An Autoclavable and Transparent Thermal Cutter for Reliably Sealing Wet Nanofibrous Membranes.

Sealing wet porous membranes is a major challenge when fabricating cell encapsulation devices. Herein, we report the development of an Autoclavable Transparent Thermal Cutter (ATTC) for reliably sealing wet nanofibrous membranes. Notably, the ATTC is autoclavable and transparent, thus enabling in situ visualization of the sealing process in a sterile environment and ensuring an appropriate seal. In addition, the ATTC could generate smooth, arbitrary-shaped sealing ends with excellent mechanical properties when sealing PA6, PVDF, and TPU nanofibrous tubes and PP microporous membranes. Importantly, the ATTC could reliably seal wet nanofibrous tubes, which can shoulder a burst pressure up to 313.2 ± 19.3 kPa without bursting at the sealing ends. Furthermore, the ATTC sealing process is highly compatible with the fabrication of cell encapsulation devices, as verified by viability, proliferation, cell escape, and cell function tests. We believe that the ATTC could be used to reliably seal cell encapsulation devices with minimal side effects.

Enhancing the Functionality of Immunoisolated Human SC-ßeta Cell Clusters through Prior Resizing.

The transplantation of immunoisolated stem cell derived beta cell clusters (SC-ß) has the potential to restore physiological glycemic control in patients with type I diabetes. This strategy is attractive as it uses a renewable ß-cell source without the need for systemic immune suppression. SC-ß cells have been shown to reverse diabetes in immune compromised mice when transplanted as ≈300 µm diameter clusters into sites where they can become revascularized. However, immunoisolated SC-ß clusters are not directly revascularized and rely on slower diffusion of nutrients through a membrane. It is hypothesized that smaller SC-ß cell clusters (≈150 µm diameter), more similar to islets, will perform better within immunoisolation devices due to enhanced mass transport. To test this, SC-ß cells are resized into small clusters, encapsulated in alginate spheres, and coated with a biocompatible A10 polycation coating that resists fibrosis. After transplantation into diabetic immune competent C57BL/6 mice, the "resized" SC-ß cells plus the A10 biocompatible polycation coating induced long-term euglycemia in the mice (6 months). After retrieval, the resized A10 SC-ß cells exhibited the least amount of fibrosis and enhanced markers of ß-cell maturation. The utilization of small SC-ß cell clusters within immunoprotection devices may improve clinical translation in the future.

Synthetic Cell Armor Made of DNA Origami.

The bioengineering applications of cells, such as cell printing and multicellular assembly, are directly limited by cell damage and death due to a harsh environment. Improved cellular robustness thus motivates investigations into cell encapsulation, which provides essential protection. Here we target the cell-surface glycocalyx and cross-link two layers of DNA nanorods on the cellular plasma membrane to form a modular and programmable nanoshell. We show that the DNA origami nanoshell modulates the biophysical properties of cell membranes by enhancing the membrane stiffness and lowering the lipid fluidity. The nanoshell also serves as armor to protect cells and improve their viability against mechanical stress from osmotic imbalance, centrifugal forces, and fluid shear stress. Moreover, it enables mediated cell-cell interactions for effective and robust multicellular assembly. Our results demonstrate the potential of the nanoshell, not only as a cellular protection strategy but also as a platform for cell and cell membrane manipulation.

Droplet-based microfluidics: an efficient high-throughput portable system for cell encapsulation.

One of the goals of tissue engineering and regenerative medicine is restoring primary living tissue function by manufacturing a 3D microenvironment. One of the main challenges is protecting implanted non-autologous cells or tissues from the host immune system. Cell encapsulation has emerged as a promising technique for this purpose. It involves entrapping cells in biocompatible and semi-permeable microcarriers made from natural or synthetic polymers that regulate the release of cellular secretions. In recent years, droplet-based microfluidic systems have emerged as powerful tools for cell encapsulation in tissue engineering and regenerative medicine. These systems offer precise control over droplet size, composition, and functionality, allowing for creating of microenvironments that closely mimic native tissue. Droplet-based microfluidic systems have extensive applications in biotechnology, medical diagnosis, and drug discovery. This review summarises the recent developments in droplet-based microfluidic systems and cell encapsulation techniques, as well as their applications, advantages, and challenges in biology and medicine. The integration of these technologies has the potential to revolutionise tissue engineering and regenerative medicine by providing a precise and controlled microenvironment for cell growth and differentiation. By overcoming the immune system's challenges and enabling the release of cellular secretions, these technologies hold great promise for the future of regenerative medicine.

Oral delivery of probiotics using single-cell encapsulation.

Adequate intake of live probiotics is beneficial to human health and wellbeing because they can help treat or prevent a variety of health conditions. However, the viability of probiotics is reduced by the harsh environments they experience during passage through the human gastrointestinal tract (GIT). Consequently, the oral delivery of viable probiotics is a significant challenge. Probiotic encapsulation provides a potential solution to this problem. However, the production methods used to create conventional encapsulation technologies often damage probiotics. Moreover, the delivery systems produced often do not have the required physicochemical attributes or robustness for food applications. Single-cell encapsulation is based on forming a protective coating around a single probiotic cell. These coatings may be biofilms or biopolymer layers designed to protect the probiotic from the harsh gastrointestinal environment, enhance their colonization, and introduce additional beneficial functions. This article reviews the factors affecting the oral delivery of probiotics, analyses the shortcomings of existing encapsulation technologies, and highlights the potential advantages of single-cell encapsulation. It also reviews the various approaches available for single-cell encapsulation of probiotics, including their implementation and the characteristics of the delivery systems they produce. In addition, the mechanisms by which single-cell encapsulation can improve the oral bioavailability and health benefits of probiotics are described. Moreover, the benefits, limitations, and safety issues of probiotic single-cell encapsulation technology for applications in food and beverages are analyzed. Finally, future directions and potential challenges to the widespread adoption of single-cell encapsulation of probiotics are highlighted.

Directing the Encapsulation of Single Cells with DNA Framework Nucleator-Based Hydrogel Growth.

Encapsulating individual mammalian cells with biomimetic materials holds potential in ex vivo cell culture and engineering. However, current methodologies often present tradeoffs between homogeneity, stability, and cell compatibility. Here, inspired by bacteria that use proteins stably anchored on their outer membranes to nucleate biofilm growth, we develop a single-cell encapsulation strategy by using a DNA framework structure as a nucleator (DFN) to initiate the growth of DNA hydrogels under cell-friendly conditions. We find that among the tested structures, the tetrahedral DFN can evenly and stably reside on cell membranes, effectively initiating hybridization chain reactions which generate homogeneously dense yet flexible single-cell encapsulation for diverse cell lines. The encapsulation persists for up to 72 hours in a serum-containing cell culture environment, representing a ~70-fold improvement compared to encapsulations mediated by single-stranded DNA nucleators. The metabolism and proliferation of the encapsulated cells are suppressed, but can be restored to the original efficiencies upon release, suggesting the superior cell compatibility of the encapsulation. We also find that compared to naked cells, the encapsulated cells exhibit a lower autophagy level after undergoing mechanical stress, suggesting the protective effect of the DNA encapsulation. This method may provide a new tool for ex vivo cell engineering.

An Outer Membrane-Inspired Polymer Coating Protects and Endows Escherichia coli with Novel Functionalities.

A bio-inspired membrane made of Pluronic L-121 is produced around Escherichia coli thanks to the simple co-extrusion of bacteria and polymer vesicles. The block copolymer-coated bacteria can withstand various harsh shocks, for example, temperature, pressure, osmolarity, and chemical agents. The polymer membrane also makes the bacteria resistant to enzymatic digestion and enables them to degrade toxic compounds, improving their performance as whole-cell biocatalysts. Moreover, the polymer membrane acts as an anchor layer for the surface modification of the bacteria. Being decorated with α-amylase or lysozyme, the cells are endowed with the ability to digest starch or self-predatory bacteria are created. Thus, without any genetic engineering, the phenotype of encapsulated bacteria is changed as they become sturdier and gain novel metabolic functionalities.

Metal-Phenolic Network Directed Coating of Single Probiotic Cell Followed by Photoinitiated Thiol-Ene Click Fortification to Enhance Oral Therapy.

Probiotics-based oral therapy has become a promising way to prevent and treat various diseases, while the application of probiotics is primarily restricted by loss of viability due to adverse conditions in the gastrointestinal (GI) tract during oral delivery. Layer-by-layer (LbL) single-cell encapsulation approaches are widely employed to improve the bioavailability of probiotics. However, they are generally time- and labor-intensive owing to multistep operation. Herein, a simple yet efficient LbL technique is developed to coat a model probiotic named Escherichia coli Nissle 1917 (EcN) through polyphenol-Ca2+ network directed allyl-modified gelatin (GelAGE) adsorption followed by cross-linking of GelAGE via photoinitiated thiol-ene click reaction to protect EcN from harsh microenvironments of GI tract. LbL single-cell encapsulation can be performed within 1 h through simple operation. It is revealed that coated EcN exhibits significantly improved viability against acidic gastric fluid and bile salts, and enhanced colonization in the intestinal tract without loss of proliferation capabilities. Furthermore, oral therapy of coated EcN remarkably relieves the pathological symptoms associated with colitis in mice including down-regulating inflammation, repairing epithelial barriers, scavenging reactive oxygen species (ROS), and restoring the homeostasis of gut microbiota. This simplified LbL coating strategy has great potential for various probiotics-mediated biomedical and nutraceutical applications.

Stem cells therapy for diabetes: from past to future.

Diabetes mellitus is a chronic disease of carbohydrate metabolism characterized by uncontrolled hyperglycemia due to the body's impaired ability to produce or respond to insulin. Oral or injectable exogenous insulin and its analogs cannot mimic endogenous insulin secreted by healthy individuals, and pancreatic and islet transplants face a severe shortage of sources and transplant complications, all of which limit the widespread use of traditional strategies in diabetes treatment. We are now in the era of stem cells and their potential in ameliorating human disease. At the same time, the rapid development of gene editing and cell-encapsulation technologies has added to the wings of stem cell therapy. However, there are still many unanswered questions before stem cell therapy can be applied clinically to patients with diabetes. In this review, we discuss the progress of strategies to obtain insulin-producing cells from different types of stem cells, the application of gene editing in stem cell therapy for diabetes, as well as summarize the current advanced cell encapsulation technologies in diabetes therapy and look forward to the future development of stem cell therapy in diabetes.

Microencapsulation of parathyroid cells via electric field and non-surgical transplantation approach.

PURPOSE: Hypoparathyroidism is a rare disease with low PTH, mostly seen as a consequence of neck surgery. Current management is the prescription of calcium and vitamin D, but the definitive treatment is parathyroid allotransplantation, which frequently triggers an immune response, thus cannot achieve the expected success. To overcome this problem, encapsulation of allogeneic cells is the most promising method. By optimizing the standard alginate cell encapsulation technique with parathyroid cells under high-voltage application, the authors reduced the size of parathyroid-encapsulated beads and evaluated these samples in vitro and in vivo. METHODS: Parathyroid cells were isolated, and standard-sized alginate macrobeads were prepared without any electrical field application, while microbeads in smaller sizes (< 500 µm), by the application of 13 kV. Bead morphologies, cell viability, and PTH secretion were evaluated in vitro for four weeks. For the in vivo part, beads were transplanted into Sprague-Dawley rats, and after retrieval, immunohistochemistry and PTH release were evaluated in addition to the assessment of cytokine/chemokine levels. RESULTS: The viability of parathyroid cells in micro- and macrobeads did not differ significantly. However, the amount of in vitro PTH secretion from microencapsulated cells was significantly lower than that from macroencapsulated cells, although it increased throughout the incubation period. Immunohistochemistry of PTH staining in both of the encapsulated cells identified as positive after retrieval. CONCLUSION: Contrary to the literature, a minimal in vivo immune response was developed for alginate-encapsulated parathyroid cells, regardless of bead size. Our findings suggest that injectable, micro-sized beads obtained using high-voltage may be a promising method for a non-surgical transplantation approach.

Building Fucoidan/Agarose-Based Hydrogels as a Platform for the Development of Therapeutic Approaches against Diabetes.

Current management for diabetes has stimulated the development of versatile 3D-based hydrogels as in vitro platforms for insulin release and as support for the encapsulation of pancreatic cells and islets of Langerhans. This work aimed to create agarose/fucoidan hydrogels to encapsulate pancreatic cells as a potential biomaterial for diabetes therapeutics. The hydrogels were produced by combining fucoidan (Fu) and agarose (Aga), marine polysaccharides derived from the cell wall of brown and red seaweeds, respectively, and a thermal gelation process. The agarose/fucoidan (AgaFu) blended hydrogels were obtained by dissolving Aga in 3 or 5 wt % Fu aqueous solutions to obtain different proportions (4:10; 5:10, and 7:10 wt). The rheological tests on hydrogels revealed a non-Newtonian and viscoelastic behavior, while the characterization confirmed the presence of the two polymers in the structure of the hydrogels. In addition, the mechanical behavior showed that increasing Aga concentrations resulted in hydrogels with higher Young's modulus. Further, the ability of the developed materials to sustain the viability of human pancreatic cells was assessed by encapsulation of the 1.1B4HP cell line for up to 7 days. The biological assessment of the hydrogels revealed that cultured pancreatic beta cells tended to self-organize and form pseudo-islets during the period studied.

Molecularly Tailored Interface for Long-Term Xenogeneic Cell Transplantation.

Encapsulation of therapeutic cells in a semipermeable device can mitigate the need for systemic immune suppression following cell transplantation by providing local immunoprotection while being permeable to nutrients, oxygen, and different cell-secreted biomolecules. However, fibrotic tissue deposition around the device has been shown to compromise the long-term function of the transplanted cells. Herein, a macroencapsulation device design that improves long-term survival and function of the transplanted cells is reported. The device is comprised of a semipermeable chitosan pouch with a tunable reservoir and molecularly engineered interface. The chitosan pouch interface decorated with 1,12-dodecanedioic acid (DDA), limits the cell adhesion and vigorous foreign body response while maintaining the barrier properties amenable to cell encapsulation. The device provides long-term protection to the encapsulated human primary hepatocytes in the subcutaneous space of immunocompetent mice. The device supports the encapsulated cells for up to 6 months as evident from cell viability and presence of human specific albumin in circulation. Solutions that integrate biomaterials and interfacial engineering such as the one described here may advance development of easy-to manufacture and retrievable devices for the transplantation of therapeutic cells in the absence of immunosuppression.

A Safe, Fibrosis-Mitigating, and Scalable Encapsulation Device Supports Long-Term Function of Insulin-Producing Cells.

Encapsulation and transplantation of insulin-producing cells offer a promising curative treatment for type 1 diabetes (T1D) without immunosuppression. However, biomaterials used to encapsulate cells often elicit foreign body responses, leading to cellular overgrowth and deposition of fibrotic tissue, which in turn diminishes mass transfer to and from transplanted cells. Meanwhile, the encapsulation device must be safe, scalable, and ideally retrievable to meet clinical requirements. Here, a durable and safe nanofibrous device coated with a thin and uniform, fibrosis-mitigating, zwitterionically modified alginate hydrogel for encapsulation of islets and stem cell-derived beta (SC-ß) cells is reported. The device with a configuration that has cells encapsulated within the cylindrical wall, allowing scale-up in both radial and longitudinal directions without sacrificing mass transfer, is designed. Due to its facile mass transfer and low level of fibrotic reactions, the device supports long-term cell engraftment, correcting diabetes in C57BL6/J mice with rat islets for up to 399 days and SCID-beige mice with human SC-ß cells for up to 238 days. The scalability and retrievability in dogs are further demonstrated. These results suggest the potential of this new device for cell therapies to treat T1D and other diseases.

Controlled generation of droplets using an electric field in a flow-focusing paper-based device.

Droplet-based microfluidics is a modular platform in high-throughput single-cell and small sample analyses. However, this droplet microfluidic system was widely fabricated using soft lithography or glass capillaries, which is expensive and technically demanding for various applications, limiting use in resource-poor settings. Besides, the variation in droplet size is also restricted due to the limitations on the operating forces that the paper-based platform is able to withstand. Herein, we develop a fully integrated paper-based droplet microfluidic platform for conducting droplet generation and cell encapsulation in independent aqueous droplets dispersed in a carrier oil by incorporating electric fields. Through imposing an electric field, the droplet size would decrease with increasing the electric field and smaller droplets can be produced at high applied voltage. The droplet diameter can be adjusted by the ratio of inner and outer flow velocities as well as the applied electric field. We also demonstrated the proof of concept encapsulation application of our paper device by encapsulating yeast cells under an electric field. Using a simple wax printing method, carbon electrodes can be integrated on the paper. The integrated paper-based microfluidic platform can be fabricated easily and conducted outside of centralized laboratories. This microfluidic system shows great potential in drug and cell investigations by encapsulating cells in resource-limited environments.

New hyaluronan-based biomatrix for 3-D follicle culture yields functionally competent oocytes.

BACKGROUND: Encapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of the natural extracellular matrix (ECM) that provides support to cells in vivo. This report describes the application of a novel tyramine-linked hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes. MATERIALS AND METHODS: Enzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml of hyaluronan gel (HA). Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Functional competence of in vitro matured (IVM) oocytes was evaluated by in vitro fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes. RESULTS: A total of 402 preantral follicles were cultured in HA gel. After hCG trigger, 314 oocyte-cumulus complexes ovulated from the embedded follicles. Meiotic maturation rate to the metaphase II stage was 73% (228/314). After insemination 83% (188/228) of IVM oocytes fertilized with a subsequent blastulation rate of 46% (87/188). A pilot transfer study with 3 recipient mice resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6-8 preantral follicles. Meiotic maturation was lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p < 0.007). Modification of the HA gel with ECMs or laminin affected antrum formation and follicle retention. Maturation rates to the metaphase II stage were however not significantly different: 74% for HA gel alone as compared to HA-LM (67%), HA-MG (56%) and HA-PM (58%). CONCLUSION: Hyaluronan gel is an effective and versatile extracellular matrix based biomaterial for 3-D culture of ovarian follicles. This culture model allowed ovulation of functionally competent metaphase II oocytes, capable of fertilization, genomic activation and blastulation. Future testing with human follicles that require longer in vitro culture times should be considered.

Reversible crosslinked assembly of a trimeric coiled-coil peptide into a three-dimensional matrix for cell encapsulation and release.

Mimicking the extracellular matrix (ECM) continues to be a goal in the field of regenerative medicine. Herein, we report a modified trimeric GCN4 coiled-coil sequence containing three ligands for metal ions specifically positioned for crosslinked assembly (TriCross). In the presence of metal ions, TriCross assembles into a three-dimensional (3D) matrix with significant cavities to accommodate cells. The matrix was found to be stable in media with serum, and mild removal of the metal leads to disassembly. By assembling TriCross with a suspension of cells in media, the matrix encapsulates cells during the assembly process leading to high cell viability. Further disassembly under mild conditions allows for the release of cells from the scaffold. As such, this peptide-based material displays many of the characteristics necessary for successful 3D cell culture.

From 3D printing to 3D bioprinting: the material properties of polymeric material and its derived bioink for achieving tissue specific architectures.

The application of 3D printing technologies fields for biological tissues, organs, and cells in the context of medical and biotechnology applications requires a significant amount of innovation in a narrow printability range. 3D bioprinting is one such way of addressing critical design challenges in tissue engineering. In a more general sense, 3D printing has become essential in customized implant designing, faithful reproduction of microenvironmental niches, sustainable development of implants, in the capacity to address issues of effective cellular integration, and long-term stability of the cellular constructs in tissue engineering. This review covers various aspects of 3D bioprinting, describes the current state-of-the-art solutions for all aforementioned critical issues, and includes various illustrative representations of technologies supporting the development of phases of 3D bioprinting. It also demonstrates several bio-inks and their properties crucial for being used for 3D printing applications. The review focus on bringing together different examples and current trends in tissue engineering applications, including bone, cartilage, muscles, neuron, skin, esophagus, trachea, tympanic membrane, cornea, blood vessel, immune system, and tumor models utilizing 3D printing technology and to provide an outlook of the future potentials and barriers.

Hydrogen-Bonded Organic Framework (HOF)-Based Single-Neural Stem Cell Encapsulation and Transplantation to Remodel Impaired Neural Networks.

Herein we present a new way to encapsulate neural stem cells (NSCs) by using hydrogen-bonded organic frameworks (HOFs) to overcome the common causes of low therapeutic efficacy during NSC transplantation: 1) loss of fundamental stem cell properties, "stemness", before transplantation, 2) cytomembrane damage during transplantation, and 3) apoptosis due to oxidative stress after transplantation. Porous carbon nanospheres (PCNs) are doped into the HOF shell during the process of mineralization to endow the cellular exoskeletons with hierarchical hydrogen bonds, and the ability to resist oxidative stress due to the catalase and superoxide dismutase-like activities of PCN. Under NIR-II irradiation, thermal-responsive hydrogen bonds dissociate to release NSCs. Stereotactic transplanting encapsulated NSC into the brain of an Alzheimer's disease (AD) mouse model further verifies that our design can enhance NSC viability, promote neurogenesis, and ameliorate cognitive impairment. As the first example of using HOFs to encapsulate NSCs, this work may inspire the design of HOF-based exoskeletons to ameliorate neurogenesis and cognitive behavioral symptoms associated with AD.

Manipulation of Multiple Cell-Cell Interactions by Tunable DNA Scaffold Networks.

Manipulation of cell-cell interactions via cell surface engineering has potential biomedical applications in tissue engineering and cell therapy. However, manipulation of the comprehensive and multiple intercellular interactions remains a challenge and missing elements. Herein, utilizing a DNA triangular prism (TP) and a branched polymer (BP) as functional modules, we fabricate tunable DNA scaffold networks on the cell surface. The responsiveness of cell-cell recognition, aggregation and dissociation could be modulated by aptamer-functionalized DNA scaffold networks with high accuracy and specificity. By regulating the DNA scaffold networks coated on the cell surface, controlled intercellular molecular transportation is achieved. Our tunable network provides a simple and extendible strategy which addresses a current need in cell surface engineering to precisely manipulate cell-cell interactions and shows promise as a general tool for controllable cell behavior.

Engineered cell-laden alginate microparticles for 3D culture.

Advanced microfabrication technologies and biocompatible hydrogel materials facilitate the modeling of 3D tissue microenvironment. Encapsulation of cells in hydrogel microparticles offers an excellent high-throughput platform for investigating multicellular interaction with their surrounding microenvironment. Compartmentalized microparticles support formation of various unique cellular structures. Alginate has emerged as one of the most dominant hydrogel materials for cell encapsulation owing to its cytocompatibility, ease of gelation, and biocompatibility. Alginate hydrogel provides a permeable physical boundary to the encapsulated cells and develops an easily manageable 3D cellular structure. The interior structure of alginate hydrogel can further regulate the spatiotemporal distribution of the embedded cells. This review provides a specific overview of the representative engineering approaches to generate various structures of cell-laden alginate microparticles in a uniform and reproducible manner. Capillary nozzle systems, microfluidic droplet systems, and non-chip based high-throughput microfluidic systems are highlighted for developing well-regulated cellular structure in alginate microparticles to realize potential drug screening platform and cell-based therapy. We conclude with the discussion of current limitations and future directions for realizing the translation of this technology to the clinic.