Cell Encapsulation in Soft,Anisometric Poly(ethylene) Glycol Microgels Using a Novel Radical‐Free Microfluidic System

Complex 3D artificial tissue constructs are extensively investigated for tissue regeneration. Frequently, materials and cells are delivered separately without benefitting from the synergistic effect of combined administration. Cell delivery inside a material construct provides the cells with a supportive environment by presenting biochemical, mechanical, and structural signals to direct cell behavior. Conversely, the cell/material interaction is poorly understood at the micron scale and new systems are required to investigate the effect of micron‐scale features on cell functionality. Consequently, cells are encapsulated in microgels to avoid diffusion limitations of nutrients and waste and facilitate analysis techniques of single or collective cells. However, up to now, the production of soft cell‐loaded microgels by microfluidics is limited to spherical microgels. Here, a novel method is presented to produce monodisperse, anisometric poly(ethylene) glycol microgels to study cells inside an anisometric architecture. These microgels can potentially direct cell growth and can be injected as rod‐shaped mini‐tissues that further assemble into organized macroscopic and macroporous structures post‐injection. Their aspect ratios are adjusted with flow parameters, while mechanical and biochemical properties are altered by modifying the precursors. Encapsulated primary fibroblasts are viable and spread and migrate across the 3D microgel structure.     

Microfluidic Templated Multicompartment Microgels for 3D Encapsulation and Pairing of Single Cells

Controlled encapsulation and pairing of single cells within a confined 3D matrix can enable the replication of the highly ordered cellular structure of human tissues. Microgels with independently controlled compartments that can encapsulate cells within separately confined hydrogel matrices would provide precise control over the route of pairing single cells. Here, a one‐step microfluidic method is presented to generate monodisperse multicompartment microgels that can be used as a 3D matrix to pair single cells in a highly biocompatible manner. A method is presented to induce microgels formation on chip, followed by direct extraction of the microgels from oil phase, thereby avoiding prolonged exposure of the microgels to the oil. It is further demonstrated that by entrapping stem cells with niche cells within separate but adjacent compartments of the microgels, it can create complex stem cell niche microenvironments in a controlled manner, which can serve as a useful tool for the study of cell–cell interactions. This microfluidic technique represents a significant step toward high‐throughput single cells encapsulation and pairing for the study of intercellular communications at single cell level, which is of significant importance for cell biology, stem cell therapy, and tissue engineering.     

A Microfluidic Strategy for Controllable Generation of Water‐in‐Water Droplets as Biocompatible Microcarriers

Droplet microfluidics has been widely applied in functional microparticles fabricating, tissue engineering, and drug screening due to its high throughput and great controllability. However, most of the current droplet microfluidics are dependent on water‐in‐oil (W/O) systems, which involve organic reagents, thus limiting their broader biological applications. In this work, a new microfluidic strategy is described for controllable and high‐throughput generation of monodispersed water‐in‐water (W/W) droplets. Solutions of polyethylene glycol and dextran are used as continuous and dispersed phases, respectively, without any organic reagents or surfactants. The size of W/W droplets can be precisely adjusted by changing the flow rate of dispersed and continuous phases and the valve switch cycle. In addition, uniform cell‐laden microgels are fabricated by introducing the alginate component and rat pancreatic islet (β‐TC6) cell suspension to the dispersed phase. The encapsulated islet cells retain high viability and the function of insulin secretion after cultivation for 7 days. The high‐throughput droplet microfluidic system with high biocompatibility is stable, controllable, and flexible, which can boost various chemical and biological applications, such as bio‐oriented microparticles synthesizing, microcarriers fabricating, tissue engineering, etc.     

Synthesis of monodisperse, covalently cross-linked, degradable "smart" microgels using microfluidics

The development of a robust method for the synthesis of highly monodisperse microgels cross-linked with degradable covalent bonds offers the potential for fabricating microgels with the highly controllable porosities, cell interactions, and degradation half-lives required for biomedical applications. A microfluidic chip is designed that enables the on-chip mixing and emulsification of two reactive polymer solutions (hydrazide and aldehyde-functionalized carbohydrates) to form monodisperse, hydrazone cross-linked microgels in the size range of ≈40-100 μm. The device can be run continuously for at least 30 h without a significant drift in particle size. The resulting microgels have a homogeneous bulk composition and can swell and deswell as the solvent conditions change in predictable ways based on the chemistry of the reactive polymers used, thereby enabling improved control over both the chemistry and morphology of the resulting microgels relative to other reported approaches. The in situ gelation chemistry used facilitates rapid microgel formation within the droplets without requiring the use of UV light or heating to initiate polymerization, thus making this approach of particular potential utility in cell encapsulation or drug delivery (as demonstrated).     

Centering Single Cells in Microgels via Delayed Crosslinking Supports Long‐Term 3D Culture by Preventing Cell Escape

Single‐cell‐laden microgels support physiological 3D culture conditions while enabling straightforward handling and high‐resolution readouts of individual cells. However, their widespread adoption for long‐term cultures is limited by cell escape. In this work, it is demonstrated that cell escape is predisposed to off‐center encapsulated cells. High‐speed microscopy reveals that cells are positioned at the microgel precursor droplets' oil/water interface within milliseconds after droplet formation. In conventional microencapsulation strategies, the droplets are typically gelled immediately after emulsification, which traps cells in this off‐center position. By delaying crosslinking, driving cells toward the centers of microgels is succeeded. The centering of cells in enzymatically crosslinked microgels prevents their escape during at least 28 d. It thereby uniquely enables the long‐term culture of individual cells within <5‐µm‐thick 3D uniform hydrogel coatings. Single cell analysis of mesenchymal stem cells in enzymatically crosslinked microgels reveals unprecedented high cell viability (>90%), maintained metabolic activity (>70%), and multilineage differentiation capacity (>60%) over a period of 28 d. The facile nature of this microfluidic cell‐centering method enables its straightforward integration into many microencapsulation strategies and significantly enhances control, reproducibility, and reliability of 3D single cell cultures.     

Microfluidic Designing Microgels Containing Highly Concentrated Gold Nanoparticles for SERS Analysis of Complex Fluids

Surface‐enhanced Raman scattering (SERS) is one of the most promising methods to detect small molecules for point‐of‐care analysis as it is rapid, nondestructive, label‐free, and applicable for aqueous samples. Here, microgels containing highly concentrated yet evenly dispersed gold nanoparticles are designed to provide SERS substrates that simultaneously achieve contamination‐free metal surfaces and high signal enhancement and reproducibility. With capillary microfluidic devices, water‐in‐oil‐in‐water (W/O/W) double‐emulsion drops are prepared to contain gold nanoparticles and hydrogel precursors in innermost drop. Under hypertonic condition, water is selectively pumped out from the innermost drops. Therefore, gold nanoparticles are gently concentrated without forming aggregates, which are then captured by hydrogel matrix. The resulting microgels have a concentration of gold nanoparticles ≈30 times higher and show Raman intensity two orders of magnitude higher than those with no enrichment. In addition, even distribution of gold nanoparticles results in uniform Raman intensity, providing high signal reproducibility. Moreover, as the matrix of the microgel serves as a molecular filter, large adhesive proteins are rejected, which enables the direct detection of small molecules dissolved in the protein solution. It is believed that this advanced SERS platform is useful for in situ detection of toxic molecules in complex mixtures such as biological fluids, foods, and cosmetics.     

Hot Brownian Motion of Thermoresponsive Microgels in Optical Tweezers Shows Discontinuous Volume Phase Transition and Bistability

Microgels are soft microparticles that often exhibit thermoresponsiveness and feature a transformation at a critical temperature, referred to as the volume phase transition temperature. Whether this transformation occurs as a smooth or as a discontinuous one is still a matter of debate. This question can be addressed by studying individual microgels trapped in optical tweezers. For this aim, composite particles are obtained by decorating  Poly-N-isopropylacrylamide (pNIPAM) microgels with iron oxide nanocubes. These composites become self-heating when illuminated by the infrared trapping laser, performing hot Brownian motion within the trap. Above a certain laser power, a single decorated microgel features a volume phase transition that is discontinuous, while the usual continuous sigmoidal-like dependence is recovered after averaging over different microgels. The collective sigmoidal behavior enables the application of a power-to-temperature calibration and provides the effective drag coefficient of the self-heating microgels, thus establishing these composite particles as potential micro-thermometers and micro-heaters. Moreover, the self-heating microgels also exhibit an unexpected and intriguing bistability behavior above the critical temperature, probably due to partial collapses of the microgel. These results set the stage for further studies and the development of applications based on the hot Brownian motion of soft particles.     

Interface‐Directed Self‐Assembly of Cell‐Laden Microgels

Cell‐laden hydrogels show great promise for creating engineered tissues. However, a major shortcoming with these systems has been the inability to fabricate structures with controlled micrometer‐scale features on a biologically relevant length scale. In this Full Paper, a rapid method is demonstrated for creating centimeter‐scale, cell‐laden hydrogels through the assembly of shape‐controlled microgels or a liquid–air interface. Cell‐laden microgels of specific shapes are randomly placed on the surface of a high‐density, hydrophobic solution, induced to aggregate and then crosslinked into macroscale tissue‐like structures. The resulting assemblies are cell‐laden hydrogel sheets consisting of tightly packed, ordered microgel units. In addition, a hierarchical approach creates complex multigel building blocks, which are then assembled into tissues with precise spatial control over the cell distribution. The results demonstrate that forces at an air–liquid interface can be used to self‐assemble spatially controllable, cocultured tissue‐like structures.     

Injectable Granular Hydrogels with Multifunctional Properties for Biomedical Applications

Injectable hydrogels are useful for numerous biomedical applications, such as to introduce therapeutics into tissues or for 3D printing. To expand the complexity of available injectable hydrogels, shear‐thinning and self‐healing granular hydrogels are developed from microgels that interact via guest–host chemistry. The microgel properties (e.g., degradation, molecule release) are tailored through their crosslinking chemistry, including degradation in response to proteases. When microgels of varied formulations are mixed, complex release and degradation behaviors are observed, including after injection to permit cellular invasion.     

Functional Microgels: Recent Advances in Their Biomedical Applications

Here, a spotlight is shown on aqueous microgel particles which exhibit a great potential for various biomedical applications such as drug delivery, cell imaging, and tissue engineering. Herein, different synthetic methods to develop microgels with desirable functionality and properties along with degradable strategies to ensure their renal clearance are briefly presented. A special focus is given on the ability of microgels to respond to various stimuli such as temperature, pH, redox potential, magnetic field, light, etc., which helps not only to adjust their physical and chemical properties, and degradability on demand, but also the release of encapsulated bioactive molecules and thus making them suitable for drug delivery. Furthermore, recent developments in using the functional microgels for cell imaging and tissue regeneration are reviewed. The results reviewed here encourage the development of a new class of microgels which are able to intelligently perform in a complex biological environment. Finally, various challenges and possibilities are discussed in order to achieve their successful clinical use in future.     

Microfluidic encapsulation of cells in polymer microgels

In this Concept article, recent advances in microfluidic platforms for the generation of cell-laden hydrogel particles (microgels) are reported. Advances in the continuous microfluidic encapsulation of cells in droplets and microgels are critically reviewed, and currently used methods for the encapsulation of cells in polymer microgels are discussed. An outlook on current applications and future directions in this field of research are also presented. This article will be of interest to chemists, materials scientists, cell biologists, bioengineers, and pharmacologists.     

Microfluidic Templating of Spatially Inhomogeneous Protein Microgels

3D scaffolds in the form of hydrogels and microgels have allowed for more native cell‐culture systems to be developed relative to flat substrates. Native biological tissues are, however, usually spatially inhomogeneous and anisotropic, but regulating the spatial density of hydrogels at the microscale to mimic this inhomogeneity has been challenging to achieve. Moreover, the development of biocompatible synthesis approaches for protein‐based microgels remains challenging, and typical gelation conditions include UV light, extreme pH, extreme temperature, or organic solvents, factors which can compromise the viability of cells. This study addresses these challenges by demonstrating an approach to fabricate protein microgels with controllable radial density through microfluidic mixing and physical and enzymatic crosslinking of gelatin precursor molecules. Microgels with a higher density in their cores and microgels with a higher density in their shells are demonstrated. The microgels have robust stability at 37 °C and different dissolution rates through enzymolysis, which can be further used for gradient scaffolds for 3D cell culture, enabling controlled degradability, and the release of biomolecules. The design principles of the microgels could also be exploited to generate other soft materials for applications ranging from novel protein‐only micro reactors to soft robots.     

Design of multicomponent microgels by selective deposition of nanomaterials

In the present paper a method for the targeted deposition of different nanomaterials on aqueous microgels is described. In the first stage poly(3,4-ethylenedioxythiophene) (PEDOT) nanorods are introduced into the microgel structure by in situ oxidative polymerization. In the second stage hydrogen tetrachloroaurate is used to transform PEDOT chains to an oxidized state in the microgel structure, leading to the fixation of chloroaurate anions on the surface of the PEDOT nanorods. The reduction of chloroaurate ions induces the formation of gold nanoparticles (AuNPs) predominantly located on the PEDOT surface. Obtained microgel/PEDOT/AuNP hybrid particles with different nanoparticle loadings exhibit superior colloidal stability and temperature sensitivity. The microgel/PEDOT/AuNP hybrid microgels exhibit extraordinary catalytic activity in aqueous media.     

Ultrathin Shell Double Emulsion Templated Giant Unilamellar Lipid Vesicles with Controlled Microdomain Formation

A microfluidic approach is reported for the high‐throughput, continuous production of giant unilamellar vesicles (GUVs) using water‐in‐oil‐in‐water double emulsion drops as templates. Importantly, these emulsion drops have ultrathin shells; this minimizes the amount of residual solvent that remains trapped within the GUV membrane, overcoming a major limitation of typical microfluidic approaches for GUV fabrication. This approach enables the formation of microdomains, characterized by different lipid compositions and structures within the GUV membranes. This work therefore demonstrates a straightforward and versatile approach to GUV fabrication with precise control over the GUV size, lipid composition and the formation of microdomains within the GUV membrane.     

Cell‐Instructive Microgels with Tailor‐Made Physicochemical Properties

A microfluidic in vitro cell encapsulation platform to systematically test the effects of microenvironmental parameters on cell fate in 3D is developed. Multiple cell types including fibroblasts, embryonic stem cells, and cancer cells are incorporated in enzymatically cross‐linked poly(ethylene glycol)‐based microgels having defined and tunable mechanical and biochemical properties. Furthermore, different approaches to prevent cell “escape” from the microcapsules are explored and shown to substantially enhance the potential of this technology. Finally, coencapsulation of microgels within nondegradable gels allows cell viability, proliferation, and morphology to be studied in different microenvironmental conditions up to two weeks in culture.     

Probing the morphology and nanoscale mechanics of single poly(N-isopropylacrylamide) microgels across the lower-critical-solution temperature by atomic force microscopy

Submicrometer-sized particles of poly(N-isopropylacrylamide) (PNIPAM) are synthesized by surfactant-free radical polymerization. The morphology and nanomechanical properties of individual, isolated PNIPAM microgel particles at the silicon/air and silicon/water interfaces, below and above the PNIPAM volume-phase-transition temperature (VPTT), are probed by atomic force microscopy. In air, and in water below the VPTT, the PNIPAM spheres are flattened and adopt a pancakelike shape. Interestingly, above the VPTT the microgels adopt a more spherical form with increased height and decreased width, which is attributed to reduced interactions of the particles with the substrate. The elastic modulus calculated from force-indentation curves obtained for individual microgel spheres reveals that the stiffness of the particle's surface decreases by two orders of magnitude upon swelling in water. Additionally, the modulus of the PNIPAM spheres in water increases by one order of magnitude when crossing the VPTT from the swollen to the collapsed states, indicating a more compact chain packing at the particle surface.     

Uniform Microgels Containing Agglomerates of Silver Nanocubes for Molecular Size‐Selectivity and High SERS Activity

Surface‐enhanced Raman scattering (SERS) is a promising technique for molecular analysis as the molecular fingerprints (Raman spectra) are amplified to detectable levels compared with common spectroscopy. Metal nanostructures localize electromagnetic field on their surfaces, which can lead to dramatic increase of Raman intensity of molecules adsorbed. However, the metal surfaces are prone to contamination, thereby requiring pretreatment of samples to remove adhesive molecules. To avoid the pretreatment and potentially achieve point‐of‐care (POC) analysis, we have developed SERS‐active microgels using the droplet‐microfluidic system. As the microgels are composed of water‐swollen network with consistent mesh size, they selectively allow diffusion of molecules smaller than the mesh, thereby excluding large adhesives. To render the microgels highly SERS‐active, we destabilize silver nanocubes to form agglomerates, which are embedded in the matrix of microgels. The nanogaps in the agglomerates provide high sensitivity in Raman measurement and size‐selective permeability of the microgel matrix obviates the pretreatment of samples. To validate the functions, we demonstrate the direct detection of Aspirin dissolved in whole blood without any pretreatment.     

Composite Microgels Created by Complexation between Polyvinyl Alcohol and Graphene Oxide in Compressed Double‐Emulsion Drops

Microgels, microparticles made of hydrogels, show fast diffusion kinetics and high reconfigurability while maintaining the advantages of hydrogels, being useful for various applications. Here, presented is a new microfluidic strategy for producing polymer‐graphene oxide (GO) composite microgels without chemical cues or a temperature swing for gelation. As a main component of microgels, polymers that are able to form hydrogen bonds, such as polyvinyl alcohol (PVA), are used. In the mixture of PVA and GO, GO is tethered by PVA through hydrogen bonding. When the mixture is rapidly concentrated in the core of double‐emulsion drops by osmotic‐pressure‐driven water pumping, PVA‐tethered GO sheets form a nematic phase with a planar alignment. In addition, the GO sheets are linked by additional hydrogen bonds, leading to a sol–gel transition. Therefore, the PVA–GO composite remains undissolved when it is directly exposed to water by oil‐shell rupture. These composite microgels can be also produced using poly(ethylene oxide) or poly(acrylic acid), instead of PVA. In addition, the microgels can be functionalized by incorporating other polymers in the presence of the hydrogel‐forming polymers. It is shown that the multicomponent microgels made from a mixture of polyacrylamide, PVA, and GO show an excellent adsorption capacity for impurities.     

High Throughput Screening of Cell Mechanical Response Using a Stretchable 3D Cellular Microarray Platform

Cells in vivo are constantly subjected to multiple microenvironmental mechanical stimuli that regulate cell function. Although 2D cell responses to the mechanical stimulation have been established, these methods lack relevance as physiological cell microenvironments are in 3D. Moreover, the existing platforms developed for studying the cell responses to mechanical cues in 3D either offer low‐throughput, involve complex fabrication, or do not allow combinatorial analysis of multiple cues. Considering this, a stretchable high‐throughput (HT) 3D cell microarray platform is presented that can apply dynamic mechanical strain to cells encapsulated in arrayed 3D microgels. The platform uses inkjet‐bioprinting technique for printing cell‐laden gelatin methacrylate (GelMA) microgel array on an elastic composite substrate that is periodically stretched. The developed platform is highly biocompatible and transfers the applied strain from the stretched substrate to the cells. The HT analysis is conducted to analyze cell mechano‐responses throughout the printed microgel array. Also, the combinatorial analysis of distinct cell behaviors is conducted for different GelMA microenvironmental stiffnesses in addition to the dynamic stretch. Considering its throughput and flexibility, the developed platform can readily be scaled up to introduce a wide range of microenvironmental cues and to screen the cell responses in a HT way.     

纳米纤维素凝胶微粒及其在Pickering泡沫中的应用

目的研究氧化纳米纤维素/乳酸链球菌素(TONCC/nisin)凝胶粒子的性质及其在环保抗菌泡沫中的应用。方法利用TONCC的表面羧基基团与nisin的表面阳离子的吸附耦合作用,制备TONCC/nisin水凝胶和微凝胶,以微凝胶作为稳定粒子,环氧大豆油丙烯酸酯(AESO)为油相,制备TONCC/nisin/AESO Pickering乳液,对水凝胶、微凝胶、乳液的稳定性进行研究;通过热固化乳液得到环保抗菌的泡沫材料,并对泡沫材料的结构和抗菌效果进行表征。结果水凝胶的结构随着在水中浸泡时长的增加而发生变化,宏观表现为坍塌变形,nisin逐渐析出,微凝胶随着静置时间的延长其粒径变化不大;以微凝胶作为界面稳定剂的AESO乳液的热稳定性较好,在90℃下加热30 min乳液液滴并未发生聚并现象,该乳液固化后形成的多孔泡沫材料对李斯特菌的抑制作用明显,当泡沫中nisin含量为2μg/g时,其抑菌率为43%。结论TONCC和nisin形成的微凝胶粒子在水中稳定性较好,可以用于乳化AESO制备Pickering泡沫,同时赋予泡沫多孔性和抗菌性,在制备环保抗菌泡沫方面有很大的应用潜力。