Vascularized subcutaneous human liver tissue from engineered hepatocyte/fibroblast sheets in mice

Subcutaneous liver tissue engineering is an attractive and minimally invasive approach used to curative treat hepatic failure and inherited liver diseases. However, graft failure occurs frequently due to insufficient infiltration of blood vessels (neoangiogenesis), while the maintenance of hepatocyte phenotype and function requires invivo development of the complex cellular organization of the hepatic lobule. Here we describe a subcutaneous human liver construction allowing for rapidly vascularized grafts by transplanting engineered cellular sheets consisting of human primary hepatocytes adhered onto a fibroblast layer. The engineered hepatocyte/fibroblast sheets (EHFSs) showed superior expression levels of vascularization-associated growth factors (vascular endothelial growth factor, transforming growth factor beta 1, and hepatocyte growth factor) invitro. EHFSs developed into vascularized subcutaneous human liver tissues contained glycogen stores, synthesized coagulation factor IX, and showed significantly higher synthesis rates of liver-specific proteins (albumin and alpha 1 anti-trypsin) invivo than tissues from hepatocyte-only sheets. The present study describes a new approach for vascularized human liver organogenesis under mouse skin. This approach could prove valuable for establishing novel cell therapies for liver diseases

Preparation of Microporous Hydrogel Sponges for 3D Perfusion Culture of Mammalian Cells

Three-dimensional (3D) perfusable organ models, primarily composed of liver cells, are expected as an efficient tool for in vitro cell-based drug screening and development. Various types of hydrogel-based 3D cell culture systems have been developed, but the lack in proper techniques to form vasculature networks in the hydrogel matrices results in inefficient supply of oxygen and nutrition to the cells. Here we propose a facile strategy to creating a perfusable hydrogel-based liver cell culture system. We utilized a bicontinuous dispersion of an aqueous two-phase system, which was composed of polyethylene glycol (PEG)-rich and gelatin methacrylate (GelMA)-rich phases, to produce cell-encapsulating microporous GelMA-based hydrogels. We successfully encapsulated HepG2 cells in the hydrogel matrix with a high cell viability, and confirmed that the spongious hydrogel was superior to homogeneous hydrogels for 3D cell culture. We performed perfusion culture for the cells encapsulated in the hydrogel sponge, to verify the usability and versatility of the presented hydrogel material for perfusion culture. The presented approach would be useful as a unique tool for developing organs-on-a-chip systems

Size effect of engineered islets prepared using microfabricated wells on islet cell function and arrangement

Pancreatic islets are heterogeneous clusters mainly composed of α and β cells, and these clusters range in diameter from 50 to several hundred micrometers. Native small islets are known to have a higher insulin secretion ability in vitro and to provide better transplantation outcomes when compared with large islets. In this study, we prepared microengineered pseudo-islets from dispersed rat islet cells using precisely-fabricated agarose gel-based microwells with different diameters (100, 300, or 500 μm) to investigate the function and survival of islet cell aggregates with well-controlled sizes. We observed that dead cells were rarely present in the small pseudo-islets with an average diameter of ∼60 μm prepared using 100 μm microwells. In contrast, we observed more dead cells in the larger pseudo-islets prepared using 300 and 500 μm microwells. The relative amount of hypoxic cells was significantly low in the small pseudo-islets whereas a hypoxic condition was present in the core region of the larger pseudo-islets. In addition, we found that the small-sized pseudo-islets reconstituted the in vivo-tissue like arrangement of the α and β cells, and restored the high insulin secretory capacity in response to high glucose. These results clearly suggest that precise size control of pseudo-islets is essential for maintaining islet cell function and survival in vitro. The small-sized pseudo-islets may be advantageous for providing a better therapeutic approach for treating type 1 diabetes mellitus via islet reorganization and transplantation

Microengineering of Collagen Hydrogels Integrated into Microfluidic Devices for Perfusion Culture of Mammalian Cells

Collagen-based hydrogels are widely used for three-dimensional (3D) culture of mammalian cells because of their high cell-activating characteristis. However, techniques for preparing of cell-embedding collagen hydrogels with micrometer-size precision in perfusable, microfluidic devices have not been fully developed. In this study, we propose a facile strategy enabling microfabrication of collagen hydrogels in microfluidic devices. We used phosphate particle-embedding polydimethylsiloxane (PP-PDMS) as a gelation agent, which neutralizes the acidic collagen soltuion. The collagen solution near the surface of the PP-PDMS is selectively gelled. We fabricated micropatterns and tubular structures made of collagen hydrogel, both of which were used for perfusion culture of mammalian cells encapsulated in the hydrogel matrix. The presented approach would be applicable to various types of cell culture experiments

The liver surface as a favorable site for islet cell sheet transplantation in type 1 diabetes model mice

Introduction: Islet transplantation is one of the most promising therapeutic approaches for patients with severe type 1 diabetes mellitus (T1DM). Transplantation of engineered islet cell sheets holds great potential for treating T1DM as it enables the creation of stable neo-islet tissues. However, a large mass of islet cell sheets is required for the subcutaneous transplantation to reverse hyperglycemia in diabetic mice. Here, we investigated whether the liver surface could serve as an alternative site for islet cell sheet transplantation. Methods: Dispersed rat islet cells (0.8 × 106 cells) were cultured on laminin-332-coated thermoresponsive culture dishes. After 2 days of cultivation, we harvested the islet cell sheets by lowering the culture temperature using a support membrane with a gelatin gel. We transplanted two recovered islet cell sheets into the subcutaneous space or onto the liver surface of severe combined immunodeficiency (SCID) mice with streptozocin-induced diabetes. Results: In the liver surface group, the non-fasting blood glucose level decreased rapidly within several days after transplantation. In marked contrast, the hyperglycemia state was maintained in the subcutaneous space transplantation group. The levels of rat C-peptide and insulin in the liver surface group were significantly higher than those in the subcutaneous space group. An immunohistological analysis confirmed that most of the islet cells engrafted on the liver surface were insulin-positive. The CD31-positive endothelial cells formed vascular networks within the neo-islets and in the surrounding tissues. In contrast, viable islet cells were not found in the subcutaneous space group. Conclusions: Compared with the subcutaneous space, a relatively small mass of islet cell sheets was enough to achieve normoglycemia in diabetic mice when the liver surface was selected as the transplantation site. Our results demonstrate that the optimization of the transplantation site for islet cell sheets leads to significant improvements in the therapeutic efficiency for T1DM. Keywords: Cell sheet, Islet cells, Type 1 diabetes mellitus, Transplantation site, Cellular therap

Collagen Microparticle-Mediated 3D Cell Organization: A Facile Route to Bottom-up Engineering of Thick and Porous Tissues

In closely packed artificial 3D cellular constructs, cells located near the center of the constructs are not functional because of the limited supply of oxygen and nutrition. Here we describe a simple, unique, and highly versatile approach to organizing cells into thick but porous 3D tissues, using cell-sized collagen microparticles as particulate scaffolds. When cells and particles are mixed and seeded in a noncell-adhesive planar chamber, they gather to form sheet-shaped structures with a thickness of 100–150 μm. In the construct, uniformly distributed particles work as a binder between cells and modulate the strong intercellular contraction. We confirmed that several factors, including the particle/cell ratio and particle size, critically affect the stability and shrink behaviors of porous tissues prepared using mouse embryonic fibroblasts (NIH-3T3 cells). Cross-sectional observation, together with cell proliferation and viability assays, revealed that the cells composing the tissues are functional primarily because interior pores between cells/particles worked as a path for efficient molecular transport. Furthermore, we prepared thick cell tissues of a liver model using human hepatocarcinoma cells (HepG2 cells), and confirmed that liver-specific functions were upregulated when composite tissues were formed using collagen microparticles prepared with several different stabilization protocols by glutaraldehyde, genipin, and methyl acetate). The process presented would be highly useful in enabling one-step production of thick cellular constructs in which porosity and morphology are tunable

Effects on coagulation factor production following primary hepatomitogen-induced direct hyperplasia

AIM: To investigate the molecular mechanisms involved in coagulation factor expression and/or function during direct hyperplasia (DH)-mediated liver regeneration