Tuning the crosslinking and degradation of hyaluronic acid/gelatin hydrogels using hydrogen peroxide for muscle cell sheet fabrication.

Cell sheets have immense potential for medical and pharmaceutical applications including tissue regeneration, drug testing, and disease modelling. In this study, composite hydrogels were prepared from a mixture of phenolated hyaluronic acid (HA-Ph) and gelatin (Gelatin-Ph), with a controlled degree of polymer crosslinking and degradation, to fabricate muscle cell sheets from myoblasts. These hydrogels were obtained via hydrogen peroxide (H2O2)-mediated crosslinking catalysed by horseradish peroxidase (HRP) and peroxide-mediated cleavage of the polymer chains. The degrees of crosslinking and degradation were modulated by altering the exposure time to air containing H2O2. The results showed that exposing a solution of 2% w/v HA-Ph, 0.75% w/v Gelatin-Ph, and 1 unit mL-1 HRP to air with 16 ppm H2O2 for 60 min yielded a stiffer hydrogel (7.16 kPa Young's modulus) than exposure times of 15 min (0.46 kPa) and 120 min (3.98 kPa). Moreover, mouse myoblast C2C12 cells cultured on a stiff hydrogel and induced to undergo myogenic differentiation formed longer and higher-density myotubes than those on softer hydrogels. The cell sheets were readily detached within 5 min by immersing the HA-Ph/Gelatin-Ph hydrogels covered with a monolayer of cells in a medium containing hyaluronidase. Our findings demonstrate that composite hydrogels with properties tuned by controlling the exposure time to H2O2, show great promise as platforms for muscle cell sheet fabrication.

Interaction of alginate with nano-hydroxyapatite-collagen using strontium provides suitable osteogenic platform.

BACKGROUND: Hydrogels based on organic/inorganic composites have been at the center of attention for the fabrication of engineered bone constructs. The establishment of a straightforward 3D microenvironment is critical to maintaining cell-to-cell interaction and cellular function, leading to appropriate regeneration. Ionic cross-linkers, Ca2+, Ba2+, and Sr2+, were used for the fabrication of Alginate-Nanohydroxyapatite-Collagen (Alg-nHA-Col) microspheres, and osteogenic properties of human osteoblasts were examined in in vitro and in vivo conditions after 21 days. RESULTS: Physicochemical properties of hydrogels illustrated that microspheres cross-linked with Sr2+ had reduced swelling, enhanced stability, and mechanical strength, as compared to the other groups. Human MG-63 osteoblasts inside Sr2+ cross-linked microspheres exhibited enhanced viability and osteogenic capacity indicated by mineralization and the increase of relevant proteins related to bone formation. PCR (Polymerase Chain Reaction) array analysis of the Wnt (Wingless-related integration site) signaling pathway revealed that Sr2+ cross-linked microspheres appropriately induced various signaling transduction pathways in human osteoblasts leading to osteogenic activity and dynamic growth. Transplantation of Sr2+ cross-linked microspheres with rat osteoblasts into cranium with critical size defect in the rat model accelerated bone formation analyzed with micro-CT and histological examination. CONCLUSION: Sr2+ cross-linked Alg-nHA-Col hydrogel can promote functionality and dynamic growth of osteoblasts.

Successful introduction of robotic-assisted percutaneous coronary intervention system into Japanese clinical practice: a first-year survey at single center.

In Japan, a robotic-assisted PCI (R-PCI) system, the CorPath GRX System (Corindus Inc.), has been approved for clinical use in 2018, which is the first introduction of R-PCI into Japan. In this study, the clinical performance of the R-PCI system in the initial year at Kurume University Hospital was evaluated comparing with conventional manual PCI (M-PCI). A total of 30 R-PCI and 77 M-PCI procedures performed between April 2019 and March 2020, were retrospectively included. The primary outcome was the rate of clinical success defined as < 30% residual stenosis without in-hospital major adverse cardiovascular events (MACE). The secondary outcomes were fluoroscopy time, dose area product (DAP), amount of radiation exposure to operators and assistants, procedural time, and contrast volume. Propensity-matching technique was used to match each R-PCI lesion to the nearest M-PCI lesion without replacement. After propensity score matching, 30 R-PCI procedures in 28 patients and 37 M-PCI procedures in 35 patients were analyzed. Clinical success rate with R-PCI was favorable and comparable to M-PCI (93.3 vs. 94.6%, p = 0.97), without any in-hospital MACE. The operator radiation exposure was significantly lower in R-PCI (0 vs. 24.5 µSV, p < 0.0001). Radiation exposure to the patients was tended to be reduced by R-PCI (DAP: 77.6 vs. 100.2 Gycm2, p = 0.07). There were no statistically significant differences in radiation exposure to the assistant, fluoroscopy time, procedural time and contrast volume between the two groups (radiation exposure to the assistant: 10.5 vs. 10.0 µSV, p = 0.64, fluoroscopy time: 27.5 vs. 30.1 min, p = 0.55, procedural time: 72.4 vs. 61.6 min, p = 0.23, and contrast volume: 93.2 vs. 102.0 ml, p = 0.36). R-PCI in selected patients demonstrated favorable clinical outcomes with dramatical reduction of radiation exposure to operators.

Horseradish peroxidase-catalyzed hydrogelation consuming enzyme-produced hydrogen peroxide in the presence of reducing sugars.

In the present work, three kinds of reducing sugars: glucose, galactose, and mannose, are applied to horseradish peroxidase (HRP)-catalyzed hydrogelation of an aqueous solution containing natural polymers modified with phenolic hydroxyl moieties. In this system, HRP consumes hydrogen peroxide that was generated from the oxidation of thiol groups in HRP in the presence of reducing sugars. Herein, we highlight the versatility of applicable sugar types and the controllable hydrogel properties. The mechanical properties and microstructures of the resultant hydrogels can be well controlled by varying the concentration and the reducing power of sugars. Moreover, reducing sugar-independent cytocompatibility of the hydrogels was confirmed by the growth of cells on them. The wide selection of sugar types provides a better understanding of the reaction mechanism and enables the characterization of hydrogels with well-controlled properties.

Differentiation potential of human adipose stem cells bioprinted with hyaluronic acid/gelatin-based bioink through microextrusion and visible light-initiated crosslinking.

Bioprinting has a great potential to fabricate three-dimensional (3D) functional tissues and organs. In particular, the technique enables fabrication of 3D constructs containing stem cells while maintaining cell proliferation and differentiation abilities, which is believed to be promising in the fields of tissue engineering and regenerative medicine. We aimed to demonstrate the utility of the bioprinting technique to create hydrogel constructs consisting of hyaluronic acid (HA) and gelatin derivatives through irradiation by visible light to fabricate 3D constructs containing human adipose stem cells (hADSCs). The hydrogel was obtained from a solution of HA and gelatin derivatives possessing phenolic hydroxyl moieties in the presence of ruthenium(II) tris-bipyridyl dication and sodium ammonium persulfate. hADSCs enclosed in the bioprinted hydrogel construct elongated and proliferated in the hydrogel. In addition, their differentiation potential was confirmed by examining the expression of pluripotency marker genes and cell surface marker proteins, and differentiation to adipocytes in adipogenic differentiation medium. Our results demonstrate the great potential of the bioprinting method and the resultant hADSC-laden HA/gelatin constructs for applications in tissue engineering and regenerative medicine.

Visible Light-Induced Hydrogelation of an Alginate Derivative and Application to Stereolithographic Bioprinting Using a Visible Light Projector and Acid Red.

Visible light-induced hydrogelation is attractive for various biomedical applications. In this study, hydrogels of alginate with phenolic hydroxyl groups (Alg-Ph) were obtained by irradiating a solution containing the polymer, ruthenium II trisbipyridyl chloride ([Ru(bpy)3]2+) and sodium persulfate (SPS), with visible light. The hydrogelation kinetics and the mechanical properties of the resultant hydrogels were tunable by controlling the intensity of the light and the concentrations of [Ru(bpy)3]2+ and SPS. With appropriate concentrations of [Ru(bpy)3]2+ and SPS, the hydrogel could be obtained following approximately 10 s of irradiation using a normal desktop lamp. The hydrogelation process and the resultant hydrogel were cytocompatible; mouse fibroblast cells enclosed in the Alg-Ph hydrogel maintained more than 90% viability for 1 week. The solution containing Alg-Ph, [Ru(bpy)3]2+ and SPS was useful as a bioink for stereolithographic bioprinting. Cell-laden hydrogel constructs could be printed using the bioprinting system equipped with a visible light projector without a significant decrease in cell viability in the presence of photoabsorbent Acid Red 18. The hydrogel construct including a perfusable helical lumen of 1 mm in diameter could be fabricated using the printing system. These results demonstrate the significant potential of this visible light-induced hydrogelation system and the stereolithographic bioprinting using the hydrogelation system for tissue engineering and regenerative medicine.

Drop-On-Drop Multimaterial 3D Bioprinting Realized by Peroxidase-Mediated Cross-Linking.

A cytocompatible inkjet bioprinting approach that enables the use of a variety of bioinks to produce hydrogels with a wide range of characteristics is developed. Stabilization of bioinks is caused by horseradish peroxidase (HRP)-catalyzed cross-linking consuming hydrogen peroxide (H2 O2 ). 3D cell-laden hydrogels are fabricated by the sequential dropping of a bioink containing polymer(s) cross-linkable through the enzymatic reaction and H2 O2 onto droplets of another bioink containing the polymer, HRP, and cells. The ≈95% viability of enclosed mouse fibroblasts and subsequent elongation of the cells in a bioprinted hydrogel consisting of gelatin and hyaluronic acid derivatives suggest the high cytocompatibility of the developed printing approach. The existence of numerous polymers, including derivatives of polysaccharides, proteins, and synthetic polymers, cross-linkable through the HRP-catalyzed reaction, means the current approach shows great promise for biofabrication of functional and structurally complex tissues.

Cryopreservation of a small number of human sperm using enzymatically fabricated, hollow hyaluronan microcapsules handled by conventional ICSI procedures.

PURPOSE: We investigated whether enzymatically fabricated hyaluronan (HA) microcapsules were feasible for use in the cryopreservation of a small number of sperm. METHODS: HA microcapsules were fabricated using a system of water-immiscible fluid under laminar flow. Three sperm were injected into a hollow HA microcapsule using a micromanipulator. Capsules containing injected sperm were incubated in a freezing medium composed of sucrose as the cryoprotectant and then placed in a Cryotop® device and plunged into liquid nitrogen. After thawing, the capsule was degraded by hyaluronidase, and the recovery rate of sperm and their motility were investigated. RESULTS: The HA microcapsule measuring 200 µm in diameter and with a 30-µm thick membrane was handled using a conventional intracytoplasmic sperm injection (ICSI) system, and the procedure involved the injection of sperm into the capsule. The HA microcapsules containing sperm were cryopreserved in a Cryotop® device and decomposed by the addition of hyaluronidase. The recovery rate of sperm after cryopreservation and degradation of HA microcapsules was sufficient for use in clinical practice (90 %). CONCLUSIONS: Hollow HA microcapsules can be used for the cryopreservation of a small number of sperm without producing adverse effects on sperm quality.

Identification of hydrogen peroxide-secreting cells by cytocompatible coating with a hydrogel membrane.

A method for identifying each cell secreting reactive oxygen species (ROS) is highly desirable to advance the understanding of the physiological and pathological processes attributed to extracellular ROS. Here, we first report a method for realizing this. The individual cells secreting hydrogen peroxide (H2O2), a common ROS, could be coated by a hydrogel membrane through a horseradish peroxidase-catalyzed reaction consuming H2O2 secreted from the cells themselves. This hydrogel membrane coating was proved to be cytocompatible. In addition, the hydrogel membrane made from an alginate derivative could be removed on demand without causing damage to the enclosed cells. These results demonstrated the feasibility of the proposed method to be an effective tool in cellular ROS studies.

Horseradish peroxidase-mediated encapsulation of mammalian cells in hydrogel particles by dropping.

We report a method for preparation of mammalian cell-enclosing hydrogel particles through horseradish peroxidase (HRP)-catalysed hydrogelation by dropping cell-suspending aqueous solution into an aqueous coagulation solution. An aqueous solution of 10% (w/v) gelatin derivative possessing phenolic hydroxyl (Ph) moieties (Gelatin-Ph), HepG2 cells and 10 U/mL HRP was dropped into an aqueous coagulation solution containing 1 mM H2O2. The resultant hydrogel formed through the HRP-catalysed reaction consuming H2O2 had a spherical shape. The sphericity decreased with decreasing concentrations of Gelatin-Ph, HRP and H2O2. The thickness of the hydrogel membrane layer of the hydrogel particles could be controlled by altering incubation time in the H2O2 solution. The cells encapsulated in the particles with a thinner hydrogel membrane grew faster. These results demonstrate that we successfully established the method of cell-encapsulation in hydrogel particles based on dropping aqueous polymer solution into aqueous coagulation solution through HRP-catalysed reaction.

Effect of cell adhesiveness of Cell Dome shell on enclosed HeLa cells.

The Cell Dome is a dome-shaped structure (diameter: 1 mm, height: 270 µm) with cells enclosed within a cavity, covered by a hemispherical hydrogel shell, and immobilized on a glass plate. Given that the cells within Cell Dome are in contact with the inner walls of the hydrogel shell, the properties of the shell are anticipated to influence cell behavior. To date, the impact of the hydrogel shell properties on the enclosed cells has not been investigated. In this study, we explored the effects of the cell adhesiveness of hydrogel shell on the behavior of enclosed cancer cells. Hydrogel shells with varying degrees of cell adhesiveness were fabricated using aqueous solutions containing either an alginate derivative with phenolic hydroxyl moieties exclusively or a mixture of alginate and gelatin derivatives with phenolic hydroxyl moieties. Hydrogel formation was mediated by horseradish peroxidase. We used the HeLa human cervical cancer cell line, which expresses fucci2, a cell cycle marker, to observe cell behavior. Cells cultured in hydrogel shells with cell adhesiveness proliferated along the inner wall of the hydrogel shell. Conversely, cells in hydrogel shells without cell adhesiveness grew uniformly at the bottom of the cavities. Furthermore, cells in non-adhesive hydrogel shells had a higher percentage of cells in the G1/G0 phase compared to those in adhesive shells and exhibited increased resistance to mitomycin hydrochloride when the cavities became filled with cells. These results highlight the need to consider the cell adhesiveness of the hydrogel shell when selecting materials for constructing Cell Dome.

3D Bioprinting of Sugar Beet Pectin through Horseradish Peroxidase-Catalyzed Cross-Linking.

Horseradish peroxidase (HRP)-mediated hydrogelation, caused by the cross-linking of phenolic groups in polymers in the presence of hydrogen peroxide (H2O2), is an effective route for bioink solidification in 3D bioprinting. Sugar beet pectin (SBP) naturally has cross-linkable phenols through the enzymatic reaction. Therefore, chemical modifications are not required, unlike the various polymers that have been used in the enzymatic cross-linking system. In this study, we report the application of SBP in extrusion-based bioprinting including HRP-mediated bioink solidification. In this system, H2O2 necessary for the solidification of inks is supplied in the gas phase. Cell-laden liver lobule-like constructs could be fabricated using bioinks consisting of 10 U/mL HRP, 4.0 and 6.0 w/v% SBP, and 6.0 × 106 cells/mL human hepatoblastoma (HepG2) cells exposed to air containing 16 ppm of H2O2 concurrently during printing and 10 min postprinting. The HepG2 cells enclosed in the printed constructs maintained their viability, metabolic activity, and hepatic functions from day 1 to day 7 of the culture, which indicates the cytocompatibility of this system. Taken together, this result demonstrates the potential of SBP and HRP cross-linking systems for 3D bioprinting, which can be applied in tissue engineering applications.

Transduction and Genome Editing of the Heart with Adeno-Associated Viral Vectors Loaded onto Electrospun Polydioxanone Nonwoven Fabrics.

In this study, we introduce electrospun polydioxanone (PDO) nonwoven fabrics as a platform for the delivery of adeno-associated virus (AAV) vectors for transduction and genome editing by adhering them to organ surfaces, including the heart. AAV vectors were loaded onto the PDO fabrics by soaking the fabrics in a solution containing AAV vectors. In vitro, the amount of AAV vectors loaded onto the fabrics could be adjusted by changing their concentration in the solution, and the number of cells expressing the green fluorescent protein (GFP) encoded by the AAV vectors increased in correlation with the increasing amount of loaded AAV vectors. In vivo, both transduction and genome editing resulted in the observation of GFP expression around AAV vector-loaded PDO fabrics attached to the surfaces of mouse hearts, indicating effective transduction and expression at the target site. These results demonstrate the great potential of electrospun PDO nonwoven fabrics carrying therapeutic AAV vectors for gene therapy.

Characterization of Chitosan Hydrogels Obtained through Phenol and Tripolyphosphate Anionic Crosslinking.

Chitosan is a deacetylated polymer of chitin that is extracted mainly from the exoskeleton of crustaceans and is the second-most abundant polymer in nature. Chitosan hydrogels are preferred for a variety of applications in bio-related fields due to their functional properties, such as antimicrobial activity and wound healing effects; however, the existing hydrogelation methods require toxic reagents and exhibit slow gelation times, which limit their application in biological fields. Therefore, a mild and rapid gelation method is necessary. We previously demonstrated that the visible light-induced gelation of chitosan obtained through phenol crosslinking (ChPh) is a rapid gelation method. To further advance this method (<10 s), we propose a dual-crosslinked chitosan hydrogel obtained by crosslinking phenol groups and crosslinking sodium tripolyphosphate (TPP) and the amino groups of chitosan. The chitosan hydrogel was prepared by immersing the ChPh hydrogel in a TPP solution after phenol crosslinking via exposure to visible light. The physicochemical properties of the dual-crosslinked hydrogels, including Young's moduli and water retentions, were subsequently investigated. Young's moduli of the dual-crosslinked hydrogels were 20 times higher than those of the hydrogels without TPP ion crosslinking. The stiffness could be manipulated by varying the immersion time, and the water retention properties of the ChPh hydrogel were improved by TPP crosslinking. Ion crosslinking could be reversed using an iron chloride solution. This method facilitates chitosan hydrogel use for various applications, particularly tissue engineering and drug delivery.

Hydrogels with Ultrasound-Treated Hyaluronic Acid Regulate CD44-Mediated Angiogenic Potential of Human Vascular Endothelial Cells In Vitro.

The development of hydrogels that allow vascular endothelial cells to form capillary-like networks is critical for advancing tissue engineering and drug discovery. In this study, we developed hydrogels composed of phenolated hyaluronic acid (HA-Ph) with an average molecular weight of 490-159 kDa via sonication in an aqueous solution. These hydrogels were synthesized by the horseradish peroxidase-catalyzed crosslinking of phenol moieties in the presence of hydrogen peroxide and phenolated gelatin. The sonication-degraded HA-Ph (198 kDa) significantly enhanced the migration ability of human umbilical vein endothelial cells (HUVECs) on cell culture plates when added to the medium compared to the original HA-Ph (490 kDa) and less-degraded HA-Ph (312-399 kDa). In addition, HUVECs cultured on these hydrogels formed networks that did not occur on hydrogels made from the original HA-Ph. CD44 expression and PI3K gene expression, both markers related to angiogenesis, were 3.5- and 1.8-fold higher, respectively, in cells cultured on sonication-degraded HA-Ph hydrogels than in those cultured on hydrogels comprising the original HA-Ph. These results highlight the potential of hydrogels containing sonication-degraded HA-Ph for tissue engineering and drug-screening applications involving human vascular endothelial cells.

Local delivery of adeno-associated viral vectors with electrospun gelatin nanofiber mats.

Adeno-associated viral (AAV) vectors play a significant role in gene therapy, yet the typical delivery methods, like systemic and local AAV injections, often lead to unintended off-target distribution and tissue damage due to injection. In this study, we propose a localized delivery approach for AAV vectors utilizing electrospun gelatin nanofiber mats, which are cross-linked with glutaraldehyde. The AAV vectors, which encoded a green fluorescent protein (GFP), were loaded onto the mats by immersing them in a solution containing the vectors. The amount of AAV vector loaded onto the mats increased as the vector concentration in the solution increased. The loaded AAV vector was steadily released into the cell culture medium over 3 days. The mats incubated for 3 days also showed the ability to transduce into the cells cultured on them. We evaluated the effectiveness of this delivery system by attaching the mats to mouse livers. GFP expression was visible on the surface of the liver beneath the attached mats, but not in areas in direct contact with the mats. These findings suggest that the attachment of AAV vector-loaded electrospun gelatin nanofiber mats to a target site present a promising solution for localized gene delivery while reducing off-target distribution.

Phototuning of Hyaluronic-Acid-Based Hydrogel Properties to Control Network Formation in Human Vascular Endothelial Cells.

In vitro network formation by endothelial cells serves as a fundamental model for studies aimed at understanding angiogenesis. The morphogenesis of these cells to form a network is intricately regulated by the mechanical and biochemical properties of the extracellular matrix. Here the effects of modulating these properties in hydrogels derived from phenolated hyaluronic acid (HA-Ph) and phenolated gelatin (Gelatin-Ph) are presented. Visible-light irradiation in the presence of tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and sodium persulfate induces the crosslinking of these polymers, thereby forming a hydrogel and degrading HA-Ph. Human vascular endothelial cells form networks on the hydrogel prepared by visible-light irradiation for 45 min (42 W cm-2 at 450 nm) but not on the hydrogels prepared by irradiation for 15, 30, or 60 min. The irradiation time-dependent degradation of HA-Ph and the changes in the mechanical stiffness of the hydrogels, coupled with the expressions of RhoA and ß-actin genes and CD44 receptors in the cells, reveal that the network formation is synergistically influenced by the hydrogel stiffness and HA-Ph degradation. These findings highlight the potential of tailoring HA-based hydrogel properties to modulate human vascular endothelial cell responses, which is critical for advancing their application in vascular tissue engineering.

Horseradish Peroxidase-Mediated Bioprinting via Bioink Gelation by Alternately Extruded Support Material.

Horseradish peroxidase (HRP)-mediated extrusion bioprinting has a significant potential in tissue engineering and regenerative medicine. However, they often face challenges in terms of printing fidelity and structural integrity when using low-viscosity inks. To address this issue, a method that alternately extrudes bioinks and support material was developed in this study. The bioinks consisting of cells, HRP, and phenolated polymers, and the support material contained hydrogen peroxide (H2O2). The support material not only prevented the collapse of the constructs but also supplied H2O2 to facilitate the enzymatic reaction. 3D constructs with tall and complex shapes were successfully printed from a low-viscosity ink containing 10 U/mL HRP and 1.0% w/v phenolated hyaluronic acid (HA-Ph), with a support material containing 10 mM H2O2. Over 90% viability of mouse fibroblasts (10T1/2) was achieved following the printing process, along with a morphology and proliferation rate similar to that of nontreated cells. Furthermore, human hepatoblastoma (HepG2) cells showed an increased spheroid size over 14 days in the printed constructs. The 10T1/2 cells adhered and proliferated on the constructs printed from inks containing both phenolated gelatin and HA-Ph. These results demonstrate the great potential of this HRP-mediated extrusion bioprinting technique for tissue engineering applications.

Visible light photocrosslinking of sugar beet pectin for 3D bioprinting applications.

Herein, we report the hydrogelation of sugar beet pectin (SBP) via visible light-mediated photocrosslinking and its applications in extrusion-based 3D bioprinting. Rapid hydrogelation (<15 s) was achieved by applying 405 nm visible light to an SBP solution in the presence of tris(bipyridine)ruthenium(II) chloride hexahydrate ([Ru(bpy)3]2+) and sodium persulfate (SPS). The mechanical properties of the hydrogel could be tuned by controlling the visible light irradiation time and concentrations of SBP, [Ru(bpy)3]2+, and SPS. High-fidelity 3D hydrogel constructs were fabricated by extruding inks containing 3.0 wt% SBP, 1.0 mM [Ru(bpy)3]2+, and 1.0 mM SPS. Human hepatoblastoma (HepG2) cells encapsulated in SBP hydrogels remained viable and metabolically active after 14 d of culture. Overall, this study demonstrates the feasibility of applying SBP and a visible light-mediated photocrosslinking system to the 3D bioprinting of cell-laden constructs for tissue engineering applications.

Fabrication of cell-laden microbeads and microcapsules composed of bacterial polyglucuronic acid.

In the past decades, the microencapsulation of mammalian cells into microparticles has been extensively studied for various in vitro and in vivo applications. The aim of this study was to demonstrate the viability of bacterial polyglucuronic acid (PGU), an exopolysaccharide derived from bacteria and composed of glucuronic acid units, as an effective material for cell microencapsulation. Using the method of dropping an aqueous solution of PGU-containing cells into a Ca2+-loaded solution, we produced spherical PGU microbeads with >93 % viability of the encapsulated human hepatoma HepG2 cells. Hollow-core microcapsules were formed via polyelectrolyte complex layer formation of PGU and poly-l-lysine, after which Ca2+, a cross-linker of PGU, was chelated, and this was accomplished by sequential immersion of microbeads in aqueous solutions of poly-l-lysine and sodium citrate. The encapsulated HepG2 cells proliferated and formed cell aggregates within the microparticles over a 14-day culture, with significantly larger aggregates forming within the microcapsules. Our results provide evidence for the viability of PGU for cell microencapsulation for the first time, thereby contributing to advancements in tissue engineering.