Functional Phenotype Flow Cytometer

Complex biological systems such as plants and animals are composed of numerous different cell types all conducting different functions. These different functions come together to make a stable functioning organism, much like an orchestra is composed of numerous different instrumentalists. However, cells are tiny and cells performing different functional roles are often indistinguishable. The aim of this project is to be able to identify and separate cells based on their differing function and their differing response to stimuli.

The proposed "Functional Phenotype Flow Cytometer", will use microfluidics (tiny plumbing devices composed of pipes and channels that allow us to control cells) to bathe a cell in different environments while at the same time reading its intracellular activity. Using previous research, on the environments that activate a specific intracellular function in a particular cell type, we can use the device to recreate an environmental cue to activate a cellular behaviour and then extract the responding cell type from a cell mixture.

To illustrate the feasibility of the device we have chosen to separate gut enteroendocrine cell types from gut epithelial cell types. These are cells present in the human digestive system that activate hormone responses and therefore orchestrate metabolic response to food digestion. Previous research has shown that intracellular calcium levels are raised in these gut enteroendocrine cell types, when the cells are bathed in an environment containing a high glucose concentration, or a high potassium chloride concentration. We will build a device which will automatically expose each cell from the mixture to a high glucose environment and sequentially pass it to the high potassium chloride environment. By identifying cells that show changes in intracellular calcium following the change in environment we will be able to identify and sort cells as enteroendocrine cells.

Excitingly, the "Functional Phenotype Flow Cytometer" can also be run as a high throughput cell analyser used to screen for the molecular links between molecules of unknown function and target cells. Here we will test the interaction between three neuropeptides with unidentified function on a library of cells each expressing on its surface candidate receptors. The "Functional Phenotype Flow Cytometer" facilitates this interaction and bathes each single cell into distinct flow lines each containing a specific peptide. Upon a successful match between the neuropeptide and a specific receptor the device records the associated increases in the intracellular calcium and identifies a successful peptide receptor match.

Successful development of a system that allows us to separate cells based on how they respond to external stimuli will drastically change how we study cells that differentiate to perform different roles and allow us to study how cells in the same organism with the same genetic blueprint perform different cellular functions. This is a key area for understanding how organisms, including humans develop, but also for understanding how disease can develop.

Historically cells have been catalogued in respect to their architecture by using fluorescent dyes to specifically label cellular and subcellular structures. Cell shape, organelle distribution and spatial localisation within the tissue have all been used to distinguish cell types. More recently, antibodies linked with fluorescent markers that can bind to individual molecules have been developed and used to differentiate cell types. However, such approaches are inaccurate, severely limited in their application to live cells and are difficult to use for classifying and separating cells based on functional phenotypic responses. Here we propose to use cell function to differentiate cell types. To our knowledge this approach is completely novel, we suspect largely because tractable tools have yet to be developed and demonstrated.

Central to the functioning of living organisms, cellular activity is linked to characteristic intracellular processes. Research has generated many optical reporters for key intracellular mechanisms in living cells such as ion indicators, ATP/ADP sensors and cyclic AMP sensors and a great volume of knowledge has been gathered about the molecular stimuli that activate the corresponding intracellular activity in specific cell types. Given that the molecular stimuli and the associated intracellular process differ between cell types, here we will develop a flexible opto-microfluidic device that sorts cells based on their functional characteristics. By analysing in real time the cell responses to specific stimuli, we will use the proposed device to isolate cells from a mixture that specifically respond to one or more stimuli and thus sort cell based on their functional phenotype. Proving the versatility of the device we will also employ the platform for real time analysis of cell function providing a high throughput platform for studying interactions of the cells with specific stimuli.

The "Functional Phenotype Flow Cytometer" (FPFC) we are proposing, has the potential for broad utility across both biological and medical research communities. The device we plan to build combines essential features from both florescence activated flow cytometry and high throughput microscopy, yet the proposed development will uniquely allow us to measure functional cellular phenotypes and conduct sorting based on these measurements. The FPFC bridges an important gap between currently available technologies and promises to have a major impact on the associated research and industrial communities in the following ways.

In the field of Cell Biology, the proposed FPFC would provide a new method for sort living cells for further experiments allowing analysis when fluorescent antibodies that specifically target the cell surface of a target cellular type are lacking (this includes many variant human cells). This opens up a wide range of applications, such as preferentially recovering immune cells, parasites or cancer cells from tissue samples. Furthermore, these methods will complement current antibody technologies and will also providing cheaper and more accurate alternatives for sorting living cells.

In the field of Molecular Biology, the FPFC would facilitate the identification of genotype-phenotype and transcriptome-phenotype relationships. Particularly with the advent of single cell DNA and RNA sequencing, the device could sort individual cells with a particular functional phenotype. Sorted cells could be further analysed with genomics and/or transcriptomes and relationships between their functional phenotype and their genome and/or transcriptome could be established. These types of studies could be used to disseminate differences in cell populations in health and disease and thus provide valuable insights for medical research and the pharmaceutical industry. Similarly when working with genetically labelled cell population single cell transcriptomics has revealed large heterogeneity among a cell type. Using the FPFC we will correlate the functional phenotype with the levels of RNA expression.

In the field of Stem Cell Biology, the proposed device would provide a method of assessing cell differentiation. Coupled with single cell transcriptomics the device will provide a powerful tool to disseminate population heterogeneity and the identification of target cells

In the field of Phenotypic Screening. High throughput phenotype screening is emerging as the emerging field for revolutionary technology developments. The proposed device will uniquely allow researchers to measure changes in the functional phenotype of a cell upon exposure to environmental metabolites and other stimuli. This is a key and hugely underdeveloped field and requires focused technological development and new commercial products.

The listed area for downstream development identify clear market space for further development and commercialisation of the proposed technology. Such endeavours will be supported by the relevant business and legal departments at the University of Exeter as detailed in the Pathways to Impact Section. In addition, our work will lead to commercial IP, which we intend to protect by IP applications. We will also explore additional commercialisation of the device to a broad range of industrial partners. Some potential projects we include the use of our technique for pharmacological drug development, which will be of benefit to public health in the UK and across the world.