Vesosomes are nested liposomal structures with high potential as advanced drug delivery vehicles, bioreactors and artificial cells. However, to date no method has been reported to prepare ...monodisperse vesosomes of controlled size. Here we report on a multistep microfluidic strategy for hierarchically assembling uniform vesosomes from dewetting of double emulsion templates. The control afforded by our method is illustrated by the formation of concentric, pericentric and multicompartment liposomes. The microfluidic route to vesosomes offers an exceptional platform to build artificial cells as exemplified by the in vitro transcription in “nucleus” liposomes and the mimicry of the architecture of eukaryotic cells. Finally, we show the transport of small molecules across the nucleic envelope via insertion of nanopores into the bilayers.
Geometrical cues have been shown to alter gene expression and differentiation on 2D substrates. However, little is known about how geometrical cues affect cell function in 3D. One major reason for ...this lack of understanding is rooted in the difficulties of controlling cell geometry in a complex 3D setting and for long periods of culture. Here, we present a robust method to control cell volume and shape of individual human mesenchymal stem cells (hMSCs) inside 3D microniches with a range of different geometries (e.g., cylinder, triangular prism, cubic, and cuboid). We find that the actin filaments, focal adhesions, nuclear shape, YAP/TAZ localization, cell contractility, nuclear accumulation of histone deacetylase 3, and lineage selection are all sensitive to cell volume. Our 3D microniches enable fundamental studies on the impact of biophysical cues on cell fate, and have potential applications in investigating how multicellular architectures organize within geometrically well-defined 3D spaces.
Microdroplets in microfluidics offer a great number of opportunities in chemical and biological research. They provide a compartment in which species or reactions can be isolated, they are ...monodisperse and therefore suitable for quantitative studies, they offer the possibility to work with extremely small volumes, single cells, or single molecules, and are suitable for high-throughput experiments. The aim of this Review is to show the importance of these features in enabling new experiments in biology and chemistry. The recent advances in device fabrication are highlighted as are the remaining technological challenges. Examples are presented to show how compartmentalization, monodispersity, single-molecule sensitivity, and high throughput have been exploited in experiments that would have been extremely difficult outside the microfluidics platform.
Cell culturing, whether for tissue engineering or cell biology studies, always involves placing cells in a non‐natural environment and no material currently exist that can mimic the entire complexity ...of natural tissues and variety of cell‐matrix interactions that is found in vivo. Here, we review the vast range of hydrogels, composed of natural or synthetic polymers that provide a route to tailored microenvironments.
This review surveys the current developments for hydrogels that are fabricated from natural and synthetic polymers for use as tailored cell culture materials. By focusing on chemical functionalization, physical manipulation, and material properties, this review serves as a guide to select hydrogel materials to answer specific questions in cell biology.
Responsive polymer materials can adapt to surrounding environments, regulate transport of ions and molecules, change wettability and adhesion of different species on external stimuli, or convert ...chemical and biochemical signals into optical, electrical, thermal and mechanical signals, and vice versa. These materials are playing an increasingly important part in a diverse range of applications, such as drug delivery, diagnostics, tissue engineering and 'smart' optical systems, as well as biosensors, microelectromechanical systems, coatings and textiles. We review recent advances and challenges in the developments towards applications of stimuli-responsive polymeric materials that are self-assembled from nanostructured building blocks. We also provide a critical outline of emerging developments.
Surface initiated atom transfer radical polymerization (SI-ATRP) was triggered after diffusion of a CuI/L activator generated at a working electrode. A stable CuIIL/CuIL ratio gradient was formed at ...the gap between the working electrode and the initiator terminated substrate due to ion diffusion. The size of the gap can be used to dictate polymer growth kinetics at different gap distances. Gradient polymer brushes were grafted when substrate was placed at a tilting angle along CuIIL/CuIL gradient.
We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A ...microfluidic device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 106 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.
Polymer brushes show great promise in next-generation antibiofouling surfaces. Here, we have studied the influence of polymer brush architecture on protein resistance. By carefully optimizing ...reaction conditions, we were able to polymerize oligoglycerol-based brushes with sterically demanding linear or dendronized side chains on gold surfaces. Protein adsorption from serum and plasma was analyzed by surface plasmon resonance. Our findings reveal a pronounced dependence of biofouling on brush architecture. Bulky yet flexible side chains as in dendronized brushes provide an ideal environment to repel proteinpossibly through formation of a hydration layer, which can be further enhanced by presenting free hydroxyl groups on the polymer brushes. A deeper understanding of how brush architecture influences protein resistance will ultimately enable fabrication of surface coatings tailored to specific requirements in biomedical applications.
The compartmentalization of cell-free gene expression systems in liposomes provides an attractive route to the formation of protocells, but these models do not capture the physical (crowded) ...environment found in living systems. Here, we present a microfluidics-based route to produce monodisperse liposomes that can shrink almost 3 orders of magnitude without compromising their stability. We demonstrate that our strategy is compatible with cell-free gene expression and show increased protein production rates in crowded liposome protocells.
Living cells are able to produce a wide variety of biological responses when subjected to biochemical stimuli. It has become apparent that these biological responses are regulated by complex chemical ...reaction networks (CRNs). Unravelling the function of these circuits is a key topic of both systems biology and synthetic biology. Recent progress at the interface of chemistry and biology together with the realisation that current experimental tools are insufficient to quantitatively understand the molecular logic of pathways inside living cells has triggered renewed interest in the bottom-up development of CRNs. This builds upon earlier work of physical chemists who extensively studied inorganic CRNs and showed how a system of chemical reactions can give rise to complex spatiotemporal responses such as oscillations and pattern formation. Using purified biochemical components,
in vitro
synthetic biologists have started to engineer simplified model systems with the goal of mimicking biological responses of intracellular circuits. Emulation and reconstruction of system-level properties of intracellular networks using simplified circuits are able to reveal key design principles and molecular programs that underlie the biological function of interest. In this Tutorial Review, we present an accessible overview of this emerging field starting with key studies on inorganic CRNs followed by a discussion of recent work involving purified biochemical components. Finally, we review recent work showing the versatility of programmable biochemical reaction networks (BRNs) in analytical and diagnostic applications.
Living cells are able to produce a wide variety of biological responses when subjected to biochemical stimuli.
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