Cell Encapsulation Research Articles

Islet cell spheroids produced by a thermally sensitive scaffold: a new diabetes treatment

The primary issues in treating type 1 diabetes mellitus (T1DM) through the transplantation of healthy islets or islet β-cells are graft rejection and a lack of available donors. Currently, the majority of approaches use cell encapsulation technology and transplant replacement cells that can release insulin to address transplant rejection and donor shortages. However, existing encapsulation materials merely serve as carriers for islet cell growth. A new treatment approach for T1DM could be developed by creating a smart responsive material that encourages the formation of islet cell spheroids to replicate their 3D connections in vivo and controls the release of insulin aggregates. In this study, we used microfluidics to create thermally sensitive porous scaffolds made of poly(N-isopropyl acrylamide)/graphene oxide (PNIPAM/GO). The material was carefully shrunk under near-infrared light, enriched with mouse insulinoma pancreatic β cells (β-TC-6 cells), encapsulated, and cultivated to form 3D cell spheroids. The controlled contraction of the thermally responsive porous scaffold regulated insulin release from the spheroids, demonstrated using the glucose-stimulated insulin release assay (GSIS), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay. Eventually, implantation of the spheroids into C57BL/6 N diabetic mice enhanced the therapeutic effect, potentially offering a novel approach to the management of T1DM.Graphical

Single-layer graphene oxide nanosheets induce proliferation and Osteogenesis of single-cell hBMSCs encapsulated in Alginate Microgels

Microfluidics cell encapsulation offers a way to mimic a 3D microenvironment that supports cell growth and proliferation, while also protecting cells from environmental stress. This technique has found extensive applications in tissue engineering and cell therapies. Several studies have demonstrated the advantages of graphene oxide (GO) as an osteogenic inducer; however, the significance of GO on stem cell fate in the single-cell state is still unclear. Here, a microfluidics-based approach is developed for continuous encapsulation of mesenchymal stem cells (MSCs) at the single-cell level using alginate microgels. So, single-layer graphene oxide (slGO) nanosheet is used to be encapsulated inside the alginate droplets. The results of AFM and SEM show that slGO can increase the roughness and reduce the stiffness of alginate hydrogels. The Young’s modulus of the alginate and alginate-slGO was obtained as 1414 kPa and 985.9 kPa, respectively. Live/dead assay and fluorescence microscopy images illustrate that slGO can maintain the viability and proliferation of microencapsulated hBMSCs. The obtained results show that slGO increases the mineralization of the encapsulated hBMSCs, so that microgels containing hBMSCs gradually became opaque during 21 days of culture. RT-qPCR results indicate that the expression of OCN, Runx2, and ALP in the alginate-slGO microgels is significantly higher than in the alginate microgels. The expression of OCN and Runx2 in the alginate-slGO microgels is 4.27 and 5.87-fold higher than in the alginate microgels, respectively. It can be concluded that low doses of slGO nanosheets have the potential to be utilized in the development of tissue engineering and bone regeneration. This finding offers a new method for creating injectable tissue transplants that are minimally invasive.

Characterization of MSC Growth, Differentiation, and EV Production in CNF Hydrogels Under Static and Dynamic Cultures in Hypoxic and Normoxic Conditions.

Mesenchymal stem cells (MSCs) hold immense therapeutic potential due to their regenerative and immunomodulatory properties. However, to utilize this potential, it is crucial to optimize their in vitro cultivation conditions. Three-dimensional (3D) culture methods using cell-laden hydrogels aim to mimic the physiological microenvironment in vitro, thus preserving MSC biological functionalities. Cellulosic hydrogels are particularly promising due to their biocompatibility, sustainability, and tunability in terms of chemical, morphological, and mechanical properties. This study investigated the impact of (1) two physical crosslinking scenarios for hydrogels derived from anionic cellulose nanofibers (to-CNF) used to encapsulate adipose-derived MSCs (adMSCs) and (2) physiological culture conditions on the in vitro proliferation, differentiation, and extracellular vesicle (EV) production of these adMSCs. The results revealed that additional Ca2+-mediated crosslinking, intended to complement the self-assembly and gelation of aqueous to-CNF in the adMSC cultivation medium, adversely affected both the mechanical properties of the hydrogel spheres and the growth of the encapsulated cells. However, cultivation under dynamic and hypoxic conditions significantly improved the proliferation and differentiation of the encapsulated adMSCs. Furthermore, it was demonstrated that the adMSCs in the CNF hydrogel spheres exhibited potential for scalable EV production with potent immunosuppressive capacities in a bioreactor system. These findings underscore the importance of physiological culture conditions and the suitability of cellulosic materials for enhancing the therapeutic potential of MSCs. Overall, this study provides valuable insights for optimizing the in vitro cultivation of MSCs for various applications, including tissue engineering, drug testing, and EV-based therapies.

WELL-POSEDNESS RESULTS FOR GENERAL REACTION–DIFFUSION TRANSPORT OF OXYGEN IN ENCAPSULATED CELLS

Abstract We provide well-posedness results for nonlinear parabolic partial differential equations (PDEs) given by reaction–diffusion equations describing the concentration of oxygen in encapsulated cells. The cells are described in terms of a core and a shell, which introduces a discontinuous diffusion coefficient as the material properties of the core and shell differ. In addition, the cells are subject to general nonlinear consumption of oxygen. As no monotonicity condition is imposed on the consumption, monotone operator theory cannot be used. Moreover, the discontinuity in the diffusion coefficient bars us from applying classical results on strong solutions. However, by directly applying a Galerkin method, we obtain uniqueness and existence of the strong form solution. These results provide the basis to study the dynamics of cells in critical states.

Covalent Self-Assembly of Bio-HCP Nanoparticles for Shell-Programmed Encapsulation of Microbial Cells.

Modifying the bacterial surface through grafting functional nanoparticles is a common strategy for programing bacteria. At this moment, the targeted nanoparticles face a dilemma of no multifunctional structure, high toxicity, and weak chemical driving forces, which restrict the broad practical applications. Like a multistage booster of a rocket, we propose a multistage covalent self-assembly strategy to protect, expand, and control the encapsulated shells of microbial cells via biocompatible hyper-cross-linked polymer nanoparticles (Bio-HCP NPs) with internal porosity and surface functional groups. The bacterial surface is enhanced with rich amino groups up to 1010 per cell for specifically grafting nanoparticles. The arming bacteria after first-stage assembly can complete biocatalysis in a highly toxic environment, and as-prepared polymer aggregates (6-20 μm) after third-stage assembly can be accurately counted in an aerosol environment. This nanoparticle encapsulation exhibits strong cell viability from pollutants and specificity from impurity particles, holding promise for various complex application scenarios.

Cardiac Matrix-Derived Granular Hydrogel Enhances Cell Function in 3D Culture.

Hydrogels derived from decellularized porcine myocardial matrix have demonstrated significant potential as therapeutic delivery platforms for promoting cardiac repair after injury. Our previous study developed a fibrin-enriched cardiac matrix hydrogel to enhance its angiogenic capacities. However, the bulk hydrogel structure may limit their full potential in cell delivery. Recently, granular hydrogels have emerged as a promising class of biomaterials, offering unique features such as a highly interconnected porous structure that facilitates nutrient diffusion and enhances cell viability. Several techniques have been developed for fabricating various types of granular hydrogels, among which extrusion fragmentation is particularly appealing due to its adaptability to many types of hydrogels, low cost, and high scalability. In this study, we first confirmed the effects of the bulk cardiac matrix hydrogel on the viability of encapsulated human umbilical vein endothelial cells and human mesenchymal stem cells. We then tested the feasibility of producing granular hydrogels from both cardiac matrix and fibrin-enriched cardiac matrix through cellular cross-linking of microgels fabricated by extrusion fragmentation. Afterward, we examined the roles of the produced granular hydrogels in the embedded cells and cell spheroids. Our in vitro data demonstrate that cardiac matrix-derived granular hydrogels support optimal viability of encapsulated cells and promote sprouting of human mesenchymal stem cell spheroids. Additionally, granular hydrogel derived from fibrin-enriched cardiac matrix accelerates angiogenic sprouting of embedded human mesenchymal stem cell spheroids. The results obtained from this study lay an important foundation for the future exploration of using cardiac matrix-derived granular hydrogels for cardiac cell therapy.

An Adhesive Hydrogel Technology for Enhanced Cartilage Repair: A Preliminary Proof of Concept.

Knee cartilage has limited natural healing capacity, complicating the development of effective treatment plans. Current non-cell-based therapies (e.g., microfracture) result in poor repair cartilage mechanical properties, low durability, and suboptimal tissue integration. Advanced treatments, such as autologous chondrocyte implantation, face challenges including cell leakage and inhomogeneous distribution. Successful cell therapy relies on prolonged retention of therapeutic biologicals at the implantation site, yet the optimal integration of implanted material into the surrounding healthy tissue remains an unmet need. This study evaluated the effectiveness of a newly developed photo-curable adhesive hydrogel for cartilage repair, focusing on adhesion properties, integration performance, and ability to support tissue regeneration. The proposed hydrogel design exhibited significant adhesion strength, outperforming commercial adhesives such as fibrin-based glues. An in vivo goat model was used to evaluate the hydrogels' adhesion properties and long-term integration into full-thickness cartilage defects over six months. Results showed that cell-free hydrogel-treated defects achieved superior integration with surrounding tissue and enhanced cartilage repair, with notable lateral integration. In vitro results further demonstrated high cell viability, robust matrix production, and successful cell encapsulation within the hydrogel matrix. These findings highlight the potential of adhesive hydrogel formulations to improve the efficacy of cell-based therapies, offering a potentially superior treatment for knee cartilage defects.

Addressing the efficiency loss and degradation of triple cation perovskite solar cells via integrated light managing encapsulation

Here, we present a holistic encapsulation method for perovskite solar cells to address both optical performance losses at the air-cell interface as well as intrinsic and extrinsic stability challenges. Our one-step method provides shielding to PSCs from oxygen and moisture-induced degradation as well as in situ patterning for light management. As a result of embedding the antireflective coating onto the front side of the cell, the power conversion efficiency (PCE) of the PSCs was increased from 14.1 ± 0.8 % to 15.6 ± 0.8 %, indicating an 8 % relative improvement. Moreover, the encapsulated devices kept their initial performance after 90 % relative humidity and water immersion tests. The outdoor exposure test showed no degradation for the encapsulated cells after 24 h of resting at −17 °C and maximum wind speeds of 7 m/s on average. Additionally, the encapsulation strategy was instrumental in mitigating oxygen and humidity-induced degradation during ISOS-LC tests, retaining up to 80 % of the initial performance for the encapsulated devices after 360 h. This research establishes in situ encapsulation and patterning as a promising solution for reducing optical losses and extrinsic instabilities in PSCs. The choice of flexible encapsulant enables it to be used for both rigid and flexible PSCs in a wide range of applications.

Biomimetic composite gelatin methacryloyl hydrogels for improving survival and osteogenesis of human adipose-derived stem cells in 3D microenvironment

Gelatin methacryloyl (GelMA) hydrogels are used for stem cell encapsulation in bone tissue engineering due to their fast and stable photo-crosslinking. However, cell viability and ability to induce osteogenesis are reduced by reactive oxygen species (ROS) produced during the crosslinking reaction. In this study, we developed biomimetic nanoparticles (TMNs) by combining tannic acid (TA) and simulated body fluid (SBF) minerals, and used them to synthesize GelMA-based composite hydrogels for addressing those limitations. The optimal concentrations of TA and SBF were investigated to create nanoparticles that can effectively scavenge ROS and induce osteogenesis. The incorporation of TMNs into composite hydrogels (G-TMN) significantly enhanced the survival and proliferation of encapsulated human adipose-derived stem cells (hADSCs) by providing resistance to oxidative conditions. In addition, the ions that were released, such as Ca2+ and PO43−, stimulated stem cell differentiation into bone cells. The hADSCs encapsulated in G-TMN had 2.0 ± 0.8-fold greater viability and 1.3 ± 1.8 times greater calcium deposition than those encapsulated in the hydrogel without nanoparticles. Furthermore, the in vivo transplantation of G-TMN into a subcutaneous mouse model demonstrated the rapid degradation of the gel-network while retaining the osteoinductive particles and cells in the transplanted area. The increased cellular activity observed in our multifunctional composite hydrogel can serve as a foundation for novel and effective therapies for bone deformities.

Exploring Calcium Alginate-Based Gels for Encapsulation of Lacticaseibacillus paracasei to Enhance Stability in Functional Breadmaking.

This study focuses on evaluating the efficiency of acid-tolerant Lacticaseibacillus paracasei bacteria encapsulated in an alginate-based gel matrix during repeated sourdough fermentation cycles, as well as their preservation during storage and throughout baking at high temperature. A double-coating procedure was applied, involving the encapsulation of bacterial cells in calcium alginate, which was further coated with chitosan. The encapsulation efficiency (EE) did not show significant difference between alginate and alginate-chitosan (97.97 and 96.71%, respectively). The higher number of L. paracasei bacteria was preserved in double-coated microbeads, with survivability rates of 89.51% and 96.90% in wet and dried microbeads, respectively. Encapsulated bacteria demonstrated effective fermentation ability, while double gel-coated cells exhibited slower acidification during sourdough fermentation, maintaining higher efficiency in the second fermentation cycle. The addition of freeze-dried, alginate-based gel-encapsulated bacteria (2-4%, w/w flour) significantly (p < 0.05) improved bread quality and extended its shelf life. A double-layer coating (alginate-chitosan) can be introduced as an innovative strategy for regulating the release of lactic acid bacteria and optimizing fermentation processes. Powdered alginate or alginate-chitosan gel-based L. paracasei microcapsules, at appropriate concentrations, can be used in the production of baked goods with acceptable quality and sensory properties, achieving a lactic acid bacteria count of approximately 106 CFU/g in the crumb, thereby meeting the standard criteria for probiotic bakery products.

Shear-Induced Cycloreversion Leading to Shear-Thinning and Autonomous Self-Healing in an Injectable, Shape-Holding Collagen Hydrogel.

In vivo injectable extracellular matrix (ECM) derived hydrogels that are suitable for cell encapsulation have always been the holy grail in tissue engineering. Nevertheless, these hydrogels still fall short today of meeting three crucial criteria: (a) flexibility on the injectability time window, (b) autonomous self-healing of the injected hydrogel, and (c) shape-retention under aqueous conditions. Here we report the development of a collagen-based injectable hydrogel, cross-linked by cycloaddition reaction between furan and maleimide groups, that (a) is injectable up to 48 h after preparation, (b) can undergo complete autonomous self-healing after injection, (c) can retain its shape and size over several years when stored in the buffer, (d) can be degraded within hours when treated with collagenase, (e) is biocompatible as demonstrated by in vitro cell-culture, and (f) is completely resorbable in vivo when implanted subcutaneously in rats without causing any inflammation.

Frozen dough steamed products: Deterioration mechanism, processing technology, and improvement strategies.

Fresh dough products lead to instability in product quality, high production costs, and more production time, which seriously affects the industrial production of the food industry. The frozen dough technology mitigates the problems of short shelf-life and easy deterioration of quality during storage and transportation. It has shown a series of advantages in large-scale industrialization, high-quality standardization, and chain operation. However, the further development of frozen dough is restricted by the deterioration of the main components (gluten, starch, and yeast) caused by freezing. This review summarizes the main production process of frozen steamed bread and buns, and the deterioration reasons for the main component of frozen dough. The improvement mechanisms of raw ingredients, processing technology, processing equipment, and additives on frozen dough quality were analyzed from the perspective of improving gluten network integrity and yeast freeze tolerance. From prefermented frozen raw to steamed products without thawing has become the preferred production process to improve production efficiency. Wheat flour mixed with other flour can maintain the gluten network continuity of frozen dough. The freeze tolerance of yeast was improved by treatment with yeast suspension, yeast cell encapsulation, screening hybridization, and genetic engineering. Process optimization and new technology-assisted fermentation and freezing effectively reduce freezing damage. Various additives improve the freeze resistance of the gluten-starch matrix by promoting protein cross-linking and inhibiting water migration. In addition, ice structural proteins and ice nucleating agents have been proven to change the growth morphology and formation temperature of ice crystals. More new technologies and additive synergies need to be further explored.

Spontaneous Formation of Solid Shell Polymeric Multicompartments at All-Aqueous Interfaces.

Multicompartmental capsules have demonstrated value in fields ranging from drug release, mimetics of artificial cells, to energy conversion and storage. However, the fabrication of devices with different compartments usually requires the use of toxic solvents, and/or the adaptation of technically demanding methods, including precision microfluidics and multistep processes. The spontaneous formation of multi-core capsules resulting from polyelectrolyte complexation at the interface of a prototypic all-aqueous two-phase system is described here. The variation of polyelectrolyte concentration and complexation time are described as simple working parameters capable of driving the formation of compartments at different yields, as well as tailoring their morphology. The mild processing technology enables the encapsulation of animal cells, which are capable of invading capsule walls for specific processing conditions.

Bacteria encapsulation into polyethylene glycol hydrogels using Michael-type addition reactions

AbstractHydrogel materials can be used to integrate bacteria cells into biohybrid systems. Here, we investigate the use of polyethylene glycol-based hydrogels that employ different Michael-type addition crosslinking chemistries, including thiol-acrylate, thiol-vinyl sulfone, and thiol-maleimide click reactions, for covalent hydrogel network formation and bacteria encapsulation. All crosslinking chemistries generated hydrogels that provided stable encapsulation and culture of Bacillus subtilis; however, significant differences in cell viability and cell morphology after encapsulation were identified. Thiol-acrylate hydrogels provided the highest cell viability and favored encapsulation of single cells, while thiol-maleimide hydrogels had the lowest cell viability and favored encapsulation of larger aggregates. These findings demonstrate the impact of crosslinking strategies for encapsulation of microorganisms into hydrogel networks and suggest that thiol-acrylate chemistries are favorable for many applications. Graphical abstract

On the transport of a millimeter-sized compound droplet in a Poiseuille flow

The compound droplet consists of the inner and outer droplets. The outer droplet's ability to isolate the inner contents from its surroundings makes applications such as drug delivery, cell encapsulation, and cell sorting possible. Here, we experimentally explore the transport of the compound droplet at different Reynolds numbers, viscosity ratios, and volume ratios. Compound droplets have a longer dimensionless transport distance along the X-axis than single-phase droplets at Reynolds numbers from 44 to 366. When the Reynolds number is increased from 81.2 to 243.7, the dimensionless maximum transport distance of the compound droplet along the X-axis becomes 2.04 times. As the Re increases, the compound droplet deflection rate decreases continuously. For a constant initial velocity of the compound droplet, an increase in the viscosity ratio leads to an increase in the dimensionless velocity along the x axis with the compound droplet during transport. The maximum transport distance along the x-axis increases, and the deflection rate decreases as the inertial and viscous forces increase with increasing viscosity and dimension ratios. The chemical droplets become more stable as a result.

Stereolithography-assisted sodium alginate-collagen hydrogel scaffold with molded internal channels

Fabricating internal vascular networks within a hydrogel scaffold is essential for facilitating the supply of nutrients, oxygen, and metabolism exchange required by the encapsulated cells. The challenges in current hydrogel scaffold fabrication involve the difficulty of building adequate internal channels, poor scaffold geometry precision, and low cell viability caused by the fabrication process and polymer material properties. Stereolithography (SLA) stands out as a 3D printing technique distinguished by its superior production efficiency, advanced precision, and remarkable resolution in crafting intricate custom geometries. These attributes establish it as an innovative approach for templates in scaffold fabrication, potentially surpassing the fused deposition modeling (FDM)-based template strategy. Meanwhile, it exerts less shear stress on the cells compared to the direct bioprinting process. This novel strategy enables the fabrication of hydrogel vascular structure within the precision of 500 µm in both channel diameter and wall thickness. In this paper, various sodium alginate and collagen (SA-Col) composite hydrogels with varying collagen concentrations have been investigated to identify the optimal ratio for fabricating hydrogel scaffolds with channels.

Droplet Microfluidics Powered Hydrogel Microparticles for Stem Cell-Mediated Biomedical Applications.

Stem cell-related therapeutic technologies have garnered significant attention of the research community for their multi-faceted applications. To promote the therapeutic effects of stem cells, the strategies for cell microencapsulation in hydrogel microparticles have been widely explored, as the hydrogel microparticles have the potential to facilitate oxygen diffusion and nutrient transport alongside their ability to promote crucial cell-cell and cell-matrix interactions. Despite their significant promise, there is an acute shortage of automated, standardized, and reproducible platforms to further stem cell-related research. Microfluidics offers an intriguing platform to produce stem cell-laden hydrogel microparticles (SCHMs) owing to its ability to manipulate the fluids at the micrometer scale as well as precisely control the structure and composition of microparticles. In this review, the typical biomaterials and crosslinking methods for microfluidic encapsulation of stem cells as well as the progress in droplet-based microfluidics for the fabrication of SCHMs are outlined. Moreover, the important biomedical applications of SCHMs are highlighted, including regenerative medicine, tissue engineering, scale-up production of stem cells, and microenvironmental simulation for fundamental cell studies. Overall, microfluidics holds tremendous potential for enabling the production of diverse hydrogel microparticles and is worthy for various stem cell-related biomedical applications.

Hydrogel microsphere stem cell encapsulation enhances cardiomyocyte differentiation and functionality in scalable suspension system

A reliable suspension-based platform for scaling engineered cardiac tissue (ECT) production from human induced pluripotent stem cells (hiPSCs) is crucial for regenerative therapies. Here, we compared the production and functionality of ECTs formed using our scaffold-based, engineered tissue microsphere differentiation approach with those formed using the prevalent scaffold-free aggregate platform. We utilized a microfluidic system for the rapid (1 million cells/min), high density (30, 40, 60 million cells/ml) encapsulation of hiPSCs within PEG-fibrinogen hydrogel microspheres. HiPSC-laden microspheres and aggregates underwent suspension-based cardiac differentiation in chemically defined media. In comparison to aggregates, microspheres maintained consistent size and shape initially, over time, and within and between batches. Initial size and shape coefficients of variation for microspheres were eight and three times lower, respectively, compared to aggregates. On day 10, microsphere cardiomyocyte (CM) content was 27 % higher and the number of CMs per initial hiPSC was 250 % higher than in aggregates. Contraction and relaxation velocities of microspheres were four and nine times higher than those of aggregates, respectively. Microsphere contractile functionality also improved with culture time, whereas aggregate functionality remained unchanged. Additionally, microspheres displayed improved β-adrenergic signaling responsiveness and uniform calcium transient propagation. Transcriptomic analysis revealed that while both microspheres and aggregates demonstrated similar gene regulation patterns associated with cardiomyocyte differentiation, heart development, cardiac muscle contraction, and sarcomere organization, the microspheres exhibited more pronounced transcriptional changes over time. Taken together, these results highlight the capability of the microsphere platform for scaling up biomanufacturing of ECTs in a suspension-based culture platform.

Boosting chondrocyte bioactivity with ultra-sulfated glycopeptide supramolecular polymers

Although autologous chondrocyte transplantation can be effective in articular cartilage repair, negative side effects limit the utility of the treatment, such as long recovery times, poor engraftment or chondrogenic dedifferentiation, and cell leakage. Peptide-based supramolecular polymers have emerged as promising bioactive systems to promote tissue regeneration through cell signaling and dynamic behavior. We report here on the development of a series of glycopeptide amphiphile supramolecular nanofibers with chondrogenic bioactivity. These supramolecular polymers were found to have the ability to boost TGFβ-1 signaling by displaying galactosamine moieties with differing degrees of sulfation on their surfaces. We were also able to encapsulate chondrocytes with these nanostructures as single cells without affecting viability and proliferation. Among the monomers tested, assemblies of trisulfated glycopeptides led to elevated expression of chondrogenic markers relative to those with lower degrees of sulfation that mimic chondroitin sulfate repeating units. We hypothesize the enhanced bioactivity is rooted in specific interactions of the supramolecular assemblies with TGFβ-1 and its consequence on cell signaling, which may involve elevated levels of supramolecular motion as a result of high charge in trisulfated glycopeptide amphiphiles. Our findings suggest that supramolecular polymers formed by the ultra-sulfated glycopeptide amphiphiles could provide better outcomes in chondrocyte transplantation therapies for cartilage regeneration. Statement of significanceThis study prepares glycopeptide amphiphiles conjugated at their termini with chondroitin sulfate mimetic residues with varying degrees of sulfation that self-assemble into supramolecular nanofibers in aqueous solution. These supramolecular polymers encapsulate chondrocytes as single cells through intimate contact with cell surface structures, forming artificial matrix that can localize the growth factor TGFβ-1 in the intercellular environment. A high degree of sulfation on the glycopeptide amphiphile is found to be critical in elevating chondrogenic cellular responses that supersede the efficacy of natural chondroitin sulfate. This work demonstrates that supramolecular assembly of a unique molecular structure designed to mimic chondroitin sulfate successfully boosts chondrocyte bioactivity by single cell encapsulation, suggesting a new avenue implementing chondrocyte transplantation with supramolecular nanomaterials for cartilage regeneration.

Label-free single-cell antimicrobial susceptibility testing in droplets with concentration gradient generation.

Bacterial communities exhibit significant heterogeneity, resulting in the emergence of specialized phenotypes that can withstand antibiotic exposure. Unfortunately, the existence of subpopulations resistant to antibiotics often goes unnoticed during treatment initiation. Thus, it is crucial to consider the concept of single-cell antibiotic susceptibility testing (AST) to tackle bacterial infections. Nevertheless, its practical application in clinical settings is hindered by its inability to conduct AST efficiently across a wide range of antibiotics and concentrations. This study introduces a droplet-based microfluidic platform designed for rapid single-cell AST by creating an antibiotic concentration gradient. The advantage of a microfluidic platform is achieved by executing bacteria and antibiotic mixing, cell encapsulation, incubation, and enumeration of bacteria in a seamless workflow, facilitating susceptibility testing of each antibiotic. Firstly, we demonstrate the rapid determination of minimum inhibitory concentration (MIC) of several antibiotics with Gram-negative E. coli and Gram-positive S. aureus, which enables us to bypass the time-consuming bacteria cultivation, speeding up the AST in 3 h from 1 to 2 days of conventional methods. Additionally, we assess 10 clinical isolates including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Staphylococcus aureus (MDRSA) against clinically important antibiotics for analyzing the MIC, compared to the gold standard AST method from the United States Clinical and Laboratory Standards Institute (CLSI), which becomes available only after 48 h. Furthermore, by monitoring single cells within individual droplets, we have found a spectrum of resistance levels among genetically identical cells, revealing phenotypic heterogeneity within isogenic populations. This discovery not only advances clinical diagnostics and treatment strategies but also significantly contributes to the field of antibiotic stewardship, underlining the importance of our approach in addressing bacterial resistance.