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Keywords:

  • bioartificial liver;
  • hepatocyte ;
  • liver;
  • liver diseases;
  • liver tissue engineering;
  • stem cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Liver tissue engineering
  5. Cell sources for liver tissue engineering
  6. Preserving hepatocyte function under in vitro culture
  7. Conclusion and perspective
  8. Acknowledgements
  9. References

Liver diseases are of major concern as they now account for millions of deaths annually. As a result of the increased incidence of liver disease, many patients die on the transplant waiting list, before a donor organ becomes available. To meet the huge demand for donor liver, alternative approaches using liver tissue engineering principles are being actively pursued. Even though adult hepatocytes, the primary cells of the liver are most preferred for tissue engineering of liver, their limited availability, isolation from diseased organs, lack of in vitro propagation and deterioration of function acts as a major drawback to their use. Various approaches have been taken to prevent the functional deterioration of hepatocytes including the provision of an adequate extracellular matrix and co-culture with non-parenchymal cells of liver. Great progress has also been made to differentiate human stem cells to hepatocytes and to use them for liver tissue engineering applications. This review provides an overview of recent challenges, issues and cell sources with regard to liver tissue engineering.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Liver tissue engineering
  5. Cell sources for liver tissue engineering
  6. Preserving hepatocyte function under in vitro culture
  7. Conclusion and perspective
  8. Acknowledgements
  9. References

The liver, the largest organ in the body, has a complex architecture and performs a myriad of functions. Even though the liver is highly regenerative, drugs and toxins or viral infections can cause extensive damage to hepatocytes, reducing function and regeneration. In developed countries, drug toxicity and alcoholic liver diseases are of major concern [1, 2], while in developing countries viral hepatitis is a major clinical concern [3, 4]. Globalization and immigration have resulted in the global spread of viral hepatitis and this disease has now become a major world problem [5]. The World Health Organization (WHO) report shows that 2.5% of total deaths in the world are because of liver disease [6], and this will become the 14th most common cause of death by 2030 [7]. In the UK, liver disease is the 5th most common cause of death [5], and continues to rise.

Extensive damage to hepatocytes can lead to liver failure. As a result of liver failure, multisystemic complications arise, leading to neurological impairment, haematological disturbance, renal dysfunction and metabolic abnormalities. Death usually occurs as a result of multiorgan failure. Liver transplantation is the only available and, usually, successful treatment option at this stage, if patient vital signs are favorable. However, the number of liver transplantations is limited because of the lack of availability of donor organs [8]. One strategy devised to cope with the shortage of donor organs is live donor organ transplantation, where partial liver grafts are being employed as an alternative to cadaveric liver transplantation. However, these surgical advancements are still not able to circumvent the requirement for donor livers [9]. Alternatives, such as hepatocyte transplantation [10-12], extracorporeal devices [13-17] and stem cell therapy [18-22], are strategies being pursued throughout the world either to prolong the life of patient until a donor liver is available or to regenerate the normal liver. Whether for liver regeneration therapy, for seeding liver support devices or for in vitro screening, there has been a constant quest for a suitable, reproducible source of hepatocytes. To date it was proved elusive.

Liver tissue engineering

  1. Top of page
  2. Abstract
  3. Introduction
  4. Liver tissue engineering
  5. Cell sources for liver tissue engineering
  6. Preserving hepatocyte function under in vitro culture
  7. Conclusion and perspective
  8. Acknowledgements
  9. References

Tissue engineering utilizes biocompatible scaffolds which provide an optimal environment for cell growth and differentiation with the aim of developing constructs that find application either in vitro or in vivo [23]. Liver tissue engineering is mainly concerned with the development of extracorporeal, liver support devices or in vivo liver regeneration using cell transplantation or implantable devices [24]. Recent trends in the field include the construction of micro tissue structures by specifically arranging the cells using of micro fabrication and nanotechnology approaches [25].

Extracorporeal temporary liver support devices

Extracorporeal temporary liver support devices are used as life-supporting bridging devices in patients awaiting a donor organ. Earlier efforts to develop artificial or non-biological devices utilized mechanical methods for removing toxic compounds [26, 27] which did not provide statistically significant improvements in patient life span. Biological support devices (bio-artificial liver) contain a cellular component, hepatocytes, combined with the bioreactor component to enhance the performance of the cells. The bioreactors are specifically designed to provide optimal conditions for the performance of the cell and have to ensure the safety of patient. As a result of the varied biochemical activities of hepatocytes, bio-artificial liver (BAL) devices were found to be much more effective than non-biological devices providing synthetic and biochemical functions [28-31]. Other BAL devices using porcine liver slices (HEPAHOPE) are under development.

It is considered that, for an effective clinical therapy, a bio-artificial liver should be able to provide at least 10% of total liver function, which requires about 1010 hepatocytes [32, 33]. A major problem is the procurement of this large number of human hepatocytes as most of these cells were previously isolated from liver tissues unacceptable for transplantation [33]. Unlike in vitro culture, conditions inside a BAL are complicated. Hepatocytes will be in contact with plasma (plasma or plasma fraction determined by the cut-off of the plasmapheresis module) which has different physio-chemical properties and poor oxygen solubility compared with culture medium. Studies have shown that hepatocytes do not perform well in plasma culture [34]. As hepatocytes have a high oxygen uptake rate and are very sensitive to oxygen concentration, a major challenge in BAL design is to provide adequate oxygen to the closely packed hepatocytes. Another major challenge is the shear stress experienced by hepatocytes in BAL devices and is mainly depended on the BAL design. Any shear stress above 5 dyn/cm2 can affect hepatocyte function [33]. As a result BAL can only be perfused very slowly, but at the same time have to provide adequate oxygen to the hepatocyte. Recent methods using modules for oxygenation and computer fluid dynamic modelling of BAL to effectively culture hepatocytes in such a device are promising [35-38]. Another major problem is the limited volume of the bioreactor, as the maximum blood/plasma that can be safely drawn out of a haemodynamically unstable liver failure patient is one litre [39]. It is a challenge to achieve 10% of liver function within a volume of one litre.

Cell transplantation

Cell transplantation has several practical advantages as compared with solid liver transplantation as: (a) several patients can be treated from a single donor organ; (b) cells can be cryopreserved and can be used later at a time of emergency; (c) it is less invasive surgery than organ transplantation; (d) it can be repeated if needed with low morbidity and; (e) the cost of treatment is less compared to whole liver transplantation [40]. Experimental studies are ongoing to assess transplantation of adult hepatocytes, fetal hepatocytes and stem cells. Even though hepatocytes can be transplanted into liver, spleen and peritoneal cavity, the therapeutic effect has been found to vary according to the site of transplantation. Intra-hepatic and splenic sites are recognized as the best sites for cell infusion [41] and the peritoneal cavity is commonly used for transplanting encapsulated hepatocytes [42-47]. There are a few reports on complications of cell infusion such as transient hemodynamic instability during cell infusion through the splenic artery and portal vein [48]. When transplanted hepatocytes do not attach, a large number of cells can reach the pulmonary circulation and can cause pulmonary emboli which can result in cardio-pulmonary compromise. Another major factor limiting the clinical use of hepatocyte transplantation is the lack of a reliable hepatocyte source. Even though cryopreserving can help to prolong the availability of cells, cryopreserved hepatocytes are less efficient in engrafting than freshly isolated hepatocytes [48]. Transplantation of hepatocytes has been found to be effective in prolonging survival in animal models of liver failure and in humans [49]. Even though the rate of engraftment of transplanted hepatocytes is often less than 30%, this procedure has been used successfully in treating inherited metabolic disorders in young patients [50-54], but the long-term function of the transplanted hepatocytes has not been demonstrated [40]. A better understanding of the factors that allow hepatocytes to engraft and integrate into the host tissue is required to avoid repeated cell infusion and possible complications. More optimization of the number of cells required to be infused at any one time is also needed.

Implantable devices

To enhance the survival and functional stability of transplanted hepatocytes, biomaterials have been developed and incorporated into implantable devices. Implantable devices are either in the form of microcarriers [55], biodegradable scaffolds [56, 57] or as encapsulation vehicles [58-60]. Since cells in microcarriers and scaffolds are open to the systemic circulation, there is the possibility of immunoreactions. Encapsulation can avoid immunological problems, but there is a requirement for a biomaterial that can provide adequate mass transfer of nutrients and the oxygen which is essential for the cells. Promising results have been shown using collagen-coated microcarriers [61, 62] and alginate encapsulation [42, 47, 63]. However, a major issue during long-term implantation is the formation of a fibrotic capsule around encapsulated structures [64-66]. These capsules can prevent the exchange of solutes between the cell and outside environment and frequently lead to cell death.

Cell sources for liver tissue engineering

  1. Top of page
  2. Abstract
  3. Introduction
  4. Liver tissue engineering
  5. Cell sources for liver tissue engineering
  6. Preserving hepatocyte function under in vitro culture
  7. Conclusion and perspective
  8. Acknowledgements
  9. References

Primary human and porcine hepatocytes, immortalized hepatocytes, human hepatic cell lines and stem cells have been utilized for liver tissue engineering (Table 1).

Table 1. Cell choices for liver tissue engineering
CellSourceApplicationAdvantagesDisadvantages
Human hepatocytesDiscarded human liver tissue

Bio-artificial liver.

Implantable devices.

Cell transplantation.

High host compatibility.

Reduced availability.

Poor in vitro proliferation.

Porcine hepatocytesPorcine liver

Bio-artificial liver.

Implantable devices.

Enhanced availability compared with human hepatocytes.

Potential zoonotic disease transmission.

Protein–protein incompatibility.

Possible immune response.

Tumour derived human hepatocyte cell linesCell lines derived from human hepatic carcinoma.

Bio-artificial liver.

Implantable devices.

Easy storage, maintenance and in vitro proliferation.

Tumorigenicity.

Reduced functional performance.

Immortalized human hepatocyte cell linesHuman hepatocytes immortalized by gene transfection.

Bio-artificial liver.

Implantable devices.

Easy in vitro proliferation, storage and maintenance.

Reduced tumorigenicity compared with tumour derived cell lines.

Potential tumorigenicity.

Long-term safety concerns.

Reduced functional performance.

Adult stem cells (MSCs)Adult tissues Cell transplantation. Implantable devices.

Unlimited availability.

Less safety concerns.

Transdifferentiation of MSCs to myofibroblasts.

Lower efficiency of the available hepatic differentiation protocols.

Longer duration of hepatic differentiation.

Oval cellsHuman fetal liverCell transplantation.

Native liver stem cells.

Can differentiate to both hepatocyte and biliary cells.

Cell isolation is difficult.

Limited availability.

Tumorigenic potential.

Human fetal hepatocytesHuman fetal liver

Bio-artificial liver.

Implantable devices. Cell transplantation.

Easy cell isolation.

Can undergo few cell divisions in vitro.

Lower functional efficiency.

Reduced availability.

Ethical concerns.

Possible tumorigenicity.

HepatoblastHuman fetal liver at early stage of gestationCell transplantation.Extensive in vitro proliferation.

Ethical issues.

Limited availability.

Embryonic/ induced pluripotent stem cellsPluripotent cell lines derived from discarded human embryo (embryonic) or genetically modified adult cells (induced pluripotent)

Bio-artificial liver.

Implantable devices.

Unlimited availability and indefinite proliferation.

Hepatic differentiation capability.

Ethical issues.

Possible teratoma formation.

Induced hepatocyte like cells (iHep)Genetically modified adult cells to express hepatic genes. Easy method for generating hepatocytes within a limited time period.Functional stability and safety.

Porcine hepatocytes

As the availability of human hepatocytes is limited and pigs have a physiology similar to humans, porcine hepatocytes were used in BAL and implantable devices [67]. Most of the BALs that entered clinical trials have utilized porcine hepatocytes [68, 69]. Development of improved cryopreservation techniques made it more convenient to use cryopreserved cells in BALs [70-74]. The main limitation of primary hepatocyte culture is the deterioration of hepatocyte functions and lack of long-term functional stability under in vitro culture conditions [75]. Various efforts have been made to maintain the functional performance of hepatocytes by providing proper extracellular matrix for attachment either as scaffolds [56, 76-81], microcarriers [82, 83] or cell entrapment/encapsulation devices [84-86]. The main concerns in using xenogenic porcine hepatocytes is the transfer of zoonotic diseases [87], protein–protein incompatibility and the possible immune responses generated during treatment [88-90]. There is therefore a compelling need to seek alternative cell sources.

Primary human hepatocytes

Primary human hepatocytes are the most preferred cell source for BAL sand implantable devices. However, the availability of primary human hepatocytes is limited, as most of them are isolated from discarded cadaveric liver samples that are not suitable for transplantation. Even though improved cryopreservation techniques have been developed to preserve these cells in an efficient way, a major concern is the deterioration of differentiated functions under in vitro culture conditions [91, 92]. In vitro models such as sandwich and spheroid culture have been developed to preserve the differentiated functions of hepatocytes [93, 94]. However, most of the in vitro cell models are not able to express all differentiated functions and often lack many of the enzymes, transporters, cytochrome P450 enzymes and cell polarization characteristics required [24].

Human hepatocyte cell lines

In an attempt to enhance cell availability various efforts have been made to produce new or to utilize existing human hepatocyte cell lines. Both tumour-derived hepatocyte cell lines and immortalized cells have been investigated.

Human tumour-derived cell lines

BALs such as Extracorporeal Liver Assist Device (ELAD) utilized the C3A cell line [14, 95]. C3A is a clone, derived from the human hepatoma cell line HepG2, which has improved hepatocyte functions compared with the parent line [14]. Studies show that C3A has an improved synthetic (albumin and alpha foetoprotein) capacity and nitrogen-metabolizing ability. However, factors limiting their usage are the low levels of ammonia removal (because of lack of complete urea enzymes), amino acid metabolism, cytochrome P450s and lack of many drug-metabolizing functions (mainly because of the reduced number of mitochondria)[96-98]. Another major concern in using cancer cell lines is the possibility of transmission of tumorogenic products or agents and possible complications arising from this [99, 100].

Immortalized human hepatocyte cell lines

Many efforts have been made to overcome the poor in vitro proliferation of primary hepatocytes by immortalizing these cells [101].Transfection of simian virus 40 Tantigen (SV40 Tantigen) has been used to develop many immortalized cell lines [102, 103]. Human hepatocytes have also been immortalized by lipofectamine-mediated co-transfection of albumin-promoter-regulated antisense constructs and genes coding for the cellular transcription factor E2F and D1 cyclin [104]. However, the risks of potential tumorigenic effects were not addressed in such studies. Recent efforts were made to develop systems which tightly control cell growth using reversible immortalization. In reversible immortalization, cells are immortalized by retrovirus-mediated transfer of oncogenes and subsequently these oncogenes are specifically excised out using the principles of Cre/loxP recombination. As an additional safety feature, to protect the patient from possible migration of the cells from BALs, some reversible immortalized cells also have a suicide gene such as HSVTK incorporated which makes the cell sensitive to anti-viral drugs such as ganciclovir [105]. The human immortalized hepatocyte cell line NKNT-3 was developed by using this advanced technology [106, 107]. Non-parenchymal cell lines such as endothelial cells (HNNT-2) [108], stellate cells (TWNT-1) [109] and cholangiocytes (MMNK-1) were also developed by this technique and may be useful for hepatocyte co-culture studies. However, possible inhibition of suicide gene transcription because of promoter methylation and DNA damage because of Cre/lox recombination are of concern. Moreover, the long-term safety and differentiated functionality of these cells have not been explored in much detail.

Stem cells

Adult, embryonic and induced pluripotent stem cells have been utilized for liver tissue engineering. The major advantage of stem cells is their proliferation capacity, which allows scalable populations of cells to be grown. In addition, pluripotent stem cells (human embryonic or induced pluripotent stem cells) can be expanded while retaining the ability to form any cell type in the human body, representing an inexhaustible cell source. Thus, stem cells are a promising cell choice for the production of hepatocytes as they can meet the great demand for these cells. The prospects for the use of different stem cell types in liver regeneration has been reviewed elsewhere [110, 111].

Adult stem cells

Adult stem cells are often the preferred cells for transplantation and implantable devices. Adult stem cells have no risk of teratoma formation and their use avoids many ethical issues compared with embryonic stem cells. Many in vitro studies have shown that mesenchymal stem cells (MSC) harvested from various sources have the capacity to transdifferentiate to hepatocyte-like cells [112, 113]. Animal studies have shown that direct injection of MSC into damaged liver can generate hepatocyte-like cells [114, 115]. However, the trans differentiation of MSC to myofibroblasts at the site of injury is of major concern [116, 117]. Therefore, recent efforts have been directed at transplantation of in vitro trans differentiated MSC to avoid the possible formation of myofibroblasts [118]. However, the differentiation of stem cells to mature adult hepatocytes in the quantities required for clinical therapy is still a challenge. None of the available differentiation protocols are able to differentiate stem cells to fully functional hepatocytes. Moreover, the process of differentiation requires complex growth factors and supplements that increase the effective costs of providing therapy.

Oval cells

Oval cells are quiescent stem cells residing in adult liver that proliferate extensively during prolonged liver injury, giving rise to both hepatocyte and cholangiocytes. Analysis of markers suggest that oval cells consist of a heterogeneous population of cells that differ in their differentiation capacity and stage of differentiation [119]. The low number of oval cells in liver limits their isolation and purification. Moreover, concern about possible tumour formation caused by oval cell predominance in hepatocellular carcinoma limits their application [120].

Human fetal hepatocytes

Unlike adult hepatocytes, fetal hepatocytes can undergo multiple cell divisions in vitro [121]. To ensure unlimited availability, human fetal hepatocytes have been immortalized by SV40 large T antigen [122]or hTERT transfection [123]. However, fetal hepatocytes show only low levels of ammonia elimination (49%) and urea production (1.1%) when compared with primary hepatocytes, limiting their application in a BAL [124]. Functional comparison of fetal hepatocytes with mature hepatocytes in AMC-BAL shows that fetal hepatocytes are not functionally mature enough to employ in clinical treatment regimes [125].

Even though fetal liver transplantation studies are showing promising results [126, 127], their availability, the ethical concerns, possible tumorigenicity and their incomplete differentiation limits their use in clinical application [128].

Hepatoblast

Hepatoblasts are progenitor cells that can be isolated from human fetal livers at early stages of gestation [129]. Unlike fetal hepatocytes, hepatoblasts can proliferate extensively under in vitro conditions and can give rise to both hepatocyte and cholangiocytes in vivo. Transplantation studies in animal models show that hepatoblast can integrate and proliferate in injured liver [130-132]. However, ethical issues and availability limit their application for regenerative therapy.

Embryonic and induced pluripotent stem cells

The main advantages of embryonic stem cells are their pluripotency, availability and their in definite proliferation in vitro [133]. Even though feeder-free cultures using matrigel have been developed, enabling ES cell culture in BAL devices, the risk of introducing murine viruses from matrigel is of concern. There are many reports on the in vitro differentiation of embryonic stem cells to hepatocyte-like cells [134-138]. However, none of the available protocols have been delivered at scale and have thereby limit the application of ES-derived hepatocytes in a BAL [139]. Major concerns for the use of embryonic stem cells for cell based therapy are the ethics, immunocompatiblty and possible teratoma formation from non-differentiated cells [140].

The development of induced pluripotent stem (iPS) cell technology has, to some extent, circumvented the ethical and immune issues. Recent efforts aimed at differentiating iPS cells to hepatocytes are promising [141, 142]. In spite of these developments, the concerns over use of viral vectors, changes in cell cycle regulators and teratoma formation are still limiting the application of iPS cells for liver regenerative therapy. However, the differentiation of embryonic stem cells and induced pluripotent stem cells to hepatocytes and other lineages will find application in drug and toxicity screening studies in the shorter term and may in the future have a role to play in cell based therapies.

Induced hepatocyte-like cells (iHep)

Recent innovative studies show that hepatocyte-like cells can be generated from adult cells by over-expressing hepatocyte lineage specific transcription factors [143, 144]. iHep cells were able to express both hepatic genes and functions and were able to engraft and reconstitute hepatic tissue in murine models. This novel method is a great advantage for liver tissue engineering as it can bypass all the complicated steps involved in generating iPS cells and their differentiation to functional hepatocytes. However, more studies are needed to further develop this technology. Moreover, the functional stability and safety of using iHep cells for liver tissue engineering applications has to be investigated.

Preserving hepatocyte function under in vitro culture

  1. Top of page
  2. Abstract
  3. Introduction
  4. Liver tissue engineering
  5. Cell sources for liver tissue engineering
  6. Preserving hepatocyte function under in vitro culture
  7. Conclusion and perspective
  8. Acknowledgements
  9. References

The major challenge in the use of primary isolated hepatocyte culture, or, indeed, hepatocytes differentiated from either ES or iPS cells, is the functional deterioration of the mature cells under in vitro conditions within a short period of culture. For a more complete hepatocyte function-appropriate microenvironment, cell–cell interactions, cell–matrix interactions and chemical gradients are required which mimic the hepatic plate of the in vivo liver [23, 145-148]. Mimicking the in vivo conditions by utilizing extracellular matrix and the co-culture of hepatocytes with non-parenchymal cells are key areas of investigation aimed at extending the life of the mature hepatocyte and preserving mature liver functions. Extracellular matrix has a profound influence on the structure and function of liver cells. Studies show that extracellular matrix resembling that of the perisinusoidal space is critical in maintaining normal liver functions [149, 150]. In vitro cultures in dishes coated with extracellular matrix such as matrigel, collagen, fibronectin and mixtures of collagen and fibronectin were only able to preserve hepatocyte functions for a short time [151]. However, collagen-sandwich systems were found to be able to maintain cellular morphology and albumin secretion for extended periods [152]. Microarray gene expression analysis demonstrates that collagen-sandwich cultures outperformed their counterpart monolayer culture in lipid, amino acid carbohydrate and alcohol metabolism, synthesis of bile acids and expression of cytochrome P450 enzymes [153, 154]. It has been shown that collagen-sandwich-cultured hepatocytes can develop well-polarized bile canaliculi and express many transport proteins [155-158].

Heterotypic interaction between hepatocytes and non-parenchymal cells has a profound role in liver physiology. Many studies have been carried out on the co-culture of hepatocytes with liver-derived cells such as biliary epithelium, stellate, endothelial and Kupffer cells. Hepatocytes have also been co-cultured with non-liver-derived cells such as human fibroblasts, aortic endothelium, lung epithelium and dermal fibroblasts. Co-culture of hepatocytes with non-parenchymal cells and the influence of these cells on liver-specific functions has been reviewed elsewhere [159]. Although the exact mechanisms by which cell–cell interactions modulate hepatocyte phenotype and function is not clear, co-culture has been found to have a profound influence on hepatocyte phenotype [158], stable long-term retention of mature liver functions such as albumin secretion [161], cytochrome P450 enzyme activity [162], phase II enzymes [163] and expression of junctional complex proteins [161, 164]. Despite the evidence of improved function using co-culture, there are no bioreactors with a co-culture component which have entered clinical trials although some are at the research stage [165]. Engineering constraints in designing such a large-scale complex bioreactor system is one of the main problems in developing this approach.

The use of biological subcellular components in BAL and implantable devices are not particularly useful because of the possible immune reactions, transfer of infections and lack of consistency. Identifying non-biological materials that can preserve hepatocyte functions will be extremely beneficial for liver tissue engineering applications. Recent approaches aimed at screening such synthetic sub-cellular matrix replacements which can support the hepatic functions of differentiated human pluripotent stem cells are promising [166]. Identification of such polymers will help to develop scalable non-biological subcellular components that can be used in BAL and implantable devices.

Conclusion and perspective

  1. Top of page
  2. Abstract
  3. Introduction
  4. Liver tissue engineering
  5. Cell sources for liver tissue engineering
  6. Preserving hepatocyte function under in vitro culture
  7. Conclusion and perspective
  8. Acknowledgements
  9. References

The major issue in liver support and regeneration therapy is the availability of suitable cell types. Approaches using cutting-edge biotechnology techniques, such as reversibly immortalizing human hepatocytes or non-viral methods of creating iPS cells are promising. The uses of stem cells are still in their infancy, and the need for a robust, completely efficient protocol for the differentiation of stem cells into functionally mature hepatocytes is still essential. A better understanding of the cellular signalling involved in the differentiation of stem cells into functional hepatocytes and maintenance of mature hepatocyte function would be extremely beneficial. Techniques using polymer library screening, microfluidics and micropatterning have helped to better understand cell–cell interactions, cell–matrix interactions and the microenvironment [166, 167]. This knowledge will not only help to improve the design of BALs for clinical use but will provide substantial benefits for physiologically relevant and high-throughput screening of drugs and new medicines.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Liver tissue engineering
  5. Cell sources for liver tissue engineering
  6. Preserving hepatocyte function under in vitro culture
  7. Conclusion and perspective
  8. Acknowledgements
  9. References
  • 1
    Larson AM, Polson J, Fontana RJ, et al. Acetaminophen-induced acute liver failure: results of a United States multicenter, prospective study. Hepatology 2005; 42: 136472.
  • 2
    Mandayam S, Jamal MM, Morgan TR. Epidemiology of alcoholic liver disease. Semin Liver Dis 2004; 24: 21732.
  • 3
    Khuroo MS, Kamili S. Aetiology and prognostic factors in acute liver failure in India. J Viral Hepat 2003; 10: 22431.
  • 4
    Acharya SK, Panda SK, Saxena A, Gupta SD. Acute hepatic failure in India: a perspective from the east. J Gastroenterol Hepatol 2000; 15: 4739.
  • 5
    Williams R. Global challenges in liver disease. Hepatology 2006; 44: 5216.
  • 6
    Lopez AD, Mathers CD, Ezzati M, Jamison DT, Murray CJ. Measuring the global burden of disease and risk factors, 1990–2001. 2006; 1: 114.
  • 7
    Mathers CD, Loncar D. Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med 2006; 3: e442.
  • 8
    Schramm C, Bubenheim M, Adam R, et al. Primary liver transplantation for autoimmune hepatitis: a comparative analysis of the European Liver Transplant Registry. Liver Transpl 2010; 16: 4619.
  • 9
    Adam R, Mcmaster P, O'grady JG, et al. Evolution of liver transplantation in Europe: report of the European Liver Transplant Registry. Liver Transpl 2003; 9: 123143.
  • 10
    Bilir BM, Guinette D, Karrer F, et al. Hepatocyte transplantation in acute liver failure. Liver Transpl 2000; 6: 3240.
  • 11
    Muraca M, Gerunda G, Neri D, et al. Hepatocyte transplantation as a treatment for glycogen storage disease type 1a. Lancet 2002; 359: 3178.
  • 12
    Strom SC, Fisher RA, Thompson MT, et al. Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure. Transplantation 1997; 63: 55969.
  • 13
    Ellis AJ, Hughes RD, Wendon JA, et al. Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure. Hepatology 1996; 24: 144651.
  • 14
    Sussman NL, Chong MG, Koussayer T, et al. Reversal of fulminant hepatic failure using an extracorporeal liver assist device. Hepatology 1992; 16: 605.
  • 15
    Mitzner SR, Stange J, Klammt S, et al. Extracorporeal detoxification using the molecular adsorbent recirculating system for critically ill patients with liver failure. J Am Soc Nephrol 2001; 12(Suppl. 17): S7582.
  • 16
    Sussman NL, Gislason GT, Conlin CA, Kelly JH. The hepatix extracorporeal liver assist device: initial clinical experience. Artif Organs 1994; 18: 3906.
  • 17
    Rifai K, Ernst T, Kretschmer U, et al. Prometheus–a new extracorporeal system for the treatment of liver failure. J Hepatol 2003; 39: 98490.
  • 18
    Habibullah CM, Syed IH, Qamar A, Taher-Uz Z. Human fetal hepatocyte transplantation in patients with fulminant hepatic failure. Transplantation 1994; 58: 9512.
  • 19
    Jang YY, Collector MI, Baylin SB, Diehl AM, Sharkis SJ. Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol 2004; 6: 5329.
  • 20
    Avital I, Inderbitzin D, Aoki T, et al. Isolation, characterization, and transplantation of bone marrow-derived hepatocyte stem cells. Biochem Biophys Res Commun 2001; 288: 15664.
  • 21
    Jones EA, Tosh D, Wilson DI, Lindsay S, Forrester LM. Hepatic differentiation of murine embryonic stem cells. Exp Cell Res 2002; 272: 1522.
  • 22
    Yamamoto Y, Teratani T, Yamamoto H, et al. Recapitulation of in vivo gene expression during hepatic differentiation from murine embryonic stem cells. Hepatology 2005; 42: 55867.
  • 23
    Griffith LG, Swartz MA. Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol 2006; 7: 21124.
  • 24
    Ananthanarayanan A, Narmada BC, Mo X, Mcmillian M, Yu H. Purpose-driven biomaterials research in liver-tissue engineering. Trends Biotechnol 2011; 29: 1108.
  • 25
    Toh YC, Zhang C, Zhang J, et al. A novel 3D mammalian cell perfusion-culture system in microfluidic channels. Lab Chip 2007; 7: 3029.
  • 26
    Omori Y. New apparatus for artificial liver support: PAT resin–removal of protein-bound substances and its effect on hepatic failure in animals. Nihon Geka Gakkai Zasshi 1985; 86: 56675.
  • 27
    Hughes R, Ton HY, Langley P, et al. Albumin-coated Amberlite XAD-7 resin for hemoperfusion in acute liver failure. Part II: in vivo evaluation. Artif Organs 1979; 3: 236.
  • 28
    Chamuleau RA. Artificial liver support in the third millennium. Artif Cells Blood Substit Immobil Biotechnol 2003; 31: 11726.
  • 29
    Rozga J, Malkowski P. Artificial liver support: quo vadis? Ann Transplant 2010; 15: 92101.
  • 30
    Rozga J. Liver support technology–an update. Xenotransplantation 2006; 13: 3809.
  • 31
    Kjaergard LL, Liu J, Als-Nielsen B, Gluud C. Artificial and bioartificial support systems for acute and acute-on-chronic liver failure: a systematic review. JAMA 2003; 289: 21722.
  • 32
    Morsiani E, Brogli M, Galavotti D, et al. Biologic liver support: optimal cell source and mass. Int J Artif Organs 2002; 25: 98593.
  • 33
    Chan C, Berthiaume F, Nath BD, et al. Hepatic tissue engineering for adjunct and temporary liver support: critical technologies. Liver Transpl 2004; 10: 133142.
  • 34
    Washizu J, Chan C, Berthiaume F, et al. Amino acid supplementation improves cell-specific functions of the rat hepatocytes exposed to human plasma. Tissue Eng 2000; 6: 497504.
  • 35
    Mareels G, Poyck PP, Eloot S, Chamuleau RA, Verdonck PR. Three-dimensional numerical modeling and computational fluid dynamics simulations to analyze and improve oxygen availability in the AMC bioartificial liver. Ann Biomed Eng 2006; 34: 172944.
  • 36
    Consolo F, Fiore GB, Truscello S, et al. A computational model for the optimization of transport phenomena in a rotating hollow-fiber bioreactor for artificial liver. Tissue Eng Part C Methods 2009; 15: 4155.
  • 37
    Sullivan JP, Gordon JE, Bou-Akl T, Matthew HW, Palmer AF. Enhanced oxygen delivery to primary hepatocytes within a hollow fiber bioreactor facilitated via hemoglobin-based oxygen carriers. Artif Cells Blood Substit Immobil Biotechnol 2007; 35: 585606.
  • 38
    Sullivan JP, Harris DR, Palmer AF. Convection and hemoglobin-based oxygen carrier enhanced oxygen transport in a hepatic hollow fiber bioreactor. Artif Cells Blood Substit Immobil Biotechnol 2008; 36: 386402.
  • 39
    Wnek GE, Bowlin GL. Encyclopedia of Biomaterials and Biomedical Engineering. New York: Marcel Dekker; 2004.
  • 40
    Fox IJ, Chowdhury JR. Hepatocyte transplantation. Am J Transplant 2004; 4(Suppl. 6): 713.
  • 41
    Gupta S, Bhargava KK, Novikoff PM. Mechanisms of cell engraftment during liver repopulation with hepatocyte transplantation. Semin Liver Dis 1999; 19: 1526.
  • 42
    Selden C, Casbard A, Themis M, Hodgson HJ. Characterization of long-term survival of syngeneic hepatocytes in rat peritoneum. Cell Transplant 2003; 12: 56978.
  • 43
    Mai G, Nguyen TH, Morel P, et al. Treatment of fulminant liver failure by transplantation of microencapsulated primary or immortalized xenogeneic hepatocytes. Xenotransplantation 2005; 12: 45764.
  • 44
    Mei J, Sgroi A, Mai G, et al. Improved survival of fulminant liver failure by transplantation of microencapsulated cryopreserved porcine hepatocytes in mice. Cell Transplant 2009; 18: 10110.
  • 45
    Mai G, Huy NT, Morel P, et al. Treatment of fulminant liver failure by transplantation of microencapsulated primary or immortalized xenogeneic hepatocytes. Transplant Proc 2005; 37: 5279.
  • 46
    Link TW, Arifin DR, Long CM, et al. Use of magnetocapsules for in vivo visualization and enhanced survival of xenogeneic HepG2 cell transplants. Cell Med 2012; 4: 7784.
  • 47
    Rahman TM, Diakanov I, Selden C, Hodgson H. Co-transplantation of encapsulated HepG2 and rat Sertoli cells improves outcome in a thioacetamide induced rat model of acute hepatic failure. Transpl Int 2005; 18: 10019.
  • 48
    Schmidt P, Kerjaschki D, Syre G, et al. Recurrence of intramembraneous glomerulonephritis in 2 consecutive kidney transplantations. Schweiz Med Wochenschr 1978; 108: 7818.
  • 49
    Gupta S, Chowdhary JR. Hepatocyte transplantation: back to the future. Hepatology 1992; 15: 15662.
  • 50
    Fox IJ, Chowdhury JR, Kaufman SS, et al. Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N Engl J Med 1998; 338: 14226.
  • 51
    Ambrosino G, Varotto S, Strom SC, et al. Isolated hepatocyte transplantation for Crigler-Najjar syndrome type 1. Cell Transplant 2005; 14: 1517.
  • 52
    Ribes-Koninckx C, Ibars EP, Agrasot MA, et al. Clinical outcome of hepatocyte transplantation in four pediatric patients with inherited metabolic diseases. Cell Transplant 2012; 21: 226782.
  • 53
    Takeda K, Tanaka K, Kumamoto T, et al. Living donor liver transplantation for Dorfman-Chanarin syndrome with 1 year follow-up: case report. Transplant Proc 2010; 42: 385861.
  • 54
    Mckiernan P. Liver transplantation and cell therapies for inborn errors of metabolism. J Inherit Metab Dis 2013; DOI: 10.1007/s10545-012-9581-z.
  • 55
    Demetriou AA, Levenson SM, Novikoff PM, et al. Survival, organization, and function of microcarrier-attached hepatocytes transplanted in rats. Proc Natl Acad Sci U S A 1986; 83: 74759.
  • 56
    Hasirci V, Berthiaume F, Bondre SP, et al. Expression of liver-specific functions by rat hepatocytes seeded in treated poly(lactic-co-glycolic) acid biodegradable foams. Tissue Eng 2001; 7: 38594.
  • 57
    Sakai Y, Furukawa K, Ushida T, Tateishi T, Suzuki M. In vitro organization of biohybrid rat liver tissue incorporating growth factor- and hormone-releasing biodegradable polymer microcapsules. Cell Transplant 2001; 10: 47983.
  • 58
    Yang MB, Vacanti JP, Ingber DE. Hollow fibers for hepatocyte encapsulation and transplantation: studies of survival and function in rats. Cell Transplant 1994; 3: 37385.
  • 59
    Gomez N, Balladur P, Calmus Y, et al. Evidence for survival and metabolic activity of encapsulated xenogeneic hepatocytes transplanted without immunosuppression in Gunn rats. Transplantation 1997; 63: 171823.
  • 60
    Yoon JJ, Nam YS, Kim JH, Park TG. Surface immobilization of galactose onto aliphatic biodegradable polymers for hepatocyte culture. Biotechnol Bioeng 2002; 78: 110.
  • 61
    Demetriou AA, Reisner A, Sanchez J, et al. Transplantation of microcarrier-attached hepatocytes into 90% partially hepatectomized rats. Hepatology 1988; 8: 10069.
  • 62
    Nagaki M, Kano T, Muto Y, et al. Effects of intraperitoneal transplantation of microcarrier-attached hepatocytes on D-galactosamine-induced acute liver failure in rats. Gastroenterol Jpn 1990; 25: 7887.
  • 63
    Haque T, Chen H, Ouyang W, et al. In vitro study of alginate-chitosan microcapsules: an alternative to liver cell transplants for the treatment of liver failure. Biotechnol Lett 2005; 27: 31722.
  • 64
    Wells GD, Fisher MM, Sefton MV. Microencapsulation of viable hepatocytes in HEMA-MMA microcapsules: a preliminary study. Biomaterials 1993; 14: 61520.
  • 65
    Rihova B. Immunocompatibility and biocompatibility of cell delivery systems. Adv Drug Deliv Rev 2000; 42: 6580.
  • 66
    Roger V, Balladur P, Honiger J, et al. Internal bioartificial liver with xenogeneic hepatocytes prevents death from acute liver failure: an experimental study. Ann Surg 1998; 228: 17.
  • 67
    Donato MT, Castell JV, Gomez-Lechon MJ. Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices: comparison with other hepatic cellular models. J Hepatol 1999; 31: 5429.
  • 68
    Park JK, Lee DH. Bioartificial liver systems: current status and future perspective. J Biosci Bioeng 2005; 99: 3119.
  • 69
    van de Kerkhove MP, Hoekstra R, Chamuleau RA, van Gulik TM. Clinical application of bio artificial liver support systems. Ann Surg 2004; 240: 21630.
  • 70
    Mitry RR, Hughes RD, Dhawan A. Progress in human hepatocytes: isolation, culture & cryopreservation. Semin Cell Dev Biol 2002; 13: 4637.
  • 71
    Lloyd TD, Orr S, Skett P, Berry DP, Dennison AR. Cryopreservation of hepatocytes: a review of current methods for banking. Cell Tissue Bank 2003; 4: 315.
  • 72
    Terry C, Dhawan A, Mitry RR, Lehec SC, Hughes RD. Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing. Liver Transpl 2006; 12: 16577.
  • 73
    Loretz LJ, Li AP, Flye MW, Wilson AG. Optimization of cryopreservation procedures for rat and human hepatocytes. Xenobiotica 1989; 19: 48998.
  • 74
    Morsiani E, Pazzi P, Puviani AC, et al. Early experiences with a porcine hepatocyte-based bioartificial liver in acute hepatic failure patients. Int J Artif Organs 2002; 25: 192202.
  • 75
    Walldorf J, Aurich H, Cai H, et al. Expanding hepatocytes in vitro before cell transplantation: donor age-dependent proliferative capacity of cultured human hepatocytes. Scand J Gastroenterol 2004; 39: 58493.
  • 76
    Naruse K, Sakai Y, Nagashima I, et al. Comparisons of porcine hepatocyte spheroids and single hepatocytes in the non-woven fabric bioartificial liver module. Int J Artif Organs 1996; 19: 6059.
  • 77
    Naruse K, Nagashima I, Sakai Y, et al. Efficacy of a bioreactor filled with porcine hepatocytes immobilized on nonwoven fabric for ex vivo direct hemoperfusion treatment of liver failure in pigs. Artif Organs 1998; 22: 10317.
  • 78
    Naruse K, Sakai Y, Lei G, et al. Efficacy of nonwoven fabric bioreactor immobilized with porcine hepatocytes for ex vivo xenogeneic perfusion treatment of liver failure in dogs. Artif Organs 2001; 25: 27380.
  • 79
    Wyss OA. Nerve stimulation with middle-frequency current pulses. Helv Physiol Pharmacol Acta 1967; 25: 85102.
  • 80
    Te velde AA, Ladiges NC, Flendrig LM, Chamuleau RA. Functional activity of isolated pig hepatocytes attached to different extracellular matrix substrates. Implication for application of pig hepatocytes in a bioartificial liver. J Hepatol 1995; 23: 18492.
  • 81
    Giri S, Acikgoz A, Pathak P, et al. Three dimensional cultures of rat liver cells using a natural self-assembling nanoscaffold in a clinically relevant bioreactor for bioartificial liver construction. J Cell Physiol 2012; 227: 31327.
  • 82
    Joly A, Desjardins JF, Fremond B, et al. Survival, proliferation, and functions of porcine hepatocytes encapsulated in coated alginate beads: a step toward a reliable bioartificial liver. Transplantation 1997; 63: 795803.
  • 83
    Gautier A, Carpentier B, Dufresne M, et al. Impact of alginate type and bead diameter on mass transfers and the metabolic activities of encapsulated C3A cells in bioartificial liver applications. Eur Cell Mater 2011; 21: 94106.
  • 84
    Sarkis R, Benoist S, Honiger J, et al. Transplanted cryopreserved encapsulated porcine hepatocytes are as effective as fresh hepatocytes in preventing death from acute liver failure in rats. Transplantation 2000; 70: 5864.
  • 85
    Wu FJ, Peshwa MV, Cerra FB, Hu WS. Entrapment of hepatocyte spheroids in a hollow fiber bioreactor as a potential bioartificial liver. Tissue Eng 1995; 1: 2940.
  • 86
    Massie I, Selden C, Hodgson H, Fuller B. Cryopreservation of encapsulated liver spheroids for a bioartificial liver: reducing latent cryoinjury using an ice nucleating agent. Tissue Eng Part C Methods 2011; 17: 76574.
  • 87
    Yang Q, Liu F, Pan XP, et al. Fluidized-bed bioartificial liver assist devices (BLADs) based on microencapsulated primary porcine hepatocytes have risk of porcine endogenous retroviruses transmission. Hepatol Int 2010; 4: 75761.
  • 88
    Baquerizo A, Mhoyan A, Kearns-Jonker M, et al. Characterization of human xenoreactive antibodies in liver failure patients exposed to pig hepatocytes after bioartificial liver treatment: an ex vivo model of pig to human xenotransplantation. Transplantation 1999; 67: 518.
  • 89
    Schulte arn Esch J, Hamann D, Soltau M, et al. Human antibody deposition, complement activation, and DNA fragmentation are observed for porcine hepatocytes in a clinically applied bioartificial liver assist system. Transplant Proc 2002; 34: 2321.
  • 90
    Te velde AA, Flendrig LM, Ladiges NC, Chamuleau RA. Immunological consequences of the use of xenogeneic hepatocytes in a bioartificial liver for acute liver failure. Int J Artif Organs 1997; 20: 22933.
  • 91
    Hewitt NJ. Optimisation of the cryopreservation of primary hepatocytes. Methods Mol Biol 2010; 640: 83105.
  • 92
    Sahi J, Grepper S, Smith C. Hepatocytes as a tool in drug metabolism, transport and safety evaluations in drug discovery. Curr Drug Discov Technol 2010; 7: 18898.
  • 93
    Guillouzo A. Liver cell models in in vitro toxicology. Environ Health Perspect 1998; 106(Suppl. 2): 51132.
  • 94
    Gomez-lechon MJ, Donato MT, Castell JV, Jover R. Human hepatocytes as a tool for studying toxicity and drug metabolism. Curr Drug Metab 2003; 4: 292312.
  • 95
    Kelly JH, Koussayer T, He D, et al. Assessment of an extracorporeal liver assist device in anhepatic dogs. Artif Organs 1992; 16: 41822.
  • 96
    el Mouelhi M, Didolkar MS, Elias EG, Guengerich FP, Kauffman FC. Hepatic drug-metabolizing enzymes in primary and secondary tumors of human liver. Cancer Res 1987; 47: 4606.
  • 97
    Wang L, Sun J, Li L, et al. Comparison of porcine hepatocytes with human hepatoma (C3A) cells for use in a bioartificial liver support system. Cell Transplant 1998; 7: 45968.
  • 98
    Tsiaoussis J, Newsome PN, Nelson LJ, Hayes PC, Plevris JN. Which hepatocyte will it be? Hepatocyte choice for bioartificial liver support systems. Liver Transpl 2001; 7: 210.
  • 99
    Mazariegos GV, Patzer JF 2nd , Lopez RC, et al. First clinical use of a novel bioartificial liver support system (BLSS). Am J Transplant 2002; 2: 2606.
  • 100
    Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 1980; 209: 4979.
  • 101
    Allen JW, Hassanein T, Bhatia SN. Advances in bioartificial liver devices. Hepatology 2001; 34: 44755.
  • 102
    Li J, Li LJ, Cao HC, et al. Establishment of highly differentiated immortalized human hepatocyte line with simian virus 40 large tumor antigen for liver based cell therapy. ASAIO J 2005; 51: 2628.
  • 103
    Schippers IJ, Moshage H, Roelofsen H, et al. Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation. Cell Biol Toxicol 1997; 13: 37586.
  • 104
    Werner A, Duvar S, Muthing J, et al. Cultivation and characterization of a new immortalized human hepatocyte cell line, HepZ, for use in an artificial liver support system. Ann N Y Acad Sci 1999; 875: 3648.
  • 105
    Kobayashi N, Okitsu T, Tanaka N. Cell choice for bioartificial livers. Keio J Med 2003; 52: 1517.
  • 106
    Kobayashi N, Fujiwara T, Westerman KA, et al. Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes. Science 2000; 287: 125862.
  • 107
    Kobayashi N, Noguchi H, Fujiwara T, Tanaka N. Establishment of a reversibly immortalized human hepatocyte cell line by using Cre/loxP site-specific recombination. Transplant Proc 2000; 32: 11212.
  • 108
    Noguchi H, Kobayashi N, Westerman KA, et al. Controlled expansion of human endothelial cell populations by Cre-loxP-based reversible immortalization. Hum Gene Ther 2002; 13: 32134.
  • 109
    Watanabe T, Shibata N, Westerman KA, et al. Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers. Transplantation 2003; 75: 187380.
  • 110
    Stutchfield BM, Forbes SJ, Wigmore SJ. Prospects for stem cell transplantation in the treatment of hepatic disease. Liver Transpl 2010; 16: 82736.
  • 111
    Szkolnicka D, Zhou W, Lucendo-villarin B, Hay DC. Pluripotent stem cell-derived hepatocytes: potential and challenges in pharmacology. Annu Rev Pharmacol Toxicol 2013; 53: 14759.
  • 112
    Stock P, Staege MS, Muller LP, et al. Hepatocytes derived from adult stem cells. Transplant Proc 2008; 40: 6203.
  • 113
    Saulnier N, Lattanzi W, Puglisi MA, et al. Mesenchymal stromal cells multipotency and plasticity: induction toward the hepatic lineage. Eur Rev Med Pharmacol Sci 2009; 13(Suppl. 1): 718.
  • 114
    Sgodda M, Aurich H, Kleist S, et al. Hepatocyte differentiation of mesenchymal stem cells from rat peritoneal adipose tissue in vitro and in vivo. Exp Cell Res 2007; 313: 287586.
  • 115
    Sato Y, Araki H, Kato J, et al. Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood 2005; 106: 75663.
  • 116
    Baertschiger RM, Serre-Beinier V, Morel P, et al. Fibrogenic potential of human multipotent mesenchymal stromal cells in injured liver. PLoS ONE 2009; 4: e6657.
  • 117
    di Bonzo LV, Ferrero I, Cravanzola C, et al. Human mesenchymal stem cells as a two-edged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential. Gut 2008; 57: 22331.
  • 118
    Piryaei A, Valojerdi MR, Shahsavani M, Baharvand H. Differentiation of bone marrow-derived mesenchymal stem cells into hepatocyte-like cells on nanofibers and their transplantation into a carbon tetrachloride-induced liver fibrosis model. Stem Cell Rev 2011; 7: 10318.
  • 119
    Fausto N. Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells. Hepatology 2004; 39: 147787.
  • 120
    Dumble ML, Croager EJ, Yeoh GC, Quail EA. Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma. Carcinogenesis 2002; 23: 43545.
  • 121
    Wege H, Le HT, Chui MS, et al. Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential. Gastroenterology 2003; 124: 43244.
  • 122
    Yoon JH, Lee HV, Lee JS, Park JB, Kim CY. Development of a non-transformed human liver cell line with differentiated-hepatocyte and urea-synthetic functions: applicable for bioartificial liver. Int J Artif Organs 1999; 22(11): 76977.
  • 123
    Deurholt T, van Til NP, Chhatta AA, et al. Novel immortalized human fetal liver cell line, cBAL111, has the potential to differentiate into functional hepatocytes. BMC Biotechnol 2009; 9: 89.
  • 124
    Poyck PP, van Wijk AC, van der Hoeven TV, et al. Evaluation of a new immortalized human fetal liver cell line (cBAL111) for application in bioartificial liver. J Hepatol 2008; 48: 26675.
  • 125
    Poyck PP, Hoekstra R, van Wijk AC, et al. Functional and morphological comparison of three primary liver cell types cultured in the AMC bioartificial liver. Liver Transpl 2007; 13: 58998.
  • 126
    Chen Y, Li J, Liu X, et al. Transplantation of immortalized human fetal hepatocytes prevents acute liver failure in 90% hepatectomized mice. Transplant Proc 2010; 42: 190714.
  • 127
    Khan AA, Habeeb A, Parveen N, et al. Peritoneal transplantation of human fetal hepatocytes for the treatment of acute fatty liver of pregnancy: a case report. Trop Gastroenterol 2004; 25: 1413.
  • 128
    Diekmann S, Bader A, Schmitmeier S. Present and Future Developments in Hepatic Tissue Engineering for Liver Support Systems: State of the art and future developments of hepatic cell culture techniques for the use in liver support systems. Cytotechnology 2006; 3: 16379.
  • 129
    Mahieu-Caputo D, Allain JE, Branger J, et al. Repopulation of athymic mouse liver by cryopreserved early human fetal hepatoblasts. Hum Gene Ther 2004; 15(12): 121928.
  • 130
    Malhi H, Irani AN, Gagandeep S, Gupta S. Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes. J Cell Sci 2002; 13: 267988.
  • 131
    Suzuki A, Zheng Y, Kondo R, et al. Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver. Hepatology 2000; 32: 12309.
  • 132
    Suzuki A, Zheng YW, Kaneko S, et al. Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver. J Cell Biol 2002; 156: 17384.
  • 133
    Oh SK, Chen AK, Mok Y, et al. Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res 2009; 2: 21930.
  • 134
    Hay DC, Fletcher J, Payne C, et al. Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling. Proc Natl Acad Sci U S A 2008; 105(34): 123016.
  • 135
    Lu H, Wang Z, Zheng Q, et al. Efficient differentiation of newly derived human embryonic stem cells from discarded blastocysts into hepatocyte-like cells. J Dig Dis 2010; 11: 37682.
  • 136
    Touboul T, Hannan NR, Corbineau S, et al. Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development. Hepatology 2010; 51: 175465.
  • 137
    Hay DC, Zhao D, Fletcher J, et al. Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo. Stem Cells 2008; 26: 894902.
  • 138
    Chamuleau RA, Deurholt T, Hoekstra R. Which are the right cells to be used in a bioartificial liver? Metab Brain Dis 2005; 20: 32735.
  • 139
    Zhou W, Hannoun Z, Jaffray E, et al. SUMOylation of HNF4alpha regulates protein stability and hepatocyte function. J Cell Sci 2012; 125 (Part 15): 36305.
  • 140
    Payne CM, Samuel K, Pryde A, et al. Persistence of functional hepatocyte-like cells in immune-compromised mice. Liver Int 2011; 31: 25462.
  • 141
    Sullivan GJ, Hay DC, Park IH, et al. Generation of functional human hepatic endoderm from human induced pluripotent stem cells. Hepatology 2010; 51: 32935.
  • 142
    Liu H, Ye Z, Kim Y, Sharkis S, Jang YY. Generation of endoderm-derived human induced pluripotent stem cells from primary hepatocytes. Hepatology 2010; 51: 18109.
  • 143
    Sekiya S, Suzuki A. Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature 2011; 475: 3903.
  • 144
    Huang P, He Z, Ji S, et al. Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature 2011; 475: 3869.
  • 145
    Malik R, Selden C, Hodgson H. The role of non-parenchymal cells in liver growth. Semin Cell Dev Biol 2002; 13: 42531.
  • 146
    Meren H, Matsumura T, Kauffman FC, Thurman RG. Relationship between oxygen tension and oxygen uptake in the perfused rat liver. Adv Exp Med Biol 1986; 200: 46776.
  • 147
    Matsumura T, Kauffman FC, Meren H, Thurman RG. O2 uptake in periportal and pericentral regions of liver lobule in perfused liver. Am J Physiol 1986; 1: G8005.
  • 148
    Matsumura T, Thurman RG. Measuring rates of O2 uptake in periportal and pericentral regions of liver lobule: stop-flow experiments with perfused liver. Am J Physiol 1983; 244: G6569.
  • 149
    Maher JJ, Bissell DM. Cell-matrix interactions in liver. Semin Cell Biol 1993; 4: 189201.
  • 150
    Fujita M, Spray DC, Choi H, et al. Extracellular matrix regulation of cell-cell communication and tissue-specific gene expression in primary liver cultures. Prog Clin Biol Res 1986; 226: 33360.
  • 151
    Castell JV, Gomez-Lechon MJ. Liver cell culture techniques. Methods Mol Biol 2009; 481: 3546.
  • 152
    Dunn JC, Yarmush ML, Koebe HG, Tompkins RG. Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB J 1989; 3: 1747.
  • 153
    Kim Y, Lasher CD, Milford LM, Murali TM, Rajagopalan P. A comparative study of genome-wide transcriptional profiles of primary hepatocytes in collagen sandwich and monolayer cultures. Tissue Eng Part C Methods 2010; 16: 144960.
  • 154
    Mathijs K, Kienhuis AS, Brauers KJ, et al. Assessing the metabolic competence of sandwich-cultured mouse primary hepatocytes. Drug Metab Dispos 2009; 37: 130511.
  • 155
    Swift B, Brouwer KL. Influence of seeding density and extracellular matrix on bile Acid transport and mrp4 expression in sandwich-cultured mouse hepatocytes. Mol Pharm 2010; 7: 491500.
  • 156
    Ansede JH, Smith WR, Perry CH, St Claire RL 3rd, Brouwer KR. An in vitro assay to assess transporter-based cholestatic hepatotoxicity using sandwich-cultured rat hepatocytes. Drug Metab Dispos 2010; 38: 27680.
  • 157
    Tuschl G, Mueller SO. Effects of cell culture conditions on primary rat hepatocytes-cell morphology and differential gene expression. Toxicology 2006; 3: 20515.
  • 158
    Treijtel N, van Helvoort H, Barendregt A, Blaauboer BJ, van Eijkeren JC. The use of sandwich-cultured rat hepatocytes to determine the intrinsic clearance of compounds with different extraction ratios: 7-ethoxycoumarin and warfarin. Drug Metab Dispos 2005; 33: 132532.
  • 159
    Bhatia SN, Balis UJ, Yarmush ML, Toner M. Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. FASEB J 1999; 13: 1883900.
  • 160
    Guguen-Guillouzo C, Clement B, Baffet G, et al. Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type. Exp Cell Res 1983; 143: 4754.
  • 161
    Mesnil M, Fraslin JM, Piccoli C, Yamasaki H, Guguen-Guillouzo C. Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to maintain differentiated functions. Exp Cell Res 1987; 173: 52433.
  • 162
    Begue JM, Guguen-Guillouzo C, Pasdeloup N, Guillouzo A. Prolonged maintenance of active cytochrome P-450 in adult rat hepatocytes co-cultured with another liver cell type. Hepatology 1984; 4: 83942.
  • 163
    Mertens K, Rogiers V, Vercruysse A. Glutathione dependent detoxication in adult rat hepatocytes under various culture conditions. Arch Toxicol 1993; 67: 6805.
  • 164
    Rojkind M, Novikoff PM, Greenwel P, et al. Characterization and functional studies on rat liver fat-storing cell line and freshly isolated hepatocyte coculture system. Am J Pathol 1995; 146: 150820.
  • 165
    Busse B, Gerlach JC. Bioreactors for hybrid liver support: historical aspects and novel designs. Ann N Y Acad Sci 1999; 875: 32639.
  • 166
    Hay DC, Pernagallo S, Diaz-Mochon JJ, et al. Unbiased screening of polymer libraries to define novel substrates for functional hepatocytes with inducible drug metabolism. Stem Cell Res 2011; 6: 92102.
  • 167
    Chang RC, Emami K, Jeevarajan A, Wu H, Sun W. Microprinting of liver micro-organ for drug metabolism study. Methods Mol Biol 2011; 671: 21938.