BIOARTIFICIAL LIVER
Halime KENAR
LIVER ANATOMY
The liver is the largest internal organ of the body, weighing about 1.3 kg in an adult [12]. It is wedge-shaped, covered by a network of connective tissue (Glisson’s capsule), and is situated in the upper right portion of the abdominal cavity [9] . Its reddish brown color is due to its great vascularity.
The liver has four lobes and two supporting ligaments (fig. 1) [12].
Figure 1. The liver and gallbladder. (a) An anterior view and (b) an inferior view.
( Adapted from Van De Graaff, Human Anatomy, Fifth Ed., McGraw-Hill, 1998,p.640)
Anteriorly, the right lobe is separated from the smaller left lobe by the falciform ligament. Inferiorly, the caudate lobe is positioned near the inferior vena cava, and the quadrate lobe is adjacent to the gallbladder [12]. The falciform ligament attaches the liver to the anterior abdominal wall and the diaphragm. The ligamentum teres extends from the falciform ligament to the umbilicus [12].
Although the liver is the largest internal organ , it is, in a sense, only one to two cells thick.This is because the liver cells, hepatocytes, form “hepatic plates” that are one to two cells thick and separated from each other by large capillary spaces called “liver sinusoids” [12]. These structures form the hexagonally shaped functional unit of the liver called a “lobule” (fig.2).
Figure 2. A cross section of a liver lobule. Blood enters a liver lobule through the vessels in a hepatic triad, passes through hepatic sinusoids, and leaves the lobule through a central vein ( Adapted from Van De Graaff, Human Anatomy, Fifth Ed., McGraw-Hill, 1998, p. 641).
Hepatocytes are separated from a fenestrated endothelium of sinusoids by the Space of Disse [5]. The sinusoids are lined with phagocytic Kupffer cells, but the large intercellular gaps between adjacent Kupffer cells make these sinusoidsmore highly permeable than other capillaries [12]. Lipocytes (Ito cells) are elaborate, extensive processes that encircle the sinusoid, well-positioned for both communication with hepatocytes and the potential to modify the extracellular space by secretion of extracellular matrix [5]. The adult liver provides a scaffold for many complex cell-cell interactions that allow for effective, coordinated organ function.
LIVER FUNCTION
The liver is a major factory in our bodies. Although many of the specific processes involved within this organ’s multiple functions are not yet fully understood, it is commonly agreed that liver’s main aim is the maintenance of homeostasis ( a constant internal environment). It is estimated that the liver has more than 200 functions, but the most vital ones are as follows:
²Detoxification: The liver transforms the potentially dangerous metabolites, toxins and excess hormones into biologically harmless water-soluble compounds. This process requires expression of a complex group of enzymes known collectively as cytochrome-oxidase P 450 system. As an example, urea, a waste product of protein breakdown, is produced by the liver, a process which removes poisonous ammonia from the body fluids.
²Metabolism: The hepatic cells assimilate carbohydrates, fats, and proteins. They convert glucose to its stored form, glycogen, which is reconverted into glucose as the body requires it for energy. The ability of the liver to maintain the proper level of glucose in the blood is called its glucose buffer function. Gluconeogenesis (production of glucose from sources other than carbohydrates) is also carried out by the liver. Excess carbohydrates and protein are also converted into fat by the liver. Digested proteins in the form of amino acids are broken down further in the liver by deamination. The liver is also capable of synthesizing certain amino acids (the so-called nonessential amino acids) from other amino acids in a process called transamination. Part of the amino acid molecule is converted into glycogen and other compounds.
²Synthesis of lipoproteins and cholesterol: The end products of fat digestion, fatty acids, are used to synthesize cholesterol and other substances needed by the body.
²Synthesis of plasma proteins: Some essential components of blood are manufactured by the liver, including about 95% of the plasma proteins and the blood-clotting substances (fibrinogen, prothrombin, and other coagulation factors). Albumin, synthesized in the liver, accounts for colloidal osmotic pressure in the plasma.
²Digestive Functions: The liver synthesizes and secretes bile acids, which are necessary for adequate digestion and absorption of fats, and secretes into the bile a bicarbonate-rich solution of inorganic ions, which helps neutralize acid in the duodenum.
²Biotransformation of pharmaceuticals and vitamins: The liver contributes to the activation of vitamin D. Drugs are transformed, making them useful to the body (conversion of prodrug to active drug).
²Storage: The liver stores essential nutrients (glycogen) and important vitamins and minerals, including vitamins A, D, K, and B12.
The liver also filters harmful substances from the blood. Phagocytic cells in the liver, Kupffer cells, remove large amounts of debris and bacteria.
Unlike most other organs, the liver has a unique regenerative property. The hepatocytes rarely replicate in healthy adults and are usually preoccupied with accomplishing their tasks. However, when the liver is injured, virtually all of the surviving hepatocytes leave their normal, growth-arrested state and replicate until the destroyed part of the liver is replaced. It is found that, as little as 10% of total liver mass is compatible with life.
LIVER DISEASES
The most common liver diseases are cirrhosis (scarred or hardened liver tissue due primarily to viral or bacterial infection, as well as alcohol abuse), hepatitis (inflammation of the liver, usually as a result of viral infection or alcohol abuse) and cancer [10]. Other causes of liver damage include drug overdoses, metabolic and autoimmune disorders, chemical toxins and trauma [10].
Despite the protections of overlapping functions and tissue regeneration, the liver can fail as a result of disease or traumatic, extensive injury. Each year over 37,000 people die of liver failure in United States, and 200,000 are treated in hospitals for liver problems [8]. Loss of liver function severely affects the entire body’s metabolism. Liver failure causes body-wide complications, which include metabolic instability; disruption of energy supply, acid-base balance, and thermoregulation; uncontrolled bleeding and sepsis; cease of organ functions due to build-up of the toxic by-products and lack of liver-synthesized nutrients [13].
The most life threatening aspect of hepatic (liver) failure is the syndrome characterized by a severe impairment of liver function, which proceeds to the development of hepatic encephalopathy (HE) (hepatic coma) within eight weeks of the first symptoms of illness [15]. Bernuau J. has divided patients with acute liver failure to fulminant hepatic failure (FHF) (appearance of HE within 2 weeks from the onset of jaundice) and subfulminant hepatic failure (HE appearance between 2 weeks and 3 months post jaundice) [15]. The important points to differentiate FHF from chronic hepatic failure (CHF) are: that in FHF, there is no evidence of existing liver disease and the mortality rate is about 70-95%[15]. In CHF, mortality is lower, evidence of chronic liver disease exists and medical management may prove successful, at least at the first stages of HE [15]. With CHF, the hepatocytes are damaged and cannot detoxify. Despite widespread disagreement, but because both acute and chronic liver failure may be complicated by HE, there is a tendency to discuss this entity under unifying pathogenic mechanisms [15].
Hepatic encephalopathy results from failure of biotransformation and excretion of toxins normally processed by the liver [4]. Raised plasma ammonia levels have been implicated; however, other chemicals involved include mercaptans, fatty acids, aromatic chain amino acids, glutamate and toxic metals (e.g. zinc, copper, manganese) [4]. Complex changes in blood-brain barrier permeability may also contribute. Hepatic encephalopathy is classified as grades I-IV, describing the progression from normal mentation to hepatic coma [4]. The grades of HE and the common clinical features are: I: slow mentation, II: inappropriate disinhibited behavior (agitation, aggression and drowsiness), III: somnolence, IV: coma [4]. Grade I/II encephalopathy has a better prognosis than those progressing to grade III/IV encephalopathy, in which development of cerebral edema is more frequent [4]. Patients with grade IV encephalopathy risk developing cerebral oedema (80%) and raised intracranial pressure (ICP), the primary cause of death [4].
Liver transplantation remains the only proven treatment modality for patients with acute liver failure and advanced hepatic encephalopathy (grades III and IV) [1]. The mortality rate of patients with FHF approaches 80% in cases in which liver transplantation is not possible [14]. Liver replacement using the orthotopic technique (OLTx; normal, whole liver) is preferred approach in patients without potential for recovery; however, because of organ shortage and patient instability, other nontraditional approaches for liver replacement have been attempted [14]. These include auxiliary liver transplantation, hepatocyte transplantation, xeno-transplantation, extracorporeal perfusion using either xenogeneic approaches or human liver perfusion, and bioartificial liver assist devices [14].
Fig.3 Approaches to cellular therapies for
the treatment of liver disease. Hepatocytes
are transplanted directly or implanted on
scaffolds. Transgenic animals are being
raised to harvest a humanized liver.
Extracorporeal devices perfuse patient’s
blood or plasma through bioreactors
containing hepatocytes.
( Allen et. al., Hepatology, Vol.34, No. 3,
p. 448, 2001)
Alternatives to whole organ transplantation for liver dysfunction are under active investigation (fig. 3) [2]. Extracorporeal support for patients suffering from liver failure has been attempted for over 40 years [2]. Temporary systems have been developed to attempt to expedite recovery from acute decompensation, facilitate regeneration in acute liver failure, or serve as a bridge to liver transplantation [2].
EXTRACORPOREAL LIVER SUPPORT SYSTEMS
Several devices for liver support have been investigated or developed, which can be split into two major categories: nonbiological and biological support systems.
1. Nonbiological Liver Support Systems: These systems are based on artificial kidney dialysis principles and rely mainly on blood detoxification[1]. For many years it was assumed that hepatic coma was due to the accumulation of small (less than 5 kDa) dialyzable toxins in the blood, and many strategies were designed to remove these toxins [1]. However, the liver is a complex organ that is capable not only of performing detoxification but also of carrying out biotransformation, synthesis of many important plasma proteins, and regulation of multiple metabolic pathways [1]. Various nonbiological approaches have met with limited success, presumably because of the role of the synthetic and metabolic functions of the liver that are inadequately replaced in these systems [2]. Hemodialysis, hemoperfusion over charcoal or resins or immobilized enzymes, plasmapheresis, and plasma exchange have all been explored [2].
2. Biological Liver Support Systems: These systems utilize viable liver tissue preparations as their active components. They are classified in two groups:
a. Purely Biological Approaches: These include whole organ perfusion, perfusion of liver slices, and cross hemodialysis [2]. They have shown encouraging results in some cases but have been difficult to implement in the clinical settings [2].
b. Biological Devices: These devices, also named as bioartificial liver (BAL), incorporate isolated cells into bioreactors to simultaneously promote cell survival and function as well as provide for a level of transport seen in vivo [2].
HISTORY OF THE BIOARTIFICIAL LIVER
The history of the extracorporeal bioartificial liver (BAL) support began in the late 1950's [1].
¨ In late 1950's Sorrentino first used the term "fegato artificiale" ("artificial liver") while describing a series of in-vitro experiments, demonstrating that fresh liver tissue homogenates can metabolize salicylic and barbituric acid, and ketone bodies, and produce urea from ammonia.
¨ In 1957, Hori introduced the principle and technique of xenogenic cross-heterohemodialysis between a patient in hepatic coma and healthy living dog.
¨In 1958, Otto et al. introduced the principle of extracorporeal isolated liver perfusion.
¨ In 1959, Mikami et al., followed by Nose and colleagues in 1963, introduced the first BAL by placing canine liver tissue homogenate, fresh liver slices, and freeze-dried granules of liver tissue in a bioreactor in which a patient 's blood was perfused through a gel-type cellophane membrane. This device was able to maintain glucose homeostasis, and metabolized excess lactate and ammonia.
With the introduction of hepatocyte isolation using enzymatic digestion by Berry and Friend in 1969, a series of new bioreactors utilizing isolated hepatocytes were developed in the mid-1970's.
¨ In 1973, Matsumara was the first to use isolated rat hepatocytes in a bioreactor with a cuprophane membrane. Around the same time, Wolf and Munkelt first introduced synthetic capillaries into an artificial liver support design. They demonstrated conjugation of bilirubin by hepatoma cells cultured in the extrafiber space of a bioreactor in which porous tubes were sealed at each end, so that the intra- and extrafiber compartments communicated with each other via the pores in the fiber wall. Using a similar system, Hager et al. described ureagenesis, protein synthesis, and drug metabolism by adult and neonatal human hepatocytes.
¨ Eiseman and colleagues carried out extensive studies using isolated hepatocyte and liver tissues placed in several devices, including a centrifuge, a dialyzer, and a perfusion chamber. This was an innovative approach and introduced two important concepts: use of plasma separation, and placement of liver cells in a chamber within a high-flow plasma recirculation loop.
¨ Uchino et al. utilized high-flow plasma re-circulation in a device in which liver cells were cultured on 200 collagen-coated borosilicated glass plates.
¨ The first clinical report utilizing a bioreactor loaded with a suspension of isolated hepatocytes was by Matsumara et al. in 1987.
Since then many hepatocyte-based BAL systems, using a variety of bioreactor designs, have been developed and tested. New strategies were designed to augment differentiated functions of isolated hepatocytes in bioreactors as understanding of cell-cell and cell-matrix improved [1].
ENGINEERING A BIOARTIFICIAL LIVER
Due to advances in cell culture and isolation technologies, as well as the availability of new and improved biomaterials for bioreactors, many BAL designs are currently under testing [1]. At present nearly all such BAL designs contain: 1) a biologic component, 2) a bioreactor, and 3) a whole blood or plasma perfusion system [1]. There are some important issues related with all these components of the BAL.
1. THE BIOLOGIC COMPONENT
ðChoice of Hepatocytes: Cell types that have been used and are currently being evaluated for use in BAL include primary hepatocytes (porcine, rabbit or human), cell lines (immortalized cells and tumor-derived cells), and stem cells (embryonic, progenitor and transdifferentiated) [2]. Each of these should be evaluated on the basis of availability, potential adverse interactions, and efficacy in providing liver-specific function [2].
Primary porcine hepatocytes are most commonly used in devices undergoing preclinical and clinical evaluation [2]. There is relatively limited information on he maintenance of liver-specific functions of porcine hepatocytes in vitro. Although some functions such as albumin secretion may be stable, others such as cytochrome P450 decline under standard culture conditions [2]. In general, primary hepatocytes are well known to require specific microenvironmental cues to maintain the hepatic phenotype in vitro, and it is likely hat a more detailed investigation of culture conditions will improve the stability of porcine hepatocytes in vitro [2]. Although the way the pig hepatocytes metabolize hormones, chemicals and toxins is similar to human's, the use of pig hepatocytes could be hampered by the risks of disease transmission and immune reactions.
Primary human cells would be ideal, but like whole organs, they are in limited supply. They have been used for BAL application, as well as for hepatocyte transplantation [2]. A persistent paradox of human hepatocytes is their facile proliferation in vivo but static nature in culture, despite significant progress in stimulating DNA synthesis of rodent hepatocytes in culture [2].
The growth limitations of primary cells has spurred attempts to develop cell lines that can proliferate in culture while maintaining liver-specific functions. Spontaneous immortalization has been documented as a result of collagen gel sandwich cultures or cocultures [2]. Cell lines derived from hepatic tumors, such as C3A (a subclone of HepG2), have already been used in clinical trials [2]. C3A cells reportedly retain normal hepatocyte differentiated function while maintaining a short population doubling time and demonstrating contact inhibition [1]. The risk of transmitting oncogenic substances or cells into the patient's circulation remains a concern [2].
Finally, stem cells are being considered for therapy of liver disease [2].
ðHepatocyte Immobilization in a Bioreactor and Stabilization of Primary Cell Phenotype:
The realization of the importance of cell-cell and cell-matrix interactions in the maintenance of hepatocyte differentiation in vitro has led to a variety of methods for seeding isolated hepatocytes in a bioreactor [1]. Some investigators using cell lines grow cells either in a tissue culture flask prior to inoculation into a bioreactor, or directly in a bioreactor until cells reach confluence [1]. Other cell immobilization techniques used include immobilization of cells on a solid matrix such as microcarriers, or porous materials such as resin, microsponges, or porous beads, hepatocyte plating on hundreds of collagen-coated glass plates, immobilization within hydrogel and gel droplets, sandwich technique, and injection of cells suspended in collagen into hollow fibers, which eventually undergo partial recanalization due to gel contraction [1].
The goal of the various seeding techniques is to encourage cell aggregation and cell-cell interaction with re-establishment of cellular polarity, which has been shown to augment and prolong the liver-specific functions of these cells [1].
Recently, there has been increased interest in using cocultures of hepatocytes and nonparenchymal cells as the biologic component for BAL systems, because liver-specific functions, as well as hepatocyte viability, have been shown to increase significantly under such a condition [1]. However, the addition of nonparenchymal cells, which are rich in antigen presenting cells, and cells with class II antigens increases the immunogenicity of the xenogeneic cells [1].
ðQuantity of Cells Required: On the basis of results from liver resections in humans, approximately 10%-30% of residual liver parenchyma is needed to support life [1]. The actual number of cells used by various investigators in BAL systems varies from 0.5* 10 8 to 2.0*10 10 [1].
ðHepatocyte Preservation and Storage: Cryopreservation of hepatocytes makes the logistics of extracorporeal liver support simpler and less costly [1]. In addition, cells are available in demand; this becomes a very important consideration in the clinical setting in which treatment of patients with FHF is carried out emergently, on short notice, and at all hours [1]. However, storage and cryopreservation of hepatocytes for subsequent use in BAL systems results in a significant loss of viable cells (approximately 10%-20%) [1]. In addition, the cryopreserved cells may not be as metabolically active as fresh cells.
Recently, Scottish scientists, Dr H. Grant and her colleagues [8], developed a simple technique that can supply pig hepatocytes for an artificial liver to treat people suffering from chronic liver disease. The device allows researchers to freeze layers of liver cells attached to membranes and to provide cells for use in BALs. These researchers have figured out what the cells need to be viable and to store them for 28 days [8]. Human liver cells, unlike other types of cells, are difficult to store in liquid nitrogen because they do not survive after thawing. The new device would solve the storage problem for BALs [8].
The use of an adequate matrix and growth factors, coculture with other types of cells, entrapment of liver cells within 3D gels, the use of a collagen sandwich configuration, and the aggregation of hepatocytes into spheroids have also been employed to improve the survival and/or function of cultured hepatocytes [1].
2. BIOREACTOR DESIGN
Continued innovation in engineering and material science has contributed greatly to the development of extracorporeal liver-assist devices. Coupled with new discoveries in cell sourcing and hepatocyte stabilization, BAL devices tailored for use with hepatocytes are becoming a reality [2]. There are 4 main types of bioreactor designs (table 1), each with inherent advantages and disadvantages: hollow fiber, flat plate and monolayer, perfused beds or scaffolds, and beds with encapsulated or suspended cells [2]. A successful and clinically effective BAL device should satisfy a few key criteria: adequate bidirectional mass transport, maintained cell viability and function, and potential for scale-up to therapeutic levels [2].
Table 1. The four main types of bioreactors that have been proposed and studied.
(Adapted from Allen et. al., Hepatology, Vol.34, No. 3, p. 450, 2001)
Bidirectional Mass Transfer: In BAL devices, bidirectional mass transfer is needed to provide nutrients to sustain cell viability and contact with the toxic substances and catabolites usually removed from the blood by the liver, and allow export of therapeutic cell products. Although most device designs address this, there are important limitations involving the use of membranes, diffusivity of key solutes, and spatial uniformity [2].
Semipermeable membranes provide selectivity for the size of biological molecules that will be exchanged between the patient and he device [2]. They are inherent in hollow fiber devices but have been used also in flat-plate and perfusion systems. The membrane in a BAL device is typically characterized by its molecular weight cutoff, which is selected both to prevent the exposure of bioreactor cells to components of he immune system and to block the transport of larger xenogenic substances into he circulation. The aim of allowing free transport of larger carrier proteins such as albumin (~60 kd) while preventing transport of immunoglobulins (~150 kd), complement (_>200kd), or viruses has led most groups to choose a membrane molecular weight cutoff of 100 to 150 kd. Membranes also prevent the migration of cells into the patient ’s
circulation, although case reports of cellular translocation exist. While transport in BAL devices is a combination of convective and diffusional phenomena, mass transfer limitation of key nutrients to and from he cellular compartment often arise because of diffusion resistances. In contrast, transport in the liver is achieved primarily by convection along the sinusoid with short diffusion distances (<5mm) across he space of Disse. Barriers to diffusive transport include membranes, collagen gels, and nonviable cells. Some designs use encapsulated cells in perfusion systems, which provide immunoisolation, but also increases diffusion resistance [2].
Oxygenation is key to hepatocyte function and may be suboptimal in current BAL devices. Hollow fiber compartments or nonwoven fabric scaffolds with fibers for gas delivery may improve oxygen delivery. Geometric constraints also may affect mass transport in a BAL. Cell
distribution and flow should be uniform. A single monolayer culture is easily perfused, but a series of stacked plates may introduce shunting through regions of low resistance [2].
Cell Viability and Function: One of the major obstacles to BAL offering long-term treatment is the inability to maintain highly functional hepatocytes in vitro [2]. Current device designs do very little to integrate an appropriate microenvironment for hepatocytes. Gel entrapment and use of spheroidal aggregates have been introduced into various membrane-based systems to provide chemical and topological ECM cues or cell-cell interaction; however, this introduces an additional diffusion barrier [2]. Single cell suspensions, used in some devices because of their desirable transport properties, quickly lose metabolic capacity [2]. Along with providing adequate attachment, future devices should consider integrating engineering strategies for efficient transport, environments that optimize cell-ECM interactions and cell-cell interactions, and relevant chemical stimuli [2].
Scale-Up: For a device to become a clinical reality, it must be scaled to a size that provides effective therapy. Clinically tested devices incorporate between 1 and 500 g of hepatocyte mass [2]. The current solution for scaling up hollow fiber devices involves increasing cartridge size and using multiple cartridges [2]. Systems using spheroids or microcarriers are easily scaled to the needed cell mass but may entrain a considerable dead volume (priming volume) [2].
Nonbiological Adjuncts: Hemoperfusion, in use since the 1960s, removes toxins but also some useful metabolites (growth factors, clotting factors, etc.) from blood or plasma circulating through a charcoal column; the column may also activate leukocytes, causing cytokine release [2]. Another method called hemodiadsorbtion minimizes direct contact with charcoal by passing he blood through a flat membrane dialyser containing a suspension of charcoal and exchange resin particles [2]. Nonspecific removal of circulating biochemical species has not resulted in a clear survival benefit. The Molecular Adsorbent Recirculating System (MARS) involves dialysis against recirculated albumin [2]. The device is more selective than charcoal hemodiadsoprtion in that it uses a membrane impregnated with albumin to facilitate the clearance of albumin-bound toxins. The device has proven especially effective in reducing blood levels of bilirubin and bile acids in cholestasis and liver failure [2].
MAIN TYPES OF BIOREACTORS PROPOSED
Hollow Fiber Bioreactor: The most commonly used design is the hollow-fiber bioreactor. In classical membrane modules, the fluid flowed either inside or outside the fiber lumen, the hepatocytes being located on the other site.
Fig 4. Schematic representation of hollow fiber based bioartificial livers relying on commercial cartridges. The hepatocytes may be located either in the lumen or the extraluminal space.
(Adapted from Legallais et al., J Membr Science, 181 (2001), p.86)
In many hollow fiber devices, the membrane must simultaneously function as a perm-selective barrier and as a scaffold for cell attachment [2]. The interaction of the hepatocyte with its microenvironment dramatically affects stability and function. Therefore, this design may not
allow for optimization of both function and transport [2]. Conversely, hollow fiber designs provide a larger surface area-to-volume ratio than flat plate designs, thus improving metabolite transport and minimizing dead volume [2]. Finally, these devices present difficulty in achieving homogeneous cell distribution during inoculation through the tight matrix of capillaries.
Flat Plate and Monolayer Bioreactor: A multi-plated porcine hepatocyte monolayer bioreactor was first proposed by Matsumara [1]. In this design, hepatocytes are cultured on multiple membranous plates in a configuration allowing blood to flow in a counter-current direction over the opposite side of a semipermeable membrane. This design was later tested by Uchino et al. and Takahashi et al., who showed a significant level of metabolic activity in animal studies[1].However, this design requires long-term hepatocyte culture, which increases the risk of cell contamination, as well as the loss of hepatocyte function. Furthermore, it would be difficult to scale-up such a system for clinical use [1].
Direct Perfusion Bioreactors: The direct perfusion of attached cells by plasma or blood into a bioreactor seems a promising and simple concept, which clearly promoted the mass transfer efficiency and neglected the immunological barrier aspects [3]. Two main approaches were under investigation [3]. The first one attempted at creating a 3D environment for the liver cells, resembling the native organ. The cells could form small aggregates, or be directly attached to a porous support. They were thus almost individually perfused, under a low diffusion gradient as in a normal liver [3]. The other approach relied on more traditional cell culture as monolayers between two collagen-coated plates [3]. The opposing sinusoidal surfaces of the hepatocytes were attached to the extracellular matrix, reproducing the in situ configuration of the intact liver. Non-parenchymal cells could be strategically added to improve the hepatocyte functions [3]. For the moment, none of the presented systems have reached the clinical trials. Though the mass transfers should be optimized, the scaling up of some systems
(especially the plates) seemed difficult to perform. In addition, hepatocytes could be subjected to high shear in some configurations, leading to cell damage or possible release to the blood stream. Only continuous culture allowed the storage of such BAL [3].
Entrapment (encapsulation)-based Bioreactors: An alternative to the above configurations was the inclusion of hepatocytes within a semi-permeable spherical structure usually called “bead” or “capsule”. The polymer bead matrix offered anchorage facilities to hepatocytes and its porous structure could act as an immunological barrier. The hepatocyte-containing beads were first developed by Tompkins et al. and Dixit for their direct implantation [3]. Hepatocyte viability was found to be maintained in such a tridimensional structure [3], even after cryopreservation. The beads might even protect the cells from shear stress damage in an extracorporeal bioreactor.
Since cell encapsulation is a widely used tool in biotechnology, several materials have been investigated to fulfill the requirements of a bioartificial liver. Several teams tested the properties of HEMA-MMA copolymer , chitosan-dextrose [3]. Calcium alginate was up to now the most popular material because of its porosity, its mechanical properties and its biocompatibility. The alginate bead external structure might be strengthened by the addition of chelating components (lysine for instance).
Most of the bioreactors designed for beads perfusion relied on fixed bed configuration , where the beads were densely packed into a column. Their major limitation for scaling-up was the perfusion velocity profile into the column: the formation of preferential channels resulted in poor perfusion for a large amount of beads and consequently limited mass transport outside the
beads. In addition, high shear stresses on the effectively perfused beads could lead to possible damage on the bead structure, and as an effect to alginate and cells release to the blood stream.
In a novel geometry, beads were subjected to a fluidized bed motion by Legallais et al. [3], lead-ing to the definition of a fluidized bed bioartificial liver (FBBAL). The results obtained with this type of bioreactor are very encouraging (bed expansion was stable and resulted in a homogeneous mixing) [3].
3. BLOOD VERSUS PLASMA PERFUSION SYSTEMS
The use of whole blood perfusion has the advantage of erythrocytes as oxygen-delivery vehicles for BAL, although leukocyte activation and cell damage may occur [2]. Conversely, plasmapheresis and plasma perfusion preserve the viability of hematopoietic cells, but the solubility of oxygen in plasma is very low. Proponents of whole-blood perfusion systems argue that plasma separation complicates liver support, increases cost, and increases the extracorporeal priming space. On the other hand, the use of whole-blood perfusion would require heparin anti-coagulation to prevent thrombosis within the circuit, greatly increasing the risk of bleeding in patients with coagulopathy from liver failure [1].
CLINICAL EXPERIENCE WITH EXTRACORPOREAL BAL DEVICES
Although no extracorporeal bioartificial liver device has received FDA approval for use in acute or chronic liver failure, a number of clinical trials are underway (table 2) [2].
Table 3. Current Clinical Trials of Extracorporeal Support Devices. ( Adapted from Allen et. al.,
Hepatology, Vol.34, No. 3, p. 452, 2001)
HepatAssist 2000" System
HepatAssist ®, is currently Circe Biomedical's leading product [13]. It is the most clinically advanced bioartificial liver support system. HepatAssist® is an extracorporeal cell-based BAL device, based on the use of an open membrane hollow fiber bioreactor. This polysulfone membrane is microporous, with a pore size of 0.2 mm. This pore size is just small enough to halt the passage of whole cells through it, but large enough to allow for free exchange of toxins and large molecular weight proteins between the hepatocytes housed outside the hollow fibers and the plasma traveling on the inside of fibers [13]. Microcarrier-attached cryopreserved porcine hepatocytes (5*10 9) are used in this device. HepatAssist ® consists of four parts: a hollow fiber bioreactor containing porcine hepatocytes, two charcoal filters, a membrane oxygenator, and a pump.
http://www.circebio.com
How does it work?
• To begin a treatment, a patient’s blood is separated into blood and plasma in a plasma separation machine
• The person's plasma is moved via the system’s tubing through charcoal filters by use of the pumping system
• These filters act as Kuppfer cells, filtering the plasma from massive bacteria and particulate matter
• The rest of the detoxification occurs within the hepatocyte lined hollow fiber column
• After this, the clean plasma is reconstituted with the blood stored in the plasmapheresis; the whole blood is reinfused into the patient
• A membrane oxygenator and heater are placed between charcoal filters and hepatocyte bioreactor. The heater keeps the plasma at body temperature and the membrane oxygenator provides the housed hepatocytes with the oxygen they require.
Phase I Trial:
The trial was finished early in 1997
• 36 patients were treated: 23 having FHF, 10 acute worsening of CLD, 3 nonfunctioning transplants
• Each patient was treated 1-5 times for 6 hr with the device
• RESULTS: 19 of FHF and nonfunctioning transplant patients were bridged to OLT, 6 recovered spontaneously, 2 patients with CLD recovered to the point of receiving an OLT, and 9 patients died
These results demonstrated safety and tolerance to the point that the FDA granted Circe Biomedical permission to proceed with a phase II/III clinical trial testing the device's efficacy on the 30-day survival of patients with acute liver failure.
Hepatix/Vitagen ELAD
Vitagen Inc. ( formerly Hepatix, Inc.) has created the first medical device to incorporate "immortalized" human liver cells, C3A cell line. This is a highly differentiated clonal population isolated from a human hepatoblastoma cell line (HepG2) [4]. Cells (200g) were originally seeded and grown in the extracapillary space of haemodialysis cartridge containing approximately 10,000 hollow fibres with a surface area of 2 m2 and perfused with whole blood [4]. The device has since been modified, now being perfused with the patient's plasma, while the cartridge contains a greater number of hepatocytes [4].
A hemodialysis-type catheter carries blood to an ultrafiltration device, with the ultrafiltrate (plasma) flowing through the ELAD being exposed to the metabolic activities of the cells, which perform some (gluconeogenesis, ureagenesis, and P 450 activity) but not all of the normal liver's metabolic function [8]. VitaGen C3A cells in the ELAD cartridge produce several human liver-specific proteins, metabolize drugs, utilize galactose and reproduce very well in culture [11]. The treated plasma is then filtered and returned to the patient. Fibres are made of cellulose acetate membrane with a pore size of 70 kDa [3].
http://www.hepatix.com
Phase I Clinical Trials:
VitaGen conducted two trials to assess the role of an artificial liver in the treatment of acute hepatic failure. The initial trial involved 11 patients all with advanced ALF, while the second trial with 12 patients, was divided into two groups [13]. Group one suffered from moderate liver disease, group two from advanced. Patients received ELAD therapy continuously for 3-168 hrs (7 days).
Results: Results showed satisfactory biocompatibility, improved hemodynamic parameters and improvement in encephalopathy, which indicates that patients could safely be removed from ELAD treatment. However, the survival outcomes of the two trials differed greatly: 1 out of 11 survived in the first trial, and 8 out of 12 survived in the second trial. The discrepancy is described as being due to the medical conditions of those in each trial. Longer-term use in patients with chronic liver failure was not addressed in this study and remains problematic [8].
Excorp Medical Bioartificial Liver Support System (BLSS)
A Phase I clinical safety evaluation of the Excorp Medical, Inc, Bioartificial Liver Support System (BLSS) is in progress[16]. Inclusion criteria are patients with acute liver failure of any etiology, presenting with encephalopathy deteriorating beyond Parson's Grade 2. The BLSS consists of a blood pump, heat exchanger to control blood temperature, oxygenator to control oxygenation and pH, bioreactor, and associated pressure and flow alarm systems. Patient liver support is provided by 70-100 g of porcine liver cells housed in the hollow fiber bioreactor. A single support period evaluation consists of 12-hour extracorporeal perfusion with the BLSS sandwiched between 12 hours of pre (baseline) and 12 hours of post support monitoring.
A catheter connects a patient to the system, which remains outside of the body, and treats blood that passes through a cylinder filled with hollow polymer fibers and a suspension containing billions of pig liver cells [7]. The fibers act as a barrier to prevent proteins and cell byproducts of the pig cells from coming in direct contact with the patient’s blood but allow the necessary contact between the cells so that the toxins in the blood can be removed [7].
Preliminary evaluation of safety considerations after enrollment of the first four patients (F, 41, acetaminophen induced, two support periods; M, 50, Wilson's disease, one support period; F, 53, acute alcoholic hepatitis, two support periods; F, 24, chemotherapy induced, one support period) is as follows [16]: All patients tolerated the extracorporeal perfusion well. All patients presented with hypoglycemia at the start of perfusion, treatable by IV dextrose. Transient hypotension at the start of perfusion responded to an IV fluid bolus. No serious or unexpected adverse events were noted. Moderate biochemical response to support was noted in all patients. Completion of the Phase I safety evaluation is required to fully characterize the safety of the BLSS.
Algenix Liver ´2000
In 1988, a multidisciplinary group was formed at the University of Minnesota for development of a BAL for human use [10]. A device was designed and subsequently tested for biochemical function in vitro. With the demonstration of successful design, biochemical function and biocompatibility were tested and confirmed in an anhepatic rabbit model [10]. In preparation for scale-up for human use, a model of FHF was developed in large dogs using D-Galactosamine administration for induction of FHF. Statistical and clinically relevant improvements were demonstrated including longer survival, coma reversal, and control of intracranial pressure [10]. Based on these results, FDA approval was given for Phase I human clinical trials [6].
How it works:
The heart of the University of Minnesota bioartificial device is a hollow fiber cartridge similar to that used for kidney dialysis [6]. Freshly harvested pig liver cells are suspended in a cold collagen solution and injected inside the fibers. The cartridge is then connected to the tubing circuit, and warm medium is perfused outside the fibers. The collagen begins to gel once it is warmed, and within 24hrs, the liver cells pull on the collagen gel to contract to 60% of its original diameter. In the resulting space, a nutrient-rich medium stream is perfused for normal functioning of the liver cells. At this point the device is ready to be hooked up to a patient. The patient's blood is circulated outside the hollow fibers. The fiber membranes allow toxins from the blood to diffuse to the cells, but prevent immune molecules from reaching the cells [6].
Two hollow fiber cartridges, each containing approximately 40 g of cells, are connected in series within an incubator maintained at 37 0C [6]. Because the patient's blood is taken from a vein, it is oxygenated to maintain a sufficient amount of oxygen for the hepatocytes. Temperature, pH and dissolved oxygen are all monitored on-line. The rest of the system consists of the necessary tubing and pumps [6].
http://hugroup.cems.umn.edu/Research/bal
For anticipated clinical applications, it is desirable to further increase the liver-specific activities in the BAL [6]. To that end, the possibility of employing hepatocyte spheroids in the BAL was explored [6]. These self-assembled spheroids formed from monolayer culture exhibit higher liver-specific functions and remained viable longer than hepatocytes in a monolayer [6]. Liver-specific function was 2-5 fold higher in the spheroid BAL than in a BAL containing isolated hepatocytes. Application of spheroids in a BAL is under further investigation [6].
FUTURE ASPECTS OF THE BAL
Despite the number of studies performed up to now, the BAL is not commercially available [3]. The existing systems still need to be improved. While the safety of BAL devices has been established, there are no uniform standards for efficacy, which may vary with the etiology of the liver failure [2]. Consensus is needed in clinical trial design, including choice of end points, use of controls, and indications for enrollment [2].
The bioreactor improvements could follow different approaches. On the biological side, it is still not clear what type and what amount of cells should be used [3]. Regarding the maintenance of hepatocyte function, use of cocultures in addition to 3D anchorage may lead to improvement. Finally, optimal blood or plasma perfusion flow rates, as well as the amount of convection to be introduced in the membrane-based systems still need to be defined [3].
REFERENCES:
1.Hui T., Rozga J., Demetriou A. A., Bioartificial liver support, J Hepatobiliary Pancreat Surg (2001) 8: 1-15
2. Allen J. W., Hassanein T., Bhatia S. N., Advances in Bioartificial Liver Devices, Hepatology (2001) Vol. 34 No.3: 447-455
3. Legallais C., David B., Dore E., Bioartificial livers (BAL): current technological aspects and future developments, Journal of Membrane Science (2001) 181:81-95
4. Rahman T., Hodgson H., Clinical management of acute hepatic failure, Intensive Care Med. (2001) 27:467-476
5. Toner M., Bhatia S. N., Balis U.J., Yarmush M.L., Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells, FASEB J. (1999) 13: 1883-1900
6. http://hugroup.cems.umn.edu/Research/bal/bal.htm
7. http://www.upmc.edu/NewsBureau/tx/bioartificial.htm
8. http://organtx.org/bioart/liver.htm
9. http://www.ariess.com/s-crina/liver-anatomy.htm
10. http://www.algenix.com
11. http://www.hepatix.com
12. Van De Graaff M. K., Human Anatomy, McGraw-Hill, New York, 1998
13.http://biomed.brown.edu/Courses/BI108/BI108_1999_Groups/Liver_Team/Liver.html
14. Shakil A.O., Mazariegos V.G., Kramer D.J., Liver transplantation: Current management,
Fulminant hepatic failure, Surgical Clinics of North America (1999) Vol. 79, No.1,February
15. Lanza R.P., Langer R., Chick W.L.,Principles of Tissue Engineering,R.G. Landes Company, Texas USA,1997
16. Mazariegos G.V., Kramer D.J., Patzer J.F., Safety observations in phase I clinical evaluation of the Excorp Medical Bioartificial Liver Support System after the first four patients, ASAIO J.,(2001) Sep-Oct; 47(5): 471-5
