Haruka Maehashi

Transplantation of Liver Organoids in the Omentum and Kidney

Liver organoids were reconstructed by mouse-immortalized hepatocytes and nonparenchymal cells (sinusoidal endothelial cells and hepatic stellate cells) in a radial-flow bioreactor (RFB). A biodegradable apatite-fiber scaffold (AFS) was used as a scaffold packed in the RFB, which enables three-dimensional cell cultures. The organoids cocultured in the RFB showed a liver-like structure with high-density layers of hepatocytes and the formation of vessel-like structures. A liver organoid consisting of three cocultured cells was transplanted under the kidney capsule (kidney group) or into the omentum (omentum group) using BALB/c nude mice. Transplanted liver organoids survived in the kidney or omentum. The expression of mRNAs of albumin, connexin 26 and 32, hepatocyte nuclear factor 4α, and glucose-6-phosphatase was increased in both groups at 8 weeks after transplantation in comparison to the pretransplant status. Tyrosine aminotransferase appeared only in the omentum group. The results suggested that the functions of liver organoids differed depending on the transplanted site in the recipient animals.

The Functional Interrelationship between Gap Junctions and Fenestrae in Endothelial Cells of the Liver Organoid

Functional intact liver organoid can be reconstructed in a radial-flow bioreactor when human hepatocellular carcinoma (FLC-5), mouse immortalized sinusoidal endothelial M1 (SEC) and A7 (HSC) hepatic stellate cell lines are cocultured. The structural and functional characteristics of the reconstructed organoid closely resemble the in vivo liver situation. Previous liver organoid studies indicated that cell-to-cell communications might be an important factor for the functional and structural integrity of the reconstructed organoid, including the expression of fenestrae. Therefore, we examined the possible relationship between functional intact gap junctional intercellular communication (GJIC) and fenestrae dynamics in M1-SEC cells. The fine morphology of liver organoid was studied in the presence of (1) irsogladine maleate (IM), (2) oleamide and (3) oleamide followed by IM treatment. Fine ultrastructural changes were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and compared with control liver organoid data. TEM revealed that oleamide affected the integrity of cell-to-cell contacts predominantly in FLC-5 hepatocytes. SEM observation showed the presence of fenestrae on M1-SEC cells; however, oleamide inhibited fenestrae expression on the surface of endothelial cells. Interestingly, fenestrae reappeared when IM was added after initial oleamide exposure. GJIC mediates the number of fenestrae in endothelial cells of the liver organoid.

A comprehensive gene expression analysis of human hepatocellular carcinoma cell lines as components of a bioartificial liver using a radial flow bioreactor

Background/Aims: The cells constituting a bioartificial liver are crucial for an effective liver support system. We compared global gene expression profiles in a radial flow bioreactor or a monolayer culture of three functional liver cell lines previously established from human hepatocellular carcinoma.

Reconstruction of Tissue-Engineered Bone Using an Apatite-Fiber Scaffold, Rat Bone Marrow Cells and Radial-Flow Bioreactor: Optimization of Flow Rate in Circulating Medium

We have successfully developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional (3D) inter-connected pores; subsequently, we have clarified that the AFSs have an excellent bioactivity on the basis of both in vitro and in vivo evaluations. In addition, we have reconstructed the tissue-engineered bone with 3D structure through 3D-cell culture of mesenchymal stem cells derived from rat bone marrow (RBMC) using the AFS settled into the radial-flow bioreactor (RFB), and examined effect of flow rate of medium in the RFB on the differentiation of osteoblasts in tissue-engineered bone. Aim in the present work is to establish of the optimal conditions of flow rate in this construction method of 3D tissue-engineered bone. The flow rates were set to 0.4, 1.3, 6.3, 11.5 and 16.5 cm3min-1; tissue-engineered bones cultured by the individual flow rates are defined as bones#1~#5. The level of differentiation of osteoblasts in all the bones#1~#5 was examined by determining the content of two kinds of differentiation maker into osteoblast, alkaline phosphatase (ALP) for initial/middle stage and osteocalcin (OC) for late stage. The ALP activity normalized for DNA content of bone#3 showed the highest value among all of them. Moreover, the OC amount normalized for DNA content of bone#3 also indicated the highest among all the examined samples. These results demonstarate that the flow rate of 6.3 cm3min-1 may promote the differentiation into osteoblast. In conclusion, we determined that this flow rate was the optimal conditions for the bone regeneratrion in RFB.

Three-dimensional culture promotes reconstitution of the tumor-specific hypoxic microenvironment under TGFβ stimulation

In vitro tumor growth in a three-dimensional (3D) architecture has been demonstrated to play an important role in biology not only for developmental organogenesis and carcinogenesis, but also for analyses on reconstitution and maintenance in a variety of biological environments surrounding the cells. In addition to providing architectural similarity to living organisms, 3D culture with a radial flow bioreactor (RFB) can also closely mimic the living hypoxic microenvironment under which specific organogenesis or carcinogenesis occurs. The findings of the present study under the RFB culture conditions show that cancer cells underwent a shift from aerobic to hypoxic energy metabolism, in addition to protein expression to maintain the 3D structure. In RFB-cultured cells, protein stability of hypoxia-inducible factor 1 (HIF1) α, a subunit of HIF1, was increased without upregulation of its mRNA. Under these conditions, PHD2, HIF-prolyl-4-hydroxy-lase 2 and a HIF1 downstream enzyme, were stabilized without affecting the mRNA levels via downregulation of FK506-binding protein 8. PHD2 accumulation, which occurred concomitant with HIF1 stabilization, may have compensated for the lack of oxygen under hypoxic conditions to regulate the HIF levels. 3D-culture-induced overexpression of carbonic anhydrase (another representative HIF downstream enzyme) was found to occur independently of cell density in RFB--cultured cells, suggesting that the RFB provided an adequately hypoxic microenvironment for the cultured cells. From these results, it was hypothesized that the key factors are regulatory molecules, which stabilize and degrade HIF molecules, thereby activating the HIF1 pathway under a hypoxic milieu.

Study of the reappearance of sieve plate-like pores in immortalized sinusoidal endothelial cells – Effect of actin inhibitor in mixed perfusion cultures

We previously reported that when the high-functioning human hepatoma cell line, FLC-5, immortalized sinusoidal endothelial cell line, M1, and immortalized hepatic stellate cell line, A7, were cultured in the 3-dimensional filled type bioreactor, tissue reorganization resembling that seen in the live liver occurred, with the appearance of pores in the sinusoidal endothelial cells (SECs) [1]. The process and mechanism of formation of these pores remain unclarified. The presence of actin at the margin of these pores has been demonstrated by electron microscopic study [2]. Swinholide-A, which is actin inhibitor synthesized from Okinawa sponge, increase the number of pores on primary culture on SECs derived from the rat [3]. In present study, we examine whether or not the pores on SECs under three-dimensional perfusion co-culture treatment with Swinholide-A behave like those in primary culture cells.

Hepatic stellate cells that coexpress LRAT and CRBP-1 partially contribute to portal fibrogenesis in patients with human viral hepatitis.

Precisely what type of cells mainly contributes to portal fibrosis, especially in chronic viral hepatitis, such as hepatic stellate cells (HSCs) in the parenchyma or myofibroblasts in the portal area, still remains unclear. It is necessary to clarify the characteristics of cells that contribute to portal fibrosis in order to determine the mechanism of portal fibrogenesis and to develop a therapeutic target for portal fibrosis. This study was undertaken to examine whether LRAT+/CRBP‐1+ HSCs contribute to portal fibrosis on viral hepatitis.

Alpha-1 antichymotrypsin is involved in astrocyte injury in concert with arginine-vasopressin during the development of acute hepatic encephalopathy

Background and aims We developed a bio-artificial liver (BAL) using a radial-flow bioreactor and rescued mini-pig models with lethal acute liver failure (ALF). The point of the rescue is the recovery from hepatic encephalopathy (HE). HE on ALF has sometimes resulted in brain death following brain edema with astrocyte swelling. Several factors, including ammonia and glutamine, have been reported to induce astrocyte swelling and injury. However, many clinicians believe that there are any other factors involved in the development of HE. Therefore, the aim of this study was to identify novel HE-inducible factors, particularly those inducing astrocyte dysfunction. Methods Mini-pig plasma samples were collected at three time points: before the administration of toxins (α-amanitin and LPS), when HE occurred after the administration of toxins, and after treatment with extracorporeal circulation (EC) by the BAL. To identify the causative factors of HE, each plasma sample was subjected to a comparative proteome analysis with two-dimensional gel electrophoresis and mass spectrometry. To assess the direct effects of candidate factors on the astrocyte function and injury, in vitro experiments with human astrocytes were performed. Results Using a proteome analysis, we identified alpha-1 antichymotrypsin (ACT), which was increased in plasma samples from mini-pigs with HE and decreased in those after treatment with EC by BAL. In in vitro experiments with human astrocytes, ACT showed growth-inhibitory and cytotoxic effects on astrocytes. In addition, the expression of water channel protein aquaporin-4, which is induced in injured astrocytes, was increased following ACT treatment. Interestingly, these effects of ACT were additively enhanced by adding arginine-vasopressin (AVP) and were canceled by adding an AVP receptor antagonist. Conclusions These results suggest that ACT is involved in astrocyte injury and dysfunction in concert with AVP during the development of acute HE.

Effect of Flow Rate of Medium in Radial-Flow Bioreactor on the Differentiation of Osteoblasts in Tissue-Engineered Bone Reconstructed Using an Apatite-Fiber Scaffold and Rat Bone Marrow Cells

Tissue engineering has been studied as a novel therapeutic technology which replaces organ transplantation. Tissue engineering consists of three factors “scaffolds”, “cells” and “growth factors”, and regenerates defecting tissue using them. We have successfully developed porous apatite-fiber scaffolds (AFSs) which have three-dimensional (3D) inter-connected pores using single-crystal apatite fibers and carbon beads; subsequently, we have clarified that the AFSs have an excellent bioactivity on the basis of both in vitro and in vivo evaluations. In addition, we have reconstructed the 3D tissue-engineered bone through 3D-cell culture of mesenchymal stem cells derived from rat bone marrow (RBMC) using the AFS settled into the radial-flow bioreactor (RFB). Aim in the present work is to examine the effect of flow rate of medium in the RFB on the differentiation of osteoblasts in tissue-engineered bone reconstructed using an AFS and RBMC. The flow rates were set to 1.3 and 6.3 cm3∙min-1; tissue-engineered bones reconstructed by the two flow rates are defined as “bone#1” and “bone#2”, respectively. The ALP activity and OC amount normalized for DNA content of bone#2 were higher than those of bone#1. These results indicate that the faster flow rate may promote the differentiation into osteoblast. Thus, the physical irritation to cells, such as flow rates, may be effective for reconstruction of tissue-engineered bone.

An efficient system for secretory production of fibrinogen using a hepatocellular carcinoma cell line

Despite an increasing demand, blood products are not always safe because most are derived from blood donations. One possible solution is the development and commercialization of recombinant fibrinogen, but this process remains poorly developed. This study aimed to develop an effective production system for producing risk‐free fibrinogen using human hepatocellular cell lines and serum‐free media.