Adenoviral transfection of hepatocytes with the thioredoxin gene confers protection against apoptosis and necrosis

A recombinant adenovirus vector containing the human thioredoxin (TRX) gene was constructed using the Cre-loxP recombination system and used to transfect rat hepatocytes with very high efficiency. The TRX gene was expressed in a dose-dependent manner and significantly modulated rat cellular functions. The TRX gene conferred resistance to oxidative stress, such as hydrogen peroxide treatment, on the host hepatocytes. FACS analysis of DNA fragmentation showed that the TRX gene suppressed hepatocyte apoptosis. It also significantly extended the life span of hepatocytes cultured conventionally on polystyrene plates. Liver-specific functions were maintained in the viability-modulated hepatocytes. Moreover, TRX expression did not affect hepatocyte spheroid formation and it extensively suppressed necrosis in the internal cells. Thus, the transfection of hepatocytes with the TRX gene successfully confers global maintenance of liver functions. These findings provide important information for the development of bioartificial liver support systems and gene therapy for liver diseases.     

Present and Future Developments in Hepatic Tissue Engineering for Liver Support Systems

The liver is the most important organ for the biotransformation of xenobiotics, and the failure to treat acute or acute-on-chronic liver failure causes high mortality rates in affected patients. Due to the lack of donor livers and the limited possibility of the clinical management there has been growing interest in the development of extracorporeal liver support systems as a bridge to liver transplantation or to support recovery during hepatic failure. Earlier attempts to provide liver support comprised non-biological therapies based on the use of conventional detoxification procedures, such as filtration and dialysis. These techniques, however, failed to meet the expected efficacy in terms of the overall survival rate due to the inadequate support of several essential liver-specific functions. For this reason, several bioartificial liver support systems using isolated viable hepatocytes have been constructed to improve the outcome of treatment for patients with fulminant liver failure by delivering essential hepatic functions. However, controlled trials (phase I/II) with these systems have shown no significant survival benefits despite the systems’ contribution to improvements in clinical and biochemical parameters. For the development of improved liver support systems, critical issues, such as the cell source and culture conditions for the long-term maintenance of liver-specific functions in vitro, are reviewed in this article. We also discuss aspects concerning the performance, biotolerance and logistics of the selected bioartificial liver support systems that have been or are currently being preclinically and clinically evaluated.     

Enhanced liver-specific functions of endothelial cell-covered hepatocyte hetero-spheroids

The performance of an extracorporeal bioartificial liver (BAL) support system depends on the functional activities of the hepatocytes immobilized in the system. One of the most promising techniques in retaining liver-specific functions is co-culturing hepatocytes with other cell types, such as epithelial cells, endothelial cells and dermal fibroblasts. Primary rat hepatocytes were suspension co-cultured with rat prostate endothelial cell line (RPEn) for 20 h in a spinner vessel to form hetero-spheroids, which contain the two types of the cells, i.e., hepatocytes and endothelial cells in the same spheroid. For the subsequent culture, the hetero-spheroids were entrapped in a Ca-alginate gel bead. From the results of incorporation efficiency test, it was found that RPEn cells have a significantly higher attachment affinity to hepatocytes than human dermal fibroblast and rat liver epithelial cells. We clearly found out that RPEn cells located on the surface of the hepatocyte spheroids from immunostained paraffin sections of the hetero-spheroids. Identical with in vivo liver tissue, laminin was stained at the surface of the hetero-spheroids. Ultrastructures of liver tissue, such as bile canaliculus-like and Disse’s space-like structures, were also found at the surface of the hetero-spheroids. In vivo liver tissue, in which hepatocytes were covered with sinusoidal endothelial cells, was partly mimicked by the endothelial cell-covered hepatocyte spheroids. And the hetero-spheroids showed significantly higher and stable albumin secretion and ammonia removal activities than pure spheroids for 12 days of observations.

Therefore, the endothelial cell-covered hepatocyte hetero-spheroids may offer a useful study model of epithelial–mesenchymal interactions and information about liver tissue engineering research as well as a substitute of a cell source of a BAL system.     

Expression and amplification of glutamine synthetase gene endows HepG2 cells with ammonia-metabolizing activity for bioartificial liver support system

To establish the ammonia-metabolizing cell lines for a bioartificial liver support system, CHO-K1 and HepG2 were transformed with pBK-CMV-GS vector that contains glutamine synthetase (gs) gene. The recombinant cell lines were selected under the various concentrations of glutamine synthetase inhibitor, methionine sulfoximine (MSX). The host CHO-K1 and HepG2 cell lines produces ammonia, but the both MSX tolerable CHO (GS-CHO) and HepG2 (GS-HepG2) cell lines endowed with the high GS activity could metabolize the ammonium from medium. The ammonia-metabolizing activity of CHO and HepG2 cell was about one-fourth of that of primary hepatocyte.     

无血清培养高密度乳猪肝细胞的形态和功能变化

本文研究了无血清培养高密度猪肝细胞的形态和功能变化。将分离的肝细胞以高密度(1×10~7/ml)培养在含激素、多种生长因子和营养成分的无血清培养基中,动态观察培养7天中肝细胞形态、活率、蛋白质合成功能、G-6-Pase活性、安定转化功能及LDH含量;同时以无血清培养低密度(5×10~5/ml)肝细胞作为对照组。研究结果表明:高密度培养的 肝细胞各项功能较低密度培养的肝细胞为低;高密度培养的肝细胞的形态、蛋白质合成功能在培养7天中保持稳定;活率随着培养时间的延长而下降,但均高于90％;安定转化功能在培养第2、3天最强;G-6-Pase活性在培养1天后明显下降,然后维持在较低水平;LDH含量在第1、2、3天较高。     

An intelligent bioreactor system for the cultivation of a bioartificial vascular graft

Cardiovascular disease is the most common cause of death, accounting for 31% of deaths worldwide. As purely synthetic grafts implicate concomitant anticoagulation and autologous veins are rare, tissue‐engineered vascular grafts are urgently needed. For successful in vitro cultivation of a bioartificial vascular graft, the suitable bioreactor should provide conditions comparable to vasculogenesis in the body. Such a system has been developed and characterized under continuous and pulsatile flow, and a variety of sensors has been integrated into the bioreactor to control parameters such as temperature, pressure up to 500 mbar, glucose up to 4.5 g/L, lactate, oxygen up to 150 mbar, and flow rate. Wireless data transfer (using the ZigBee specification based on the IEEE 802.15.4 standard) and multiple corresponding sensor signal processing platforms have been implemented as well. Ultrasound is used for touchless monitoring of the growing vascular structure as a quality control before implantation (maximally achieved ultrasound resolution 65 μm at 15 MHz). To withstand the harsh conditions of steam sterilization (120°C for 20 min), all electronics were encapsulated. With such a comprehensive physiologically conditioning, sensing, and imaging bioreactor system, all the requirements for a successful cultivation of vascular grafts are available now.     

Hepatocyte growth factor and epidermal growth factor promote spheroid formation in polyurethane foam/hepatocyte culture and improve expression and maintenance of albumin production

In our previous pre-clinical study with pig hepatic failure, an artificial liver with polyurethane foam (PUF)/primary porcine hepatocyte spheroids had superior curative effect. We examined the effect of hepatocyte growth factor (HGF), also known as scatter factor, on the quick formation of hepatocyte spheroids and albumin production. Spheroids were formed in the pores of PUF within 3 days regardless of addition of growth factors. In particular, spheroids were formed within 1 day in medium containing 100 ng/ml HGF and 50 ng/ml epidermal growth factor (EGF). 10,000 ng/ml HGF was effective for albumin production, but the activity dramatically decreased after 6 days in EGF-free medium. On the other hand, 100 ng/ml HGF was effective for albumin production in EGF-containing medium. Albumin production rate with ≥1000 ng/ml HGF was about 1.5 times higher than that with 100 ng/ml HGF. Furthermore, albumin production rate at 3 weeks was about 1.5 times higher than that at 2 days with 1000 ng/ml HGF. The maintenance of albumin production rate depended on the activity of the individual cell and not cell growth. In other words, we were able to show the effectiveness of HGF for functional hepatocyte organoid formation in PUF pores.     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.     

增强微胶囊在血液中稳定性的研究

目的:通过改进成膜工艺,减少血液中HCO3-对海藻酸钠-ε-聚赖氨酸(ε-AP)微胶囊稳定性的影响,从而增强ε-AP微胶囊在血液中的稳定性,使其能够应用于生物人工肝血浆/血液灌注。方法:改进成膜工艺:在成膜液中添加10 mmol/L的Na HCO3并增加ε-聚赖氨酸反应浓度来制备ε-AP微胶囊,用膨胀度表征其机械强度,荧光标记的牛血清白蛋白(FITC-BSA)检测其通透性能,并以微胶囊的膨胀度变化作为衡量指标,考察微胶囊在Na HCO3溶液和血清中的稳定性。结果:改进成膜工艺制备的ε-AP微胶囊与常规条件制备的微胶囊具有接近的机械强度和通透性;改进成膜工艺能够增强的ε-AP微胶囊在Na HCO3溶液和血清中的稳定性;常规条件制备的ε-AP微胶囊在血清中膨胀度增加了约560%,而改进成膜工艺制备的微胶囊膨胀度仅增加160%,其稳定性显著增强。结论:改进成膜工艺能够增强ε-AP微胶囊在血液中的稳定性。     

Cryopreservation of hepatocytes: a review of current methods for banking

Cryopreservation, the freezing of hepatocytes in liquid nitrogen for storage, has been investigated for many years, as a method of long-term storage for hepatocytes. Unfortunately an agreed acceptable protocol has been elusive, in part due to the susceptibility of hepatocytes to the freeze thaw process involved. A method for long-term storage (months, possibly years) of human hepatocytes, in particular, is desirable for the development of a clinically applicable bioartificial liver, hepatocyte transplantation and for pharmacotoxicological research. The sources of human liver tissue from which hepatocytes can be derived are limited. Many groups have modified and improved the process of cryopreservation and many new techniques have been published, including the incorporation of such cryopreserved cells in clinically based studies. Further evaluation is still required to develop a universally acceptable protocol. This article reviews the difficulties involved in cryopreserving hepatocytes for banking and examines recent technical advances within this field.