Microfluidics - Driving Innovation and Streamlining Single Cell Analysis
Microfluidic platforms that harness picodroplet technology (picolitre volume aqueous droplets in stabilised oil emulsions) are unlocking the potential of single cell analysis to enable exciting new discoveries and advances with the potential to transform scientific research, drug development and precision medicine.
These systems compartmentalise single cells and trap their released molecules, enabling tens of millions of novel single cell tests to be performed on each biochip per day.
Our scientific understanding of cellular processes and signalling pathways has historically been informed by analyses of cellular populations, providing a global appreciation of molecular responses to external stimuli. However, this approach is inherently limited as it provides information only regarding the average response of the population and doesn’t account for heterogeneity (1).
Bulk assays also mask the many complex multifunctional processes occurring at a single cellular level that allow fine tuning of specific biological responses, such as immunological pathways where the quality of an individual cell’s response may be more important than the overall level (1-3).
The introduction of single cell analysis technologies has opened up new possibilities for the study of rare variant cells, found in both naturally occurring and engineered populations (4). Examples of rare cells include circulating tumour cells (CTCs), antigen-specific lymphocytes within the immune system and haematopoietic stem cells (HSCs) (4).
These rare variants, occurring at rates of one in a million or one in a billion, can reveal insights concerning cancer-causing mutations, novel approaches to treatment, drug resistance and discovery of new viable medicines (5). Single cell analysis has driven a rapid evolution in scientific understanding regarding cellular variability and differentiation between tissue types as well as variants within individual tissues.
These advances are providing a deeper appreciation of important variations in gene expression, cell cycle processes and responses to specific environmental factors (5). Projects such as the Human Cell Atlas Project are using single cell techniques to map and explore critical cellular differences within individuals and comparing these variations across populations (6). This work will highlight critical modifications associated with disease to aid the development of highly efficacious medicines with improved toxicity profiles (6).
In an industry that is becoming increasingly focused on precision medicines, the biopharmaceutical community has embraced the opportunity of single cell analysis in both naturally occurring and engineered cell lines. Molecules such as cytokines and antibodies represent novel and extremely commercially attractive therapeutic options for the management of cancers, inflammatory disorders and immunological diseases. Monoclonal antibodies are now the fastest growing therapeutic class (7,8).
Forecasts suggest that this market will be worth approximately $125 billion by 2020 (7,8). Unsurprisingly, many commercial developers are now seeking the most efficient and precise methods of monoclonal antibody identification and generation to meet the growing demand and advance this field of medicine (7,8). These types of treatment support a more targeted approach to disease management, but identification of those rare cells producing the highest quality and/or yield of a specific molecule may require analysis of millions or billions of cells.
Optimising analysis: a delicate balance of throughput and sensitivity
For single cell analysis to be truly commercially viable, systems and procedures must be optimised to ensure maximum throughput, reliable automation of complex processes and high quality results. This represents a costly technical challenge.
Measurement of secretory proteins, such as antibodies, that are released from single cells in a population can be difficult as these molecules quickly become lost in the ‘molecular soup’ surrounding the cells. Primary cells (from the human body) can be fragile and require careful handling. Conventional techniques are generally unable to offer the optimal balance of high throughput and sensitivity required for analysis in such cells.
Flow cytometry or fluorescence-activated cell sorting (FACS) techniques offer very high throughput levels but can be harsh on delicate primary cell lines as cell suspensions are usually pressure driven through a flow cell (13). Manual techniques such as limiting dilution (used in commercial monoclonal antibody production for decades) and clone picking tend to be less abrasive than flow cytometry but offer relatively inefficient solutions in terms of throughput (13). Clone picking typically allows approximately 10,000 cellular tests to be completed over a three-week period.
Ongoing frustrations in the biopharmaceutical industry have led commercial stakeholders to seek fully-automated, integrated systems that provide very high throughput alongside delicate handling of cells with the potential to measure difficult targets, such as secretory proteins.
In many cases, industrial laboratories may use up to four different single cell processing systems, each offering a partial solution, with samples being passed between systems to obtain an overall result. Commercial partners need one system that delivers on every level; simplifying the process, streamlining resources and cutting down on waste.
The promise of picodroplet microfluidics
Microfluidic platforms have provided a welcome solution for single cell separation, isolation and analysis, and this approach is becoming increasingly popular within the biopharmaceutical industry (13,14). Traditional microfluidic platforms comprise small plastic biochips in which fluids are pumped through channels, each around 1μm in depth (14). These systems facilitate automated sample testing and innovations have brought various advances in terms of flow rate regulation, channel numbers and throughputs (14).
Isolating single cells and/or secreted proteins from the stream of fluid as it is pumped through the channels can be challenging. However, a recent wave of microfluidic advances (over the past 10-15 years) has seen the development of droplet technology, allowing individual cells to be compartmentalised within a smaller (picolitre volume) droplet or ‘picodroplet’ (15).
This exceptionally powerful and sensitive technique allows single cells to be isolated from a population of millions and enables accurate detection of rare cell types. Secreted proteins (eg cytokines, monoclonal antibodies) may be trapped or compartmentalised within the picodroplets for analysis (1,16,17). The picodroplets provide a defined and controlled environment for each cell that can be manipulated for research or manufacturing purposes (18).
The core technology underpinning picodroplet microfluidics cleverly fuses established molecular and bacterial encapsulation techniques with antibody recognition platforms. Sphere Fluidics was among the first companies to recognise the potential of this approach and to design fully-automated systems that harness this technology for scientific and commercial purposes.
Areas of study such as antibiotic resistance, hybridoma screening, drug resistance in cancer cells and cell signalling are already benefiting from picodroplet platforms (2,5,16-18). Diagnostics, including point-of-care systems, are also showing promise (19,20). These advances will support earlier diagnosis, improve the accuracy of testing and increase the potential for personalised treatment of disease (19,20).
The speed of these systems is remarkable; up to 1,000 times faster than manual clone picking procedures, which take a few weeks to analyse 10,000 cells. Modern microfluidic picodroplet systems can identify novel antibodies in as little as 15 minutes and are able to analyse 40 million cells within six hours. The overall quality of the system and the results obtained are also improved because the platform is much gentler, compared with flow cytometry; the picodroplet package acts as a cushion, protecting the cells as they are pumped around the system.
The cells leave the microfluidic system with a minimal impact on cell viability, unlike the significantly-decreased viability often seen with FACS, providing better recovery rates. Cell suspensions and adherent cells can be maintained within a picodroplet for several days with comparable viability to cells cultured in conventional flask environments (18). Antibody measurement is highly specific and extremely fast using the latest, easy-to-use, integrated and automated systems, which also appeals to biopharmaceutical partners.
New horizons in microfluidics
Genome editing and tissue engineering are emerging areas that are also embracing microfluidic innovations (21,23). Genetic reprogramming of cell lines, such as stem cells, has generated a number of excellent in vitro models for research concerning signalling pathways, cellular behaviour and human disease (24). With traditional methodologies, this area of research faced similar challenges (in terms of throughput and specificity) to that of rare cell detection and isolation.
Manual identification of targets among hundreds of thousands of cells was limited by excessive costs and resulted in scientists constraining analyses to lower numbers, making recognition of specific cell types extremely difficult. Microfluidic techniques have been shown to improve efficiency of genetic editing and cellular reprogramming processes by as much as 50-fold (24). The confined and easily regulated microenvironment also results in production of high quality and durable cell lines.
Pioneering concepts that are taking microfluidic technology to new levels include the development of picodroplet fusion that will provide insights concerning specific cell-cell interactions and pairing. This concept is being adapted for single cell genome editing studies (25,26).
Picodroplet fusion may also facilitate parallel detection procedures (eg mass spectrometry and functional assays), offering further efficiencies and allowing in-depth analysis of precious cellular/tissue samples. Picodroplet splitting or fission also has the potential to enhance the range of technologies in which microfluidics may be applied (26). Splitting picodroplets may further increase the throughput of picodroplet production and increase assay sensitivity (25).
In addition to human cell analysis, picodroplet microfluidic systems are demonstrating increasing value in the exploration of microbial resistance and biodiversity (25). One of the issues associated with metagenomic analysis of microbial colonies is that virulent, fast-growing strains can out-grow slower growing cultures and this diversity is lost when examining samples using traditional whole genome amplification (WGA) techniques (27). Picodroplet microfluidic systems separate the different microbial strains for analysis so that insights can be drawn from each strain independently (27).
The applications and possibilities for this technology are expanding rapidly as it becomes adopted more extensively across the academic research and commercial biopharmaceutical sectors. Stripping away much of the complexity associated with multi-process analysis and speeding up delivery of results has opened up new opportunities to improve understanding of cellular biology and its application in medicine.
Productive partnerships in microfluidics
A number of exciting collaborations are ongoing in this area. For example, we are currently working with researchers to develop a machine that implements high quality, automated, single cell engineering/ genome editing using an integrated system within a disposable biochip. Tools such as this will free researchers from lengthy, manual procedures, allowing them to focus instead on experimental design and interpretation of results. This tool should also cut consumable costs by minimally 50-fold.
Many government and commercial stakeholders have seen the potential of single cell analysis technology and forged partnerships that have nurtured and driven the development of microfluidic innovations. Much of this work has been supported by grants from national funding bodies, such as Innovate UK, as well as biopharmaceutical companies, fuelling the field with the rich and diverse range of expertise and experience that each partner brings.
Partners have been able to shape the way in which this technology has developed by sharing insights concerning the most useful applications for picodroplet microfluidics in their area of work. This has led to the development of platforms that are tailored to the needs of the end user, with enhanced features and functionality that provide optimal results.
Practical benefits of microfluidics for commercial users and scientists
In addition to broadening scientific horizons and revealing novel research applications, this cutting-edge technology can undoubtedly simplify and improve the working lives of many people within the biopharmaceutical and molecular biology industries. Automated and fully-integrated microfluidic systems remove much of the complexity associated with multi-process analysis.
User friendly interfaces have been designed and adapted with the specific needs of commercial scientists in mind, resulting in a streamlined solution and removing laborious processes that require multiple pieces of equipment (each with limited capabilities) and complicated protocols.
Users are essentially able to initiate analyses at the touch of button and turn their attention to other important areas of research with the reassurance that output quality will be maintained, while the length of time required to complete analysis is substantially reduced.
This broadens the possibilities for scientists working in this area and releases capacity so that they are empowered to explore new and interesting areas of research, without the frustrations or limitations associated with traditional techniques. Companies are also able to make more effective and strategic use of their scientist’s expertise, as their time is no longer dominated by mundane tasks and processes. Significant environmental savings can also be made through reduction in the use of plastics.
Conclusion - Microfluidics: making time, realising savings and benefiting patients
Microfluidic innovations provide many practical solutions; precious time, money and resources can be saved through the application of these systems. The potential benefits of picodroplet microfluidic platforms are multi-dimensional.
As application of these systems expands, advantages will extend to those working across the commercial pharmaceutical and biotechnology sectors, scientific/academic researchers, healthcare providers and patients.
Although the possibilities for this technology are vast from a scientific and commercial perspective, the ultimate goal remains the identification and acceleration of innovative treatments and diagnostic tools with the potential to save or improve the lives of those with debilitating disease.
New and interesting disease biomarkers or biotherapeutic targets can be identified in a fraction of the time, compared with conventional platforms, advancing scientific research and improving our ever-evolving knowledge of the molecular signalling pathways underpinning disease.
These insights will elevate drug discovery and development well beyond current limitations, removing the widespread application of one-size-fits all therapeutics; bringing an exciting and ground-breaking new generation of potent precision medicines and accurate diagnostics with the potential to transform disease management. DDW
Dr Frank F. Craig MBA has 20 years of international, general management experience gained from GlaxoSmithKline, Amersham Biosciences and several start-up firms. Dr Craig co-founded Aurora Biosciences (San Diego, USA), Smart Holograms (Cambridge, UK) and Sphere Fluidics (Cambridge, UK). He is now CEO and Director of Sphere Fluidics Limited (Cambridge, UK) and President of Sphere Fluidics Incorporated (New Jersey, USA). He has a PhD in Cell Biology and Microbiology from Glasgow University and an MBA from Warwick Business School.
1 Seah et al. Microfluidic single-cell technology in immunology and antibody screening. Mol Aspects Med. 2018;59:47-61.
2 Chokkalingam et al. Probing cellular heterogeneity in cytokine-secreting immune cells using droplet-based microfluidics. Lab Chip. 2013;13(24):4740-4744.
3 Yuan et al. Challenges and emerging directions in single-cell analysis. Genome Biology. 2017;18:84.
4 Proserpio et al. Single-cell technologies are revolutionizing the approach to rare cells. Immunol Cell Biol. 2016;94(3):225-229.
5 Yeo et al. Microfluidic enrichment for the single cell analysis of circulating tumor cells. Sci Rep. 2016;29(6):22076.
6 Regev et al. The Human Cell Atlas: from vision to reality. Nature. 2017;550:451-453.
7 Liu et al. The history of monoclonal antibody development – Progress, remaining challenges and future innovations. Annals of Medicine and Surgery.2014;3:113-116.
8 Ecker et al. The therapeutic monoclonal antibody market. MAbs. 2015;7(1):9-14.
9 Shalek et al. Single-cell analyses to tailor treatments. Sci Transl Med. 2017;9(408).
10 Editorial. What happened to personalized medicine? Nat Biotechnol. 2012;30(1):1.
11 Borsu et al. Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples. J Mol Diagn. 2016;18(6):903- 911.
12 Watanabe et al. Multiplex Ultrasensitive Genotyping of Patients with Non-Small Cell Lung Cancer for Epidermal Growth Factor Receptor (EGFR) Mutations by Means of Picodroplet Digital PCR. EBioMedicine. 2017;21:86-93.
13 Gross et al. Technologies for Single-Cell Isolation. Int. J. Mol. Sci. 2015;16:16897-16919.
14 Whitesides. The origins and the future of microfluidics. Nature. 2006;442(7101):368-373.
15 Smith et al. Sensitive, high throughput detection of proteins in individual, surfactantstabilized picoliter droplets using nanoelectrospray ionization mass spectrometry. Anal Chem. 2013;85(8):3812-3816.
16 Liu et al. High-throughput screening of antibiotic-resistant bacteria in picodroplets. Lab Chip. 2016;16(9):1636-1643.
17 El Debs et al. Functional single-cell hybridoma screening using droplet-based microfluidics. Proc Natl Acad Sci USA. 2012;109(29):11570-11575.
18 Theberge et al. Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and biology. Angew Chem Int Ed Engl. 2010;49(34):5846-5868.
19 Chin et al. Microfluidics-based diagnostics of infectious diseases in the developing world. Nat Med. 2011 Jul 31;17(8):1015-1019.
20 Pandey et al. Microfluidics Based Point-of- Care Diagnostics. Biotechnol J. 2018;13(1).
21 Han et al. CRISPR-Cas9 delivery to hard-totransfect cells via membrane deformation. Sci Adv. 2015;1(7):e1500454.
22 van Duinen et al. Microfluidic 3D cell culture: from tools to tissue models. Curr Opin Biotechnol. 2015;35:118-126.
23 Inamdar et al. Microfluidic cell culture models for tissue engineering. Curr Opin Biotechnol. 2011;22(5):681-689.
24 Luni et al. High-efficiency cellular reprogramming with microfluidics. Nat Methods. 2016;13(5):446-452.
25 Simon et al. in Day et al (eds). Microdroplet Technology: Principles and Emerging Applications in Biology and Chemistry, Integrated Analytical Systems. Springer Science+Business Media. 2012.
26 Schoeman et al. Electrofusion of single cells in picoliter droplets. Sci Rep. 2017;8(1):3714.
27 Hammond et al. Picodroplet partitioned whole genome amplification of low biomass samples preserves genomic diversity for metagenomic analysis. Microbiome. 2016 Oct 6;4(1):52.