Lotus-root-shaped cell-encapsulated construct as a retrieval graft for long-term transplantation of human iPSC-derived ß-cells.

Cell therapy using human-stem-cell-derived pancreatic beta cells (hSC-ßs) is a potential treatment method for type 1 diabetes mellitus (T1D). For therapeutic safety, hSC-ßs need encapsulation in grafts that are scalable and retrievable. In this study, we developed a lotus-root-shaped cell-encapsulated construct (LENCON) as a graft that can be retrieved after long-term hSC-ß transplantation. This graft had six multicores encapsulating hSC-ßs located within 1 mm from the edge. It controlled the recipient blood glucose levels for a long-term, following transplantation in immunodeficient diabetic mice. LENCON xenotransplanted into immunocompetent mice exhibited retrievability and maintained the functionality of hSC-ßs for over 1 year after transplantation. We believe that LENCON can contribute to the treatment of T1D through long-term transplantation of hSC-ßs and in many other forms of cell therapy.

Millimeter-thick xenoislet-laden fibers as retrievable transplants mitigate foreign body reactions for long-term glycemic control in diabetic mice.

Transplantation technologies of pancreatic islets as well as stem cell-derived pancreatic beta cells encapsulated in hydrogel for the induction of immunoprotection could advance to treat type 1 diabetes mellitus, if the hydrogel transplants acquire retrievability through mitigating foreign body reactions after transplantation. Here, we demonstrate that the diameter of the fiber-shaped hydrogel transplants determines both in vivo cellular deposition onto themselves and their retrievability. Specifically, we found that the in vivo cellular deposition is significantly mitigated when the diameter is 1.0 mm and larger, and that 1.0 mm-thick xenoislet-laden fiber-shaped hydrogel transplants can be retrieved after being placed in the intraperitoneal cavities of immunocompetent diabetic mice for more than 100 days, during which period the hydrogel transplants can normalize the blood glucose concentrations of the mice. These findings could provide an innovative concept of a transplant that would promote the clinical application of stem cell-derived functional cells through improving their in vivo efficacy and safety.

3D culture of functional human iPSC-derived hepatocytes using a core-shell microfiber.

Human iPSC-derived hepatocytes hold great promise as a cell source for cell therapy and drug screening. However, the culture method for highly-quantified hepatocytes has not yet been established. Herein, we have developed an encapsulation and 3D cultivation method for iPSC-hepatocytes in core-shell hydrogel microfibers (a.k.a. cell fiber). In the fiber-shaped 3D microenvironment consisting of abundant extracellular matrix (ECM), the iPSC-hepatocytes exhibited many hepatic characteristics, including the albumin secretion, and the expression of the hepatic marker genes (ALB, HNF4α, ASGPR1, CYP2C19, and CYP3A4). Furthermore, we found that the fibers were mechanically stable and can be applicable to hepatocyte transplantation. Three days after transplantation of the microfibers into the abdominal cavity of immunodeficient mice, human albumin was detected in the peripheral blood of the transplanted mice. These results indicate that the iPSC-hepatocyte fibers are promising either as in vitro models for drug screening or as implantation grafts to treat liver failure.

Biofabrication Using Electrochemical Devices and Systems.

Biofabrication is roughly defined as techniques producing complex 2D and 3D tissues and organs from raw materials such as living cells, matrices, biomaterials, and molecules. It is useful for tissue engineering, regenerative medicine, drug screening, and organs-on-a-chip. Biofabrication could be carried out by microfluidic techniques, optical methods, microfabrication, 3D bioprinting, etc. Meanwhile, electrochemical devices and/or systems have also been reported. In this progress report, the recent advances in applying these devices/systems for biofabrication are summarized. After introducing the concept of biofabrication, biofabrication strategies using electrochemical approaches are summarized. Then, various electrochemical systems such as probes and chip devices are described. Next, the biofabrication of hydrogels for 3D cell culture, electrochemical modification on cell culture surfaces, electrodeposition of conductive materials in hydrogels for cell culture, and biofabrication of cell aggregates using dielectrophoresis is discussed. In addition, electrochemical stimulation methods such as electrotaxis are mentioned as promising techniques for biofabrication. Finally, future research directions in this field and the application prospects are highlighted.

Improvement in the Mechanical Properties of Cell-Laden Hydrogel Microfibers Using Interpenetrating Polymer Networks.

Microencapsulation of cells is a promising technique in biomedical applications such as cell therapy. Recently, cell-laden hydrogel microfibers have been proposed as another shape microcapsule instead of microbeads; however, these are brittle with little stretching capability. This paper describes a cell-laden hydrogel microfiber that showed enhanced mechanical properties and handleability by using a double-network (DN) hydrogel consisting of alginate and polyacrylamide. The DN hydrogel microfiber supported approximately 6-fold higher strain and exhibited 10-fold higher tensile strength than the conventional alginate form. The DN hydrogel microfiber could also encapsulate pancreatic ß cells while maintaining cell viability and function. The in vivo functionality of the DN hydrogel microfiber was demonstrated by transplanting 3D assemblies of the microfibers into the intraperitoneal or subcutaneous space of diabetic mice, which successfully decreased their blood glucose levels. Thus, cell-laden DN hydrogel microfibers may represent a promising material for various biomedical applications.

Electrochemical Hydrogel Lithography of Calcium-Alginate Hydrogels for Cell Culture.

Here we propose a novel electrochemical lithography methodology for fabricating calcium-alginate hydrogels having controlled shapes. We separated the chambers for Ca2+ production and gel formation with alginate with a semipermeable membrane. Ca2+ formed in the production chamber permeated through the membrane to fabricate a gel structure on the membrane in the gel formation chamber. When the calcium-alginate hydrogels were modified with collagen, HepG2 cells proliferated on the hydrogels. These results show that electrochemical hydrogel lithography is useful for cell culture.

Alginate gel microwell arrays using electrodeposition for three-dimensional cell culture.

In this study, we developed a novel method for fabricating microwell arrays constructed from alginate gels, and the alginate gel microwells were used for three-dimensional (3D) cell culture. The alginate gel microwells were fabricated on a patterned ITO electrode using alginate gel electrodeposition. Embryonic stem (ES) cells or hepatocellular carcinoma cells (HepG2) were cultured in the alginate gel microwells containing 3T3 cells. During the culture, embryoid bodies (EBs) or HepG2 spheroids were successfully fabricated in the alginate gel microwells. The oxygen consumption of the EBs indicated that they were successfully cultured. Liver-specific gene expressions of the HepG2 spheroids apparently increased by performing 3D co-culture in the microwell arrays with 3T3 cells. These results show that the alginate gel microwells are a useful 3D culture system.

Electrodeposition of alginate gels for construction of vascular-like structures.

In this study, tubular hydrogel structures were constructed via electrodeposition using alginate gels. Electrolysis of water in alginate solutions with calcium carbonate particles induced gel aggregation around Pt wire electrodes, forming tubular alginate gel structures. The simple method is a promising approach for construction of multi-layer tubular hydrogel structures for tissue engineering.

Manipulation of microparticles for construction of array patterns by negative dielectrophoresis using multilayered array and grid electrodes.

In this study, a useful method was developed to fabricate array patterns of microparticles not on electrode surfaces, but on arbitrary surfaces, using negative-dielectrophoresis (n-DEP). First, electrodes were designed and electric field simulations were performed to manipulate microparticles toward target areas. Based on the simulation results, multilayered array and grid (MLAG) electrodes, consisting of array electrodes surrounded by insulated regions and a grid electrode, were fabricated for the formation of localized, non-uniform electric fields. The MLAG electrode was mounted to a target substrate in a face-to-face configuration with a spacer. When an AC voltage (4.60 Vrms and 1 MHz) was applied to the MLAG electrode, array patterns of 6 and 20 microm diameter microparticles were rapidly fabricated on the target substrate with ease. The results suggest that MLAG electrodes can be widely applied for the fabrication of biochips including cell arrays.