Deterministic scRNA-seq captures variation in intestinal crypt and organoid composition.

Single-cell RNA sequencing (scRNA-seq) approaches have transformed our ability to resolve cellular properties across systems, but are currently tailored toward large cell inputs (>1,000 cells). This renders them inefficient and costly when processing small, individual tissue samples, a problem that tends to be resolved by loading bulk samples, yielding confounded mosaic cell population read-outs. Here, we developed a deterministic, mRNA-capture bead and cell co-encapsulation dropleting system, DisCo, aimed at processing low-input samples (<500 cells). We demonstrate that DisCo enables precise particle and cell positioning and droplet sorting control through combined machine-vision and multilayer microfluidics, enabling continuous processing of low-input single-cell suspensions at high capture efficiency (>70%) and at speeds up to 350 cells per hour. To underscore DisCo's unique capabilities, we analyzed 31 individual intestinal organoids at varying developmental stages. This revealed extensive organoid heterogeneity, identifying distinct subtypes including a regenerative fetal-like Ly6a+ stem cell population that persists as symmetrical cysts, or spheroids, even under differentiation conditions, and an uncharacterized 'gobloid' subtype consisting predominantly of precursor and mature (Muc2+) goblet cells. To complement this dataset and to demonstrate DisCo's capacity to process low-input, in vivo-derived tissues, we also analyzed individual mouse intestinal crypts. This revealed the existence of crypts with a compositional similarity to spheroids, which consisted predominantly of regenerative stem cells, suggesting the existence of regenerating crypts in the homeostatic intestine. These findings demonstrate the unique power of DisCo in providing high-resolution snapshots of cellular heterogeneity in small, individual tissues.

Localized Ionic Reinforcement of Double Network Granular Hydrogels.

Nature produces soft materials with fascinating combinations of mechanical properties. For example, the mussel byssus embodies a combination of stiffness and toughness, a feature that is unmatched by synthetic hydrogels. Key to enabling these excellent mechanical properties are the well-defined structures of natural materials and their compositions controlled on lengths scales down to tens of nanometers. The composition of synthetic materials can be controlled on a micrometer length scale if processed into densely packed microgels. However, these microgels are typically soft. Microgels can be stiffened by enhancing interactions between particles, for example through the formation of covalent bonds between their surfaces or a second interpenetrating hydrogel network. Nonetheless, changes in the composition of these synthetic materials occur on a micrometer length scale. Here, 3D printable load-bearing granular hydrogels are introduced whose composition changes on the tens of nanometer length scale. The hydrogels are composed of jammed microgels encompassing tens of nm-sized ionically reinforced domains that increase the stiffness of double network granular hydrogels up to 18-fold. The printability of the ink and the local reinforcement of the resulting granular hydrogels are leveraged to 3D print a butterfly with composition and structural changes on a tens of nanometer length scale.

Integrated Conversion of Lignocellulosic Biomass to Bio-Based Amphiphiles using a Functionalization-Defunctionalization Approach.

Concerns over the sustainability and end-of-life properties of fossil-derived surfactants have driven interest in bio-based alternatives. Lignocellulosic biomass with its polar functional groups is an obvious feedstock for surfactant production but its use is limited by process complexity and low yield. Here, we present a simple two-step approach to prepare bio-based amphiphiles directly from hemicellulose and lignin at high yields (29 % w/w based on the total raw biomass and >80 % w/w of these two fractions). Acetal functionalization of xylan and lignin with fatty aldehydes during fractionation introduced hydrophobic segments and subsequent defunctionalization by hydrogenolysis of the xylose derivatives or acidic hydrolysis of the lignin derivatives produced amphiphiles. The resulting biodegradable xylose acetals and/or ethers, and lignin-based amphiphilic polymers both largely retained their original natural structures, but exhibited competitive or superior surface activity in water/oil systems compared to common bio-based surfactants.

Influence of Sphericity on Surface Termination of Icosahedral Colloidal Clusters.

Colloids self-organize into icosahedral clusters composed of a Mackay core and an anti-Mackay shell under spherical confinement to minimize the free energy. This study explores the variation of surface arrangements of colloids in icosahedral clusters, focusing on the determining factors behind the surface arrangement. To efficiently assemble particles in emulsion droplets, droplet-to-droplet osmotic extraction from particle-laden droplets to salt-containing droplets is used, where the droplets are microfluidically prepared to guarantee a high size uniformity. The icosahedral clusters are optimally produced during a 24-h consolidation period at a 0.04 m salt concentration. The findings reveal an increase in the number of particle layers from 10 to 15 in the icosahedral clusters as the average number of particles increases from 3300 to 11 000. Intriguingly, the number of layers in the anti-Mackay shells, or surface termination, appears to more strongly depend on the sphericity of the clusters than on the deviation in the particle count from an ideal icosahedral cluster. This result suggests that the sphericity of the outermost layer, formed by the late-stage rearrangement of particles to form an anti-Mackay shell near the droplet interface, may play a pivotal role in determining the surface morphology to accommodate a spherical interface.

Influence of the Degree of Swelling on the Stiffness and Toughness of Microgel-Reinforced Hydrogels.

The stiffness and toughness of conventional hydrogels decrease with increasing degree of swelling. This behavior makes the stiffness-toughness compromise inherent to hydrogels even more limiting for fully swollen ones, especially for load-bearing applications. The stiffness-toughness compromise of hydrogels can be addressed by reinforcing them with hydrogel microparticles, microgels, which introduce the double network (DN) toughening effect into hydrogels. However, to what extent this toughening effect is maintained in fully swollen microgel-reinforced hydrogels (MRHs) is unknown. Herein, it is demonstrated that the initial volume fraction of microgels contained in MRHs determines their connectivity, which is closely yet nonlinearly related to the stiffness of fully swollen MRHs. Remarkably, if MRHs are reinforced with a high volume fraction of microgels, they stiffen upon swelling. By contrast, the fracture toughness linearly increases with the effective volume fraction of microgels present in the MRHs regardless of their degree of swelling. These findings provide a universal design rule for the fabrication of tough granular hydrogels that stiffen upon swelling and hence, open up new fields of use of these hydrogels.

Recycling of Load-Bearing 3D Printable Double Network Granular Hydrogels.

Sustainable materials, such as recyclable polymers, become increasingly important as they are often environmentally friendlier than their one-time-use counterparts. In parallel, the trend toward more customized products demands for fast prototyping methods which allow processing materials into 3D objects that are often only used for a limited amount of time yet, that must be mechanically sufficiently robust to bear significant loads. Soft materials that satisfy the two rather contradictory needs remain to be shown. Here, the authors introduce a material that simultaneously fulfills both requirements, a 3D printable, recyclable double network granular hydrogel (rDNGH). This hydrogel is composed of poly(2-acrylamido-2-methylpropane sulfonic acid) microparticles that are covalently crosslinked through a disulfide-based percolating network. The possibility to independently degrade the percolating network, with no harm to the primary network contained within the microgels, renders the recovery of the microgels efficient. As a result, the recycled material pertains a stiffness and toughness that are similar to those of the pristine material. Importantly, this process can be extended to the fabrication of recyclable hard plastics made of, for example, dried rDNGHs. The authors envision this approach to serve as foundation for a paradigm shift in the design of new sustainable soft materials and plastics.

Hydroelastomers: soft, tough, highly swelling composites.

Inspired by the cellular design of plant tissue, we present an approach to make versatile, tough, highly water-swelling composites. We embed highly swelling hydrogel particles inside tough, water-permeable, elastomeric matrices. The resulting composites, which we call hydroelastomers, combine the properties of their parent phases. From their hydrogel component, the composites inherit the ability to highly swell in water. From the elastomeric component, the composites inherit excellent stretchability and fracture toughness, while showing little softening as they swell. Indeed, the fracture properties of the composite match those of the best-performing, tough hydrogels, exhibiting fracture energies of up to 10 kJ m-2. Our composites are straightforward to fabricate, based on widely-available materials, and can easily be molded or extruded to form shapes with complex swelling geometries. Furthermore, there is a large design space available for making hydroelastomers, since one can use any hydrogel as the dispersed phase in the composite, including hydrogels with stimuli-responsiveness. These features make hydroelastomers excellent candidates for use in soft robotics and swelling-based actuation, or as shape-morphing materials, while also being useful as hydrogel replacements in other fields.

Does the Size of Microgels Influence the Toughness of Microgel-Reinforced Hydrogels?

Rapid advances in the biomedical field increasingly often demand soft materials that can be processed into complex 3D shapes while being able to reliably bear significant loads. Granular hydrogels have the potential to serve as artificial tissues because they can be 3D printed into complex shapes and their composition can be tuned over short length scales. Unfortunately, granular hydrogels are typically soft such that they cannot be used for load-bearing applications. To address this shortcoming, individual microgels can be connected through a percolating network, such that they introduce the double network toughening mechanism into granular hydrogels. However, the influence of the microgel size and concentration on the processing and toughness of microgel-reinforced hydrogels (MRHs) remains to be elucidated. Here, it is demonstrated that processing and toughness depend on the inter-microgel connectivity, while the stress at break is solely dependent on the microgel size. These findings offer an in-depth understanding of how liquid- and paste-like precursors containing soft, deformable microgels can be processed into bulk microstructured soft materials and how the size and concentration of these microgels influence the mechanical properties of microgel-reinforced hydrogels.

Temperature-induced liquid crystal microdroplet formation in a partially miscible liquid mixture.

Liquid-in-liquid droplets are typically generated by the partitioning of immiscible fluids, e.g. by mechanical shearing with macroscopic homogenisers or microfluidic flow focussing. In contrast, partially miscible liquids with a critical solution temperature display a temperature-dependent mixing behaviour. In this work, we demonstrate how, for a blend of methanol (MeOH) and the thermotropic liquid crystal (LC) 4-Cyano-4'-pentylbiphenyl (5CB), cooling from a miscible to an immiscible state allows the controlled formation of microdroplets. A near-room-temperature-induced phase separation leads to nucleation, growth and coalescence of mesogen-rich droplets. The size and number of the droplets is tunable on the microscopic scale by variation of temperature quench depth and cooling rate. Further cooling induces a phase transition to nematic droplets with radial configuration, well-defined sizes and stability over the course of an hour. This temperature-induced approach offers a scalable and reversible alternative to droplet formation with relevance in diagnostics, optoelectronics, materials templating and extraction processes.

Linear triglycerol-based fluorosurfactants show high potential for droplet-microfluidics-based biochemical assays.

Fluorosurfactants have expanded the landscape of high-value biochemical assays in microfluidic droplets, but little is known about how the spatial geometries and polarity of the head group contribute to the performance of fluorosurfactants. To decouple this, we design, synthesize, and characterize two linear and two dendritic glycerol- or tris-based surfactants with a common perfluoropolyether tail. To reveal the influence of spatial geometry, we choose inter-droplet cargo transport as a stringent test case. Using surfactants with linear di- and triglycerol, we show that the inter-droplet cargo transport is minimal compared with their dendritic counterparts. When we encapsulated a less-leaky sodium fluorescent dye into the droplets, quantitatively, we find that the mean fluorescence intensity of the PFPE-dTG stabilized PBS-only droplets after 72 h was ∼3 times that of the signal detected in PBS-only droplets stabilized by PFPE-lTG. We also demonstrate that the post-functionalization of PFPE-lTG having a linear geometry and four hydroxy groups enables the 'from-Droplet' fishing of the biotin-streptavidin protein complex without the trade-off between fishing efficiency and droplet stability. Thus, our approach to design user-friendly surfactants reveals the aspects of spatial geometry and facile tunability of the polar head groups that have not been captured or exploited before.

From vesicles to materials: bioinspired strategies for fabricating hierarchically structured soft matter.

Certain organisms including species of mollusks, polychaetes, onychophorans and arthropods produce exceptional polymeric materials outside their bodies under ambient conditions using concentrated fluid protein precursors. While much is understood about the structure-function relationships that define the properties of such materials, comparatively less is understood about how such materials are fabricated and specifically, how their defining hierarchical structures are achieved via bottom-up assembly. Yet this information holds great potential for inspiring sustainable manufacture of advanced polymeric materials with controlled multi-scale structure. In the present perspective, we first examine recent work elucidating the formation of the tough adhesive fibres of the mussel byssus via secretion of vesicles filled with condensed liquid protein phases (coacervates and liquid crystals)-highlighting which design principles are relevant for bio-inspiration. In the second part of the perspective, we examine the potential of recent advances in drops and additive manufacturing as a bioinspired platform for mimicking such processes to produce hierarchically structured materials. This article is part of the theme issue 'Bio-derived and bioinspired sustainable advanced materials for emerging technologies (part 1)'.

Shear Stress-Responsive Polymersome Nanoreactors Inspired by the Marine Bioluminescence of Dinoflagellates.

Some marine plankton called dinoflagellates emit light in response to the movement of surrounding water, resulting in a phenomenon called milky seas or sea sparkle. The underlying concept, a shear-stress induced permeabilisation of biocatalytic reaction compartments, is transferred to polymer-based nanoreactors. Amphiphilic block copolymers that carry nucleobases in their hydrophobic block are self-assembled into polymersomes. The membrane of the vesicles can be transiently switched between an impermeable and a semipermeable state by shear forces occurring in flow or during turbulent mixing of polymersome dispersions. Nucleobase pairs in the hydrophobic leaflet separate when mechanical force is applied, exposing their hydrogen bonding motifs and therefore making the membrane less hydrophobic and more permeable for water soluble compounds. This polarity switch is used to release payload of the polymersomes on demand, and to activate biocatalytic reactions in the interior of the polymersomes.

Reversible Oxygen Sensing Based on Multi-Emission Fluorescence Quenching.

Oxygen is ubiquitous in nature and it plays a key role in several biological processes, such as cellular respiration and food deterioration, to name a few. Currently, reversible and non-destructive oxygen sensing is usually performed with sensors produced by photosensitization of phosphorescent organometallic complexes. In contrast, we propose a novel route of optical oxygen sensing by fluorescence-based quenching of oxygen. We hereby developed for the first time a set of multi-emissive purely organic emitters. These were produced through a one-pot hydrothermal synthesis using p-phenylenediamine (PPD) and urea as starting materials. The origin of the multi-emission has been ascribed to the diversity of chemical structures produced as a result of oxidative oligomerization of PPD. A Bandrowski's base (BB, i.e., trimer of PPD) is reported as the main component at reaction times higher than 8 h. This indication was confirmed by electrospray-ionization quadrupole time-of-flight (ESI-QTOF) and liquid chromatography-mass spectrometry (LC-MS) analysis. Once the emitters are embedded within a high molecular weight poly (vinyl alcohol) matrix, the intensities of all three emission centers exhibit a non-linear quenching provoked by oxygen within the range of 0-8 kPa. The detection limit of the emission centers are 0.89 kPa, 0.67 kPa and 0.75 kPa, respectively. This oxygen-dependent change in fluorescence emission is reversible (up to three tested 0-21% O2 cycles) and reproducible with negligible cross-interference to humidity. The cost-effectiveness, metal-free formulation, cross-referencing between each single emission center and the relevant oxygen range are all appealing features, making these sensors promising for the detection of oxygen, e.g., in food packaged products.

Water: How Does It Influence the CaCO3 Formation?

Nature produces biomineral-based materials with a fascinating set of properties using only a limited number of elements. This set of properties is obtained by closely controlling the structure and local composition of the biominerals. We are far from achieving the same degree of control over the properties of synthetic biomineral-based composites. One reason for this inferior control is our incomplete understanding of the influence of the synthesis conditions and additives on the structure and composition of the forming biominerals. In this Review, we provide an overview of the current understanding of the influence of synthesis conditions and additives during different formation stages of CaCO3 , one of the most abundant biominerals, on the structure, composition, and properties of the resulting CaCO3 crystals. In addition, we summarize currently known means to tune these parameters. Throughout the Review, we put special emphasis on the role of water in mediating the formation of CaCO3 and thereby influencing its structure and properties, an often overlooked aspect that is of high relevance.

Selectively Permeable Double Emulsions.

Natural organisms are made of different types of microcompartments, many of which are enclosed by cell membranes. For these organisms to display a proper function, the microcompartments must be selectively permeable. For example, cell membranes are typically permeable toward small, uncharged molecules such as water, selected nutrients, and cell signaling molecules, but impermeable toward many larger biomolecules. Here, it is reported for the first time dynamic compartments, namely surfactant-stabilized double emulsions, that display selective and tunable permeability. Selective permeability is imparted to double emulsions by stabilizing them with catechol-functionalized surfactants that transport molecules across the oil shell of double emulsions only if they electrostatically or hydrophobically attract encapsulants. These double emulsions are employed as semipermeable picoliter-sized vessels to controllably perform complexation reactions inside picoliter-sized aqueous cores. This thus far unmet level of control over the transport of reagents across oil phases opens up new possibilities to use double emulsion drops as dynamic and selectively permeable microcompartments to initiate and maintain chemical and biochemical reactions in picoliter-sized cell-mimetic compartments.

Black Lipid Membranes: Challenges in Simultaneous Quantitative Characterization by Electrophysiology and Fluorescence Microscopy.

Horizontal black lipid membranes (BLMs) enable optical microscopy to be combined with the electrophysiological measurements for studying ion channels, peptide pores, and ionophores. However, a careful literature review reveals that simultaneous fluorescence and electrical recordings in horizontal BLMs have been rarely reported for an unclear reason, whereas many works employ bright-field microscopy instead of fluorescence microscopy or perform fluorescence imaging and electrical measurements one after another separately without truly exploiting the advantage of the combined setup. In this work, the major causes related to the simultaneous electrical and fluorescence recordings in horizontal BLMs are identified, and several solutions to counteract the issue are also proposed.

Microfluidic fabrication of vesicles with hybrid lipid/nanoparticle bilayer membranes.

Hybrid lipid/nanoparticle membranes are suitable model systems both to study the complex interactions between nanoparticles and biological membranes, and to demonstrate technological concepts in cellular sensing and drug delivery. Unfortunately, embedding nanoparticles into the bilayer membrane of lipid vesicles is challenging due to the poor control over the vesicle fabrication process of conventional methodologies and the fragility of the modified lipid bilayer assembly. Here, the utility of water-in-oil-in-water double emulsion drops with ultrathin oil shells as templates to form vesicles with hybrid lipid/nanoparticle membranes is reported. Moreover, upon bilayer formation, which occurs through dewetting of the oil solvent from the double emulsion drops, a phase separation is observed in the vesicle membrane, with solid-like nanoparticle-rich microdomains segregated into a continuous fluid-like nanoparticle-poor phase. This phase coexistence evidences the complex nature of the interactions between nanoparticles and lipid membranes. In this context, this microfluidic-assisted fabrication strategy may play a crucial role in thoroughly understanding such interactions given the uniform membrane properties of the resulting productions. Furthermore, the high encapsulation efficiency of both the vesicle membrane and core endows these vesicles with great potential for sensing applications and drug delivery.

The interfacial structure of nano- and micron-sized oil and water droplets stabilized with SDS and Span80.

In this work, we provide a comparison between the stability and the interfacial structure of micrometer-sized and nanometer-sized droplets by employing a multi-instrumental approach comprised of the surface-sensitive technique of sum frequency scattering as well as dynamic light scattering and microscopy. We monitor the stability of oil-in-water and water-in-oil emulsions and the structure of surfactants at the oil/water nano-interface, when stabilized with an oil-soluble neutral surfactant (Span80), a water-soluble anionic surfactant (sodium dodecyl sulfate, SDS), or with a combination of the two. Micron-sized droplets are found to be stabilized only when a surfactant soluble in the continuous phase is present in the system, in agreement with what is traditionally observed empirically. Surprisingly, the nanodroplets behave differently. Both oil and water nanodroplets can be stabilized by the same (neutral Span80) surfactant but with different surface structures. A combination of SDS and Span80 also suffices, but for the case of water droplets, the strongly amphiphilic SDS molecules are not detected at the interface. For the case of oil droplets, both surfactants are at the interface but do not structurally affect one another. Thus, it appears that, in this study, empirical rules such as the Bancroft rule, the hydrophile-lipophile-balance scale, and the surfactant affinity difference predict the stability of the micrometer-sized droplets better than the nanometer-sized ones, probably due to a different balance of interactions on different length scales.

Nacre-inspired Hard and Tough Materials.

Nature fabricates materials with properties that are difficult to reproduce with manmade counterparts. For example, nacre, composed of layers of CaCO3 crystals that are interspaced with small quantities of organic components, is one of the toughest known biomaterials. To produce materials with such fascinating proper- ties, nature has established processes that offer an excellent control over their structure and local composition. Inspired by nacre, a lot of work has been devoted to the fabrication and characterization of composites with similar structures that nevertheless display distinctly different mechanical properties. The first part of this review summarizes methods used to produce nacre-inspired layered composites, their influence on the composition, structure, and mechanical properties. A key difference between the formation of nacre and that of nacre-inspired materials is the mechanism and kinetics of the formation of the inorganic components. In an endeavor to gain a better control over the mechanical properties of the inorganic platelets contained in nacre-inspired composites, the second part of this review describes methods to control the shape, structure, and orientation of CaCO3 formed in organic scaffolds.

Amorphous CaCO3: Influence of the Formation Time on Its Degree of Hydration and Stability.

Calcium carbonate (CaCO3) is one of the most abundant biominerals that is prevalent in rocks and often used as a structural material in marine animals. Many of these natural CaCO3-based materials display excellent mechanical properties that are difficult to reproduce by man-made counterparts. This difficulty arises from the incomplete understanding of the influence of processing conditions on the structure and composition of CaCO3. To gain a better understanding of the evolution of the structure and composition of amorphous CaCO3 (ACC) particles during early stages, we introduce a new, organic solvent-free method that quenches this process with a high temporal resolution. We produce ACC particles inside small airborne drops that are formed with a microfluidic spray-dryer. These drops dry within 100 ms to 10 s and thereby arrest the formation of CaCO3 particles on that time scale. Using the microfluidic spray-dryer, we demonstrate that the amount of mobile water contained in ACC particles increases with increasing formation time and hence with increasing particle size. As a result of the higher concentration of mobile water, larger particles are less stable against temperature-induced solid-state crystallization and electron beam-induced decomposition than smaller counterparts. The amount of mobile water contained in ACC can be substantially reduced, and hence their kinetic stability against solid-state transformations increased, if certain organic additives, such as poly(acrylic acid) (PAA), are incorporated. These insights might open up new opportunities to fabricate biomimetic CaCO3-based materials with tunable structures and hence with properties that can be adapted to the needs of specific applications.