Transplantation of hepatocytes cultured on hydroxyapatite into Nagase analbuminemia rats

doi:10.1016/S1389-1723(03)90102-2

Journal of Bioscience and Bioengineering

Volume 96, Issue 1, 2003, Pages 83-85

https://doi.org/10.1016/S1389-1723(03)90102-2 Get rights and content

Abstract

Rat hepatocytes were seeded in porous hydroxyapatite (HA) disks for hepatocyte transplantation to Nagase analbuminemic rat (NAR). To increase the cell density in the HA, cell suspension in the tube containing the disks were centrifuged down. The centrifugation increased the viable cell number in HA disk approximately 4-fold over the static method. The HA disks were intraperitoneally transplanted into NARs and harvested three weeks after the transplantation. Angiogenesis with no obvious inflammatory reaction was found inside the pore areas of HA. The ectopic transplantation well supported the viability of hepatocytes seeded in HA disks evidenced by significant increases in the host rats' serum albumin level as time passed after the transplantation.

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The bioreactors used in tissue engineering can be categorized into two main groups: stirred tank bioreactors where cells or cell aggregates can move in an excess of media and perfusion bioreactors in which cells are immobilized in a reactor core and media is flowed past them. Here, emphasis will be placed on the advantages of perfusion over static culture and the use of bioreactors in culturing stem cells, especially hematopoietic stem cells (HSC) and liver cells. In each case the need for large-scale cell culturing will be described and the experience with bioreactors reviewed. Finally, the concept of the stem cell niche will be considered in the context of core materials for larger bioreactors.
Preparation of immunogen-reduced and biocompatible extracellular matrices from porcine liver
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These small scaffolds could be plausible for various research purposes related to regenerative medical science such as cell culture, stem cell differentiation and migration (23–25), in vitro drug testing and biological assays (26), and bio-absorbable tissue engineering (17). Furthermore, it also could be applied to in vivo transplantation studies for disease treatment (27–29) and host immune response of ECM (16,17). Recently, some investigators have demonstrated whole organ decellularization techniques.
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Safety evaluation of stem cells used for clinical cell therapy in chronic liver diseases; with emphasize on biochemical markers
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Citation Excerpt :
These procedures resulted in clearance by the native liver and led to significant improvement in cell survival. Higashiyama et al. (2003), showed that transplanted rat hepatocytes seeded in porous hydroxyapatite (HA) disks into the peritoneal cavity of Nagase analbuminemia rats (NARs), was accompanied with angiogenesis inside the pores in HA disks and hepatocyte viability was found to be about 3 weeks [132]. Besides, transplantation of HA disks seeded with co-cultured bone marrow MSCs and hepatocytes into NARs and liver damaged mice could improve the efficacy of cell therapy as shown by elevation of serum albumin and IL-6 [133].
There are several issues to be considered to reduce the risk of rejection and minimize side effects associated with liver cell transplantation in chronic liver diseases. The source and the condition of stem cell proliferation and differentiation ex vivo and the transplantation protocols are important safety considerations for cell based therapy. The biochemical and molecular markers are important tools for safety evaluation of different processes of cell expansion and transplantation. Studies show that hepatocytes differentiated from adult and embryonic stem cells exhibit biochemical and metabolic properties resembling mature hepatocytes. Therefore these assays can help to assess the biological and metabolic performance of hepatocytes and progenitor stem cells. The assays also help in testing the contribution of transplanted hepatocytes in improving the repair and function of damaged liver in the recipient.
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Liver transplantation is an important therapeutic option for many individuals with metabolic liver disease. Nevertheless, the invasive nature of surgery and limitations of donor organ availability have led to the search for alternatives to whole-organ transplantation. Cell-based therapies have been a particularly active area of investigation in recent years. Hepatocyte transplantations have been performed for a variety of indications, including acute liver failure, end-stage liver disease, and inborn errors of metabolism. Individuals with inborn errors of metabolism who have undergone hepatocyte transplantation have shown clinical improvement and partial correction of the underlying metabolic defect. In most cases, sustained benefits have not been observed. This may be related to inadequate cell dose, variations in the quality of hepatocyte preparations, rejection of the transplanted cells, or senescence of transplanted hepatocytes. Though initial proof of concept with hepatocyte transplantation has been demonstrated by a number of investigators, wide application of this technology has been hindered by the inability to secure a reliable and well-characterized cell source(s) for transplantation and by the challenges of sustained engraftment and expansion of transplanted cells in vivo. Cell-based therapies, including those based on stem cells or more differentiated progenitor cells, may represent the future of cell transplantation for treatment of metabolic liver disease.
Three-dimensional high-density culture of HepG2 cells in a 5-ml radial-flow bioreactor for construction of artificial liver
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A three-dimensional high-density cell culture is essential for the construction of an artificial tissue. Many researchers have reported that three-dimensional cell culture enhances cell function. The use of a radial-flow bioreactor (RFB) has enabled the cultivation of cells at high density for constructing a three-dimensional tissue. In this study, we have developed a novel, small RFB, which has a bed volume of 5 ml and is equipped with a porous support as an immobilized scaffold; its performance was tested using the hepatoblastoma cell line, HepG2. Among the other supports tested here, hydroxyl apatite was selected from the viewpoint of its ability to support good cell growth at high density with uniform distribution in a bioreactor. The HepG2 cells grew well in the scaffold under a sufficient supply of nutrients by radial flow and were used to construct a three-dimensional tissue in the scaffold. The concentration of the cells cultivated in this 5-ml RFB reached 10⁸ cells/ml and the glucose consumption rate was almost similar to that obtained when using a 30-ml RFB, which has already been reported previously. This high glucose consumption continued over 7 d after the growth phase. Furthermore, albumin production was maintained in the stable phase. Gene expression profiles of cells obtained from long-term cultures in the 5-ml RFB were analyzed. It was found that the expressions of genes encoding the cell cycle-related proteins, cyclins, and cell cycle division 2 (cdc2) were suppressed in the stable phase. In addition, the number of cells incorporating 5′-bromo-2′-deoxyuridine (BrdU) in the stable phase markedly decreased compared with that in the growth phase. These results indicated that the majority of cells in the stable phase remain in the G0/G1 phase. Furthermore, this implies that the three-dimensional tissue constructed in the 5-ml RFB showed the high function similar to a normal liver in the human body. Therefore, the 5-ml RFB was considered as a useful tool and a substitute method for animal experiments.
Availability of bone marrow stromal cells in three-dimensional coculture with hepatocytes and transplantation into liver-damaged mice
2005, Journal of Bioscience and Bioengineering
Rat bone marrow stromal cells (BMSCs) were cultured in porous hydroxyapatite (HA) disks for 2 weeks to form a cell layer on the surface. Freshly isolated hepatocytes were then inoculated into both BMSC-cultured and non-treated HA disks. Hepatocytes cocultured with BMSCs secreted significantly more albumin than those in monoculture in vitro. The cell-packed HA disks were implanted into the peritoneal cavity of Nagase analbuminemia rats (NARs), and 4 weeks later, blood samples were collected to measure the albumin concentration. The cotransplantation of BMSCs with hepatocytes significantly increased the serum albumin concentration in NARs. The HA disks coculturing mice hepatocytes and BMSCs were also implanted into mice, in which liver damage had been induced using carbon tetrachloride and phenobarbital. The decreased serum albumin level in liver-damaged mice was completely recovered by the transplantation of hepatocytes and BMSCs. The serum level of IL-6 in liver-damaged mice was also increased by the cotransplantation of BMSCs and hepatocytes. Thus, the transplantation of BMSCs appears to have a systemic effect on recipients through the increase in the serum cytokine level as well as a local effect on cotransplanted hepatocytes.

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Abstract

References (19)

Preparation of isolated rat liver cells

Methods Cell Biol.

Hydroxyapatite ceramics as a carrier of gene-transduced bone marrow cells

J. Orthop. Sci.

Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation

N. Engl. J. Med.

Hepatocyte transplantation for the treatment of human disease

Semin. Liver Dis.

Defining hepatocellular chimerism in a liver failure patient bridged with hepatocyte infusion

Transplantation

Prolonged function of hepatocytes transplanted into the spleens of Nagase analbuminemic rats

Eur. Surg. Res.

Restoration of serum albumin levels in Nagase analbuminemic rats by hepatocyte transplantation

Hepatology

Comparison of two methods of autologous intrasplenic hepatocellular transplantation in partially hepatectomized dogs

Eur. Surg. Res.

Studies on the safety of intrasplenic hepatocyte transplantation: relevance to ex vivo gene therapy and liver repopulation in acute hepatic failure

Hum. Gene Ther.

Cited by (8)

Materials for perfusion bioreactors used in tissue engineering

Preparation of immunogen-reduced and biocompatible extracellular matrices from porcine liver

Safety evaluation of stem cells used for clinical cell therapy in chronic liver diseases; with emphasize on biochemical markers

Cell-based therapies for metabolic liver disease

Three-dimensional high-density culture of HepG2 cells in a 5-ml radial-flow bioreactor for construction of artificial liver

Availability of bone marrow stromal cells in three-dimensional coculture with hepatocytes and transplantation into liver-damaged mice