Hepatic tissue engineering for adjunct and temporary liver support: Critical technologies


  • Christina Chan,

    1. Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Boston, MA
    2. Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI
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    • The first two authors contributed equally to this work.

  • François Berthiaume,

    1. Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Boston, MA
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    • The first two authors contributed equally to this work.

  • Bharath D. Nath,

    1. Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Boston, MA
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  • Arno W. Tilles,

    1. Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Boston, MA
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  • Mehmet Toner,

    1. Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Boston, MA
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  • Martin L. Yarmush M.D., Ph.D.

    Corresponding author
    1. Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and the Shriners Hospitals for Children, Boston, MA
    • Shriners Hospital for Children, 51 Blossom St., Boston, MA 02114
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    • Telephone: 617-371-4882; FAX: 617-371-4950


The severe donor liver shortage, high cost, and complexity of orthotopic liver transplantation have prompted the search for alternative treatment strategies for end-stage liver disease, which would require less donor material, be cheaper, and less invasive. Hepatic tissue engineering encompasses several approaches to develop adjunct internal liver support methods, such as hepatocyte transplantation and implantable hepatocyte-based devices, as well as temporary extracorporeal liver support techniques, such as bioartificial liver assist devices. Many tissue engineered liver support systems have passed the “proof of principle” test in preclinical and clinical studies; however, they have not yet been found sufficiently reliably effective for routine clinical use. In this review we describe, from an engineering perspective, the progress and remaining challenges that must be resolved in order to develop the next generation of implantable and extracorporeal devices for adjunct or temporary liver assist. (Liver Transpl 2004;10:1331–1342.)

While many patients' lives are saved each year by orthotopic liver transplantation (OLT), the limitations of this therapeutic approach are well known: (a) severe shortage of organ donors; (b) high cost; and (c) requirement for life-long immunosuppressive drugs. Expansion of the donor pool to include living donors, marginal and domino livers, as well as using split livers has been a major focus of transplant surgeons in the past few years. Although this may alleviate the donor organ shortage, there still remains a great potential benefit to developing alternatives that could be more cost effective and less invasive.

Several potential avenues are currently being explored to provide adjunct and temporary liver support as alternatives to OLT. These approaches may find applications in the treatment of acute liver failure by allowing endogenous liver regeneration, as well as in chronic liver failure by ameliorating complications arising from the disease. A situation in which the native liver retains some functional capabilities but is afflicted by a chronic deficiency is most amenable to adjunct liver support. Temporary liver support is meant to replace a greater spectrum of liver functions over a short period of time (days to weeks) and may also serve as a bridge to OLT by allowing more time to find a better match between donor and recipient or stabilize the patient prior to surgery. In this review we delineate, from an engineering perspective, the progress and remaining challenges in the development of technologies that are critical for reliable adjunct and temporary liver support systems.


OLT, orthotopic liver transplantation; BAL, bioartificial liver.

Adjunct Internal Liver Support With Implantable Devices

The concept of adjunct liver support has been validated by the success of auxiliary partial liver transplantation.1, 2 Hepatocyte transplantation and hepatocyte-based implantable devices are an appealing alternative to auxiliary partial liver transplantation for several reasons: (a) several patients could be treated with one single donor liver; (b) the implantation procedure could be performed using less invasive surgery; (c) isolated liver cells can be cryopreserved for long periods; and (d) the liver cells could be genetically engineered in vitro to upregulate specific functions.

Hepatocyte Transplantation

Hepatocyte transplantation is the simplest form of adjunct internal liver support and has been investigated for over 25 years. In general, the efficiency of engraftment has been found to be quite low and a lag time, which may be as much as 48 hours, is necessary before any clinical benefit occurs.3 Thus, this approach offers an attractive prospect for correcting most nonemergency conditions such as inherited metabolic defects of the liver.4 In early studies the choice of the transplantation site was dictated by accessibility and ease of procedure, as well as by spatial considerations: the pulmonary vascular bed, dorsal and inguinal fat pads, and peritoneal cavity. However, expression of liver-specific functions by transplanted hepatocytes could not be achieved in most of these ectopic sites. A microenvironment resembling that of liver, including a basement substrate to promote hepatocyte anchorage and a venous blood supply mimicking the mechanical and biochemical environment of the hepatic sinusoid is required.5 The splenic pulp and the host liver itself are now the preferred sites for transplantation of hepatocytes.6 When implanted into the spleen, hepatocytes may engraft locally or migrate into the liver. Some of the successes with hepatocyte transplantation in experimental animals, although often not very dramatic, have prompted clinical studies. The best results have been obtained in the treatment of specific metabolic disorders; however, except for one case with Crigler-Najjar syndrome type I, there was no detectable long-term function of transplanted human hepatocytes.7

Implantable Devices

To improve the survival and function of implanted hepatocytes, the latter have been incorporated into biocompatible support materials, effectively constituting an implantable device. There are two major types of implantables devices (Fig. 1) : (a) hepatocytes in open matrices that allow tissue — especially blood vessels — ingrowth from the host, thus leading to integration with the surrounding tissue, and (b) hepatocytes isolated from the surrounding environment in the host by a selective membrane barrier.

Figure 1.

Types of implantable devices with hepatocytes. (A) Cells are in an open matrix, which is often biodegradable. Ingrowth of surrounding tissue, especially blood vessels, is encouraged. This provides no protection from the immune system from the host. (B) Cells are encapsulated so that they are protected by a barrier that prevents immune cells and factors from reaching the cells while allowing small (usually < 50 kD) metabolites to go across. Metabolite transport to and from the nearest vascular bed is chiefly by diffusion and may be adversely affected by the presence of a fibrotic layer, which often develops around such implants.

In early studies, isolated hepatocytes attached to collagen-coated dextran microcarriers were transplanted by intraperitoneal injection in two rat models of liver dysfunction: (a) the Nagase analbuminemic rat, and (b) the Gunn rat, which has an inherited deficiency of bilirubin-uridine disphosphate glucruronosyltransferase activity causing a lack of conjugated bilirubin in the bile.8 In both models, microcarriers promoted cell attachment, survival, and function of the transplanted hepatocytes.

Prevascularizing the cell polymer devices in combination with hepatotrophic stimulation have been used to encourage liver tissue regeneration around the implant.9 Furthermore, materials that biodegrade at controlled rates in vivo (such as collagen and poly-lactic-glycolic copolymers) can be used,10, 11 and novel techniques, such as solid freeform fabrication can be used to reproducibly manufacture three-dimensional porous materials of well-defined pore size, distribution, and interconnectivity.12, 13 Recently, novel biomaterials that are bioactive as well as resorbable have been developed.14 For example, biomaterials are being designed to stimulate tissue repair through the release of factors that elicit specific cellular responses, such as cell proliferation, differentiation, and synthesis of extracellular matrix. Thus far, one of the more common approaches is to incorporate growth factors into tissue engineering scaffolds.15, 16 There is also an interest in using “smart” materials consisting of stimuli-responsive polymers that change their properties in response to changes in the external environment.17

Encapsulated Hepatocytes

A limitation of hepatocyte-based devices using open matrices is the need to use the host's own cells or at the very least an allogeneic cell source, both of which are very difficult to obtain, which seriously limits the usefulness of these devices. To circumvent this problem, there is great interest in using hepatocytes from xenogeneic sources. Since there is no immunosuppressive regimen that currently exists to prevent rejection of xenografts, hepatocytes have been encapsulated into small microspheres as well as into hollow fibers. In theory, encapsulating with a synthetic, permeable membrane provides a physical separation that protects the cells from the immune system of the host by excluding high molecular weight immunocompetent proteins (e.g., antibodies and complement) as well as leukocytes, while allowing free exchange of nutrients and oxygen. Nevertheless, if the microcapsule causes complement activation after implantation, the breakdown complement products could be small enough to enter the microcapsules and damage the transplanted cells. Initial applications of semi-permeable microcapsules contained hemoglobin as blood substitute, enzymes to treat inborn errors of metabolism, or absorbents to treat drug overdoses.18 With advances in genetically engineered cells, microencapsulated cells have been used to remove ammonia in liver failure and amino acids such as phenylalanine in phenylketonuria.19 Numerous studies have been performed with encapsulated hepatocytes without immunosuppressive drugs. Transplantation of microencapsulated xenogeneic hepatocytes into Gunn rats without immunosuppression reduced serum bilirubin levels for up to 9 weeks before returning to control rat levels, possibly due to the deterioration of the biomaterial.20 The viability and function of encapsulated hepatocytes is highly dependent on the composition of the hollow fiber material.21 Better results may be possible if angiogenesis near the capsule surface can be promoted,22 and the formation of a fibrotic layer around the capsule can be avoided.23

Extracorporeal Temporary Liver Support

Extracorporeal temporary liver support systems are life-support systems that are analogous in concept to kidney dialysis machines, but specifically designed for liver failure patients. Since the liver has the ability to regenerate, temporary liver support may be sufficient to prevent patient death during the most severe phase of the illness, and allow regeneration of the host liver. The other main purpose of liver support systems is to provide a bridge to transplantation while awaiting a suitable donor.

The first attempts at developing devices for temporary and adjunct liver support consisted of nonbiological devices incorporating hemodialysis, hemofiltration, and/or plasma exchange units aimed at removing toxins accumulating in the patient's blood. Charcoal perfusion, the most extensively characterized nonbiological method, showed benefits in various animal models, but no survival benefit was reported in the only one reported randomized clinical trial.21 Recently, there has been renewed interest in further refining these approaches, with three different systems at various stages of clinical assessment. Although it fell out of favor due to the development of OLT, extracorporeal whole liver perfusion has also experienced renewed interest in recent years, and it has successfully been used as a bridge to transplantation. A comprehensive listing of systems and approaches used for extracoporeal liver assist is provided in Table 1. Our discussion below is restricted to hepatocyte- and liver cell–based extracorporeal devices.

Table 1. Clinical Trials for Temporary Extracorporeal Liver Support
Device*ConfigurationCell Mass and SourcePerfusate and Treatment ProtocolTrial PhaseRefs.
  • *

    ELAD. Extracorporeal liver assist device; BLSS, bioartificial liver support system; MELS, modular extracorporeal liver system; AMC-BAL, Amsterdam bioartificial liver system; MARS, molecular adsorbent recycling system; FDA, Food and Drug Administration.

  • Based on 108 hepatocytes/g liver tissue.

  • Clinical data not yet published, reference describes device design.

Whole Liver Perfusion
Whole pig, baboon or human liver  Whole blood, 5 hours median perfusion time, most patients received 1 or 2 perfusionsI/II(85, 86)
Dialysis and Filtration Systems
MARS (Teraklin AG, Rostock, Germany)Albumin-loaded hemofilter, 60-kD cut-offNoneWhole blood, 12–132 hoursI/II(87, 88)
Liver Dialysis Unit (HemoCleanse Technologies, West Layfayette, IN)Hemodiabsorption across 5-kD cut-off cellulosic membranesNoneWhole blood, 6 hours/day; up to 5 daysFDA Approved(89, 90)
Prometheus (Fresenius Medical Care AG, Bad Hornburg, Germany)Hemofilter, 250-kD cut-off, connected to two absorber cartridges, in series with conventional dialyzerNoneWhole blood, up to 12 hours divided into 2 treatments over 2 daysI(91, 92)
Bioartificial Livers
HepatAssist (Circe Biomedical, Lexington, MA)Hollow-fiber, polysulphone, 0.15–0.20 μm pore size50 g cryopreserved primary porcine hepatocytes on microcarrier beads*Plasma, 6 hours/session; up to 14 sessionsII/III(93–95)
BLSS (Excorp Medical, Oakdale, MN)Hollow-fiber, cellulose acetate, 100-kD cut-off70–100 g primary porcine hepatocytesWhole blood, 12 hours/session; up to 2 sessionsI/II(96, 97)
MELS (Charité Virchow, Berlin, Germany)Hollow-fiber (Interwoven, multicompartment)250–500 g primary porcine or human hepatocytesPlasma, continuous up to 3 daysI/II(98)
ELAD (Vital Therapies, La Jolla, CA)Hollow-fiber, cellulose acetate, 120-kD cut-off100 g human hepatoblastous CJA cells per cartridge, up to 4 cartridges/devicePlasma, continuous up to 107 hoursII (due to resume in 2004)(99)
AMC-BAL (Hep-Art Medical Devices, B.V., Amsterdam, The Netherlands)Spirally wound, non-woven polyester matrix, no membrane70–150 g* primary porcine hepatocytesPlasma, up to 18 hours/sessions up to 2 sessionsI(52, 100)
Radial-flow bioreactor (Sant'Anna University Hospital, Italy)Radial-flow bioreactor230 g primary porcine hepatocytesPlasma, 6–24 hour treatments, mostly in one sessionI/II(101)
LiverX-2000 (Algenix, Inc., Minneapolis, MN)Cells embedded in collagen matrix within hollow-fibers40 g primary porcine hepatocytes per cartridge, 2 cartridges/deviceBloodI/II(102)
Hybrid bioartificial liver (Hepatobiliary Institute of Nanjing University, China)Polysulfone hollow-fiber cartridge with 100-kD cut-off combined with adsorption column100 g primary porcine hepatocytesPlasma, one 6-hour treatment, except one patient with 2 × 6 hour treatmentsI(103)

Although such devices are in principle more complex than dialysis and filtration systems, they could provide biochemical and synthetic functions that are not available in the systems containing no cells.24 The mechanisms of liver failure are not yet well understood and the most critical hepatic functions in patients undergoing liver failure not known; therefore, it is yet unclear whether dialysis and filtration systems, which are likely to be cheaper, will supplant hepatocyte- or cell-based bioartificial livers.

As an engineering problem, the main design constraints for bioartificial livers (BAL) are summarized in Table 2. Several hurdles must be overcome for the development of BAL systems. Some of the issues that the field continues to wrestle with are: (a) how to maximize long-term functional stability of hepatocytes in inhospitable environments; (b) how to manufacture a liver functional unit that is scalable without creating transport limitations or excessive priming volume that must be filled by blood or plasma from the patient; (c) how to procure the large number of cells that is needed for a clinically effective device.

Table 2. Engineering Design Constraints for Bioartificial Livers
Parameter/ComponentDesired Value/Property
Minimum functional capacity1%–10% of liver
Maximum priming volume1 L
Maximum distance between cells and nutrient supply1 mm
Maximum size of cell aggregates100 μm
Scaffold materialBiocompatible and supports hepatocyte differentiated function

Maximizing Liver-Specific Functions in Plasma

The availability of stable long-term liver cell culture systems that express high levels of liver-specific functions is an essential step in the development of liver-assist devices using hepatocytes. Three types of long-term culture techniques for adult hepatocytes have been used for bioartificial liver development: (a) coculture of hepatocytes with a “feeder” cell line, such as fibroblasts, (b) three-dimensional network of collagen or other matrix, and (c) hepatocyte aggregates or spheroids. Furthermore, hepatoma cell lines, which do not require specific substrate configurations, have been used as well. Some of these techniques can be combined; for example, Takezawa et al. used thermally responsive polymer substrates to develop multicellular spheroids of fibroblasts and hepatocytes.25, 26

It is important to point out that although cultured hepatocytes may function well in culture medium, it is likely that the cells will change their phenotype once they are exposed to the potentially inhospitable environment contributed by plasma or blood from a sick patient, as well as other factors that occur in bioreactors, such as fluid shear stress. In prior studies, we have shown that the culture conditions used prior to placing the hepatocytes in contact with human plasma as well as during plasma exposure, can dramatically affect hepatocellular metabolism. For example, hepatocytes cultured in standard hepatocyte culture medium containing supraphysiological levels of insulin become fatty once they are exposed to plasma, and this can be prevented by “preconditioning” the cells in a medium containing physiological levels of insulin.27, 28 It is also possible to profoundly affect the expression of liver-specific functions by hepatocytes by changing a number of environmental conditions in the bioreactor environment (summarized in Table 3). For example, urea synthesis dramatically increases with increasing oxygen tension while cytochrome P450 decreases.28 Amino acid supplementation to human plasma increases urea and albumin synthesis, as well as cytochrome P450 activities.29 Coculture with mouse 3T3 cells also increases albumin and urea secretion to levels that exceed in vivo rates several-fold.30, 31 While albumin and urea secretion decrease at higher fluid shear rates, the latter tend to increase cytochrome P450 detoxification rates, at least in the short term.32 Sophisticated optimization techniques that can tackle the large number of adjustable environmental variables may be helpful for optimizing the bioreactor environment.33, 34 Since varying one specific environmental condition increases the expression of liver-specific functions many times, it is reasonable to assume that optimization of several such parameters simultaneously may yield an order of magnitude or more in improvement.

Table 3. Effect of Environmental Conditions on Specific Hepatocellular Functions
Environmental ParameterEffect on Metabolic Functions
Insulin levels during medium culture prior to plasma exposureHigh insulin favors intracellular lipid accumulation, low insulin favors lipid oxidation
Oxygen tensionIncreasing oxygen tension increases urea synthesis and decreases cytochrome P450 activity
Amino acid levelsAmino acid supplementation to the plasma increases both albumin and urea synthesis
XenobioticsInduce specific cytochrome P450 activities
Fluid shear rate on cell surfaceDecreases albumin and urea synthesis but increases cytochrome P450-mediated detoxification
Co-culture with mouse embryonic fibroblasts (3T3 cells)Increases both albumin and urea synthesis

In the hepatic lobule, blood flows from the periportal outer region towards the central hepatic vein. Hepatocytes in the periportal, intermediate or centrilobular, and perivenous zones exhibit different morphological and functional characteristics. Spatial heterogeneity in the hepatic lobule is clearly important for some aspects of hepatic function. For example, urea synthesis is a process with high capacity to metabolize ammonia but low affinity for the substrate. Ammonia removal by glutamine synthesis is a high affinity process that removes traces of ammonia that cannot be metabolized by the urea cycle.35 Coexpression of both enzyme systems would not be productive because the higher affinity process (glutamine synthesis) would be saturated under most operating conditions, leading to a reduced efficiency in ammonia extraction. On the other hand, replicating the functional heterogeneity of hepatocytes in the lobule would likely enhance the performance at the tissue level.

Functional heterogeneity also has important implications in the metabolism of hepatotoxins such as acetaminophen. Acetaminophen is normally degraded by glucuronidation and sulfation reactions, which are uniformly distributed along the acinus. After acetaminophen overdose, these processes are saturated and cytochrome P450 activities primarily located in the centrilobular region metabolize significant amounts to toxic metabolites causing oxidative stress and protein cross-linking. Although these metabolites can be detoxified by glutathione-dependent reactions, centrilobular hepatocytes do not have an efficient glutathione recycling system, and as a result are the main target of acetaminophen-induced hepatotoxicity. Repeat exposure to incremental doses of acetaminophen increases the tolerance to hepatic damage by partially shifting the expression of cytochrome P450 towards the periportal region,36 which has the most active glutathione recycling metabolism in the liver.37

The maintenance of functional heterogeneity in the liver is dependent on several factors, including gradients of hormones, substrates, oxygen, and extracellular matrix composition, although the relative importance of each one of these factors is currently unknown. In one study where hepatocytes were chronically exposed to increasing oxygen tensions within the physiological range of about 5 mm Hg (perivenous) to 85 mm Hg (periportal), urea synthesis increased about 10-fold, while P450IA1 activity decreased slightly and albumin secretion was unchanged.38 These data suggest that by creating environmental conditions that emulate certain parts of the liver sinusoid, it is possible to modulate hepatocyte metabolism in a way that is consistent with in vivo behavior. Spatial control of the layout of the cells in the device may be achieved using micropatterning and microfabrication techniques,39, 40 or using separate bioreactor modules that are optimized to perform a subset of hepatocellular functions, as illustrated in Figure 2.

Figure 2.

Potential bioreactor configurations in a bioartificial liver. (A) Single unit optimized to perform all functions. (B) Two subunits, the first one optimized for ammonia conversion to urea, a high capacity but low affinity process, and the second one optimized for ammonia conversion to glutamine, a high affinity process that scavenges ammonia not metabolized in the first subunit. (C) Three subunits, the first two designed to clear ammonia under high oxygen tension, and a third subunit operating at lower oxygen tension that is optimized for efficient P450 detoxification pathways.

Bioreactor Designs and Oxygen Transport

The most popular bioreactor designs are shown in Figure 3 and discussed in greater detail below. Most devices tested clinically consist of hollow fiber cartridges containing either porcine hepatocytes or human hepatoblastoma cells. In most cases, cells are loaded into the extraluminal compartment and patient plasma or blood is perfused through the fiber lumens.41–43 Similar hollow fiber cartridges have also been used in animal studies with hepatocytes seeded inside the fibers and the plasma flowing over the outer surface of the fibers.44, 45 Because of the relatively large diameter of the fibers as well as transport limitations associated with the fiber wall, these systems are prone to substrate transport limitations.46

Figure 3.

Most common bioreactor designs for bioartificial livers. (A) Hepatocyte aggregates or seeded on microcarriers are placed on the outside of hollow fibers. Oxygenated plasma is flown through the hollow fibers. (B) Hepatocyte aggregates in a supporting matrix are inside hollow fibers and oxygenated plasma is flown outside the hollow fibers. (C) Similar to panel A, although separate hollow fibers are used to deliver hepatocyte culture medium, plasma, and oxygen into the system. Circle with O2 is a hollow fiber perpendicular to the plane of the paper. (D) Hepatocyte aggregates are in a supporting matrix next to hollow fibers that deliver oxygen. Oxygenated plasma is flown in the space outside of the hollow fibers and percolates through the matrix-hepatocyte network. (E) Hepatocytes are seeded as a monolayer on the bottom surface of a flat plate and placed within a parallel-plate flow chamber. Oxygenated plasma is flown directly above the cells. (F) System is similar to panel E, except that oxygen is delivered through a permeable membrane directly above the flow channel with the hepatocytes.

The high oxygen uptake rate of hepatocytes and the relatively low solubility of oxygen in aqueous media deprived of oxygen carriers makes oxygen transport the most constraining parameter in the design of bioartificial liver devices.47, 48 Thus, to improve oxygen delivery, novel designs using additional fibers that carry gaseous oxygen straight into the device have been used. Using this approach, Gerlach et al. were able to demonstrate that hepatocytes could express differentiated functions over several weeks.49 Using a device consisting of hepatocytes seeded onto a woven polyester substrate with integrated hollow fibers for oxygen supply, Flendrig et al. showed that the survival time of pigs undergoing total hepatic ischemia was significantly increased over the control group50, 51; more recently, this device was successfully used to treat 7 acute liver failure patients, of which 6 were bridged to a transplant and one spontaneously recovered.52 An alternative topology in bioartificial liver development is based on a flat surface geometry.53–55 In this configuration, it is easier to control the internal flow distribution and ensure that all cells are adequately perfused. Its main drawback is that it is difficult to build a system containing a sufficient cell concentration. For example, a channel height of 1 mm would result in a 10-L reactor if one assumes that about 20 × 109 hepatocytes cultured on an area of 10 m2 are needed to support a patient. This is an unacceptably high volume for a patient, which is likely hemodynamically unstable.

The volume of the device in the flat-plate geometry is directly proportional to the inverse of the channel height. However, fluid has to flow through a smaller gap, which rapidly increases the drag force (shear stress) imparted by the flow on the cells. Our recent data suggest that albumin and urea synthesis decreases significantly at shear stresses >5 dy/cm2.56 To reduce the deleterious effects of high shear, it may be possible to use grooved surfaces as previously done for blood cells.57, 58 Cells lodge inside the grooves where they are less exposed to the shear stress, which allows for faster flow without causing cell damage. The grooves may on the other hand significantly increase the fluid hold-up volume.

In an attempt to provide to cells adequate oxygenation and protection from shear in perfused bioreactors, De Bartolo et al. incorporated two membranes into the flat-plate geometry.53 The first membrane is gas-permeable and minimizes the oxygen transport limitations in the system. The second membrane separates cells from plasma and adds a significant barrier to the transport of protein-bound toxins that need to be processed by the cells.46 Others have reported the design of a radial flow bioreactor with an internal membrane oxygenator for the culture of hematopoietic cells.59 Based on a theoretical analysis, the proposed design would have removed oxygen transport limitations in the bioreactor, but no experimental data were shown. Recently, we developed a flat-plate microchannel bioreactor where cells directly contact the circulating medium.56, 60 The channel is closed by a gas-permeable membrane on one surface, which decouples oxygen transport from the flow rate in the device. Comparing this with a similar flat-plate design where a nonpermeable glass surface is substituted to the membrane, we showed that internal membrane oxygenation removes the oxygen limitations that occur at low volumetric flow rates.61

Minimum Cell Mass and Functional Capacity

The cell mass required to support an animal model of hepatic failure has not been systematically determined. Prior studies have shown significant improvements in various parameters using as low as 2%–3% of the normal liver mass of the animal.53, 62 Devices that have undergone clinical testing have used 6 × 109 to 1 × 1011 porcine hepatocytes49, 63 or 4 × 1010 C3A cells.64 Recently, in an experimental pig model of hepatic failure, treatment with a bioartificial liver containing 6 × 108 pig hepatocytes (about 3%–5% of the liver mass) significantly improved survival.65 Recently, there have been efforts to improve cell viability in large-scale devices. Hepatocytes have been transfected with an anti-apoptotic gene (nitric oxide synthase) or exposed to an anti-apoptotic drug (ZVAD-fmk) to increase their resistance to what appears to be mainly hypoxic injury.66, 67

Clinical improvements have also been seen in hepatocyte transplantation studies using less than 10% of the host's liver mass. Intrasplenic transplantation of 2.5 × 107 allogeneic rat hepatocytes (about 5% of the rat liver mass) prolonged the survival, improved blood chemistry, and lowered blood transforming growth factor-β1 (an inhibitor of hepatocyte growth) levels in anhepatic rats.68 In another study using reversibly transformed human hepatocytes, 50 × 106 cells were injected intrasplenically into rats subjected to a 90% hepatectomy.69 In a recent study on humans with acute liver failure, intrasplenic and intra-arterial injections of human hepatocytes (ranging from 109 − 4 × 1010 per patient, i.e., 1%–10% of the total liver mass) transiently improved several blood chemistry parameters and brain function after a lag time of about 48 hours, but did not improve survival.3 The lag time before any benefit is observed may reflect the time required for the engraftment of the cells in the host liver. Better survival of the injected cells may be possible if the cells are seeded in prevascularized polymeric scaffolds.70 The relatively low number of hepatocytes needed to effect a therapeutic benefit may be due, in part, to the fact that the exogenously supplied hepatocytes may aid the regeneration of the native liver.62

Assuming that the minimum cell mass necessary to support a patient undergoing acute liver failure is about 5%–10% of the total liver weight, this yields a bioartificial liver containing about 1010 cells. Designing this system with a priming volume not exceeding about 1 L is still a daunting challenge. Knowing which functions are most critical would help to rationally improve the efficacy of bioartificial liver systems and dramatically reduce the minimum therapeutic cell mass. For example, it is well known that hepatocytes exhibit a metabolic zonation along the acinus.38 Periportal and centrilobular hepatocytes express high levels of urea cycle enzymes and low levels of glutamine synthetase while pericentral hepatocytes are the opposite.35 Another example is the reduction in albumin synthesis during the acute phase response, a process which may help sustain the increased level of acute phase proteins.71

Cell Procurement For Liver-Assist Devices

While several technical difficulties remain to be addressed with respect to the design of implantable and extracorporeal liver-assist devices, clearly a major hurdle for both approaches is the procurement of a sufficient number of cells that are required to achieve a therapeutic effect. A summary of the various potential cell sources for liver-assist devices (both implantable and extracorporeal) is in Table 4 and discussed below.

Table 4. Characteristics of Various Cell Sources Available for Bioartificial 0Livers
Cell SourceSupplyLiver-Specific FunctionHost CompatibilityOther Risks to Patient
Primary human hepatocytesScarceGenerally unstable, but limited experience with cell cultureExcellentSmall
Stem-cell derived human hepatocytesUnlimitedLow efficiency of differentiation to hepatocytesExcellentSmall
Immortalized human hepatocytesUnlimitedFunction poorly characterizedExcellentTransmission of tumorigenic products
Human hepatoma cell lineUnlimitedReduced level compared to primary cellsExcellentTransmission of tumorigenic products
Primary xenogenic (porcine) hepatocytesUnlimitedCan express high levels but generally decreases over timeMetabolic mismatch, potential severe immune complicationsTransmission of zoonoses

Human hepatocytes appear to be the “natural” choice for hepatocyte transplantation, internal and external liver assist devices, however, they are scarce due to a competing demand of OLT. Whether adult human hepatocytes can be induced to replicate in vitro and the daughter cells express high levels of liver-specific functions remains to be shown. Human hepatocyte cell lines have been developed via spontaneous transformation,72 as well as via retroviral transfection of the simian virus 40 large T antigen.73 Recently, a novel technology, which uses a reversible transformation strategy with the SV40 T antigen and Cre-Lox recombination, was used to grow human hepatocytes in vitro.69 These cells, when transplanted into the spleen of 90% hepatectomized rats, improved biochemical and clinical parameters. In bioartificial liver devices tested so far, the only human cells used have been the cancer-derived C3A line.42, 64 However, one study suggests that C3A cells have lower levels of P450IA1 activity, ammonia removal, and amino acid metabolism that adult porcine hepatocytes.74 Furthermore, when using immortalized human cell lines, there are concerns with the possibility of transmission of tumorigenic products into the patient. Xenogeneic hepatocytes offer no risk of transmitting malignancies to the patient, but pose other problems, including the risk of hyperacute rejection,75 transmission of zoonoses,76 and potential mismatch between xenogeneic and human liver functions. The first two could be addressed by dedicated breeding programs of transgenic animals. On the other hand, there is little known about the third factor.

While no one has achieved the goal of generating a safe, fully functional yet clonal, immortalized, or genetically engineered human cell that can be substituted for primary hepatocytes, a new promising avenue is the discovery of liver stem cells. The existence of hepatic stem cells was hypothesized over 40 years ago,77 and recent data suggest that there are stem cells present within7880 as well as outside the liver,81 which can differentiate into fully mature hepatocytes. In vitro studies suggest the presence of a subpopulation of small hepatocytes in rat liver with a high proliferative potential.82 Three independent studies in rats, mice, and humans have shown that a major extrahepatic source is stem cells of the bone marrow which may take part in normal tissue renewal as well as in liver regeneration after severe experimentally induced hepatic injury.81, 83, 84


New technologies for hepatic tissue engineering are being developed to generate various treatment options that could be useful adjuncts to allogeneic OLT for treating end stage liver disease. Biomaterials can be used to improve the engraftment efficiency of transplanted hepatocytes. Furthermore, immunoisolation techniques, by placing cells behind selective membranes that block the passage of large molecules, such as in microcapsules or hollow fibers, could eventually be used to implant allogeneic and xenogeneic hepatocytes without the need for immunosuppression. However, none of these techniques have yet been able to maintain, in a reliable fashion, long-term function of implanted hepatocytes.

Extracorporeal bioartificial liver support devices containing liver cells have passed the “proof of principle” test in preclinical and clinical studies, although tangible clinical benefits have not yet been demonstrated. To become a clinically feasible treatment approach, bioreactor designs that can hold a large cell mass in a relatively small volume must be developed.

A significant practical hurdle for all hepatocyte-based liver support systems that must be surmounted is the identification of a reliable cell source. Thus, the advantages and disadvantages of this treatment modality must be evaluated along with other similar options, namely extracorporeal whole liver perfusion and dialysis and filtration systems with no cells, the latter of which are currently ahead with respect to clinical testing and gaining regulatory approval. Ultimately, several temporary and adjunct treatment approaches may be available, and the best choice may depend on the etiology of liver failure in each individual patient.