Granular Biomaterials as Bioactive Sponges for the Sequestration and Release of Signaling Molecules.

A major challenge for the regeneration of chronic wounds is an underlying dysregulation of signaling molecules, including inflammatory cytokines and growth factors. To address this, we propose to use granular biomaterials composed of jammed microgels, to enable the rapid uptake and delivery of biomolecules, and provide a strategy to locally sequester and release biomolecules. Sequestration assays on model biomolecules of different sizes demonstrated that granular hydrogels exhibit faster transport than comparable bulk hydrogels due to enhanced surface area and decreased diffusion lengths. To demonstrate the potential of modular granular hydrogels to modulate local biomolecule concentrations, we engineered microgel scaffolds that can simultaneously sequester excess pro-inflammatory factors and release pro-healing factors. To target specific biomolecules, microgels were functionalized with affinity ligands that bind either to interleukin 6 (IL-6) or to vascular endothelial growth factor A (VEGF-A). Finally, disparate microgels were combined into a single granular biomaterial for simultaneous sequestration of IL-6 and release of VEGF-A. Overall, we demonstrate the potential of modular granular hydrogels to locally tailor the relative concentrations of pro- and anti-inflammatory factors. This article is protected by copyright. All rights reserved.

Deterministic Single-Cell Encapsulation in PEG Norbornene Microgels for Promoting Anti-Inflammatory Response and Therapeutic Delivery of Mesenchymal Stromal Cells.

Tissue engineering at single-cell resolution has enhanced therapeutic efficacy. Droplet microfluidics offers a powerful platform that allows deterministic single-cell encapsulation into aqueous droplets, yet the direct encapsulation of cells into microgels remains challenging. Here, the design of a microfluidic device that is capable of single-cell encapsulation within polyethylene glycol norbornene (PEGNB) hydrogels on-chip is reported. Cells are first ordered in media within a straight microchannel via inertial focusing, followed by the introduction of PEGNB solution from two separate, converging channels. Droplets are thoroughly mixed by passage through a serpentine channel, and microgels are formed by photo-photopolymerization. This platform uniquely enables both single-cell encapsulation and excellent cell viability post-photo-polymerization. More than 90% of singly encapsulated mesenchymal stromal cells (MSCs) remain alive for 7 days. Notably, singly encapsulated MSCs have elevated expression levels in genes that code anti-inflammatory cytokines, for example, IL-10 and TGF-ß, thus enhancing the secretion of proteins of interest. Following injection into a mouse model with induced inflammation, singly encapsulated MSCs show a strong retention rate in vivo, reduce overall inflammation, and mitigate liver damage. These translational results indicate that deterministic single-cell encapsulation could find use in a broad spectrum of tissue engineering applications.

Injectable liposome-containing click hydrogel microparticles for release of macromolecular cargos.

Hydrogel microparticles ranging from 0.1-100 µm, referred to as microgels, are attractive for biological applications afforded by their injectability and modularity, which allows facile delivery of mixed populations for tailored combinations of therapeutics. Significant efforts have been made to broaden methods for microgel production including via the materials and chemistries by which they are made. Via droplet-based-microfluidics we have established a method for producing click poly-(ethylene)-glycol (PEG)-based microgels with or without chemically crosslinked liposomes (lipo-microgels) through the Michael-type addition reaction between thiol and either vinyl-sulfone or maleimide groups. Unifom spherical microgels and lipo-microgels were generated with sizes of 74 ± 16 µm and 82 ± 25 µm, respectively, suggesting injectability that was further supported by rheological analyses. Super-resolution confocal microscopy was used to further verify the presence of liposomes within the lipo-microgels and determine their distribution. Atomic force microscopy (AFM) was conducted to compare the mechanical properties and network architecture of bulk hydrogels, microgels, and lipo-microgels. Further, encapsulation and release of model cargo (FITC-Dextran 5 kDa) and protein (equine myoglobin) showed sustained release for up to 3 weeks and retention of protein composition and secondary structure, indicating their ability to both protect and release cargos of interest.

One Step Encapsulation of Mesenchymal Stromal Cells in PEG Norbornene Microgels for Therapeutic Actions.

Cell therapies require control over the cellular response under standardized conditions to ensure continuous delivery of therapeutic agents. Cell encapsulation in biomaterials can be particularly effective at providing cells with a uniformly supportive and permissive cell microenvironment. In this study, two microfluidic droplet device designs were used to successfully encapsulate equine mesenchymal stromal cells (MSCs) into photopolymerized polyethylene glycol norbornene (PEGNB) microscale (∼100-200 µm) hydrogel particles (microgels) in a single on-chip step. To overcome the slow cross-linking kinetics of thiol-ene reactions, long dithiol linkers were used in combination with a polymerization chamber customized to achieve precise retention time for microgels while maintaining cytocompatibility. Thus, homogeneous cell-laden microgels could be continuously fabricated in a high-throughput fashion. Varying linker length mediated both the gel formation rate and material physical properties (stiffness, mass transport, and mesh size) of fabricated microgels. Postencapsulation cell viability and therapeutic indicators of MSCs were evaluated over 14 days, during which the viability remained at least 90%. Gene expression of selected cytokines was not adversely affected by microencapsulation compared to monolayer MSCs. Notably, PEGNB-3.5k microgels rendered significant elevation in FGF-2 and TGF-ß on the transcription level, and conditioned media collected from these cultures showed robust promotion in the migration and proliferation of fibroblasts. Collectively, standardized MSC on-chip encapsulation will lead to informed and precise translation to clinical studies, ultimately advancing a variety of tissue engineering and regenerative medicine practices.

Dual carbon sequestration with photosynthetic living materials.

Natural ecosystems offer efficient pathways for carbon sequestration, serving as a resilient approach to remove CO2 from the atmosphere with minimal environmental impact. However, the control of living systems outside of their native environments is often challenging. Here, we engineered a photosynthetic living material for dual CO2 sequestration by immobilizing photosynthetic microorganisms within a printable polymeric network. The carbon concentrating mechanism of the cyanobacteria enabled accumulation of CO2 within the cell, resulting in biomass production. Additionally, the metabolic production of OH- ions in the surrounding medium created an environment for the formation of insoluble carbonates via microbially-induced calcium carbonate precipitation (MICP). Digital design and fabrication of the living material ensured sufficient access to light and nutrient transport of the encapsulated cyanobacteria, which were essential for long-term viability (more than one year) as well as efficient photosynthesis and carbon sequestration. The photosynthetic living materials sequestered approximately 2.5 mg of CO2 per gram of hydrogel material over 30 days via dual carbon sequestration, with 2.2 ± 0.9 mg stored as insoluble carbonates. Over an extended incubation period of 400 days, the living materials sequestered 26 ± 7 mg of CO2 per gram of hydrogel material in the form of stable minerals. These findings highlight the potential of photosynthetic living materials for scalable carbon sequestration, carbon-neutral infrastructure, and green building materials. The simplicity of maintenance, coupled with its scalability nature, suggests broad applications of photosynthetic living materials as a complementary strategy to mitigate CO2 emissions.

Optimizing Immunofunctionalization and Cell Capture on Micromolded Hydrogels via Controlled Oxygen-Inhibited Photopolymerization.

With circulating tumor cells (CTCs) playing a critical role in cancer metastasis, the quantitation and characterization of CTCs promise to provide precise diagnostic and prognostic information in service of personalized therapies. However, as CTCs are extremely rare, high yield, high purity strategies are required to target and isolate CTCs from patient samples. Recently, we demonstrated the selective capture of CTCs upon antibody-functionalized polyethylene glycol diacrylate (PEGDA) hydrogels photopolymerized within polydimethylsiloxane (PDMS) microfluidic molds. Isolated CTC purity was subsequently enriched by selectively releasing desired cells from photodegradable hydrogel capture surfaces. However, the fabrication of these acrylate-based hydrogels by photopolymerization is subject to oxygen inhibition, which dramatically affects the physical and chemical properties of hydrogel interfaces formed in proximity to PDMS boundaries. To evaluate how antibody conjugation density and cell capture is impacted by fabrication parameters affected by oxygen inhibition, PEGDA hydrogel features were polymerized within PDMS micromolds under different UV exposure conditions and linker (acrylate-PEG-biotin) concentrations. Predictions of acrylate conversion throughout the hydrogel feature were performed using a 1D reaction-diffusion model that describes oxygen-inhibited photopolymerization. The functional consequences of photopolymerization parameters and solution stoichiometry on CTC capture were experimentally quantified and evaluated. Results show that hydrogel surfaces polymerized under shorter exposure times and with higher linker concentrations display superior functionalization and higher CTC capture efficiency. Conversely, highly cross-linked hydrogel surfaces polymerized under longer exposure times are insensitive to functionalization and display poor capture, regardless of linker concentration. By highlighting the importance of oxygen-inhibited photopolymerization, these findings provide guidelines to design micromolded hydrogels with controlled ligand expression. In addition to enhancing the selective cell capture capacity of immunofunctional hydrogels, the ability to quantifiably design hydrogel interfaces described here will improve the sensitivity of hydrogel biosensors, provide a platform to finely screen cell-matrix interactions, and generally enhance the fidelity of micromolded hydrogel features.

Intracellular connections between basal bodies promote the coordinated behavior of motile cilia.

Hydrodynamic flow produced by multiciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cell's cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by focused ion beam scanning electron microscopy imaging that cilia are coupled both longitudinally and laterally in the ciliate Tetrahymena thermophila by the underlying BB and cortical cytoskeletal network. To visualize the behavior of individual cilia in live, immobilized Tetrahymena cells, we developed Delivered Iron Particle Ubiety Live Light (DIPULL) microscopy. Quantitative and computer analyses of ciliary dynamics reveal that BB connections control ciliary waveform and coordinate ciliary beating. Loss of BB connections reduces cilia-dependent fluid flow forces.

Microgels Formed by Spontaneous Click Chemistries Utilizing Microfluidic Flow Focusing for Cargo Release in Response to Endogenous or Exogenous Stimuli.

Protein therapeutics have become increasingly popular for the treatment of a variety of diseases owing to their specificity to targets of interest. However, challenges associated with them have limited their use for a range of ailments, including the limited options available for local controlled delivery. To address this challenge, degradable hydrogel microparticles, or microgels, loaded with model biocargoes were created with tunable release profiles or triggered burst release using chemistries responsive to endogenous or exogeneous stimuli, respectively. Specifically, microfluidic flow-focusing was utilized to form homogenous microgels with different spontaneous click chemistries that afforded degradation either in response to redox environments for sustained cargo release or light for on-demand cargo release. The resulting microgels were an appropriate size to remain localized within tissues upon injection and were easily passed through a needle relevant for injection, providing means for localized delivery. Release of a model biopolymer was observed over the course of several weeks for redox-responsive formulations or triggered for immediate release from the light-responsive formulation. Overall, we demonstrate the ability of microgels to be formulated with different materials chemistries to achieve various therapeutic release modalities, providing new tools for creation of more complex protein release profiles to improve therapeutic regimens.

Slip slidin' away: Bristle-driven gliding by Tetradesmus deserticola (Chlorophyta) in microfluidic chambers.

Microalgae within the Scenedesmaceae are often distinguished by spines, bristles, and other wall characteristics. We examined the dynamic production and chemical nature of bristles extruded from the poles of Tetradesmus deserticola previously isolated from microbiotic crust. Rapidly growing cells in a liquid growth medium were established in polydimethylsiloxane microfluidic chambers specially designed to maintain aerobic conditions over time within a chamber 6-12 µm deep. This geometry enabled in-focus imaging of single cells over long periods. Differential interference contrast (DIC) imaging revealed that after multiple fission of mother cells, the newly released, lemon-shaped daughter cells began extruding bristles from each pole. In some instances, the bristles became stuck to either the glass floor or polydimethylsiloxane (PDMS) walls of the chamber, and the force by which the new bristle was extruded was sufficient to propel the cells across the field of view at ~1.2 µm · h-1 . Confocal fluorescence and DIC imaging of cells stained with pontamine fast scarlet and calcofluor, and treated with proteinase K, suggested that bristles are proteinaceous and may also host carbohydrate modifications. The polar bristles extruded by this desert-derived T. deserticola may simply be relics of bristles produced by an aquatic ancestor for flotation or predator deterrence. But, their tendency to attach to glass (silicate) and/or PDMS surfaces suggests a potential role in tethering cells in place or binding soil particles. T. deserticola is closely related to T. obliquus, which is of interest for biofuels development; extruded bristles in T. deserticola may offer tethers for industrial use of these stress-tolerant algae.

Investigating low salinity waterflooding via glass micromodels with triangular pore-throat architectures.

Glass micromodels have been extensively used to simulate and investigate crude oil, brine, and surface interactions due to their homogeneous wettability, rigidity, and ability to precisely capture a reservoir's areal heterogeneity. Most micromodels are fabricated via two-dimensional patterning, implying that feature depths are constant despite varying width, which sub-optimally describes a three-dimensional porous architecture. We have successfully fabricated micromodels with arbitrary triangular cross sections via femtosecond pulsed laser direct writing resulting in depth-dependent channel width. As such, we have achieved arbitrary geometric control over device fabrication and thus a more accurate recapitulation of a geological porous media. With this fabrication technique, we are now able to directly observe pore-level, depth-dependent multiphase flow phenomena. This platform was used to study the low salinity effect (LSE) by simulating waterflooding processes using various brine solutions that differ in cation type and salinity. Patterned pore-throat structures were created to investigate displacement behavior during waterflooding. Real-time monitoring of the displacement processes, combined with a comparison of the brine chemistry before and after waterflooding provides an insight into realistic interactions occurring between crude oil and brine. The results indicate that produced emulsions were prone to coalesce in the presence of lower salinity brine. Combined with previous work, the LSE was interpreted as favored coalescence and resisted breakup that resulting in a more continuous aqueous phase during waterflooding therefore improving the displacement efficiency.

Microfluidic encapsulation of Xenopus laevis cell-free extracts using hydrogel photolithography.

Cell-free extract derived from the eggs of the African clawed frog Xenopus laevis is a well-established model system that has been used historically in bulk aliquots. Here, we describe a microfluidic approach for isolating discrete, biologically relevant volumes of cell-free extract, with more expansive and precise control of extract shape compared with extract-oil emulsions. This approach is useful for investigating the mechanics of intracellular processes affected by cell geometry or cytoplasmic volume, including organelle scaling and positioning mechanisms. For complete details on the use and execution of this protocol, please refer to Geisterfer et al. (2020).

Fabrication of sealed sapphire microfluidic devices using femtosecond laser micromachining.

Due to its hardness, strength, and transparency, sapphire is an attractive material for the construction of microfluidic devices intended for high-pressure applications, but its physiochemical properties resist traditional microfabrication and bonding techniques. Here a femtosecond pulsed laser was used to directly machine fluidic channels within sapphire substrates and to form bonds between machined and flat sapphire windows, resulting in the creation of sealed microfluidic devices. Sapphire-sapphire bond strength was determined by destructive mechanical testing, and the integrity of the bond was verified by the capillary filling of the channel with air and ethanol. This combination of optical micromachining and bonding establishes a fully integrated approach to the fabrication of sapphire-based microfluidic systems.

Microtubule-dependent pushing forces contribute to long-distance aster movement and centration in Xenopus laevis egg extracts.

During interphase of the eukaryotic cell cycle, the microtubule (MT) cytoskeleton serves as both a supportive scaffold for organelles and an arborized system of tracks for intracellular transport. At the onset of mitosis, the position of the astral MT network, specifically its center, determines the eventual location of the spindle apparatus and ultimately the cytokinetic furrow. Positioning of the MT aster often results in its movement to the center of a cell, even in large blastomeres hundreds of microns in diameter. This translocation requires positioning forces, yet how these forces are generated and then integrated within cells of various sizes and geometries remains an open question. Here we describe a method that combines microfluidics, hydrogels, and Xenopus laevis egg extract to investigate the mechanics of aster movement and centration. We determined that asters were able to find the center of artificial channels and annular cylinders, even when cytoplasmic dynein-dependent pulling mechanisms were inhibited. Characterization of aster movement away from V-shaped hydrogel barriers provided additional evidence for a MT-based pushing mechanism. Importantly, the distance over which this mechanism seemed to operate was longer than that predicted by radial aster growth models, agreeing with recent models of a more complex MT network architecture within the aster.

Convection-driven microfabricated hydrogels for rapid biosensing.

A microscale biosensing platform using rehydration-mediated swelling of bio-functionalized hydrogel structures and rapid target analyte capture is described. Induced convective flow mitigates diffusion limited incubation times, enabling model assays to be completed in under three minutes. Assay design parameters have been evaluated, revealing fabrication criteria required to tune detection sensitivity.

Microtubule Growth Rates Are Sensitive to Global and Local Changes in Microtubule Plus-End Density.

The microtubule cytoskeleton plays critically important roles in numerous cellular functions in eukaryotes, and it does so across a functionally diverse and morphologically disparate range of cell types [1]. In these roles, microtubule assemblies must adopt distinct morphologies and physical dimensions to perform specific functions [2-5]. As such, these macromolecular assemblies-as well as the dynamics of the individual microtubule polymers from which they are made-must scale and change in accordance with cell size, geometry, and function. Microtubules in cells typically assemble to a steady state in mass, leaving enough of their tubulin subunits soluble to allow rapid growth and turnover. This suggests some negative feedback that limits the extent of assembly, for example, decrease in growth rate, or increase in catastrophe rate, as the soluble subunit pool decreases. Although these ideas have informed the field for decades, they have not been observed experimentally. Here, we describe the application of an experimental approach that combines cell-free extracts with photo-patterned hydrogel micro-enclosures as a means to investigate microtubule dynamics in cytoplasmic volumes of defined size and shape. Our measurements reveal a negative correlation between microtubule plus-end density and microtubule growth rates and suggest that these rates are sensitive to the presence of nearby growing ends.

The Perinuclear ER Scales Nuclear Size Independently of Cell Size in Early Embryos.

Nuclear size plays pivotal roles in gene expression, embryo development, and disease. A central hypothesis in organisms ranging from yeast to vertebrates is that nuclear size scales to cell size. This implies that nuclei may reach steady-state sizes set by limiting cytoplasmic pools of size-regulating components. By monitoring nuclear dynamics in early sea urchin embryos, we found that nuclei undergo substantial growth in each interphase, reaching a maximal size prior to mitosis that declined steadily over the course of development. Manipulations of cytoplasmic volume through multiple chemical and physical means ruled out cell size as a major determinant of nuclear size and growth. Rather, our data suggest that the perinuclear endoplasmic reticulum, accumulated through dynein activity, serves as a limiting membrane pool that sets nuclear surface growth rate. Partitioning of this local pool at each cell division modulates nuclear growth kinetics and dictates size scaling throughout early development.

Crosslinker length dictates step-growth hydrogel network formation dynamics and allows rapid on-chip photoencapsulation.

Hydrogels formed via free radical-mediated thiol-ene step-growth photopolymerization have been developed for a broad range of tissue engineering and regenerative medicine applications. While the crosslinking mechanism of thiol-ene hydrogels has been well-described, there has been only limited work exploring the physical differences among gels arising from variations in crosslinker properties. Here, we show that the character of linear polyethylene glycol (PEG) dithiols used to crosslink multi-arm polyethylene glycol norbornene (PEGNB) can be used as a facile strategy to tune hydrogel formation kinetics, and therefore the equilibrium hydrogel network architecture. Specifically, we report the dramatic effect of crosslinker length on PEGNB hydrogel formation kinetics and the formed hydrogel properties. It is shown that the hydrogel formation kinetics and formed hydrogel properties can be tuned by solely varying the crosslinker length. It was hypothesized that under identical reaction conditions, a more accessible 3.5 k PEG dithiol crosslinker would improve network ideality relative to a shorter 1.5 k crosslinker. Longer linkers consequently promote significantly more rapid macromer crosslinking and therefore gelation. Accelerated gel formation satisfies an urgent unmet need for rapid polymerization in droplet microfluidics. Using long linkers, we demonstrate the ability to photopolymerize PEGNB microgels under flow on a microfluidic chip, with reliable control over microgel size and shape in a high-throughput manner. To further validate the potential of this platform to produce novel, microstructured cell carrier vehicles, 3T3 fibroblasts were successfully encapsulated and cultured over 14 days with excellent cell viability. This study demonstrates that PEGNB hydrogel dynamics could be readily customized to fulfill a variety of needs in tissue engineering, controlled cell delivery, or drug release applications.

Nucleoplasmin is a limiting component in the scaling of nuclear size with cytoplasmic volume.

How nuclear size is regulated relative to cell size is a fundamental cell biological question. Reductions in both cell and nuclear sizes during Xenopus laevis embryogenesis provide a robust scaling system to study mechanisms of nuclear size regulation. To test if the volume of embryonic cytoplasm is limiting for nuclear growth, we encapsulated gastrula-stage embryonic cytoplasm and nuclei in droplets of defined volume using microfluidics. Nuclei grew and reached new steady-state sizes as a function of cytoplasmic volume, supporting a limiting component mechanism of nuclear size control. Through biochemical fractionation, we identified the histone chaperone nucleoplasmin (Npm2) as a putative nuclear size effector. Cellular amounts of Npm2 decrease over development, and nuclear size was sensitive to Npm2 levels both in vitro and in vivo, affecting nuclear histone levels and chromatin organization. We propose that reductions in cell volume and the amounts of limiting components, such as Npm2, contribute to developmental nuclear size scaling.

Light-inducible activation of cell cycle progression in Xenopus egg extracts under microfluidic confinement.

Cell-free Xenopus egg extract is a widely used and biochemically tractable model system that allows recapitulation and elucidation of fundamental cellular processes. Recently, the introduction of microfluidic extract manipulation has enabled compartmentalization of bulk extract and a newfound ability to study organelles on length scales that recapitulate key features of cellular morphology. While the microfluidic confinement of extracts has produced a compelling platform for the in vitro study of cell processes at physiologically-relevant length scales, it also imposes experimental limitations by restricting dynamic control over extract properties. Here, we introduce photodegradable polyethylene glycol (PEG) hydrogels as a vehicle to passively and selectively manipulate extract composition through the release of proteins encapsulated within the hydrogel matrix. Photopatterned PEG hydrogels, passive to both extract and encapsulated proteins, serve as protein depots within microfluidic channels, which are subsequently flooded with extract. Illumination by ultraviolet light (UV) degrades the hydrogel structures and releases encapsulated protein. We show that an engineered fluorescent protein with a nuclear localization signal (GST-GFP-NLS) retains its ability to localize within nearby nuclei following UV-induced release from hydrogel structures. When diffusion is considered, the kinetics of nuclear accumulation are similar to those in experiments utilizing conventional, bulk fluid handling. Similarly, the release of recombinant cyclin B Δ90, a mutant form of the master cell cycle regulator cyclin B which lacks the canonical destruction box, was able to induce the expected cell cycle transition from interphase to mitosis. This transition was confirmed by the observation of nuclear envelope breakdown (NEBD), a phenomenological hallmark of mitosis, and the induction of mitosis-specific biochemical markers. This approach to extract manipulation presents a versatile and customizable route to regulating the spatial and temporal dynamics of cellular events in microfluidically confined cell-free extracts.

Engineering functional hydrogel microparticle interfaces by controlled oxygen-inhibited photopolymerization.

Functional poly(ethylene glycol) diacrylate (PEGDA) hydrogel microparticles for the detection of bioactive macromolecules were fabricated via oxygen-inhibited photopolymerization in a droplet microfluidic device. Hydrogel network functionalization and architecture were characterized using a biotin-avidin binding assay, which revealed radial network inhomogeneities dependent on exposure conditions. Empirical results were corroborated using a reaction-diffusion model, describing the effects of exposure intensity on the spatial photopolymerization kinetics and resulting polymeric mesh network. The combination of finely controlled exposure conditions and predictive simulations enables the generation of tailored particles with microengineered interfaces and gradients in crosslinking density, which dictate solute diffusivity and elasticity, augmenting the utility of this approach in engineering multifunctional, size-excluding hydrogel particles for multiplexed biomolecular sensing.