Effects of Transglutaminase Concentration and Drying Method on Encapsulation of Lactobacillus plantarum in Gelatin-Based Hydrogel.

Lactobacillus plantarum is a kind of probiotic that benefits the host by regulating the gut microbiota, but it is easily damaged when passing through the gastrointestinal tract, hindering its ability to reach the destination and reducing its utilization value. Encapsulation is a promising strategy for solving this problem. In this study, transglutaminase (TGase)-crosslinked gelatin (GE)/sodium hexametaphosphate (SHMP) hydrogels were used to encapsulate L. plantarum. The effects of TGase concentration and drying method on the physiochemical properties of the hydrogels were determined. The results showed that at a TGase concentration of 9 U/gGE, the hardness, chewiness, energy storage modulus, and apparent viscosity of the hydrogel encapsulation system were maximized. This concentration produced more high-energy isopeptide bonds, strengthening the interactions between molecules, forming a more stable three-dimensional network structure. The survival rate under the simulated gastrointestinal conditions and storage stability of L. plantarum were improved at this concentration. The thermal stability of the encapsulation system dried via microwave vacuum freeze drying (MFD) was slightly higher than that when dried via freeze drying (FD). The gel structure was more stable, and the activity of L. plantarum decreased more slowly during the storage period when dried using MFD. This research provides a theoretical basis for the development of encapsulation technology of probiotics.

Microfiber-reinforced hydrogels prolong the release of human induced pluripotent stem cell-derived extracellular vesicles to promote endothelial migration.

Extracellular vesicle (EV)-based approaches for promoting angiogenesis have shown promising results. Yet, further development is needed in vehicles that prolong EV exposure to target organs. Here, we hypothesized that microfiber-reinforced gelatin methacryloyl (GelMA) hydrogels could serve as sustained delivery platforms for human induced pluripotent stem cell (hiPSC)-derived EV. EV with 50-200 nm size and typical morphology were isolated from hiPSC-conditioned culture media and tested negative for common co-isolated contaminants. hiPSC-EV were then incorporated into GelMA hydrogels with or without a melt electrowritten reinforcing mesh. EV release was found to increase with GelMA concentration, as 12 % (w/v) GelMA hydrogels provided higher release rate and total release over 14 days in vitro, compared to lower hydrogel concentrations. Release profile modelling identified diffusion as a predominant release mechanism based on a Peppas-Sahlin model. To study the effect of reinforcement-dependent hydrogel mechanics on EV release, stress relaxation was assessed. Reinforcement with highly porous microfiber meshes delayed EV release by prolonging hydrogel stress relaxation and reducing the swelling ratio, thus decreasing the initial burst and overall extent of release. After release from photocrosslinked reinforced hydrogels, EV remained internalizable by human umbilical vein endothelial cells (HUVEC) over 14 days, and increased migration was observed in the first 4 h. EV and RNA cargo stability was investigated at physiological temperature in vitro, showing a sharp decrease in total RNA levels, but a stable level of endothelial migration-associated small noncoding RNAs over 14 days. Our data show that hydrogel formulation and microfiber reinforcement are superimposable approaches to modulate EV release from hydrogels, thus depicting fiber-reinforced GelMA hydrogels as tunable hiPSC-EV vehicles for controlled release systems that promote endothelial cell migration.

Hydrogel-encapsulation to enhance bacterial diagnosis of colon inflammation.

Bacteria can be genetically programmed to sense and report the presence of disease biomarkers in the gastrointestinal (GI) tract. However, diagnostic bacteria are typically delivered via oral administration of liquid cultures, resulting in poor survival and high dispersal in vivo. These limitations confound recovery and analysis of engineered bacteria from GI or stool samples. Here, we demonstrate that encapsulating bacteria inside of alginate core-shell particles enables robust survival, containment, and diagnostic function in vivo. We demonstrate these benefits by encapsulating a strain engineered to report the presence of the biomarker thiosulfate via fluorescent protein expression in order to diagnose dextran sodium sulfate-induced colitis in rats. Hydrogel-encapsulated bacteria engineered to sense and respond to physiological stimuli should enable minimally invasive monitoring of a wide range of diseases and have applications as next-generation smart therapeutics.

Toxicity of per- and polyfluoroalkyl substances to microorganisms in confined hydrogel structures.

Per- and polyfluoroalkyl substances (PFAS) as a group of environmentally persistent synthetic chemicals has been widely used in industrial and consumer products. Bioaccumulation studies have documented the adverse effects of PFAS in various living organisms. Despite the large number of studies, experimental approaches to evaluate the toxicity of PFAS on bacteria in a biofilm-like niche as structured microbial communities are sparse. This study suggests a facile approach to query the toxicity of PFOS and PFOA on bacteria (Escherichia coli K12 MG1655 strain) in a biofilm-like niche provided by hydrogel-based core-shell beads. Our study shows that E. coli MG1655 upon complete confinement in hydrogel beads exhibit altered physiological characteristics of viability, biomass, and protein expression, compared to their susceptible counterpart cultivated under planktonic conditions. We find that soft-hydrogel engineering platforms may provide a protective role for microorganisms from environmental contaminants, depending on the size or thickness of the protective/barrier layer. We expect our study to provide insights on the toxicity of environmental contaminants on organisms under encapsulated conditions that could potentially be useful for toxicity screening and in evaluating ecological risk of soil, plant, and mammalian microbiome.

Transplantation of encapsulated autologous olfactory ensheathing cell populations expressing chondroitinase for spinal cord injury: A safety and feasibility study in companion dogs.

Spinal cord injury (SCI) can cause irreversible paralysis, with no regenerative treatment clinically available. Dogs with natural SCI present an established model and can facilitate translation of experimental findings in rodents to people. We conducted a prospective, single arm clinical safety study in companion dogs with chronic SCI to characterize the feasibility of intraspinal transplantation of hydrogel-encapsulated autologous mucosal olfactory ensheathing cell (mOEC) populations expressing chondroitinase ABC (chABC). mOECs and chABC are both promising therapies for SCI, and mOECs expressing chABC drive greater voluntary motor recovery than mOECs alone after SCI in rats. Canine mOECs encapsulated in collagen hydrogel can be matched in stiffness to canine SCI. Four dogs with complete and chronic loss of function caudal to a thoraco-lumbar lesion were recruited. After baseline measures, olfactory mucosal biopsy was performed and autologous mOECs cultured and transduced to express chABC, then hydrogel-encapsulated and percutaneously injected into the spinal cord. Dogs were monitored for 6 months with repeat clinical examinations, spinal MRI, kinematic gait and von Frey assessment. No adverse effects or significant changes on neurological examination were detected. MRI revealed large and variable lesions, with no spinal cord compression or ischemia visible after hydrogel transplantation. Owners reported increased pelvic-limb reflexes with one dog able to take 2-3 unsupported steps, but gait-scoring and kinematic analysis showed no significant improvements. This novel combination approach to regeneration after SCI is therefore feasible and safe in paraplegic dogs in a clinical setting. A randomised-controlled trial in this translational model is proposed to test efficacy.

Polymer Encapsulation of Bacterial Biosensors Enables Coculture with Mammalian Cells.

Coexistence of different populations of cells and isolation of tasks can provide enhanced robustness and adaptability or impart new functionalities to a culture. However, generating stable cocultures involving cells with vastly different growth rates can be challenging. To address this, we developed living analytics in a multilayer polymer shell (LAMPS), an encapsulation method that facilitates the coculture of mammalian and bacterial cells. We leverage LAMPS to preprogram a separation of tasks within the coculture: growth and therapeutic protein production by the mammalian cells and l-lactate biosensing by Escherichia coli encapsulated within LAMPS. LAMPS enable the formation of a synthetic bacterial-mammalian cell interaction that enables a living biosensor to be integrated into a biomanufacturing process. Our work serves as a proof-of-concept for further applications in bioprocessing since LAMPS combine the simplicity and flexibility of a bacterial biosensor with a viable method to prevent runaway growth that would disturb mammalian cell physiology.

Hydrogel-Assisted 3D Volumetric Hotspot for Sensitive Detection by Surface-Enhanced Raman Spectroscopy.

Effective hotspot engineering with facile and cost-effective fabrication procedures is critical for the practical application of surface-enhanced Raman spectroscopy (SERS). We propose a SERS substrate composed of a metal film over polyimide nanopillars (MFPNs) with three-dimensional (3D) volumetric hotspots for this purpose. The 3D MFPNs were fabricated through a two-step process of maskless plasma etching and hydrogel encapsulation. The probe molecules dispersed in solution were highly concentrated in the 3D hydrogel networks, which provided a further enhancement of the SERS signals. SERS performance parameters such as the SERS enhancement factor, limit-of-detection, and signal reproducibility were investigated with Cyanine5 (Cy5) acid Raman dye solutions and were compared with those of hydrogel-free MFPNs with two-dimensional hotspots. The hydrogel-coated MFPNs enabled the reliable detection of Cy5 acid, even when the Cy5 concentration was as low as 100 pM. We believe that the 3D volumetric hotspots created by introducing a hydrogel layer onto plasmonic nanostructures demonstrate excellent potential for the sensitive and reproducible detection of toxic and hazardous molecules.

Thiol-ene cross-linked alginate hydrogel encapsulation modulates the extracellular matrix of kidney organoids by reducing abnormal type 1a1 collagen deposition.

Differentiated kidney organoids from induced pluripotent stem cells hold promise as a treatment for patients with kidney diseases. Before these organoids can be translated to the clinic, shortcomings regarding their cellular and extracellular compositions, and their developmental plateau need to be overcome. We performed a proteomic analysis on kidney organoids cultured for a prolonged culture time and we found a specific change in the extracellular matrix composition with increased expression of types 1a1, 2 and 6a1 collagen. Such an excessive accumulation of specific collagen types is a hallmark of renal fibrosis that causes a life-threatening pathological condition by compromising key functions of the human kidney. Here we hypothesized the need for a three-dimensional environment to grow the kidney organoids, which could better mimic the in vivo surroundings of the developing kidney than standard culture on an air-liquid interface. Encapsulating organoids for four days in a soft, thiol-ene cross-linked alginate hydrogel resulted in decreased type 1a1 collagen expression. Furthermore, the encapsulation did not result in any changes of organoid structural morphology. Using a biomaterial to modulate collagen expression allows for a prolonged kidney organoid culture in vitro and a reduction of abnormal type 1a1 collagen expression bringing kidney organoids closer to clinical application.

Dimethyl sulfoxide-free cryopreservation for cell therapy: A review.

Cell-based therapeutics promise to transform the treatment of a wide range of diseases including cancer, genetic and degenerative disorders, or severe injuries. Many of the commercial and clinical development of cell therapy products require cryopreservation and storage of cellular starting materials, intermediates and/or final products at cryogenic temperature. Dimethyl sulfoxide (Me2SO) has been the cryoprotectant of choice in most biobanking situations due to its exceptional performance in mitigating freezing-related damages. However, there is concern over the toxicity of Me2SO and its potential side effects after administration to patients. Therefore, there has been growing demand for robust Me2SO-free cryopreservation methods that can improve product safety and maintain potency and efficacy. This article provides an overview of the recent advances in Me2SO-free cryopreservation of cells having therapeutic potentials and discusses a number of key challenges and opportunities to motivate the continued innovation of cryopreservation for cell therapy.

Hydrogel-Encapsulated Enzyme Facilitates Colorimetric Acute Toxicity Assessment of Heavy Metal Ions.

Conventional analysis of heavy metal ions in water requires highly skilled staff and sophisticated equipment. These limitations make conventional approaches difficult to perform analysis on-site without delay. Herein, we report a facile colorimetric sensing system developed for acute toxicity assessment of heavy metal ions. A bioactive enzyme, ß-galactosidase, was used as sensing agent rather than bacteria or other higher organisms to improve selectivity and response time. The developed bioassay is capable of assessing the toxicity of heavy metal ions such as Hg(II), Cd(II), Pb(II), and Cu(II). The effects of enzyme concentration on the assessing performances (i.e., sensitivity and response time) of bioassay were explored and illustrated. Generally, low enzyme concentration facilitates sensitivity enhancement, achieving a 50% inhibiting concentration (IC50) of 0.76 µM (=152 ppb) Hg(II), and high enzyme concentration ensures quick response, enabling a response time down to 9 min. Moreover, the enzyme and substrate were respectively encapsulated by hydrogel to further simplify the assay procedure and enhance the stability of the enzyme. The hydrogel-encapsulated enzyme worked well even when heated up to 60 °C and retained ca. 90% activity after storage for 5 months. Moreover, the developed toxicity-assessing system is feasible for assessing toxicity of actual water samples. This assay approach is low cost and time effective and has no potential ethic issues. In addition, this work paves the way for the development of toxicity assessment kits for on-site analysis based on functional bioactive molecules.

Ectopic models for endochondral ossification: comparing pellet and alginate bead culture methods.

Key aspects of native endochondral bone development and fracture healing can be mimicked in mesenchymal stem cells (MSCs) through standard in vitro chondrogenic induction. Exploiting this phenomenon has recently emerged as an attractive technique to engineer bone tissue, however, relatively little is known about the best conditions for doing so. The objective of the present study was to compare the bone-forming capacity and angiogenic induction of hypertrophic cell constructs containing human adipose-derived stem cells (hASCs) primed for chondrogenesis in two different culture systems: high-density pellets and alginate bead hydrogels. The hASC constructs were subjected to 4 weeks of identical chondrogenic induction in vitro, encapsulated in an agarose carrier, and then implanted subcutaneously in immune-compromised mice for 8 weeks to evaluate their endochondral potential. At the time of implantation, both pellets and beads expressed aggrecan and type II collagen, as well as alkaline phosphatase (ALP) and type X collagen. Interestingly, ASCs in pellets formed a matrix containing higher glycosaminoglycan and collagen contents than that in beads, and ALP activity per cell was higher in pellets. However, after 8 weeks in vivo, pellets and beads induced an equivalent volume of mineralized tissue and a comparable level of vascularization. Although osteocalcin and osteopontin-positive osteogenic tissue and new vascular growth was found within both types of constructs, all appeared to be better distributed throughout the hydrogel beads. The results of this ectopic model indicate that hydrogel culture may be an attractive alternative to cell pellets for bone tissue engineering via the endochondral pathway. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of Gelatin Microparticles as Adherent-Substrates for Mesenchymal Stem Cells in a Hydrogel Composite.

Due to the lack of cell-adhesive moieties in traditional synthetic hydrogels, the present work investigated the use of degradable gelatin microparticles (GMPs) as temporary adherent substrates for anchorage-dependent mesenchymal stem cells (MSCs). MSCs were seeded onto GMPs of varying crosslinking densities and sizes to investigate their role on influencing MSC differentiation and aggregation. The MSC-seeded GMPs were then encapsulated in poly(ethylene glycol)-based hydrogels and cultured in serum-free, growth factor-free osteochondral medium. Non-seeded MSCs co-encapsulated with GMPs in the hydrogels were used as a control for comparison. Over the course of 35 days, MSCs seeded on GMPs exhibited more cell-cell contacts, greater chondrogenic potential, and a down-regulation of osteogenic markers compared to the controls. Although the factors of GMP crosslinking and size had nominal influence on MSC differentiation and aggregation, GMPs demonstrate potential as an adherent-substrate for improving cell delivery from hydrogel scaffolds by facilitating cell-cell contacts and improving MSC differentiation.