Periodic Reporting for period 4 - HybCell (Engineering of hybrid cells using lab-on-chip technology)
Periodo di rendicontazione: 2021-01-01 al 2021-12-31
How can we create an artificial cell and mimic intracellular biochemical processes?
A living cell has an amazing metabolism allowing it to grow and divide, and to respond and adapt to changes that occur in the environment. Moreover, cells produce compounds, which are secreted out of the cell, e.g. for cell-cell communication and other purposes. In this regard, described with a technical wording, living cells can be considered as very impressive machinery that consists of multiple reaction compartments, each of which has its particular role and provides particular reaction conditions. When considering that some cell types are able to synthesize complex organic molecules in large yields, it becomes obvious that the efficiency of this machinery is unmet in any macroscopic man-made (bio)reaction system. However, since decades the aim of many chemists, material scientists, synthetic biologist, biotechnologists and others is to investigate, understand and copy principles from natural systems including a single living cell and exploit their potential for similar purposes such as protein production, but also in a completely different context, e.g. the realization of highly specific cell-free sensors. Moreover, simple mimics could be the key to understand early forms of life and hence, the very early evolution of life. For cell mimics, also referred to as protocells, minimal cells, or artificial cells, some of the key challenges are not yet solved, which are (i) the fascinating way a natural cell can form various compartments; (ii) the signalling and regulatory systems of a cell that allows for signal transmission as well as exchange of compounds in and out of the cell and cell organelles, which is highly sophisticated so that mimicking and bottom-up assembling seem currently far out of reach.
The overall aim of the here described projects is to learn fundamental characteristics of cellular organization and compartmentalization, in particular to mimic the lipid membrane, and to exploit this knowledge for engineering minimal cells with a great impact in the context of synthetic biology and also pharmaceutical and medical applications.
The key methods to address these challenges are based on lab-on-chip technology (also referred to as microfluidics technology) that provides the unique potential to systematically investigate membrane properties by allowing precise formation, positioning, manipulation and analysis of the membranes; together with many more advantages such as the fast and controlled fluid supply, the possibility of tailoring the chemical surface patterns and surface topology and the application of electrical fields. Microfluidic platforms will allow going far beyond the existing methods in membrane research, so that controlled bottom-up formation of simple to more and more complex systems becomes possible.
A living cell has an amazing metabolism allowing it to grow and divide, and to respond and adapt to changes that occur in the environment. Moreover, cells produce compounds, which are secreted out of the cell, e.g. for cell-cell communication and other purposes. In this regard, described with a technical wording, living cells can be considered as very impressive machinery that consists of multiple reaction compartments, each of which has its particular role and provides particular reaction conditions. When considering that some cell types are able to synthesize complex organic molecules in large yields, it becomes obvious that the efficiency of this machinery is unmet in any macroscopic man-made (bio)reaction system. However, since decades the aim of many chemists, material scientists, synthetic biologist, biotechnologists and others is to investigate, understand and copy principles from natural systems including a single living cell and exploit their potential for similar purposes such as protein production, but also in a completely different context, e.g. the realization of highly specific cell-free sensors. Moreover, simple mimics could be the key to understand early forms of life and hence, the very early evolution of life. For cell mimics, also referred to as protocells, minimal cells, or artificial cells, some of the key challenges are not yet solved, which are (i) the fascinating way a natural cell can form various compartments; (ii) the signalling and regulatory systems of a cell that allows for signal transmission as well as exchange of compounds in and out of the cell and cell organelles, which is highly sophisticated so that mimicking and bottom-up assembling seem currently far out of reach.
The overall aim of the here described projects is to learn fundamental characteristics of cellular organization and compartmentalization, in particular to mimic the lipid membrane, and to exploit this knowledge for engineering minimal cells with a great impact in the context of synthetic biology and also pharmaceutical and medical applications.
The key methods to address these challenges are based on lab-on-chip technology (also referred to as microfluidics technology) that provides the unique potential to systematically investigate membrane properties by allowing precise formation, positioning, manipulation and analysis of the membranes; together with many more advantages such as the fast and controlled fluid supply, the possibility of tailoring the chemical surface patterns and surface topology and the application of electrical fields. Microfluidic platforms will allow going far beyond the existing methods in membrane research, so that controlled bottom-up formation of simple to more and more complex systems becomes possible.
We developed methods to form, capture and analyse artificial cells, so-called giant unilamellar vesicles (GUVs) formed by lipid membranes. We extensively studied the diverse interactions of membranes with small molecules and peptides. Therefore, we manufactured microfluidic devices that enable immobilization of GUVs. Subsequently, peptides or other molecules can be easily supplied to the GUVs and mechanistic and kinetic studies can be performed. We could show how membrane composition as well as membrane charges influence permeation or partitioning of molecules. We mimicked the asymmetry of the plasma membrane in living cells, which resulted in faster permeation of short peptides. Other types of peptides did not permeate across the membrane but partitioned into it, aggregated there and disintegrated the membrane by formation of pores. Moreover, one of the major difficulties in the bottom-up assembly of artificial cells is the reconstitution of membrane proteins. Here, we demonstrated a new process to integrate membrane proteins in artificial cells by fuding cell-derived lipid vesicles with the synthetic lipid membrane. In this way, membrane proteins from the cell of origin is transfered into the new artificial environment.
Membranes are often exposed to flow, and can undergo visible deformation, when the flow rates are high. Besides this effect, mechanical forces can be transferred across the membrane, which is referred to as mechanotransduction. We investigated this effect by use of GUVs with enclosed tracer particles, and could observe the process by means of a specialized imaging technique and custom-made particle-tracking software. The exposure to shear forces on GUVs resulted in a flow of tracer particles within the vesicle, which reflected the external flow with respect to strength and symmetry/asymmetry. In addition, mechanotransduction was found to be dependent on the composition of the membrane.
For the construction of more complex and cell-like models with intracellular compounds and organelles, resembling the intracellular architecture, the traditional methods for vesicles formation are limited. In particular, the encapsulation efficiency is very low and particles, cells or small liposomes cannot be encapsulated. Therefore, we developed microfluidic systems to form water-in-oil (w/o) droplets of nL volume water-in oil-water (w/o/w) droplets and giant unilamellar vesicles at rates of several Hz, with monodisperse size distribution and defined composition. Using these methods, we have built simple multi-compartment systems and conducted reactions across these artificial organelles. In the next step, we also integrated cell-free protein synthesis in these advanced artificial cells, mimicking the most important biosynthesis pathways in the cells, i.e. the gene transcription and translation. Most important, we demonstrated various applications of these methods. Noteworthy are the high-throughput screening approaches based on double-emulsion droplets, which can be formed at kHz frequency and subsequently analyzed in flow cytometers. We underlined their high benefit and potential for high throughput screening such as directed evolution.
Membranes are often exposed to flow, and can undergo visible deformation, when the flow rates are high. Besides this effect, mechanical forces can be transferred across the membrane, which is referred to as mechanotransduction. We investigated this effect by use of GUVs with enclosed tracer particles, and could observe the process by means of a specialized imaging technique and custom-made particle-tracking software. The exposure to shear forces on GUVs resulted in a flow of tracer particles within the vesicle, which reflected the external flow with respect to strength and symmetry/asymmetry. In addition, mechanotransduction was found to be dependent on the composition of the membrane.
For the construction of more complex and cell-like models with intracellular compounds and organelles, resembling the intracellular architecture, the traditional methods for vesicles formation are limited. In particular, the encapsulation efficiency is very low and particles, cells or small liposomes cannot be encapsulated. Therefore, we developed microfluidic systems to form water-in-oil (w/o) droplets of nL volume water-in oil-water (w/o/w) droplets and giant unilamellar vesicles at rates of several Hz, with monodisperse size distribution and defined composition. Using these methods, we have built simple multi-compartment systems and conducted reactions across these artificial organelles. In the next step, we also integrated cell-free protein synthesis in these advanced artificial cells, mimicking the most important biosynthesis pathways in the cells, i.e. the gene transcription and translation. Most important, we demonstrated various applications of these methods. Noteworthy are the high-throughput screening approaches based on double-emulsion droplets, which can be formed at kHz frequency and subsequently analyzed in flow cytometers. We underlined their high benefit and potential for high throughput screening such as directed evolution.
In the time of the ERC Consolidator Grant "HybCell", we have developed a large microfluidic toolbox for formation of cell-like vessels with defined volume (nanoliters and sub-nanoliters), composition and content, with tailored lipid membranes or hydrophobic shells. Additionally, microfluidic methods were employed to enable advanced manipulation procedures such as immobilization, rapid fluid exchange, incubation for defined periods and exposure to electrical fields. We adopted various analytical methods including optical and fluorescence microscopy, cytometry and mass spectrometry.
These new tools hpave the way to create improved cell mimics, important in numerous applications. For example, we have established a platform with sessile droplets for the monitoring of drug permeation with and without fluorescent labels across artificial membranes, which is ready for further automation. New and future applications of this platform include massively parallel drug tests and single-cell analysis. Moreover, we exploited double emulsion droplets for high-throughput applications (drug screening, directed evolution) and we will continue this direction after finishing the ERC Consoldator grant.
These new tools hpave the way to create improved cell mimics, important in numerous applications. For example, we have established a platform with sessile droplets for the monitoring of drug permeation with and without fluorescent labels across artificial membranes, which is ready for further automation. New and future applications of this platform include massively parallel drug tests and single-cell analysis. Moreover, we exploited double emulsion droplets for high-throughput applications (drug screening, directed evolution) and we will continue this direction after finishing the ERC Consoldator grant.