Divide et Impera
Our ambition is to understand and harness single cells for both fundamental biological insights and to discover high-value molecules. The approach we take is to break up populations of biological entities into individual constituents. We use micro-encapsulation technologies to isolate, assay in-situ and characterize these populations across several scales from 3D cell cultures, unicellular organisms, down to cells and proteins. In the process, we develop advanced microfluidics-based platforms for high-throughput biological screening.
The themes we are interested in encompass: microfluidics, protein engineering, 3D cell cultures and single-cell biology
Microfluidics
Engineering and technical development of microfluidics/lab-on-a-chip platforms include the introduction of AI and image-based approaches for microdroplet manipulation, novel read-outs for high-throughput molecular and cellular screens and massively parallel multidimensional screens. Key enabling features are: the possibility to automatically generate increasingly complex (bio)chemical mixtures, the ability to track them in high spatio-temporal resolution and using feedback mechanisms to refine their content over time.
We deposit published microfluidic CAD designs in the public database Dropbase
● Anagnostidis V, Sherlock B, Metz J, Mair P, Hollfelder F, Gielen F (2020). Deep learning guided image-based droplet sorting for on-demand selection. and analysis of single cells and 3D cell cultures. Lab Chip 2020, 20, 889.. Author URL. Full text.
● Gielen F, Buryska T, Van Vliet L, Butz M, Damborsky J, Prokop Z, Hollfelder F (2015). Interfacing microwells with nanoliter compartments: a sampler generating high-resolution concentration gradients for quantitative biochemical analyses in droplets. Anal Chem, 87(1), 624-632. Abstract. Author URL.
● Gielen F, van Vliet L, Koprowski BT, Devenish SRA, Fischlechner M, Edel JB, Niu X, deMello AJ, Hollfelder F (2013). A fully unsupervised compartment-on-demand platform for precise nanoliter assays of time-dependent steady-state enzyme kinetics and inhibition. Anal Chem, 85(9), 4761-4769. Abstract. Author URL.
Protein Engineering
How much and how fast can we improve protein function?
Microdroplet technology has already shown outstanding potential for the exploration of sequence space and biological diversity at ultra-high-throughput (10^8/day) leading to reduced screening time, reagent consumption (50 µL per library) and costs. We are interested in using UHTS directed evolution for a range of enzymes including therapeutic enzymes such as lysins from bacteriophages which can degrade the peptidoglycan wall of bacteria and act as antimicrobials.
● Gielen F, Hours R, Emond S, Fischlechner M, Schell U, Hollfelder F (2016). Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS). Proc Natl Acad Sci U S A, 113(47), E7383-E7389. Abstract. Author URL. Full text.
● Mair P, Gielen F, Hollfelder F (2017). Exploring sequence space in search of functional enzymes using microfluidic droplets. Curr Opin Chem Biol, 37, 137-144. Abstract. Author URL. Full text.
3D cell cultures
How does the micro-environment influence the fate of isogenic cell cultures?
Three-dimensional (3D) cell cultures provide a physiologically relevant model to study disease modelling and can be used for high-throughput drug testing. We develop methods for the high-throughput generation of 3D cell cultures with micro-encapsulation and screen large spheroid populations for drug response followed by isolation of rare phenotypes. We are working on applications from epithelial-mesenchymal transitions to stem cells differentiation.
● Anagnostidis V, Sherlock B, Metz J, Mair P, Hollfelder F, Gielen F (In Press). Deep learning guided image-based droplet sorting for on-demand selection. and analysis of single cells and 3D cell cultures. Abstract. Author URL. Full text.
● Kleine-Brüggeney H, van Vliet LD, Mulas C, Gielen F, Agley CC, Silva JCR, Smith A, Chalut K, Hollfelder F (2019). Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation. Small, 15(5). Abstract. Full text.
Single-cell biology
How different are cells and unicellular organisms?
By encapsulating single cells into microdroplets, we can precisely control and interrogate their genotypes, phenotypes and individual behaviours. Insights learned from large number of single cells help define their uniqueness in new ways, knowledge used for quantitative modelling approaches.