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Cell encapsulation is a widely used technique in the field of Tissue Engineering and Regenerative Medicine (TERM). However, for the particular case of liquefied compartmentalised systems, only a limited number of studies have been reported in the literature. We have been exploring a unique cell encapsulation system composed by liquefied and multilayered capsules. This system transfigured the concept of 3D scaffolds for TERM, and was already successfully applied for bone and cartilage regeneration. Due to a number of appealing features, we envisage that it can be applied in many other fields, including in advanced therapies or as disease models for drug discovery. In this review, we intend to highlight the advantages of this new system, while discussing the methodology, and sharing the protocol optimization and results. The different liquefied systems for cell encapsulation reported in the literature will be also discussed, considering the different encapsulation matrixes as core templates, the types of membranes, and the core liquefaction treatments.

Hydrogels constructed from naturally derived polymers provide an aqueous environment that encourages cell growth, however, mechanical properties are poor and degradation can be difficult to predict. Whilst, synthetic hydrogels exhibit some improved mechanical properties, these materials lack biochemical cues for cells growing and have limited biodegradation. To produce hydrogels that support 3D cell cultures to form tissue mimics, materials must exhibit appropriate biological and mechanical properties. In this study, novel organic-inorganic hybrid hydrogels based on chitosan and silica were prepared using the sol-gel technique. The chemical, physical and biological properties of the hydrogels were assessed. Statistical analysis was performed using One-Way ANOVAs and independent-sample t-tests. Fourier transform infrared spectroscopy showed characteristic absorption bands including amide II, Si-O and Si-O-Si confirming formation of hybrid networks. Oscillatory rheometry was used to characterise the sol to gel transition and viscoelastic behaviour of hydrogels. Furthermore, in vitro degradation revealed both chitosan and silica were released over 21 days. The hydrogels exhibited high loading efficiency as total protein loading was released in a week. There were significant differences between TC2G and C2G at all-time points (p < 0.05). The viability of osteoblasts seeded on, and encapsulated within, the hydrogels was >70% over 168 h culture and antimicrobial activity was demonstrated against Pseudomonas aeruginosa and Enterococcus faecalis. The hydrogels developed here offer alternatives for biopolymer hydrogels for biomedical use, including for application in drug/cell delivery and for bone tissue engineering.

The encapsulation of single cells has emerged as a promising field in recent years, owing to its potential applications in cell‐based therapeutics, bioprinting, in vitro cell culture, high‐throughput screening, and diagnostics. Single‐cell units offer several advantages, including compatibility with standard imaging techniques, superior diffusion rates, and lower material‐to‐cell volume ratios. They also serve as effective carriers for targeted drug delivery, allowing precise administration of therapeutics in cell‐mediated quantities. Moreover, single‐cell units exhibit improved circulation potential throughout the vasculature, with a reduced likelihood of entrapment compared to multicell strategies. However, the production of single‐cell units from random dispersion of cells follows the Poisson distribution, requiring the separation of empty and multicell units from single‐cell ones. Various methods have been developed to address this challenge; nevertheless, the majority of these strategies are either expensive or time‐consuming. This review provides an in‐depth analysis of the advantages and limitations of single‐cell units and their applications, as well as a comprehensive overview of the most used techniques for single‐cell encapsulation and sorting strategies.

Single-cell encapsulation, by constructing cell-scale microenvironments, enables precise protection, regulation, and functional enhancement of individual cells, holding significant importance in biomedical fields such as bioanalysis and cell therapy. Although various materials—including polymers, nanoparticles, hydrogels, polyphenols, and inorganic minerals—have been explored for single-cell encapsulation, limitations in controllability, biocompatibility, and multifunctional integration remain. In contrast, DNA nanomaterials offer unique advantages, including programmable architecture, high biocompatibility, precise spatial control, and modular functionality, making them highly suitable for the development of intelligent single-cell encapsulation systems. In this review, a systematic summary of recent advances in DNA nanomaterial-based single-cell encapsulation is presented. The fundamental encoding and assembly principles underlying the engineered encapsulation of cells at the membrane interface using DNA nanostructures are elucidated. Subsequently, the distinctive merits of DNA-based cell encapsulation and its applications in biomedical research are comprehensively summarized. Finally, the prevailing challenges and future directions in this burgeoning field are critically discussed, aiming to provide novel insights and perspectives for the advancement of advanced functional materials in both academic and clinical research pertaining to single-cell encapsulation.

Extracellular matrix (ECM) properties affect multiple cellular processes such as cell survival, proliferation, and protein synthesis. Thus, a polymeric‐cell delivery system with the ability to manipulate the extracellular environment can act as a fundamental regulator of cell function. Given the promise of stem cell therapeutics, a method to uniformly enhance stem cell function, in particular trophic factor release, can prove transformative in improving efficacy and increasing feasibility by reducing the total number of cells required. Herein, a click‐chemistry powered 3D, single‐cell encapsulation method aimed at synthesizing a polymeric coating with the optimal thickness around neural progenitor cells is introduced. Polymer encapsulation of neural stem cells significantly increases the release of neurotrophic factors such as VEGF and CNTF. Cell encapsulation with a soft extracellular polymer upregulates the ADCY8‐cAMP pathway, suggesting a mechanism for the increase in paracrine factors. Hence, the described single‐cell encapsulation technique can emerge as a translatable, nonviral cell modulation method and has the potential to improve stem cells' therapeutic effect. Through the use of cell glycoengineering techniques and click chemistry, single‐cell polymer encapsulation is possible. This polymeric extracellular matrix can modify the trophic factor production of neural progenitor cells. The cyclic adenosine monophosphate pathway and actin interactions are important in trophic factor regulation, specifically for VEGFB. The ability to encapsulate cells has implications for optimizing stem cell therapeutics.

Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive “smart” hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) proteins using genetically encoded SpyTag-SpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarHC hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.

Cell encapsulation has been shown to hold promise for effective, long-term treatment of type 1 diabetes (T1D). However, challenges remain for its clinical applications. For example, there is an unmet need for an encapsulation system that is capable of delivering sufficient cell mass while still allowing convenient retrieval or replacement. Here,we report a simple cell encapsulation design that is readily scalable and conveniently retrievable. The key to this design was to engineer a highly wettable, Ca2+-releasing nanoporous polymer thread that promoted uniform in situ cross-linking and strong adhesion of a thin layer of alginate hydrogel around the thread. The device provided immunoprotection of rat islets in immunocompetent C57BL/6 mice in a short-term (1-mo) study, similar to neat alginate fibers. However, the mechanical property of the device, critical for handling and retrieval, was much more robust than the neat alginate fibers due to the reinforcement of the central thread. It also had facile mass transfer due to the short diffusion distance. We demonstrated the therapeutic potential of the device through the correction of chemically induced diabetes in C57BL/6 mice using rat islets for 3 mo as well as in immunodeficient SCID-Beige mice using human islets for 4 mo. We further showed, as a proof of concept, the scalability and retrievability in dogs. After 1 mo of implantation in dogs, the device could be rapidly retrieved through a minimally invasive laparoscopic procedure. This encapsulation device may contribute to a cellular therapy for T1D because of its retrievability and scale-up potential.

Protein-based hydrogels have emerged as promising alternatives to synthetic hydrogels for biomedical applications, owing to the precise control of structure and function enabled by protein engineering. Nevertheless, strategies for assembling 3D molecular networks that carry the biological information encoded in full-length proteins remain underdeveloped. Here we present a robust protein gelation strategy based on a pair of genetically encoded reactive partners, SpyTag and SpyCatcher, that spontaneously form covalent isopeptide linkages under physiological conditions. The resulting “network of Spies” may be designed to include cell-adhesion ligands, matrix metalloproteinase-1 cleavage sites, and full-length globular proteins [mCherry and leukemia inhibitory factor (LIF)]. The LIF network was used to encapsulate mouse embryonic stem cells; the encapsulated cells remained pluripotent in the absence of added LIF. These results illustrate a versatile strategy for the creation of information-rich biomaterials.

Monitoring biological systems can be costly, destructive, lack robustness, and is hindered by limited real-time capabilities.Advancements in synthetic biology towards the construction of efficient whole-cell biosensors (WCB) offer a promising alternative to conventional analytical methods.The implementation of WCB requires containment to promote biosafety and the coexistence of different cell types to ensure functionality in real-world applications.Encapsulation methods can enable safety and effective communication between cells within complex biological systems. The emerging field of biosensors exploits the abilities of cells to identify specific molecules, presenting improved sensitivity, specificity, and limit of detection. Whole-cell biosensors (WCB) are organisms specifically engineered to detect a target analyte and express a reporter in response. In biomanufacturing, they can be used for monitoring of key substrate and metabolite concentrations or strain engineering, while in medicine, they can be used to diagnose disease or report on human–microbe interactions. Many applications require WCB to coexist with mammalian cells where a key challenge is to keep separate cell populations viable while still allowing them to interact. In this review, we highlight key considerations when encapsulating WCB to engineer controlled microenvironments that enable collaboration and coexistence of different populations. The emerging field of biosensors exploits the abilities of cells to identify specific molecules, presenting improved sensitivity, specificity, and limit of detection. Whole-cell biosensors (WCB) are organisms specifically engineered to detect a target analyte and express a reporter in response. In biomanufacturing, they can be used for monitoring of key substrate and metabolite concentrations or strain engineering, while in medicine, they can be used to diagnose disease or report on human–microbe interactions. Many applications require WCB to coexist with mammalian cells where a key challenge is to keep separate cell populations viable while still allowing them to interact. In this review, we highlight key considerations when encapsulating WCB to engineer controlled microenvironments that enable collaboration and coexistence of different populations.

Mesenchymal stem cell (MSC) therapies demonstrate particular promise in ameliorating diseases of immune dysregulation but are hampered by short in vivo cell persistence and inconsistencies in phenotype. Here, we demonstrate that biomaterial encapsulation into alginate using a microfluidic device could substantially increase in vivo MSC persistence after intravenous (i.v.) injection. A combination of cell cluster formation and subsequent cross-linking with polylysine led to an increase in injected MSC half-life by more than an order of magnitude. These modifications extended persistence even in the presence of innate and adaptive immunity-mediated clearance. Licensing of encapsulated MSCs with inflammatory cytokine pretransplantation increased expression of immunomodulatory-associated genes, and licensed encapsulates promoted repopulation of recipient blood and bone marrow with allogeneic donor cells after sublethal irradiation by a ∼2-fold increase. The ability ofmicrogel encapsulation to sustain MSC survival and increase overall immunomodulatory capacity may be applicable for improving MSC therapies in general.