Artificial and Bio Artificial Liver

Artificial and bioartificial liverdevices: present and futureB Carpentier, A Gautier, C Legallais

University of Technology ofCompiegne, UMR CNRS 6600 –Biomechanics andBioengineering, Compiegne,France

Correspondence to:Dr C Legallais, UTC, UMR CNRS6600 Biomechanics andBioengineering, BP 20529,60205 Compiegne Cedex,France; [email protected]

ABSTRACTLiver failure is associated with high morbidity andmortality without transplantation. There are two types ofdevice for temporary support: artificial and bioartificiallivers. Artificial livers essentially use non-living compo-nents to remove the toxins accumulated during liverfailure. Bioartificial livers have bioreactors containinghepatocytes to provide both biotransformation andsynthetic liver functions. We review here the operatingprinciples, chemical effects, clinical effects and compli-cations of both types, with specific attention paid tobioartificial systems. Several artificial support systemshave FDA marketing authorisation or are CE labelled, butthe improvement they provide in terms of patient clinicaloutcome has not yet been fully demonstrated. At present,different bioartifical systems are being investigatedclinically on the basis of their promises and capacity toprovide and replace most liver functions. However,important issues such as cost, cell availability, main-tenance of cell viability and functionality throughouttreatment, and regulatory issues, as well as difficultchallenges, including implementing cell-housing devices atthe patient’s bedside on an emergency basis, havedelayed their appearance in intensive care units and onthe market. Bioreactors are, nevertheless, when com-bined with artificial components, a pragmatic approach forfuture treatment of liver failure.

Liver failure results from the liver’s inability toperform its normal functions. It is a severe clinicalsyndrome in which the liver’s metabolic functions– detoxification, biotransformation, excretion andsynthesis – are severely impaired, leading to theaccumulation of lethal toxins in the patient andthe onset of life-threatening complications andmanifestations. The main detoxification and bio-transformation functions of the liver are detailed inthe Appendix.

Liver failure is associated with a rise in numerousendogenous substances such as bilirubin, ammo-nia, glutamine, lactate, aromatic amino acids, freefatty acids, phenol, mercaptans, benzodiazepinesand proinflammatory cytokines. These toxins areknown to play an important role in the pathogen-esis of liver failure.1–5 The loss of liver functionappears to result from an overload of hepatotoxicsubstances that progressively saturate availabledetoxification pathways, leading to the accumula-tion of other toxins and the production ofcytokines. Moreover, the accumulation of toxinsmay further impair the patient’s liver as a result ofhepatocellular apoptosis and necrosis.

The most frequent complications of liver failureare hepatic encephalopathy (HE), coagulopathy,

jaundice, cholestasis, pruritus, ascites, immunedisorders, sepsis and kidney failure.1 6

In acute liver failure (ALF), liver function isnormal 2–8 weeks before the onset of the disease.In acute-on-chronic liver failure (A-on-C LF), liverfunction decreases abruptly in a patient alreadysuffering from chronic liver insufficiency.1 6 Themost frequent causes of ALF are intoxications,especially acetaminophen intoxication, viral infec-tions and hepatic ischaemia. Sometimes the causeremains unknown.7 8 A-on-C LF usually occurs inpatients with cirrhosis, following infectious dis-orders, toxin exposure or gastrointestinal bleeding.Both are associated with high morbidity andmortality without transplantation.

Extracorporeal liver support devices have there-fore been developed in the last few decades in orderto either bridge patients to liver transplantation orallow the native liver to recover from injury. Theymay also be valuable when primary non-functionoccurs after liver transplantation or when a largehepatic resection leaves too little liver in reserve.3 9

There are two types of liver assist devices:artificial and bioartificial livers. Artificial liver(AL) devices use non-living components to cleansethe blood or plasma of its toxins. Removal is basedon physical/chemical gradients and adsorption.Bioartificial liver (BAL) devices contain a cell-housing bioreactor, the role of which is to replacethe primary and most important liver functions(oxidative detoxification, biotransformation, excre-tion and synthesis).

In this paper, the first part focuses on the currenttherapies available in intensive care units (ICUs)for treating or supporting patients with liverfailure. The operating principles of ALs, the effectsof ALs on chemical and clinical parameters andtheir market status will be noted and discussed.

The second part will focus on future treatmentsfor liver failure patients. Special attention will begiven to BAL devices that are currently included inclinical trials. Their operating principles, chemicaland clinical effects and side effects and complica-tions will be reviewed.

LIVER SUPPORT FOR PATIENTS WITH LIVERFAILURE: STATE OF THE ART

Social and economic data on liver support therapiesThe World Health Organization estimates that10% of the world’s population has chronic liverdisease, including 25 million Americans. Fulminanthepatic failure generally ends in death within 96 hwithout transplantation. This pathology affects

Recent advances in clinical practice

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around five people per million in Western coun-tries; ie, approximately 2500 people per year.

Liver transplantation is the only efficient treat-ment for patients with acute or fulminant organfailure. The shortage of liver donors has resulted in ahigh death rate in potential graft patients on awaiting list for a suitable donor (fig 1). In the last twodecades, the expanding gap between the number ofpatients on the waiting list for transplantation andthe number of transplants available has highlightedthe need for a temporary liver support system thatcould be used on an emergency basis.

Moreover, not all patients are candidates fortransplants and substitute solutions must be foundto face this issue. Liver transplantation is a riskytreatment that requires life-long immunosuppres-sion treatment. Xenotransplantation is not yet wellcontrolled and is subject to ethical questions.10

Partial transplantation of the liver within peoplefrom the same family (living donors) or fromcadavers has nevertheless led to substantial progress.

Approaches, clinical results and market status ofthe main artificial liver devicesOperating principlesMost ALs aim to replace detoxification functionsand use membrane separation associated withcolumns or suspensions of sorbents – includingcharcoal, anion-exchange or cation-exchange resins– that selectively remove toxins and/or regeneratedialysate or plasma filtrate. The treatment units(TUs) that may be included in liver assist devicesare shown in fig 2. ALs do not contain bioreactors.TUs are implemented either in parallel or in serieswithin the extracorporeal circuit (figs 3 and 4).

The Liver Dialysis deviceLiver Dialysis (HemoCleanse, Lafayette, Indiana,USA) – originally called Biologic-DT – was devel-oped by Ash et al.11 12 The patient’s blood is movedfrom a central vein via a single lumen multi-holedcatheter using a push–pull procedure, resulting in aflow rate of 200–250 ml/min.13 Simultaneously, a2-litre suspension of powdered activated charcoaland cation exchange resin (TU 7) is pumped from asorbent bag through the dialysate side of a flat-plate membrane dialyser (TU 4: 5 kDa molecularweight cut-off (MWCO) cellulosic membranes)and returned to the sorbent bag.

Liver Dialysis is currently being redesigned. Amethod for immobilising the powdered charcoal ina block is being evaluated for increased toxinbinding capacity and better regeneration of dialy-sate.14 15 The next system should be able to supportany type of hollow fibre (TUs 1 to 4).

The Molecular Adsorbent Recirculating SystemThe Molecular Adsorbent Recirculating System(MARS; Gambro Lundia, Lund, Sweden) was initi-ally developed by Stange and Mitzner.1 16 Thepatient’s blood is drawn from the femoral vein to ahigh-flux albumin-coated polysulfone haemodialyser(TU 6: surface area of 2.1 m2).9 17 Toxins (both

albumin-bound and soluble) are transferred to thecounter-current human albumin-enriched dialysate(TU 5).18 The exogenous albumin dialysate is thenregenerated in a closed loop by dialysis (TU 4 using alow-flux polysulfone membrane) against a conven-tional bicarbonate-buffered dialysate and adsorptionthrough charcoal and anion-exchange resin columns(TU 7).9 Blood and albumin dialysate flow rates areusually set between 150 and 250 ml/min, andbicarbonate dialysate between 300 and 500 ml/min.

The Prometheus devicePrometheus was designed by Fresenius MedicalCare (Bad Homburg, Germany).19 20 The patient’sblood is drawn at a flow rate of 200 ml/min to analbumin-permeable polysulfone filter (TU 2: Albu-Flow, MWCO 250 kDa), generating an albumin-containing filtrate. The endogenous albumin fil-trate subsequently flows through a neutral resinand an anion exchanger in series (TU 7). The bloodleaving the Albu-Flow is consequently dialysed in aconventional high-flux polysulfone dialyser (TU 4),against a 500 ml/min bicarbonate dialysate.

Clinical results and complications associated with thetreatmentLiver Dialysis, MARS and Prometheus treatmentsare usually performed 6–8 h daily for several daysor until the patient’s blood pressure and liverfunction have improved and the encephalopathyhas cleared.1 9 21 However, the optimal timing forthe initiation, frequency and duration of thetreatments, plus the biochemical and clinicalparameters on which to base the decision, remainpartially unanswered issues. The most importantrandomised controlled trials to have been con-ducted on the safety and efficacy of Liver Dialysis,MARS and Prometheus are summarised in table 1.

The Liver Dialysis deviceThe largest prospective randomised trial with LiverDialysis was carried out on 56 patients.11 22

Compared with controls and standard medicaltherapy (SMT), Liver Dialysis was responsible forimproved neurological status and blood pressure inpatients with either A-on-C LF or ALF and a higheroccurrence of positive outcome in patients with A-on-C LF and stage III–IV encephalopathy (suffi-cient recovery of liver function to allow hospitaldischarge or adequate clinical improvement toallow transplantation). In patients with liver failurecomplicated with kidney failure (type I hepatorenalsyndrome), no continuous veno-venous haemofil-tration was required as Liver Dialysis was able toremove up to 3 litres of fluid throughout eachtreatment without causing hypotension.

Liver Dialysis was associated with certain risks,including an increased degree of bleeding inpatients with active bleeding and disseminatedintravascular coagulation – due to activation andaggregation of the patients’ platelets – and clottingof the blood circuit during treatment.


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The Molecular Adsorbent Recirculating SystemSignificant reductions in serum bilirubin, bile acids,ammonia, urea, lactate and creatinine levels haverepeatedly been documented with MARS ther-apy.1 3 4 6 9 17 31–35 Total bilirubin and conjugatedbilirubin are reduced, whereas no changes inunconjugated bilirubin levels are observed.34 MARSdoes not interfere with valuable molecules such asalbumin,17 36 coagulation factors and electrolytes.6 33

MARS does not alter blood cell counts and arterialblood gases and does not generate haemodynamicinstability.6 Improvement in mean arterial pressure(MAP), systemic vascular resistance, cardiac outputand cerebral blood flow (due to reduced cerebraloedema) have repeatedly been demon-strated.17 31 32 37 38 Improvement in HE grade duringMARS treatment has frequently been observedregardless of the aetiology of the liver fail-ure.6 17 21 31 38–43 Improvement in HE was not neces-sarily associated with significant changes in cytokineand ammonia levels.24

Clinical outcome depends greatly on the aetiol-ogy of the liver failure. Survival rates at the time ofdischarge vary between 60% and 70% in patientswith either ALF or A-on-C LF of various aetiolo-gies.32 43 In one study in which MARS therapy wasused to treat 56 patients with ALF of variousaetiologies, the survival rate was 88% at 6 monthsand 84% at 1 year. The highest incidence of liverrecovery was observed in patients with ALF due tointoxication.3 Some authors have reported thesuccessful support of patients with ALF beforeliver transplantation.44 The impact of MARS onthe outcome of patients with A-on-C LF iscontroversial. Heeman et al conducted a prospec-tive randomised controlled study in 23 patientswith cirrhosis and with A-on-C LF.27 Renaldysfunction, HE and 30-day survival improvedwith MARS compared to SMT.

A prospective, randomised, controlled, multi-centre trial – the largest published so far – has

recently been completed to assess the efficacy,safety and tolerability of MARS treatment inpatients with advanced cirrhosis associated withsevere HE.23 Besides safety and tolerability, higher,earlier and more frequent improvements in HEgrade could be demonstrated when compared toSMT.

Further randomised studies are necessary beforeany strong conclusions can be drawn. Even thoughmany case-report studies have shown encouragingpatient outcome, the effects of MARS on clinicaloutcome is not yet proven for ALF and A-on-C LFpatients. However, trends for improved survivalrates have shown up in several randomised studies.

Randomised, controlled trials in patients withALF (the FULMAR trial; preliminary data pre-sented at the American Association for the Studyof Liver Diseases meeting in November 2008) andA-on-C LF (the MARS RELIEF trial) are ongoing.

Adverse events or complications have sometimesbeen noted, such as mild thrombocytopenia,9 17 45

and disseminated intravascular coagulation withsignificant bleeding.42 MARS is associated with thesame risks as any other extracorporeal filtrationprocedure requiring catheterisation.32 MARS seemsto be relatively safe but should be used withcaution in patients with a coagulopathy.

The Prometheus deviceA significant decrease in both albumin-bound andwater-soluble toxins has repeatedly been demon-strated throughout treatment withPrometheus.2 19 46 47 The Prometheus treatmentimproves serum levels of conjugated bilirubin, bileacids, ammonia, creatinine, urea and bloodpH.2 19 46–48 Krisper et al have compared the effectsof MARS and Prometheus on toxin removal. Theyobserved a higher efficiency and delivered dosewith Prometheus compared to MARS. However,this was not responsible for significant differencesin plasma levels.2 Substances such as albumin,fibrinogen, coagulation factors, other plasma pro-teins, electrolytes and cytokines (interleukin 6 andtumour necrosis factor a) remained unchangedduring the treatment with Prometheus.20 46 48

Even though no randomised, controlled studieshave been reported yet, treatment withPrometheus seems to have beneficial effects onpatient outcome.19 46 Further investigations mustbe completed, especially in the course of a multi-centre randomised study named HELIOS (ongoingstudy).

Decreased blood pressure, clotting of the sec-ondary circuit and a slight increase in leucocytecounts were the only side effects or complicationsreported for the Prometheus therapy.19 48

Market status of the major artificial liver devicesIn 1997, Liver Dialysis was the first AL to receiveapproval from the US Food and DrugAdministration (FDA) for use in the treatment ofencephalopathy secondary to liver failure, underthe name of Biologic-DT. Liver Dialysis is not

Figure 1 1994 to 2005 statistics from the European Liver Transplant Registry: timecourse of cadaveric and living organ transplants, persons on the waiting list andsubsequent death while on the waiting list.


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currently marketed in the USA as it is beingredesigned. MARS and Prometheus have receivedthe CE label to allow their sale in Europe. Althoughthey are used in many countries, they are not yetavailable in the USA.

FUTURE TREATMENT OF LIVER FAILURE:BIOARTIFICIAL LIVER SYSTEMSThe aim of BALs is to provide both liver detoxifica-tion and synthetic functions, using a combinationof physical and chemical procedures, and bioreac-tors hosting cells (fig 2). The advantages and

disadvantages of the various most commonly usedcell sources are summarised in table 2.

Four configurations of BAL are currently underinvestigation: hollow fibre cartridges or chambers(ELAD, HepatAssist, MELS), monolayer cultures,perfused matrices (BLSS, AMC-BAL) within dedi-cated devices and microencapsulation-based sys-tems.49–51 Only the devices currently under clinicalinvestigation are described in detail in this paper.

The treatment units that make up a BAL areimplemented either in parallel or in series withinthe extracorporeal circuit (figs 3 and 4).

Figure 2 Treatment units (TUs): the principal unitary treatment blocks that may be included within an extracorporeal liversupport system. Each extracorporeal liver support system is based on a combination of three or more blocks that are addedeither in series or in parallel within the circuit. The aim and methods on which each block is based are indicated. TU 1:plasmapheresis – based either on centrifugation or microporous membrane filtration. TU 2: plasma fractionation – filtrationbased on a very high permeability membrane (MWCO .70–100 kDa). TU 3: haemofiltration – filtration based on a mediumpermeability membrane (15 kDa , MWCO ,70 kDa). TU 4: haemodialysis – diffusion through a low-to-mediumpermeability membrane. A buffered dialysate present in the extracapillary space is used for the removal of water solubletoxins. TU 5: albumin dialysis – same as TU 4, with albumin solution on the dialysate side. TU 6: aided transfer – same as TU5, with an albumin-coated membrane to help the albumin-bound toxins cross the membrane. TU 7: adsorption on varioussubstrates to remove protein-bound toxins, such as bilirubin or bile acids. TU 8: bioreactor hosting cells for biotransformationand synthesis functions. TU 9: oxygen supply. MWCO, molecular weight cut-off.


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Bioreactor specifications for bioartificial liverapplicationsSeveral requirements must be satisfied in order toensure full efficiency of the bioreactor. Functionalliver cells (table 3) must be isolated from theexternal environment and immobilised on orwithin a favourable substrate (fig 5).

The substrate should allow the preservation ofcell morphology and metabolism throughout thetreatment. Oxygen and nutrients should be acces-sible to the cells in appropriate concentrations. Aporous material (not necessarily the substrate)should act as a barrier between the patient’s bloodor plasma and the hepatocytes located within the

Figure 3 Schematicprinciples of extracorporealsystems for the treatmentof liver failure patients.Liver support systems aredesigned in such a waythat liver failure toxins canbe removed from thepatient – andbiotransformation andsynthesis can occur whenbioartificial livers (BALs)are considered – bycombining essential andoptional treatment unitstogether. Blood is drawnfrom a central vein anddirected towards thetreatment units (TUs) (seefig 2) before being returnedto the patient. Differentstrategies have beenchosen for each device.Treatment units are eitherconnected in series or inparallel to the others,depending on the device.

Figure 4 Summary of thetreatment units integratedwithin each artificial orbioartificial liver supportdevice. Refer to fig 2 toidentify each treatment unitblock.


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bioreactor in order to isolate the cells from theimmune factors (immunoglobulins, MW 150 kDa)and leucocytes, and to avoid immune rejection(table 4). Smaller particles such as toxins andmetabolites or synthesised proteins (albumin andcoagulation factors for instance) should be free tocross the barrier.

The perfusion system is more or less com-plex (table 5). An initial detoxification stepusing charcoal and/or ion exchange resins could beperformed prior to the passage of the blood orplasma through the bioreactor in order to avoid

the death of the pool of supporting cells.An oxygenator can also be included upstream in thecircuit to improve oxygen delivery (fig 4).

Operating principles, and the chemical and clinicaleffects of bioartificial liversHere, we focus only on the devices that arecurrently under clinical investigation. Their marketstatus and stage of advancement are detailed laterin the paper. HepatAssist and ELAD are the onlyBAL systems on which randomised and controlledtrials have been performed (table 1).

Table 1 Randomised controlled trials on the safety and efficacy of artificial livers and bioartificial livers

Liver assist deviceNumber ofpatients Inclusion criteria Treatment Clinical results Authors

Liver Dialysis 56 A-on-C LF Liver Dialysis vs SMT Improved neurological status and MAP Ash et al11 22

ALF Increased degree of bleeding in patientswith active bleeding and DIVC

MARS 70 HE grade 3 or 4 MARS (6 h per day, 5 days) +SMT or SMT alone

Higher, earlier and more frequentimprovement of HE compared to SMT

Hassanein et al23

18 A-on-C LF due to alcohol abuse MARS (four sessions of 8 h, Improvement of HE Sen et al24

7 days) + SMT or SMT alone Unchanged MAP and renal function

No significant change in plasmacytokines and ammonia levels

27 Hypoxic liver failure aftercardiogenic shock

MARS (three consecutivesessions at least) vs SMT

Improved survival El Banayosy et al25

Bilirubin levels greater than8 mg/ml

Further study with larger patient cohortneeded

18 A-on-C LF MARS + SMT Decreased serum bilirubin levels Laleman et al26

Alcoholic cirrhosis and or Prometheus + SMT or Improvement in MAP and SVRI

superimposed alcoholichepatitis

SMT alone – threeconsecutive days (6 hsessions)

Reduction of the hyperdynamiccirculation

24 Decompensated cirrhosis MARS (2 weeks, up to 10 Improved 30-day survival Heemann et al27

Bilirubin greater than 20 mg/dlnot responding to prior SMT

sessions) + SMT vs SMTalone

Decrease in plasma bile acids andbilirubin

Improvement in renal dysfunction and HE

Prometheus 24 Decompensated cirrhosis Single 6 h treatment withPrometheus (study group),

No differences in systemichaemodynamics

Dethloff et al28

MARS or haemodialysis No improvement in MAP

No adverse effects

Decrease in platelet count

18 A-on-C LFAlcoholic cirrhosis andsuperimposed alcoholichepatitis

MARS + SMT or Prometheus+ SMT orSMT alone – threeconsecutive days (6 hsessions)

Decreased serum bilirubin levels (moreeffective than MARS)No improvement in MAP, SVRI andhyperdynamic circulation

Laleman et al26

HepatAssist 171 Fulminant or subfulminanthepatic failure and primary non-function following livertransplantation

HepatAssist (6 h daily) + SMTvs SMT alone

Improved survival at 30 days (71% vs62%). Good safety

Demetriou et al29

ELAD 24 Acute liver failure with ELAD (median period of 72 h) Good biocompatibility Ellis et al30

potentially recoverable lesion orfulfilment of criteria fortransplantation

vs SMT DIVC or hypersensitivity reaction (2cases)Survival comparable to SMTBetter indices of prognosis required

BLSS None conducted yet

AMC-BAL None conducted yet

MELS None conducted yet

A-on-C LF, acute-on-chronic liver failure; ALF, acute liver failure; AMC-BAL, Academic Medical Center – Bioartificial Liver; BLSS, Bioartificial Liver Support System; DIVC, disseminatedintravascular coagulation; ELAD, Extracorporeal Liver Assist Device; HE, hepatic encephalopathy; MAP, mean arterial pressure; MARS, Molecular Adsorbent Recirculating System; MELS,Molecular Extracorporeal Liver Support; SMT, standard medical therapy; SVRI, systemic vascular resistance index.


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The HepatAssist deviceThe HepatAssist device developed by Demetriou etal initially used 56109 to 76109 cryopreservedporcine hepatocytes constrained within the extra-capillary compartment of a hollow fibre bioreactor(TU 1 and 8).52 The cells are attached to micro-carriers. The patient’s blood is separated intoplasma (TU 1) collected in a special bag used as abuffer reservoir. The plasma passes through acolumn of activated charcoal (TU 7) and anoxygenator (TU 9) before it is circulated withinthe bioreactor (fig 6a).

In a phase I clinical trial, 39 patients with ALFwere treated with HepatAssist. Thirty-twopatients were successfully bridged to liver trans-plantation and six recovered without requiring agraft. Survival rate at 1 month was 90%.53 Based onthese results, a multicentre phase II/III prospectiverandomised controlled trial was initiated in whichHepatAssist therapy was compared to standardmedial therapy in patients with ALF or primarynon-function.29 One hundred and seventy-onepatients with ALF (fulminant or sub-fulminant)or primary non-function after liver transplantationwere enrolled in this two-armed study. Eighty-sixpatients received SMT, whereas 85 patients weretreated with the HepatAssist system. The survivalrate at 30 days was 71% and 62% in the entirepopulation (p = 0.26) and 73% and 59% in the ALFsubgroup (p = 0.12) with HepatAssist treatmentand SMT, respectively. It was thus possible todemonstrate the safety of the system, but notimproved survival.

In another controlled study,54 10 out of 13selected patients with ALF were treated withHepatAssist as a bridge to transplantation. Twopatients improved spontaneously and one wastransplanted before the HepatAssist treatmentcould be initiated. Ten patients received one tothree HepatAssist treatment sessions. A significantimprovement in neurological status was observedusing the Glasgow Coma Scale (6.5 (SD 3.7) and9.6 (SD 4.4) before and after treatment, respec-tively). Meanwhile, a significant decrease inbilirubin and transaminase levels was observed.

HepatAssist was relatively well tolerated.HepatAssist-related complications were transientepisodes of haemodynamic instability and bleedingin some patients. Two patients died after beingtransplanted, whereas eight survived with a meanfollow-up of 18–32 months.

Due to the use of a microporous membrane,mass transfers between the plasma and theimmobilised hepatocytes were high. No immuno-logical response was reported despite the pore sizewhich should not prevent immunoglobulin trans-fer. In a retrospective study carried out in 28patients previously treated with the HepatAssistsystem,55 absence of porcine endogenous retrovirus(PERV) infection was observed. All patients werenegative for PERV up to 5 years after treatmentwith HepatAssist as demonstrated by polymerasechain reaction analysis of peripheral blood mono-nuclear cells.

The Extracorporeal Liver Assist DeviceThe Extracorporeal Liver Assist Device (ELAD)developed by Sussman et al56 uses 200 g of C3Ahuman hepatoblastoma cells located in the extra-capillary compartment of hollow fibre cartridges(TUs 3 and 8). Blood is first separated into plasma.Prior to entering the cell-containing bioreactor,plasma is directed towards activated charcoal (TU7) and an oxygenator (TU 9) (fig 6a).

The first clinical applications have recently beenstudied to demonstrate the safety of the system. Inone case study,57 a patient with fulminant liverfailure of unknown aetiology was treated with theELAD system. Continuous treatment for 6 dayswas associated with improved neurological statusand clinical parameters. Treatment was discontin-ued when evidence of native liver functionrecovery occurred. However, the patient died fromseptic shock a few days later.

The short-term safety of the ELAD system wasassessed in another clinical pilot study in whicheleven patients were enrolled.56 No safety problemssuch as haemodynamic instability, complementactivation or deterioration of vital organ function

Table 2 Advantages and disadvantages of potential cell sources for bioartificial livers

Cell source Advantages Disadvantages

Autologous cells harvestedfrom the patient

i. No immune reactions

ii. Low risk of infection withpathogens

i. Limited availability

ii. Heterogeneity in quality and behaviour

Allogenic cells collected formdonors

i. High availability (above all, inemergencies)

ii. Pooling possible from differentdonors

i. Risk of disease transmission

ii. Risk of immune response

Allogenic cell lines (aftergenetic mutation making indefiniteproliferationpossible)

i. High availability

ii. Production of a large number ofcells

iii. Infinite growth capability

i. Loss of functions

ii. Potential tumorigenicity

Xenogenic cells isolated fromdifferent species

i. High availabilityii. Production of a large number ofcells

i. Risk of animal pathogen transmissionii. Risk of immunogenic rejectioniii. Regulatory issues


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were associated with ELAD treatment. Metabolicsupport was observed in 10 patients suffering fromlate-stage liver failure.

A pilot, controlled, two-armed study was per-formed to evaluate ELAD treatment in ALFpatients with a significant chance of survival (17patients) or fulfilling criteria for transplantation(seven patients).30 The two groups were formedbased on predicted outcome. In each group,patients were either treated with ELAD plusSMT or with SMT alone. No acceleration inplatelet consumption and haemodynamic instabil-ity were associated with ELAD treatment.However, two patients were withdrawn from thestudy because of exacerbated pre-existing dissemi-nated intravascular coagulation or hypersensitivityreaction. Encephalopathy grade worsened in 58%of the control patients versus 25% of the patientstreated with ELAD, showing some benefit withELAD support. Survival rates for the control andELAD-treated patients were similar in ALF patientswith potentially recoverable lesions (75% vs 78%,respectively). In patients who satisfied the trans-plantation criteria, survival was higher in the ELADgroup compared to the controls (33% vs 25%,respectively). This study did not show anyimprovement in either the chemical parametersor the clinical outcome.

Based on these results, the device was modifiedand used in a clinical trial to assess safety.58 The70 kDa cut-off membrane was replaced by a120 kDa cut-off membrane (TU 2). Four hollowfibre cartridges with 100 g of C3A hepatoblastomacells were used for each treatment. Compared tothe initial settings, the flow rate through eachcartridge was set at 500 instead of 150–200 ml/min. Five patients with fulminant liver failurereceived continuous ELAD therapy with themodified version of the system as a bridge to andthroughout liver transplantation. They were allsuccessfully bridged and transplanted. Survival rateat 30 days was 80%. No adverse events orcomplications were reported. Patients’ haemody-namic conditions did not worsen in the course ofthe treatment. The cells in the bioreactors weremetabolically active throughout the treatment, asevidenced by oxygen consumption. Larger rando-mised multicentre trials should now therefore beperformed.

The Bioartificial Liver Support SystemThe Bioartificial Liver Support System (BLSS)59 60

developed by Patzer et al59 is a haemofiltration

device (TUs 3 and 8) that contains approximately100 g of primary porcine hepatocytes within theextracapillary compartment of a hollow fibrebioreactor (membrane cut-off 100 kDa). Wholeblood is perfused through the fibres after beingheated through a heat exchanger and oxygenatedusing an oxygenator (TU 9). Treatment with BLSSlasts for approximately 12 h (fig 6a).

The first clinical use of the BLSS was performedin a patient with fulminant liver failure.61 Fourpatients with ALF were treated with the BLSSdevice in a phase I clinical trial.60 Inclusion criteriawere ALF of any aetiology associated with ence-phalopathy deteriorating beyond grade 2. A singlesupport session consisted of a 12-h extracorporealBLSS treatment. A second treatment session couldbe given if necessary. Ammonia and total bilirubinlevels decreased by 33% and 6%, respectively,compared with pre-treatment values. Kidney andneurological functions did not improve signifi-cantly during or after BLSS therapy. Transienthypotension in one patient was the only adverseevent observed. No PERV infection could bedetected up to 1 year post-therapy.

The Academic Medical Center – Bioartificial LiverThe AMC-BAL developed by Chamuleau’s team62

consists of a plasmapheresis system (TU 1) and aseparate polycarbonate housing that contains athree-dimensional non-woven hydrophilic poly-ester matrix (TU 8). Ten billion (106109) primaryporcine hepatocytes are seeded within the matrixwhich is wound around a polycarbonate core.Hollow fibres are regularly distributed amid thematrix layers for oxygen supply and CO2 removal(TU 9). Plasma is perfused through the chamber,between the matrix layers.

Because of legislation issues on xenotransplanta-tion in European countries, the system is beingredesigned with a human-derived hepatocyte cellline (fig 6b).63 However, the system has been tested

Table 3 Biological components in bioartificial liver devices

System Cell type Cell source Cell amount

HepatAssist Porcine Cryopreserved 56109 to 76109

ELAD Human, tumour cell line C3A 200–400 g

BLSS Porcine Freshly isolated 70–120 g

MELS Porcine/human Freshly isolated Up to 600 g

AMC-BAL Porcine Freshly isolated 106109

AMC-BAL, Academic Medical Center – Bioartificial Liver; BLSS, Bioartificial Liver Support System; ELAD,Extracorporeal Liver Assist Device; MELS, Modular Extracorporeal Liver Support.

Figure 5 Principle of bioartificial livers. Nutrients andoxygen cross the membrane from the plasma to thehepatocyte-housing compartment to maintain cellactivities. Plasma toxins are transferred to thehepatocytes for elimination or further transformation intometabolites. Metabolites and synthesised substances arereturned to the plasma stream.


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for safety and efficacy. In one case study, a patientwas successfully bridged to liver transplantationafter 35 h of treatment with the AMC-BAL.64

Biochemical and clinical parameters improvedduring therapy. No severe adverse events or porcineendogenous retrovirus activity in the patient’sblood or blood cells were reported.

In a phase I clinical trial, the AMC-BAL was usedas a bridge to liver transplantation in patients withALF.65 Seven patients with grade III or IVencephalopathy and meeting the criteria fortransplantation were treated with AMC-BALtreatment sessions of 8–35 h. Three patientsreceived two additional treatment sessions withtwo different BALs. Six patients were successfullybridged to transplantation. One patient recoveredliver function after two treatment sessions and didnot need transplantation. No severe adverse eventswere reported.

The Modular Extracorporeal Liver Support deviceThe Modular Extracorporeal Liver Support (MELS)device was developed by Sauer et al.66 It is made ofseveral units: a bioreactor (TU 8), a detoxificationmodule performing single pass albumin dialysis(SPAD) (TU 5) and a haemodiafiltration module(TUs 3 and 4). The bioreactor itself contains threebundles of hollow fibre membrane interwoven intoa three-dimensional capillary network. Two bun-dles of polysulfone membrane with a cut-off of400 kDa are used to perfuse the cells with thepatient’s plasma (TU 2) (fig 6a). The third set offibres is used for in situ oxygen supply (TU 9). Theextracapillary space is loaded with up to 500–600 gof human hepatocytes. The cells attach to thefibres and form aggregates. Prior to therapy, astand-by phase of 21 days allows the cells to adaptto the new environment, tissue reformation andquality controls.

In one case study, a patient with primary non-function after transplantation was treated withthe MELS system as a bridge towards a newtransplantation.67 The bioreactor was loaded with470 g from a discarded liver (viability 60%; 5–10%of non-parenchymal cells) and integrated into acircuit including continuous single pass albumindialysis and continuous veno-venous haemodiafil-tration. MELS therapy was performed for 79 h.Total plasma bilirubin levels were significantlyreduced (21.1 mg/dl at the initiation of therapy vs

10.1 mg/dl after treatment). Ammonia decreasedfrom 100 mmol/l to 22.7 mmol/l. The patient’sneurological status improved significantly fromcoma stage IV to coma stage I. Coagulation factorswere improved. Kidney function was also recov-ered after treatment. No adverse events werereported.

Market status of major bioartificial liver devicesHepatAssist is now presented as HepaMate(HepaLife Technologies, Boston, Massachusetts,USA). It contains 146109 cryopreserved hepato-cytes. A new monitor (HepaDrive) is proposed bythe company. It is now in a new phase III clinicaltrial in the USA.

ELAD is manufactured by Vital Therapies,which was founded in 2003 and is based in SanDiego, California, USA, with a subsidiary inBeijing, China. ELAD manufacturing is carriedout in San Diego in a facility that is compliantwith good manufacturing practice (GMP). Patientenrolment has begun for a randomised, controlled,multi-centre, phase II clinical trial that willinvestigate ELAD as a treatment for patients withALF, with three protocols. The study is planned tobe expanded to 15 sites in the USA and Europewithin 2009.

Excorp Medical (Minneapolis, Minnesota, USA),manufactures BLSS. This corporation is headquar-tered and registered in the state of Minnesota,USA. It has a Wholly Foreign Owned Entity inJiangSu Province, China and a Wholly ForeignOwned Subsidiary in Hong Kong, China. Inassociation with the University of PittsburghMedical Center, patients with ALF or A-on-C LF– regardless of underlying cause or aetiology – areto be enrolled in the phase I/II clinical trial. Thecompany anticipates that the phase III efficacystudy will be conducted in six centres in the USAand will involve up to 150 patients. Clinical trialsare also planned in China.

Hep-Art Medical Devices is a small spin-offcompany of the Academic Medical Center inAmsterdam which develops the AMC-BAL. Tocomply with EU standards, several functional first-grade human liver cell lines are currently beingdeveloped.

How can bioartificial livers be implemented inclinical practice?Cell-housing devices are obliged to meet severalrequirements before being used in everyday med-ical practice. First, they must not be harmful to thepatient and must be relatively safe. Any side effectsmust be minor compared to the clinical benefits.The regulatory issues and international rules arestated and explained in the next section.

The use of living components within extracor-poreal assist devices results in major challenges:preservation of sustained cell metabolism andfunction, safety and applicability at the bedsideon an emergency basis are the main issues thatneed to be addressed before a bioartificial liver can

Table 4 Membrane types in bioartificial liver devices

System Membrane type Membrane cut-offRetained cells/substances

HepatAssist Polysulfone 3000 kDa Blood cells

ELAD Cellulose acetate 70 kDa Above albumin

BLSS Cellulose acetate 100 kDa Above albumin/largemolecules

MELS Polyethersulfone 400 kDa Very large molecules

AMC-BAL Polysulfone Hepatocytes in direct contactwith plasma

–

AMC-BAL, Academic Medical Center – Bioartificial Liver; BLSS, Bioartificial Liver Support System; ELAD,Extracorporeal Liver Assist Device; MELS, Modular Extracorporeal Liver Support.


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reach the market. The first point brings us back tothe cell source.

Safety regarding cell preparation could beachieved provided all operations are conducted inaccordance with good manufacturing practice(GMP). If primary cells are to be used, specialattention must be paid with regard to the harvest-ing and isolation procedures. Nowadays, cell banksare able to meet such requirements.

The bioartificial device should be readily avail-able to clinicians. This is one of the most difficultand costly issues that has to be addressed. Thereare several options: cell-containing bioreactors canbe supplied with culture medium for several daysbefore use or frozen and thawed right before use.When needed, the bioreactor is removed from itsfeeding circuit or thawed and included within theextracorporeal device to support patients with liverfailure. The first solution is extremely expensivesince the bioreactors have to be fed with culturemedium and looked after. There are risks, andcareful attention would have to be paid tomaintaining the aseptic conditions throughoutthe whole process. Cryopreservation may seemthe ideal solution; however, many cells may dieduring freezing or/and defrosting. Cell metabolism

and detoxification capacities must remain highenough to treat the patient.

Regulatory issuesThe World Health Organization (WHO) recentlystated that a harmonised international regulatoryapproach would be ideal for the successful transla-tion of tissue engineering research to the clinic andmarket place. In the USA, BALs, considered asTissue-Engineered Medical Products (TEMP), areexamined by the Office of Combination Products(OCP), or by the Office of Orphans Products(OOP). The Public Health Service (PHS) requestsproof of the safety, purity and potency ofbiological products before introduction into inter-state commerce. However, pre-market clinicalstudies are allowed and include three phases: phaseI, feasibility; phase II, proper and safe dosing, andpotential efficacy; and phase III, pivotal study withwell controlled clinical trial design.68

In the EU, BALs would be treated as AdvancedTherapies Medicinal Products (ATMP). On 30December 2008, new European legislation laiddown the rules on how ATMP are to be authorised,supervised and monitored to ensure that they aresafe and effective.69 The legislation establishes

Table 5 Perfusion characteristics in bioartificial liver devices

System Perfusion

Bloodfiltration rate(ml/min)

Plasmafiltration rate(ml/min)

Bioreactorflow rate(ml/min)

Oxygenationlocation Anticoagulation

HepatAssist Plasma 90–100 50 400 Pre-bioreactor Citrate

ELAD Blood/plasmaultrafiltrate

150–200 – 15–200 Pre-bioreactor Heparin

BLSS Blood 100–200 – 100–250 Pre-bioreactor Heparin

MELS Plasma 150–300 31 100–200 Inside bioreactor Heparin

AMC-BAL Plasma 100 40–50 150 Inside bioreactor Heparin

AMC-BAL, Academic Medical Center – Bioartificial Liver; BLSS, Bioartificial Liver Support System; ELAD, Extracorporeal Liver Assist Device; MELS,Modular Extracorporeal Liver Support.

Figure 6 Exchange principles between the hepatocytes and plasma or blood in the (a) HepatAssist, MELS, ELAD,BLSS and (b) AMC-BAL systems. In (a), plasma or blood is perfused through the lumen of hollow fibres whose structureacts as a selective barrier for the hepatocytes placed in the outer compartment. In the MELS device, another hollow fibrenetwork is specifically added for cell oxygenation. In (b), plasma is in direct contact with the hepatocytes. The hollowfibre membrane is dedicated to oxygen supply. AMC-BAL, Academic Medical Center – Bioartificial Liver; BLSS,Bioartificial Liver Support System; ELAD, Extracorporeal Liver Assist Device; MELS, Modular Extracorporeal LiverSupport.


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within the European Medicines Agency a newcommittee dedicated to advanced therapies. TheCommittee for Advanced Therapies (CAT) plays acentral role in the scientific assessment of advancedtherapy products.

CONCLUSIONArtificial liver and bioartificial liver devices eachrepresents a potentially useful option for thetreatment of patients with liver failure (table 6).

Both ALs and BALs have been intensely studiedin the last few decades. ALs have proven to be auseful option in many cases for improving bio-chemical parameters and clinical symptoms butthe benefits in terms of patient outcome have notyet been fully demonstrated. Detoxification alone,as provided by an AL device, may improve thecondition of patients with liver failure if appliedearly. As demonstrated with the limited success ofplasma exchange, detoxification alone may not besufficient in most cases. BAL devices have not yetreached the market and are still subject tocumbersome studies.

Some patients could be efficiently treated withBAL systems, proving the potential for the concept.However, their use is still very confidential. To us,their transfer from the laboratory bench to thepatient’s bedside is hindered by three types ofobstacle: (1) scientific challenges, including theabsence of a readily available functional cell source,the loss of cell viability and functionality beforeand throughout treatment, problematic large-scalecell culture in a dynamic setting, and the currentlimitations of cryopreservation procedures (toprovide ICUs with ready-to-use devices on anemergency basis); (2) regulatory matters; and (3)cost issues due to inappropriate storage procedures,complexity of use and the number of patients toinclude in clinical studies before authorisation.

These important challenges delay the appear-ance of BAL systems in ICUs and on the market.Bioreactors combined with artificial componentsnevertheless represent a pragmatic approach forfuture treatment of liver failure patients andshould be investigated further. On the one hand,special attention needs to be paid to identifying/isolating a readily available and functional sourceof cells and to improving hepatocyte entrapment.Bioreactor configurations that are not hollow-fibre-based should be considered to improve both large-scale cell culture prior to therapy and masstransfers during treatment. On the other hand,

regulatory issues need to be reconsidered. A betterchance to develop should be given to these newand promising products. This could be achieved byencouraging multi-disciplinary academic teams, aswell as small and/or leading companies, toaccompany such developments.

Acknowledgements: Certain parts of several of the figures wereproduced using Servier Medical Art (www.servier.com).

Competing interests: None.

Provenance and peer review: Commissioned; externally peerreviewed.

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Table 6 Advantages and disadvantages of artificial and bioartificial liver support

Type of device Advantages Disadvantages

Artificial systems i. Relatively easy to use

ii. Limited cost for system conception andpatient treatment

i. Only detoxification

ii. Limited efficacy

Bioartificial systems i. All hepatic functions ensuredii. Expected clinical results morepromising

i. Cell source still under discussionii. Complexity of implementing living componentsiii. High cost for design, operation and patients treatmentiv. Heavy logistics


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APPENDIX: BIOTRANSFORMATION FUNCTIONS OFTHE LIVERThe liver is a highly complex metabolic organ that participates innutrient absorption, storage and delivery according to the body’sdemands. It also plays a key role in the biotransformation,detoxification and elimination of endogenous waste, medicines andexogenous toxins (fig A1). Furthermore, it contributes to blood flowcontrol and antibacterial immune defence.Ammonia metabolism – the main toxic waste resulting from proteincatabolism – mainly takes place in the liver. Ammonia is capturedand transformed into urea by periportal hepatocytes before beingeliminated by the kidney. Perihepatic (or centrolobular) hepatocytes,localised close to the hepatic venules, are able to transform ammoniainto glutamine.Bile acids – synthesised by the liver from cholesterol – are liberatedinto the intestine in the bile. The liver is also able to capture bile acidscirculating within the blood after their intestinal reabsorption. A smallamount (5%) of the secreted bile acids is eliminated in the stool. Thisis the main way of eliminating cholesterol from the body.Bilirubin is the degradation product of haem, a prosthetic groupincluded within haemoglobin and haemoproteins such as myoglobinand cytochromes. Haem is transformed into biliverdin and then intobilirubin by haem oxygenase, the activity of which is predominantly in

the spleen, Kupffer cells and, to a lesser degree, hepatocytes.Bilirubin molecules are insoluble and thus albumin-bound in the plasma.In the liver, bilirubin is captured, conjugated with glucuronic acids tomake it soluble and eliminated in the bile. Within the intestine, bilirubinmolecules are deconjugated and hydrogenated into urobilinogen bycolon bacteria. A small amount is reabsorbed by the intestine to beeliminated once again in the bile and, to a lesser degree, in urine. Mostof it is eliminated in the stool. The oxidation of urobilinogen to urobilin isresponsible for the coloration of stools and urine.Xenobiotics such as exotoxins and drugs must also be eliminated.Most hydrosoluble xenobiotics are eliminated by the kidneys. Incontrast, liposoluble toxins must be solubilised before beingeliminated. These modifications are mainly made by the liver and,to a lesser degree, by the lungs, kidneys and intestine through twotypes of reaction: phase I or oxidation reactions (super-family of P450cytochromes or CYP) and phase II or conjugation reactions (such asUDP-glucuronyl-transferases, for example). The former increase themolecule’s solubility through the addition of polar groups, whereasthe latter enable the linkage of hydrosoluble endogenous molecules –such as glucuronic acids, glutathione, sulfates and amino acids – tooxidised molecules (simultaneously reducing their pharmacologicalactivity and increasing their hydrosolubility). Those molecules cansubsequently be eliminated in the bile or by the kidneys.

Figure A1 The mainbiotransformation anddetoxification functions ofthe liver, which, incoordination with thekidneys and intestine, playsa central role in theelimination of endogenousand exogenous toxins.


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Artificial and Bio Artificial Liver

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