) Graduate training
UoM (Kielty) is approved to submit a full proposal for an EPSRC-MRC Centre for Doctoral Training (CDT) in Regenerative Medicine. It will offer integrated graduate training by research that connects physical, chemical, biological and medical sciences to clinical application, all on our integrated state-of-the-art biomedical campus. The CDT will (with co-supervision model) train the next generation of interdisciplinary researchers to the highest standards in stem cell biology and cell-matrix biology, and their clinical applications. Importantly, it will be fully integrated with our prospective MRC Centre for Regenerative Medicine (the Centre graduate training programme was deemed excellent by MRC international panel).
Realising the benefits: Impact will be achieved by training graduates in scientific methods and translational applications required to: analyse and select stem cells, investigate their phenotype, determine the role of niche in directing stem cell fate; design niche-based biomaterials to control stem cell fate. Training will encompass stem cell biology; tissue engineering and biomaterials; pre-clinical models, Good Manufacturing Practice.
(4) General Public
Our stem cell research and regenerative medicine activities in UoM and UKRMP hubs are powerful platforms for informing the public about regenerative medicine, e.g. through public meetings and internet fora.
Realising the benefits: We will write review articles for student science magazines; train PhD students in public engagement; develop a range of outreach materials.