Droplet-based microfluidic platform for intracellular ion channel drug discovery

New technology is widening the chance of developing new pharmacological compounds and has the potential to create new jobs and have economic and societal impact on healthcare. Economic impact: The average expenditure to develop and bring to market a new drug is estimated to be approximately $2 billion, with target identification, discovery and the development of a new chemical compound before clinical trials accounting for 30% of the total cost. Commercialization of the developed platform has potential to add considerable information over the gold standard obtained by current electrophysiological tests with automated patch-clamp technology and impact the pre-screening stages of compounds, most of which is currently provided as a service by CROs and/or CMOs. Therefore, outcomes from this project have potential to carve a unique sector in the market of drug screening instrumentation. To assess this impact and available opportunities, we have secured the participation of members from academia, healthcare technology and industry to act as an advisory board for consultancy regarding knowledge transfer and commercialisation. Industrial impact: The developed system will be tested in collaboration with project partner Apconix for the development of pharmacological assays based on combinatorial chemistry approaches. Outside the immediate benefit in identifying new active compounds for the CLIC4 channel, the potential for translating the developed procedures for other pharmacologically relevant eukaryotic ion channels present cost-saving arguments against current live-cell based, large-screening assay in the Pharmaceutical industry. In addition, the collaboration with project partner Smartox will explore a further application of the technology for use with limited resources of venom samples only available in extremely small quantities that currently elude industrial automated screening. The proposed technology has the potential to greatly impact drug discovery by identifying new efficient venom-derived drugs. Together, this double partnership will enable us to address different screening needs for both SME and the Pharmaceutical Industry. Societal impact: Identification of new drug candidates from the developed technology will expand the panel of drugs with implications in cancer, mitochondrial dysfunction and neurodegenerative diseases. Improving the efficacy of identified drugs at preclinical level is expected to improve outcomes during clinical trials. Therefore, wide societal benefits would be in the form of new medicine and their improved efficacy. Furthermore, the applicability of the proposed technology to the other identified applications (see academic beneficiaries) and healthcare related fields could impact the screening of vaccines and environmental toxins, all of which will produce massive societal benefits

Droplet-based microfluidic platform for intracellular ion channel drug discovery

New technology is widening the chance of developing new pharmacological compounds and has the potential to create new jobs and have economic and societal impact on healthcare. Economic impact: The average expenditure to develop and bring to market a new drug is estimated to be approximately $2 billion, with target identification, discovery and the development of a new chemical compound before clinical trials accounting for 30% of the total cost. Commercialization of the developed platform has potential to add considerable information over the gold standard obtained by current electrophysiological tests with automated patch-clamp technology and impact the pre-screening stages of compounds, most of which is currently provided as a service by CROs and/or CMOs. Therefore, outcomes from this project have potential to carve a unique sector in the market of drug screening instrumentation. To assess this impact and available opportunities, we have secured the participation of members from academia, healthcare technology and industry to act as an advisory board for consultancy regarding knowledge transfer and commercialisation. Industrial impact: The developed system will be tested in collaboration with project partner Apconix for the development of pharmacological assays based on combinatorial chemistry approaches. Outside the immediate benefit in identifying new active compounds for the CLIC4 channel, the potential for translating the developed procedures for other pharmacologically relevant eukaryotic ion channels present cost-saving arguments against current live-cell based, large-screening assay in the Pharmaceutical industry. In addition, the collaboration with project partner Smartox will explore a further application of the technology for use with limited resources of venom samples only available in extremely small quantities that currently elude industrial automated screening. The proposed technology has the potential to greatly impact drug discovery by identifying new efficient venom-derived drugs. Together, this double partnership will enable us to address different screening needs for both SME and the Pharmaceutical Industry. Societal impact: Identification of new drug candidates from the developed technology will expand the panel of drugs with implications in cancer, mitochondrial dysfunction and neurodegenerative diseases. Improving the efficacy of identified drugs at preclinical level is expected to improve outcomes during clinical trials. Therefore, wide societal benefits would be in the form of new medicine and their improved efficacy. Furthermore, the applicability of the proposed technology to the other identified applications (see academic beneficiaries) and healthcare related fields could impact the screening of vaccines and environmental toxins, all of which will produce massive societal benefits

Droplet-based microfluidic platform for intracellular ion channel drug discovery

New technology is widening the chance of developing new pharmacological compounds and has the potential to create new jobs and have economic and societal impact on healthcare. Economic impact: The average expenditure to develop and bring to market a new drug is estimated to be approximately $2 billion, with target identification, discovery and the development of a new chemical compound before clinical trials accounting for 30% of the total cost. Commercialization of the developed platform has potential to add considerable information over the gold standard obtained by current electrophysiological tests with automated patch-clamp technology and impact the pre-screening stages of compounds, most of which is currently provided as a service by CROs and/or CMOs. Therefore, outcomes from this project have potential to carve a unique sector in the market of drug screening instrumentation. To assess this impact and available opportunities, we have secured the participation of members from academia, healthcare technology and industry to act as an advisory board for consultancy regarding knowledge transfer and commercialisation. Industrial impact: The developed system will be tested in collaboration with project partner Apconix for the development of pharmacological assays based on combinatorial chemistry approaches. Outside the immediate benefit in identifying new active compounds for the CLIC4 channel, the potential for translating the developed procedures for other pharmacologically relevant eukaryotic ion channels present cost-saving arguments against current live-cell based, large-screening assay in the Pharmaceutical industry. In addition, the collaboration with project partner Smartox will explore a further application of the technology for use with limited resources of venom samples only available in extremely small quantities that currently elude industrial automated screening. The proposed technology has the potential to greatly impact drug discovery by identifying new efficient venom-derived drugs. Together, this double partnership will enable us to address different screening needs for both SME and the Pharmaceutical Industry. Societal impact: Identification of new drug candidates from the developed technology will expand the panel of drugs with implications in cancer, mitochondrial dysfunction and neurodegenerative diseases. Improving the efficacy of identified drugs at preclinical level is expected to improve outcomes during clinical trials. Therefore, wide societal benefits would be in the form of new medicine and their improved efficacy. Furthermore, the applicability of the proposed technology to the other identified applications (see academic beneficiaries) and healthcare related fields could impact the screening of vaccines and environmental toxins, all of which will produce massive societal benefits

From emulsion to single-phase microfluidics : an integrated approach to culture and perfusion of multicellular spheroids

This study presents a novel microfluidic approach for developing large scale screening assays of anticancer compounds on 3D multicellular spheroids. We have developed a microfluidic device with associated protocols that combine the high-throughput characteristics of droplet microfluidics for spheroid formation and aggregation with those of single-phase microfluidics for substance exchange, long term culture and drug perfusion

Transitioning from multi-phase to single-phase microfluidics for long-term culture and treatment of multicellular spheroids

When compared to methodologies based on low adhesion or hanging drop plates, droplet microfluidics offers several advantages for the formation and culture of multicellular spheroids, such as the potential for higher throughput screening and the use of reduced cell numbers, whilst providing increased stability for plate handling. However, a drawback of the technology is its characteristic compartmentalisation which limits the nutrients available to cells within an emulsion and poses challenges to the exchange of the encapsulated solution, often resulting in short-term cell culture and/or viability issues. The aim of this study was to develop a multi-purpose microfluidic platform that combines the high-throughput characteristics of multi-phase flows with that of ease of perfusion typical of single-phase microfluidics. We developed a versatile system to upscale the formation and long-term culture of multicellular spheroids for testing anticancer treatments, creating and array of fluidically addressable, compact spheroids that could be cultured in either medium or within a gel scaffold. The work provides proof-of-concept results for using this system to test both chemo- and radio-therapeutic protocols using in vitro 3D cancer models

Microwell arrays for monitoring phenotypic heterogeneity in vascular cell populations

Significant remodeling of the vascular wall underlies cardiovascular disease resulting in the formation of atherosclerotic plaques populated with macrophage and smooth muscle cells (SMCs). These SMCs are thought to arise from the vessel wall, as mature SMCs de-differentiate from a contractile to a migratory, proliferate phenotype. However, the remodeling process is not fully understood and uncertainties remain over plaque cell origins and the plasticity of cells within the vascular wall. Both drug development and regenerative medicine have been restricted by these uncertainties. Recently, through a combination of time-lapse, high-speed fluorescence and 3D reconstruction microscopy, we demonstrated unambiguously [1] that freshly isolated mature, contractile SMCs can rapidly transform into not only a migratory but a phagocytic phenotype, a characteristic behaviour of macrophage. Results also showed strong heterogeneity in the proliferative capacity of SMCs [2] and the presence of other highly proliferative cell types in vascular wall that readily interact with SMCs. To better understand vascular call fate, including characterizing the phenotype of cell subpopulations, we employed SU-8 microfabrication to create a series of addressable microwell arrays that enable screening at the single cell level of large numbers of freshly isolated vascular cells. By incorporating microwells of different areas (from 60x60 to 180x180) and seeding with a cell suspension of appropriate density (either a pure SMC population or a mixed vascular population), cells sedimented stochastically across the microwell arrays such that many wells contained single cells. These cells were characterized by imaging in situ prior to tracking them for >1 week as they were induced to de-differentiate in culture. To validate this approach, variation in the proliferation of individual cells was tracked and the expression of SMC markers (e.g. SMA) following phenotypic modulation quantified. This microwell array approach, which is amenable to drug screening applications, will enable detailed characterization of phenotypic changes in vascular cell sub-populations, providing new insights to inform tissue engineering applications

Emulsion technologies for multicellular tumour spheroid radiation assays

A major limitation with current in vitro technologies for testing anti-cancer therapies at the pre-clinical level is the use of 2D cell culture models which provide a poor reflection of the tumour physiology in vivo. Three dimensional cell culture models, such as the multicellular spheroid, provide instead a more accurate representation. However, existing spheroid-based assessment methods are generally labour-intensive and low-throughput. Emulsion based technologies offer enhanced mechanical stability during multicellular tumour spheroid formation and culture and are scalable to enable higher-throughput assays. The aim of this study was to investigate the characteristics of emulsion-based techniques for the formation and long term culture of multicellular UVW glioma cancer spheroids and apply these findings to assess the cytotoxic effect of radiation on spheroids. Our results showed that spheroids formed within emulsions had similar morphological and growth characteristics to those formed using traditional methods. Furthermore, we have identified the effects produced on the proliferative state of the spheroids due to the compartmentalised nature of the emulsions and applied this for mimicking tumour growth and tumour quiescence. Finally, proof of concept results are shown to demonstrate the scalability potential of the technology for developing high-throughput screening assays

Real-time assessment of nanoparticle-mediated antigen delivery and cell response

Nanomaterials are increasingly being developed for applications in biotechnology, including the delivery of therapeutic drugs and of vaccine antigens. However, there is a lack of screening systems that can rapidly assess the dynamics of nanoparticle uptake and their consequential effects on cells. Established in vitro approaches are often carried out on a single time point, rely on time-consuming bulk measurements and are based primarily on populations of cell lines. As such, these procedures provide averaged results, do not guarantee precise control over the delivery of nanoparticles to cells and cannot easily generate information about the dynamics of nanoparticle-cell interactions and/or nanoparticle-mediated compound delivery. Combining microfluidics and nanotechnology with imaging techniques, we present a microfluidic platform to monitor nanoparticle uptake and intracellular processing in real-time and at the single-cell level. As proof-of-concept application, the potential of such a system for understanding nanovaccine delivery and processing was investigated and we demonstrate controlled delivery of ovalbumin-conjugated gold nanorods to primary dendritic cells. Using time-lapse microscopy, our approach allowed monitoring of uptake and processing of nanoparticles across a range of concentrations over several hours on hundreds of single-cells. This system represents a novel application of single-cell microfluidics for nanomaterial screening, providing a general platform for studying the dynamics of cell-nanomaterial interactions and representing a cost-saving and time-effective screening tool for many nanomaterial formulations and cell types

A novel microfluidic drug testing platform for studying communication between independent neural networks

Many in-vitro systems used during pre-clinical trials fail to recreate the biological complexity of the in-vivo neural microenvironment.Taking advantage of recent advances in microfluidic technology, we seek to develop a perfusion based drug discovery platform that is capable of high-throughput pharmacological profiling. This in turn will allow us to better understand how drugs influence the communication between functionally connected neural networks

Functionalisation of human chloride intracellular ion channels in microfluidic droplet-interface bilayers

Profiling ion flux through human intracellular chloride ion channels using live-cell based techniques, such as patch-clamp electrophysiology, is laborious and time-consuming. The integration of scalable microfluidic systems with automatable protocols based on droplet-interface-bilayers (DIBs) within which ion channels are incorporated circumvents several limitations associated with live-cell measurements and facilitates testing in controllable in vitro conditions. Here, we have designed and tested novel microfluidic layouts for the formation of arrays of DIBs in parallel and developed the first example of a miniaturised, DIB-based, fluorescence assays for Clfluxing, allowing the investigation of the functional properties of the human chloride intracellular ion channel 1 (CLIC1). The microfluidic protocols relied on passive geometries for droplet pairing and DIB formation. Using recombinantly expressed CLIC1, we identified the best conditions to maximise protein integration into a lipid bilayer and the oligomerisation of the protein into functional ion channels. Finally, CLIC1 ion channel functionality was assessed relative to α-Haemolysin into microfluidic DIBs using the same Clfluxing assay