Final Report Summary - EXCELL (Exploring Cellular Dynamics at Nanoscale)
Executive summary:
EXCELL involves a cross-disciplinary and design-based research to generate an innovative, biologically inspired technological platform, capable to monitor real time cellular dynamics. The strategy is to explore and understand how the insertion of nanostructures inside cells or exposure of cells / tissue to nano-structured surfaces, affect the cellular function and the molecular machinery, aiming at creating an integrated approach by ensuring a biocompatible cellular environment.
The consortium combines expertise in cell and molecular biology, neuroscience, micro and nanotechnology, electronics, biophysics, biotechnology, bioengineering, bioinformatics, mathematical modelling, analytical chemistry, and industrial end-use to address the targeted goals, thus clearly going beyond the state of the art methods used in traditional biotechnology or system biology. The approach proposed by EXCELL has the capacity to drive new discoveries and theories, and to show concrete applicability not only in neurophysiology and medicine (as exemplified in EXCELL), but also in biomedical technologies and bioinformatics, material science and engineering. Although the main target of EXCELL is the clinical use of stem cells in the treatment of neurodegenerative and metabolic disorder, the expected results will open up industrial applications and the opportunity of new start-ups in several fields ranging from medical devices, to novel bioresponsive materials, medical textiles, etc.
To explore biointeractions at nanoscale and to understand individual selected phenomena, the development of a microfluidic array Lab-in-a-cell (LIC) platform (a motherboard) with different types of exchangeable / disposable LIC chips, has been central and designed to: (i) address a whole cell population, single cells in a cell population or a tissue grown in the LIC platform; (ii) precisely control the biochemical environment for cell and tissue culturing with the possibility of time controlled multiple stimulations in order to test the effect of different compounds and their concentrations on cell growth and differentiation; (iii) to monitor real time kinetics of multiple interdependent intra and extra-cellular analytes, ranging from the level of expression of genes to the release of metabolites, and cell-to-cell communication processes (transport, secretion, exocytosis, endocytosis, signal transduction, ion channels); (iv) to understand stem cell differentiation at the molecular level in order to derive strategies to influence the differentiation process itself; (v) to advance and improve the understanding and consequently the therapies of diseases such as neurodegenerative disorders (Parkinson's, Alzheimer's) epilepsy, Amyotrophic lateral sclerosis (ALS), and demyelinating disorders like multiple sclerosis, and metabolic illnesses (diabetes, osteoporosis).
The main EXCELL achievements are the following: (1) a polymer based biocompatible 16-channel microfluidic array LIC motherboards complete with miniaturised pumping / valving system designed for easy plug-in of different types of microfluidic LIC chips connected to electronics. Two types of motherboards are available and also delivered to different partners; one more simple system intended for inverted optical microscopy of cell and tissue cultures and one more complex for upright microscopy with integrated electronic potentiostat for combined optical and electrochemical detction; (2) different types of biocompatible exchangeable / disposable LIC chips with or without integrated two-dimensional (2D) and 3D electrode array structures have been developed in different materials; (3) a miniaturised multichannel potentiostat for electrochemical measurements (impedance, fast scan voltammetry, amperometry and potentiometry) custom made for the LIC-platform and that individually can address single electrodes in the electrochemical array of LIC chips; (4) a data acquisition software (Lab View environment) for the different types of sensing schemes has successfully been tested in combination with the potentiostat and LIC chips; (5) protocols for functionalising micro and nanostructures and immobilisation of different biomolecules as well as for biocompatible growth and differentiation of stem cells and tissues have been achieved; and (6) bioanalysis protocols for monitoring cell growth (time-laps microscopy and impedance spectroscopy) and exocytosis of dopamine (amperometry) from growing and differentiated PC12 cells and stem cells have been established. Protocol for measuring the intra-cellular redox environment, Green fluorescent protein (GFP) labelled neuronal stem cells indicating Tyrosine hydroxylase (TH) transporter appearance, and oxygen sensitive intracellular probes for metabolism studies have been established. Optogenetic protocols and logistics have moreover been developed and applied in vitro. (Bio)sensor protocols for measurement of molecular beacon probe encoding for TH-micro-Ribonucleic acid (mRNA), gamma-aminobutyric acid (GABA), glutathione, and alkaline phosphatase have been achieved.
Project context and objectives:
Our first European application pursuing the basic EXCELL idea was called 'Mapping the dynamics of cellular processes' with the acronym MAD. The proposal was not successful and as someone pointed out, maybe because of how some evaluators may have perceived the referral to 'the MAD idea, the MAD scientists, and the MAD project'. When we decided to resubmit a modified version of MAD, we found that the title 'Exploring cellular dynamics at nanoscale' with the acronym EXCELL, was more appropriate. The proposal was favourably evaluated and started officially on 1 September 2008. The idea The idea of EXCELL was born in 2004 at a brainstorming session in the European network of excellence Nano2Life in which chemists, biologists, physiologists, and engineers were asked to brainstorm around their 'unlimited scientific dream'. A medical doctor raised his hand and asked 'Can I really wish anything I want?', and I said 'sure of course'. 'Then, instead of putting the cell on a chip, I would like to put the chip inside a cell', he said with a smile. This was of course not the first time people had been thinking in these terms or pursuing this scientific dream, however, it was the first time for this man and many of those present to realise that maybe this was not only science fiction made up in Hollywood, but something that technology might actually be able to achieve. More importantly, a true cross-disciplinary dialogue had started that proved immensely valuable for many of us that took part in this brainstorming session, and those to follow. Coming from the field of chip-based electrochemical biosensors, the chip in a cell idea, or as we refer to it, the LIC, could easily be visualised as a chip with very tiny 3D nanoelectrode arrays, where each individual electrode could penetrate inside a cell and potentially monitor unexplored intracellular reactions. In principle this was not a new idea either, since detection of intracellular activity had been pursued already in the mid 1980's and 1990's by Wightman and Ewing. They placed microelectrode fibres inside cells for detection of e.g. dopamine, however considering the size of a mammalian cell (10-20 um), these electrodes are huge (2-10 um) and likely detrimental to the cell. When considering the advancement in the field of nanoscience and nanotechnology, there was a real opportunity to explore and pave new avenues in the construction of nanosized structures for a multitude of applications that previously had been impossible to achieve, partly due to technological limitations. The vision Transplantation of multipotent NSC lines is widely used as an experimental tool to understand the therapeutic approach for cell replacement in the central nervous system, as well as a research technique for exploring basic development. However, studies on functional integration of these cells into the host tissue are lagging behind. The fundamental problem, yet still unsolved, is to learn how the differentiation of these initially immature cells can be diverted into certain neuronal phenotypes. In this regard, transplantation of NSC lines, which differentiate into certain neuronal phenotype, producing specific neurotransmitters and neuromodulators (e.g. catecholamines, amino acids, neuropeptides and proteins), and their functional integration is particularly useful, since they could be used both for cell replacement and / or gene therapy e.g. in neurodegenerative disorders. Major efforts are made worldwide to identify the factors and conditions that could direct human NSC differentiation into a particular neuronal phenotype, a major aspect being to reliably determine the release of neurotransmitters from single differentiated neurons first in culture conditions and then from transplanted cells in brain slices. The EXCELL vision has therefore been to develop a unique platform that enables to address these fundamental problems of the cell and molecular biology of stem cells. The idea being to develop a number of completely novel nano(bio)sensors incorporated into a microfluidic LIC array platform for monitoring a completely new set of intra and extra cellular parameters and the possibility for probing the kinetics and dynamics of different interdependent reactions as they appear in the cell, creating a sort of an integrated systems biology chip.
The visionary LIC platform: A schematic representation of some targeted analytes associated to the differentiation of stem cells into dopminergic neurons (from expression of genes and proteins to release of metabolites): Tyrosine hydroxylase (TH) is the rate limiting enzyme for catecholamine (dopamine is one) production in such cells. Some possible intra- and extra-cellular placed nanobiosensors for detection of :A) mRNA; B) cellular redox status, C) TH detection, and D) extra-cellular release of catecholamines. A LIC platform seen from the side, comprising a transparent microfluidic cell culture chip that incorporates different types of nano structures (e.g. extra-cellular planar Interdigitated electrode (IDEs) arrays, intracellular probes, such as pillar electrodes, cantilevers, and / or Quantum dots (QDs).
The scientific and technological development of EXCELL has been conducted within seven scientific and technological work packages, using a step-by-step approach with tasks displaying an increasing degree of complexity, to minimise risk and to assure success beyond the state-of-the-art. The objectives were to:
- develop a basic microfluidic LIC structure (LIC-B) - a transparent polymeric cell and tissue culture array with multiple microfluidic streams (8x8 or 16x16);
- construct extra-cellular placed micro and nano-sized interdigitated electrode (IDE) arrays (IDAs) with different geometries that will be used for in vitro real time monitoring of cell morphological changes (growth, division, migration, differentiation), cytotoxicity, G-protein coupled receptor (GPCR)-ligand interactions, and / or selective and sensitive amperometric detection of different secreted compounds, e.g. catecholamines, amino and acids;
- construct intracellular nano-sized pillar electrode arrays that will be used for in vitro real time sensing of intracellular m-RNA, proteins and small molecules and intra cellular redox state. The pillars will be individually addressable and movable perpendicularly so that they mechanically can penetrate into individual cells, using individually controlled actuators;
- develop electronics that can address each individual sensor in the IDE and pillar arrays;
- integrate the basic LIC-B structure and the sensor array chips with electronics, at first one at a time and later with several platforms, incorporating different types of LIC chips with various integrated electrode structures;
- develop chemical and biochemical protocols for (i) nanostructure functionalisation and biological modification of nanobiosensors; (ii) biocompatible intra and extra cellular sensing of various biomarkers; and (iii) bioassisted insertion of nanopillars into individual cells;
- develop strategies for real time intracellular optical imaging on the LICs, using: (i) oxygen sensitive near-infrared phosphorescent probes for studying cell viability / cytotoxitcity, and mitochondrial functions; (ii) genetically engineered cells with green fluorescent protein (GFP) / luciferase (LUC) reporters for detection of intracellular proteins and GPCR-ligand interactions; (iii) and molecular beacons-quantum dots (MB-QD) for mRNA detection;
- genetically engineer cells for the identification of: (i) specific neuronal phenotypes generated from human NSCs or other alternative sources of neuronal-like cells; and (ii) differentiated progeny-derived from SKSCs. In both cases, vectors will be engineered to express reporter genes from a cell-type specific gene promoter;
- show the applicability of the LIC platforms, i.e. first the individual sensing LIC platforms and later with several sensing platforms integrated in the LIC in medical research;
- develop data acquisition software and background noise reduction based on the generated multidimensional LIC data;
- develop pattern recognition protocols to interpret the kinetics, dynamics and interdependence of the sensor responses generated in the LIC;
- compare gene expression profiles and cytotoxic effects on cells that are exposed respective unexposed to nano(bio)structures and nano(bio)structured surfaces as well as for cells exposed to chemical and electrical stimulation / provocation;
- validate and verify generated data and results when possible with conventional culture plate based bioassays and biochemical assays;
- gain new insight into stem cell differentiation mechanisms and dynamics at the molecular level and develop capability to chemically guide the differentiation process in specific directions into certain neuronal phenotypes (e.g. dopaminergic or GABAergic phenotypes);
- correlate and validate complex nano-measurements obtained with different nano-sensors with conventional whole-cell patch-clamp electrophysiological measurements of specific stem-cell derived differentiated neurons, and to develop multi-modal combinations of these novel nano- and more conventional electrophysiological measurements;
- communicate and disseminate the EXCELL-LIC.
EXCELL involves a cross-disciplinary and design-based research to generate an innovative, biologically inspired technological platform, capable to monitor real time cellular dynamics. The strategy is to explore and understand how the insertion of nanostructures inside cells or exposure of cells / tissue to nano-structured surfaces, affect the cellular function and the molecular machinery, aiming at creating an integrated approach by ensuring a biocompatible cellular environment.
The consortium combines expertise in cell and molecular biology, neuroscience, micro and nanotechnology, electronics, biophysics, biotechnology, bioengineering, bioinformatics, mathematical modelling, analytical chemistry, and industrial end-use to address the targeted goals, thus clearly going beyond the state of the art methods used in traditional biotechnology or system biology. The approach proposed by EXCELL has the capacity to drive new discoveries and theories, and to show concrete applicability not only in neurophysiology and medicine (as exemplified in EXCELL), but also in biomedical technologies and bioinformatics, material science and engineering. Although the main target of EXCELL is the clinical use of stem cells in the treatment of neurodegenerative and metabolic disorder, the expected results will open up industrial applications and the opportunity of new start-ups in several fields ranging from medical devices, to novel bioresponsive materials, medical textiles, etc.
To explore biointeractions at nanoscale and to understand individual selected phenomena, the development of a microfluidic array Lab-in-a-cell (LIC) platform (a motherboard) with different types of exchangeable / disposable LIC chips, has been central and designed to: (i) address a whole cell population, single cells in a cell population or a tissue grown in the LIC platform; (ii) precisely control the biochemical environment for cell and tissue culturing with the possibility of time controlled multiple stimulations in order to test the effect of different compounds and their concentrations on cell growth and differentiation; (iii) to monitor real time kinetics of multiple interdependent intra and extra-cellular analytes, ranging from the level of expression of genes to the release of metabolites, and cell-to-cell communication processes (transport, secretion, exocytosis, endocytosis, signal transduction, ion channels); (iv) to understand stem cell differentiation at the molecular level in order to derive strategies to influence the differentiation process itself; (v) to advance and improve the understanding and consequently the therapies of diseases such as neurodegenerative disorders (Parkinson's, Alzheimer's) epilepsy, Amyotrophic lateral sclerosis (ALS), and demyelinating disorders like multiple sclerosis, and metabolic illnesses (diabetes, osteoporosis).
The main EXCELL achievements are the following: (1) a polymer based biocompatible 16-channel microfluidic array LIC motherboards complete with miniaturised pumping / valving system designed for easy plug-in of different types of microfluidic LIC chips connected to electronics. Two types of motherboards are available and also delivered to different partners; one more simple system intended for inverted optical microscopy of cell and tissue cultures and one more complex for upright microscopy with integrated electronic potentiostat for combined optical and electrochemical detction; (2) different types of biocompatible exchangeable / disposable LIC chips with or without integrated two-dimensional (2D) and 3D electrode array structures have been developed in different materials; (3) a miniaturised multichannel potentiostat for electrochemical measurements (impedance, fast scan voltammetry, amperometry and potentiometry) custom made for the LIC-platform and that individually can address single electrodes in the electrochemical array of LIC chips; (4) a data acquisition software (Lab View environment) for the different types of sensing schemes has successfully been tested in combination with the potentiostat and LIC chips; (5) protocols for functionalising micro and nanostructures and immobilisation of different biomolecules as well as for biocompatible growth and differentiation of stem cells and tissues have been achieved; and (6) bioanalysis protocols for monitoring cell growth (time-laps microscopy and impedance spectroscopy) and exocytosis of dopamine (amperometry) from growing and differentiated PC12 cells and stem cells have been established. Protocol for measuring the intra-cellular redox environment, Green fluorescent protein (GFP) labelled neuronal stem cells indicating Tyrosine hydroxylase (TH) transporter appearance, and oxygen sensitive intracellular probes for metabolism studies have been established. Optogenetic protocols and logistics have moreover been developed and applied in vitro. (Bio)sensor protocols for measurement of molecular beacon probe encoding for TH-micro-Ribonucleic acid (mRNA), gamma-aminobutyric acid (GABA), glutathione, and alkaline phosphatase have been achieved.
Project context and objectives:
Our first European application pursuing the basic EXCELL idea was called 'Mapping the dynamics of cellular processes' with the acronym MAD. The proposal was not successful and as someone pointed out, maybe because of how some evaluators may have perceived the referral to 'the MAD idea, the MAD scientists, and the MAD project'. When we decided to resubmit a modified version of MAD, we found that the title 'Exploring cellular dynamics at nanoscale' with the acronym EXCELL, was more appropriate. The proposal was favourably evaluated and started officially on 1 September 2008. The idea The idea of EXCELL was born in 2004 at a brainstorming session in the European network of excellence Nano2Life in which chemists, biologists, physiologists, and engineers were asked to brainstorm around their 'unlimited scientific dream'. A medical doctor raised his hand and asked 'Can I really wish anything I want?', and I said 'sure of course'. 'Then, instead of putting the cell on a chip, I would like to put the chip inside a cell', he said with a smile. This was of course not the first time people had been thinking in these terms or pursuing this scientific dream, however, it was the first time for this man and many of those present to realise that maybe this was not only science fiction made up in Hollywood, but something that technology might actually be able to achieve. More importantly, a true cross-disciplinary dialogue had started that proved immensely valuable for many of us that took part in this brainstorming session, and those to follow. Coming from the field of chip-based electrochemical biosensors, the chip in a cell idea, or as we refer to it, the LIC, could easily be visualised as a chip with very tiny 3D nanoelectrode arrays, where each individual electrode could penetrate inside a cell and potentially monitor unexplored intracellular reactions. In principle this was not a new idea either, since detection of intracellular activity had been pursued already in the mid 1980's and 1990's by Wightman and Ewing. They placed microelectrode fibres inside cells for detection of e.g. dopamine, however considering the size of a mammalian cell (10-20 um), these electrodes are huge (2-10 um) and likely detrimental to the cell. When considering the advancement in the field of nanoscience and nanotechnology, there was a real opportunity to explore and pave new avenues in the construction of nanosized structures for a multitude of applications that previously had been impossible to achieve, partly due to technological limitations. The vision Transplantation of multipotent NSC lines is widely used as an experimental tool to understand the therapeutic approach for cell replacement in the central nervous system, as well as a research technique for exploring basic development. However, studies on functional integration of these cells into the host tissue are lagging behind. The fundamental problem, yet still unsolved, is to learn how the differentiation of these initially immature cells can be diverted into certain neuronal phenotypes. In this regard, transplantation of NSC lines, which differentiate into certain neuronal phenotype, producing specific neurotransmitters and neuromodulators (e.g. catecholamines, amino acids, neuropeptides and proteins), and their functional integration is particularly useful, since they could be used both for cell replacement and / or gene therapy e.g. in neurodegenerative disorders. Major efforts are made worldwide to identify the factors and conditions that could direct human NSC differentiation into a particular neuronal phenotype, a major aspect being to reliably determine the release of neurotransmitters from single differentiated neurons first in culture conditions and then from transplanted cells in brain slices. The EXCELL vision has therefore been to develop a unique platform that enables to address these fundamental problems of the cell and molecular biology of stem cells. The idea being to develop a number of completely novel nano(bio)sensors incorporated into a microfluidic LIC array platform for monitoring a completely new set of intra and extra cellular parameters and the possibility for probing the kinetics and dynamics of different interdependent reactions as they appear in the cell, creating a sort of an integrated systems biology chip.
The visionary LIC platform: A schematic representation of some targeted analytes associated to the differentiation of stem cells into dopminergic neurons (from expression of genes and proteins to release of metabolites): Tyrosine hydroxylase (TH) is the rate limiting enzyme for catecholamine (dopamine is one) production in such cells. Some possible intra- and extra-cellular placed nanobiosensors for detection of :A) mRNA; B) cellular redox status, C) TH detection, and D) extra-cellular release of catecholamines. A LIC platform seen from the side, comprising a transparent microfluidic cell culture chip that incorporates different types of nano structures (e.g. extra-cellular planar Interdigitated electrode (IDEs) arrays, intracellular probes, such as pillar electrodes, cantilevers, and / or Quantum dots (QDs).
The scientific and technological development of EXCELL has been conducted within seven scientific and technological work packages, using a step-by-step approach with tasks displaying an increasing degree of complexity, to minimise risk and to assure success beyond the state-of-the-art. The objectives were to:
- develop a basic microfluidic LIC structure (LIC-B) - a transparent polymeric cell and tissue culture array with multiple microfluidic streams (8x8 or 16x16);
- construct extra-cellular placed micro and nano-sized interdigitated electrode (IDE) arrays (IDAs) with different geometries that will be used for in vitro real time monitoring of cell morphological changes (growth, division, migration, differentiation), cytotoxicity, G-protein coupled receptor (GPCR)-ligand interactions, and / or selective and sensitive amperometric detection of different secreted compounds, e.g. catecholamines, amino and acids;
- construct intracellular nano-sized pillar electrode arrays that will be used for in vitro real time sensing of intracellular m-RNA, proteins and small molecules and intra cellular redox state. The pillars will be individually addressable and movable perpendicularly so that they mechanically can penetrate into individual cells, using individually controlled actuators;
- develop electronics that can address each individual sensor in the IDE and pillar arrays;
- integrate the basic LIC-B structure and the sensor array chips with electronics, at first one at a time and later with several platforms, incorporating different types of LIC chips with various integrated electrode structures;
- develop chemical and biochemical protocols for (i) nanostructure functionalisation and biological modification of nanobiosensors; (ii) biocompatible intra and extra cellular sensing of various biomarkers; and (iii) bioassisted insertion of nanopillars into individual cells;
- develop strategies for real time intracellular optical imaging on the LICs, using: (i) oxygen sensitive near-infrared phosphorescent probes for studying cell viability / cytotoxitcity, and mitochondrial functions; (ii) genetically engineered cells with green fluorescent protein (GFP) / luciferase (LUC) reporters for detection of intracellular proteins and GPCR-ligand interactions; (iii) and molecular beacons-quantum dots (MB-QD) for mRNA detection;
- genetically engineer cells for the identification of: (i) specific neuronal phenotypes generated from human NSCs or other alternative sources of neuronal-like cells; and (ii) differentiated progeny-derived from SKSCs. In both cases, vectors will be engineered to express reporter genes from a cell-type specific gene promoter;
- show the applicability of the LIC platforms, i.e. first the individual sensing LIC platforms and later with several sensing platforms integrated in the LIC in medical research;
- develop data acquisition software and background noise reduction based on the generated multidimensional LIC data;
- develop pattern recognition protocols to interpret the kinetics, dynamics and interdependence of the sensor responses generated in the LIC;
- compare gene expression profiles and cytotoxic effects on cells that are exposed respective unexposed to nano(bio)structures and nano(bio)structured surfaces as well as for cells exposed to chemical and electrical stimulation / provocation;
- validate and verify generated data and results when possible with conventional culture plate based bioassays and biochemical assays;
- gain new insight into stem cell differentiation mechanisms and dynamics at the molecular level and develop capability to chemically guide the differentiation process in specific directions into certain neuronal phenotypes (e.g. dopaminergic or GABAergic phenotypes);
- correlate and validate complex nano-measurements obtained with different nano-sensors with conventional whole-cell patch-clamp electrophysiological measurements of specific stem-cell derived differentiated neurons, and to develop multi-modal combinations of these novel nano- and more conventional electrophysiological measurements;
- communicate and disseminate the EXCELL-LIC.