tissue engineering cell and tissue engineering for liver...

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TISSUE ENGINEERING Cell and tissue engineering for liver disease Sangeeta N. Bhatia, 1,2 * Gregory H. Underhill, 3 Kenneth S. Zaret, 4 Ira J. Fox 5 Despite the tremendous hurdles presented by the complexity of the livers structure and function, advances in liver physiology, stem cell biology and reprogramming, and the engineering of tissues and devices are accel- erating the development of cell-based therapies for treating liver disease and liver failure. This State of the Art Review discusses both the near- and long-term prospects for such cell-based therapies and the unique chal- lenges for clinical translation. INTRODUCTION Liver disease and the subsequent loss of liver function is an enormous clinical challenge, and is currently the 12th most frequent cause of death in the United States and the 4th most frequent for middle-aged adults (1). The situation is progressively worsening, prompted by sev- eral factors including the emergence of new liver diseases such as non- alcoholic fatty liver disease and steatohepatitis, the lack of a hepatitis C vaccine, and an aging population of hepatitis patients at risk for pro- gression to hepatocellular carcinoma (2, 3). Liver transplantation is the primary treatment for liver failure and is the only therapy shown to directly alter mortality. To expand the supply of available livers for transplant, numerous surgical options have been pursued, including split liver transplants and living-related partial donor procedures (4). In spite of these surgical advances and improvements in organ alloca- tion, organ shortages remain acute, suggesting that it is unlikely that liver transplantation procedures alone will ever meet the increasing de- mand. Cell-based therapies have long-held promise as an alternative to organ transplantation. In this State of the Art Review, we will de- scribe both near- and long-term prospects for cell-based treatments, including the use of stem cells and other nonhepatocyte sources and tissue engineering, within the context of clinical manifestations of liver disease. We will discuss the unique potential and big challenges that exist for cell-based approaches and will provide an overview of funda- mental biological questions, technological tools, and future directions for the field. THE LIVER IN HEALTH AND DISEASE The liver is the largest internal organ in the body, accounting for 2 to 5% of body weight, and performs a complex array of more than 500 func- tions including metabolic, synthetic, immunologic, and detoxification processes. The liver also exhibits a unique capacity for regeneration, with the potential for full restoration of liver mass and function even after massive damage in which less than one-third of the cells remain uninjured (5, 6). In fact, procedures such as partial liver transplants take advantage of this significant regenerative potential combined with the bodys finely tuned homeostatic regulation of liver mass. However, the potential for liver regeneration is often difficult to predict clinically, and criteria for identifying patients that may resolve liver failure complications due to regenerative responses remain poorly de- fined. As a result, efforts have been made toward the development of liver support technologies that could provide temporary function for patients with liver failure, thereby enabling sufficient time for regen- eration of the native liver tissue or serving as a bridge to transplantation. These measures include extracorporeal support devices that act in a manner analogous to kidney dialysis systems, processing the blood or plasma of liver failure patients (7, 8). Initial designs based on nonbio- logical exchange/filtering systems have shown limited clinical success, likely due to the insufficient level of hepatocellular functions exhibited by these devices. To provide a larger complement of important liver functions, including synthetic and regulatory processes, support devices incorporating living hepatic cells have been developed, although these systems remain primarily experimental to date (9). In addition to tem- porary extracorporeal platforms, the development of cell-based thera- pies aimed at the replacement of damaged or diseased liver tissue is an active area of research. For instance, the transplantation of isolated liver cell types, such as mature hepatocytes, has been extensively ex- plored (10) and has potential as an attractive therapeutic option partic- ularly for inherited single gene metabolic deficiencies. Moreover, liver tissue engineering approaches, wherein preformed cellular constructs are implanted as therapeutics, are under development. Finally, these en- gineered tissues are also being explored as in vitro model systems for fundamental and applied studies of liver function in healthy and diseased states. The development of liver cellbased therapies poses unique chal- lenges, largely stemming from the scale and complexity of liver structure and function. The organ displays a repeated, multicellular architecture, in which hepatocytes, the main parenchymal cell of the liver, are ar- ranged in cords that are sandwiched by extracellular matrix in the space of Disse (Fig. 1). The space between cords is also home to a mul- titude of supporting cell types such as sinusoidal endothelial cells, Kupffer cells, biliary ductal cells, and stellate cells. Because of this ar- chitectural arrangement and cellular heterogeneity, the hepatocytes are exposed to gradients of nutrients, hormones, and growth factors delivered via the combined blood supply of the portal vein and hepatic artery. In particular, a major challenge that has hindered the advance- ment of cell-based therapeutic strategies is the propensity of hepatocytes to lose liver-specific functions and the ability to replicate when iso- lated from the normal in vivo microenvironment. Microenvironmental signals including soluble factors, extracellular matrix components, 1 Institute for Medical Engineering & Science at MIT, Department of Electrical Engineering and Computer Science, David H. Koch Institute at MIT, and the Howard Hughes Medical Institute, Cambridge, MA 02139, USA. 2 Division of Medicine, Brigham and Womens Hospital, Boston, MA 02115, USA. 3 Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. 4 Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 5 Department of Surgery, Childrens Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, and McGowan Institute for Regenerative Medicine, Pittsburgh, PA 15224, USA. *Corresponding author. E-mail: [email protected] STATE OF THE ART REVIEW www.ScienceTranslationalMedicine.org 16 July 2014 Vol 6 Issue 245 245sr2 1 on July 17, 2014 stm.sciencemag.org Downloaded from

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  • S TAT E OF THE ART R EV I EW

    T I S SUE ENG INEER ING

    Cell and tissue engineering for liver diseaseSangeeta N. Bhatia,1,2* Gregory H. Underhill,3 Kenneth S. Zaret,4 Ira J. Fox5

    Despite the tremendous hurdles presented by the complexity of the liver’s structure and function, advances inliver physiology, stem cell biology and reprogramming, and the engineering of tissues and devices are accel-erating the development of cell-based therapies for treating liver disease and liver failure. This State of the ArtReview discusses both the near- and long-term prospects for such cell-based therapies and the unique chal-lenges for clinical translation.

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    INTRODUCTION

    Liver disease and the subsequent loss of liver function is an enormousclinical challenge, and is currently the 12th most frequent cause ofdeath in the United States and the 4th most frequent for middle-agedadults (1). The situation is progressively worsening, prompted by sev-eral factors including the emergence of new liver diseases such as non-alcoholic fatty liver disease and steatohepatitis, the lack of a hepatitis Cvaccine, and an aging population of hepatitis patients at risk for pro-gression to hepatocellular carcinoma (2, 3). Liver transplantation is theprimary treatment for liver failure and is the only therapy shown todirectly alter mortality. To expand the supply of available livers fortransplant, numerous surgical options have been pursued, includingsplit liver transplants and living-related partial donor procedures (4).In spite of these surgical advances and improvements in organ alloca-tion, organ shortages remain acute, suggesting that it is unlikely thatliver transplantation procedures alone will ever meet the increasing de-mand. Cell-based therapies have long-held promise as an alternativeto organ transplantation. In this State of the Art Review, we will de-scribe both near- and long-term prospects for cell-based treatments,including the use of stem cells and other nonhepatocyte sources andtissue engineering, within the context of clinical manifestations of liverdisease. We will discuss the unique potential and big challenges thatexist for cell-based approaches and will provide an overview of funda-mental biological questions, technological tools, and future directionsfor the field.

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    THE LIVER IN HEALTH AND DISEASE

    The liver is the largest internal organ in the body, accounting for 2 to 5%of body weight, and performs a complex array of more than 500 func-tions including metabolic, synthetic, immunologic, and detoxificationprocesses. The liver also exhibits a unique capacity for regeneration,with the potential for full restoration of liver mass and function evenafter massive damage in which less than one-third of the cells remainuninjured (5, 6). In fact, procedures such as partial liver transplants

    1Institute for Medical Engineering & Science at MIT, Department of Electrical Engineeringand Computer Science, David H. Koch Institute at MIT, and the Howard Hughes MedicalInstitute, Cambridge, MA 02139, USA. 2Division of Medicine, Brigham and Women’sHospital, Boston, MA 02115, USA. 3Department of Bioengineering, University of Illinoisat Urbana-Champaign, Urbana, IL 61801, USA. 4Institute for Regenerative Medicine,Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.5Department of Surgery, Children’s Hospital of Pittsburgh of UPMC, University ofPittsburgh School of Medicine, and McGowan Institute for Regenerative Medicine,Pittsburgh, PA 15224, USA.*Corresponding author. E-mail: [email protected]

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    take advantage of this significant regenerative potential combinedwith the body’s finely tuned homeostatic regulation of liver mass.However, the potential for liver regeneration is often difficult to predictclinically, and criteria for identifying patients that may resolve liverfailure complications due to regenerative responses remain poorly de-fined. As a result, efforts have been made toward the development ofliver support technologies that could provide temporary function forpatients with liver failure, thereby enabling sufficient time for regen-eration of the native liver tissue or serving as a bridge to transplantation.These measures include extracorporeal support devices that act in amanner analogous to kidney dialysis systems, processing the blood orplasma of liver failure patients (7, 8). Initial designs based on nonbio-logical exchange/filtering systems have shown limited clinical success,likely due to the insufficient level of hepatocellular functions exhibitedby these devices. To provide a larger complement of important liverfunctions, including synthetic and regulatory processes, support devicesincorporating living hepatic cells have been developed, although thesesystems remain primarily experimental to date (9). In addition to tem-porary extracorporeal platforms, the development of cell-based thera-pies aimed at the replacement of damaged or diseased liver tissue isan active area of research. For instance, the transplantation of isolatedliver cell types, such as mature hepatocytes, has been extensively ex-plored (10) and has potential as an attractive therapeutic option partic-ularly for inherited single gene metabolic deficiencies. Moreover, livertissue engineering approaches, wherein preformed cellular constructsare implanted as therapeutics, are under development. Finally, these en-gineered tissues are also being explored as in vitro model systems forfundamental and applied studies of liver function in healthy anddiseased states.

    The development of liver cell–based therapies poses unique chal-lenges, largely stemming from the scale and complexity of liver structureand function. The organ displays a repeated, multicellular architecture,in which hepatocytes, the main parenchymal cell of the liver, are ar-ranged in cords that are sandwiched by extracellular matrix in thespace of Disse (Fig. 1). The space between cords is also home to a mul-titude of supporting cell types such as sinusoidal endothelial cells,Kupffer cells, biliary ductal cells, and stellate cells. Because of this ar-chitectural arrangement and cellular heterogeneity, the hepatocytesare exposed to gradients of nutrients, hormones, and growth factorsdelivered via the combined blood supply of the portal vein and hepaticartery. In particular, a major challenge that has hindered the advance-ment of cell-based therapeutic strategies is the propensity of hepatocytesto lose liver-specific functions and the ability to replicate when iso-lated from the normal in vivo microenvironment. Microenvironmentalsignals including soluble factors, extracellular matrix components,

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    and heterotypic cell-cell interactions have all been implicated in theregulation of hepatocyte survival and phenotypic stability. The successof cell-based therapies hinges on the capacity to replace or overcomesuch necessary signals to promote the survival and function of hepa-tocytes transferred to patients in a clinical setting. Research efforts toachieve this goal have included studies at the interface of microtech-nology and cell biology, and have led to the ability to manipulate thehepatocyte phenotype by the controlled presentation of environmentalcues. Such systems exhibit long-term stabilized hepatocyte functionand also enable fundamental studies investigating drug-induced liverinjury and hepatotropic infections, in addition to providing insight

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    into how clinically transplanted hepatocytesmight be supported in an in vivo setting. Thecontinued evolution of in vitro liver platformsthrough the incorporation of additional liver celltypes and capabilities, such as perfusion and con-trol over tissue architecture in three dimensions,will be critical for the improved understand-ing of mechanisms underlying hepatocyteprocesses and the enhanced functionality ofcell-based therapeutics.

    Another obstacle in the progress of cell-based approaches is the limited availability ofhuman hepatocytes. Only a small supply of hu-man hepatocytes is currently available fromorgans determined to be inappropriate for trans-plantation. Despite the significant proliferativecapacity exhibited during regeneration in vivo,as noted above, mature human hepatocyte pro-liferation in culture is limited. Hence, the eluci-dation of molecular mediators that regulatehepatocyte proliferation and that could poten-tially promote expansion in vitro is an activearea of investigation. In addition, significantresearch efforts are focused on the potential ofalternative cell sources, most notably leveragingstem cell differentiation and reprogramming.On the basis of knowledge of developmentalmechanisms, recent advances in pluripotent stemcell differentiation protocols have illustratedthat highly proliferative pluripotent stem cellscan give rise to hepatocyte-like cells derived froma single donor (that is, normal/disease geno-type) (11–14). Yet, it remains to be seen whetherit will be feasible to produce these cells on aclinically relevant scale and whether lingeringsafety concerns will be overcome for transplan-tation purposes.

    In the case of hepatic failure, in particularhepatic encephalopathy, which is classified bygraded alterations in mental status, the livercommunity faces a greater challenge—at leastin some ways—than in other cases of organfailure, in that the clinical priorities for achiev-ing functional improvement (that is, cardiacoutput for heart and filtration rate for kidney)are not available because of our lack of funda-mental understanding of the preexisting dis-

    ease state. That is, we have limited “biomarkers” that are predictive ofclinical response in a liver failure patient. For example, despite the use ofmetabolic readouts and parameters, such as timing of jaundice, bilirubinlevels, prothrombin time, and age, the causes of hepatic encephalopathyare not completely understood, with distinct clinical indicators in acuteversus chronic liver disease and marked patient-to-patient variability.Thus, any animal models used to assess candidate therapeutics mustbe very carefully chosen given that etiologies of liver failure fromtrauma, metabolic liver disease, and cirrhotic disease are each verydistinct, and any clinical trial design must be tailored on the basis ofthe particular clinical setting in question. Despite the many challenges

    Hepatic arterybranchPortal vein

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    Fig. 1. Structure of the liver. The liver is the largest internal organ in the body and performsmore than 500 functions, including numerous metabolic, synthetic, immunologic, and detox-

    ification processes. (Top) The liver exhibits a hierarchical structure consisting of repeated func-tional tissue units (liver lobules). Within a lobule, oxygenated blood enters through branches ofthe hepatic artery and portal vein and flows in specialized sinusoidal vessels toward the centralvein. Bile, which is produced and excreted by hepatocytes, flows in the counter direction towardthe intrahepatic bile duct. (Bottom) Hepatocytes are polarized epithelial cells that interact closelywith a number of nonparenchymal cell types along the sinusoidal tracts of the liver lobule. Col-lectively, these cellular components and multiscale tissue structures contribute to the diversefunctional roles of the liver.

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    that remain, substantial progress has been made in the understanding ofliver development, regeneration, and the development of instructiveanimal models. These insights, together with recent innovations in en-gineered in vitro culture platforms and in vivo transplantation approaches,form a strong foundation for future advancements and the ultimateimplementation of cell-based therapeutics for liver disease.

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    CLINICAL MANIFESTATIONS OF LIVER DISEASE

    The vast majority of liver functions are mediated by the hepatocyte,the functional metabolic unit of the liver that constitutes about two-thirds of its cell mass (15). Although liver disease does not routinelyresult in abdominal pain, it does lead to a variety of life-threateningmetabolic and physiologic abnormalities. For example, the absence ofthese functions leads to bleeding abnormalities, accumulation of neu-rotoxins causing altered consciousness, low blood sugar and accumula-tion of serum ammonia, and jaundice from elevation of serum bilirubin.Unfortunately, although patients with liver disease can be medicallysupported through therapies targeted at features such as portal hyper-tension and coagulopathy, there are no therapeutic strategies that col-lectively augment the range of affected functions, and thus, an organtransplant has been the only permanently successful therapy to date.This approach contrasts with other organ systems, such as the heartand the kidney, in which patients with failing tissues can be given iono-tropes to improve contractility or diuretics to improve fluid balance,without the need for immediate transplantation.

    The most appropriate approaches to future treatment design for pa-tients with liver damage depend largely on the particular etiology of theorgan damage in an individual case. Types of liver disease can be broad-ly grouped into three categories: chronic liver disease due to metabolicdysfunction, that is, in the absence of trauma or tissue scarring; acuteliver failure that does not damage normal tissue architecture but is as-sociated with direct injury and loss of hepatocytes; and chronic liverfailure that is accompanied by widespread tissue damage and scar-basedremodeling, or cirrhosis (Fig. 2).

    Metabolic-based chronic liver diseasePatients with life-threatening, inborn liver-based metabolic disorders,often caused by defects in single enzymes or transport proteins, are nottypically accompanied by changes in liver architecture. However, evenin the absence of structural change to the liver, these inborn deficits oftencause injury to other organ systems, such as the brain. This is the casewith urea cycle disorders, Crigler-Najjar syndrome, and phenylketonuria.In oxalosis, the liver-based genetic abnormality leads to accumulation ofoxalate crystals in the kidney and renal failure. To date, although patientssuffering from this category of liver diseases are treated by organ trans-plantation, their clinical manifestations offer an ideal scenario for celltransplantation therapy. Given that the tissue architecture is unscarredand patients can be identified early and treated over time—before anacute episode—transferred hepatocytes may have both the time andthe opportunity to engraft and replace endogenous, functionally in-adequate cells.

    Acute liver injuryAcute liver failure is defined as a rapid deterioration of liver functionover a period of less than 26 weeks in patients who have no preexist-ing liver disease (16). Acute injury initially leads to necrosis of hepa-

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    tocytes, which causes release of liver-specific enzymes, such as alaninetransaminase (ALT) and aspartate transaminase (AST) into the blood.Increases in ALT and AST indicate hepatocyte injury but are not in-dicators of hepatic failure in and of themselves. Acute liver failurecarries a very high mortality in many, but not all, cases; thus, it is im-portant to identify its cause because some forms can be treated or willresolve spontaneously. Clinically, the dominant findings are onset offatigue and jaundice, a transient increase in serum concentrations of

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    Fig. 2. The liver in health and disease. Mechanisms that lead to he-patocyte damage and reduce liver function include drug-mediated tox-

    icity, alcohol-induced and nonalcoholic fatty liver disease, hepatotropicinfections, and hereditary disorders. Fatty liver disease, resulting fromboth chronic alcohol exposure and nonalcoholic mechanisms, is in-creasingly common and leads to the chronic accumulation of fat drop-lets within the liver. Liver cirrhosis can be caused by hepatitis virusinfection, autoimmune processes, chronic alcohol abuse, chronic in-flammation, and fat accumulation. Cirrhosis is characterized by altera-tions in the sinusoidal structure and function of the liver and theaccumulation of extracellular matrix, which is commonly referred toas scarring. These alterations lead to a reduction in hepatic functionthat can progress to hepatic failure and increased risk of hepatocel-lular carcinoma.

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    ALT and AST, followed by progressive increases in serum bilirubin,worsening coagulation, and development of altered consciousness (he-patic encephalopathy), leading to coma and brain swelling. Histolog-ically, the liver architecture is intact except for loss or necrosis of thehepatocytes. Because it is often difficult to know which patients willrecover spontaneously, liver transplantation is currently the only avail-able treatment option for severe, even if transient, acute hepatic failure.It remains unclear if hepatocyte transplantation procedures could besufficiently optimized such that cell engraftment would occur on a clini-cally relevant time scale because of the urgent, acute status. The adventof extracorporeal artificial liver devices may eventually address this need(see next section) in that they could offer a clinical “bridge” to addressacute symptoms during a period in which the endogenous liver couldbe given an opportunity to recover spontaneously.

    Chronic liver failure with accompanying cirrhosisLiver failure more commonly occurs in the setting of cirrhosis, as anacute decompensation or abrupt loss of liver function, in patients withchronic liver disease. Cirrhosis of the liver caused about 1 million deathsworldwide in 2010 (17). Histologically, cirrhosis is characterized by ex-pansion of the extracellular matrix with capillarization of the sinusoidalendothelium and loss of fenestrae with production of regenerative he-patic nodules (18) (Fig. 2). In addition to producing symptoms of he-patic failure, the scarring from cirrhosis results in resistance to flow inthe portal circulation. The increased pressure in what is normally a low-pressure conduit leads to gastrointestinal bleeding and severe accumu-lation of abdominal ascites, and can lead to secondary dysfunction ofthe kidneys (hepatorenal syndrome) and lungs (hepatopulmonary syn-drome). Some patients with cirrhosis are completely asymptomatic,whereas others with a similar histological picture exhibit severe symp-toms of hepatic failure. Cirrhosis may be caused by hepatitis B or C in-fection, autoimmune processes, chronic alcohol abuse, or inflammationand fat accumulation from chronic metabolic syndromes. Additionally,in contrast to examples discussed above, some inborn errors of metab-olism can lead to structural liver damage with cirrhosis and liver failure.Examples include a1-antitrypsin deficiency, hemochromatosis, Wilson’sdisease, hereditary tyrosinemia, and cystic fibrosis. As is currently thecase for acute liver failure, the only definitive therapy for end-stage cir-rhosis and liver failure is orthotopic (that is, in the natural position) livertransplantation. While it may one day be possible for these nonacutepatients to benefit from cell transplantation or implantable tissue-engineered grafts, given that their symptoms should provide sufficienttime for both engraftment and vascularization, there are significantbarriers to overcome that may preclude these therapeutic advancesfrom being applied. Specifically, neither site of implantation—in this case,the high-pressure portal system—or the scarred microenvironment maybe amenable to accepting infiltrating cells or engineered grafts. Thus,ectopic sites of transplantation are being considered for these sorts ofinterventions.

    DESIGN AND DEVELOPMENT OF EXISTING CELL-BASEDTHERAPEUTIC INTERVENTIONS

    Clinical effectiveness of hepatocyte transplantationIn the clinical scenarios of liver damage presented above, there is avariety of cases in which hepatocyte transplantation—either in the con-text of an extracorporeal device, a tissue-engineered graft, or as individ-

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    ually engrafted cells—may offer a clinical alternative to orthotopic organtransplantation (Fig. 3). Since the development of techniques for iso-lation of individual hepatocytes by collagenase digestion (19), investi-gators have studied whether hepatocyte transplantation could be usedto treat liver diseases, first in the laboratory and then in patients. Ashepatocyte transplant would classically involve simple infusion ofisolated cells into the liver through the portal vein, this form of ther-apy is far less invasive than orthotopic liver transplantation and couldbe performed safely in severely ill patients. In the presence of normalhost liver architecture, some fraction of the transplanted cells shouldcross the endothelium to integrate into the host liver (20–22). Becausethe native liver is not removed, the transplanted hepatocytes only needto provide replacement of defective enzyme activity or enough hepaticfunction to overcome the liver dysfunction. Other sites of transplan-tation have been explored in animal models including the mesentery,spleen, and the renal capsule (23–25). In these settings, the survivaland persistence of cells at ectopic sites seems to depend minimallyon nutrient availability through local vascular beds; however, cellsmay not require perfusion specifically with portal blood containing“hepatotropic” factors, as was once believed. Unexpectedly, the lymphnode, a highly vascularized site, has been a useful location for promotingremarkable growth and function of transplanted hepatocytes in animalmodels (26), adding further support for the potential clinical utility ofadult hepatocyte transplantation. Regardless, in acute liver failure, about40% of patients with advanced symptoms recover spontaneously with

    Liver transplantation

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    Fig. 3. Organ transplantation and cell-based therapies. Currently,liver transplantation is the primary treatment for patients with liver fail-

    ure and is the only therapy shown to improve survival. Because of thelimited number of livers suitable for transplantation, advanced surgicalprocedures including split liver and partial donor transplants have beenpursued clinically. Additionally, a diverse range of cell-based therapiesare currently being explored to treat liver disease and liver failure. Theseinclude the transplantation of various adult and stem cell–derived cellpopulations, the development of extracorporeal BAL devices, and theimplantation of engineered tissues.

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    only medical management (27). Because there is no effective means todistinguish patients who will survive without transplantation from thosewho will not (16), treatment with transplanted hepatocytes could poten-tially obviate the need to perform an irreversible organ transplant on apatient who could recover spontaneously. However, in an acute setting, itremains to be established whether functional engraftment could beaccomplished on a clinically relevant time scale.

    Numerous studies in rodents done over the last 30 years indicatethat adult hepatocyte transplantation can reverse hepatic failure andcan correct various metabolic deficiencies of the liver (28). Althoughclinical trials of hepatocyte transplantation have demonstrated thelong-term safety of the procedure, only partial correction of metabolicdisorders has been achieved, and the degree to which donor hepato-cytes have restored failing livers has not yet been adequate to circum-vent the need for organ replacement (10, 29, 30). Because of thelimited availability of fresh donor hepatocytes, or effective alternatives,such as cryopreserved hepatocytes or in vitro expanded cells, trialshave been limited to case reports or anecdotes involving few patientsand no untreated control patients (23, 24, 29, 31–35). However, thislimited clinical experience with hepatocyte transplantation has helpedto identify barriers to its successful application and potential pathwaysfor overcoming these challenges. One explanation for the failure totranslate laboratory studies to the clinic is the limited number of ani-mal models that fully recapitulate human disease and the challenges ofmapping animal model data to clinical outcomes and action plans. Areevaluation of the results of cell transplantation in animal studies hasshown that attaining adequate levels of engraftment and technical is-sues such as cell availability will need further attention. In addition, im-munological graft rejection is more difficult to manage in patients thanexpected because it is not possible to diagnose rejection of donor hepa-tocytes by conventional biopsy techniques. This gap most likely explainswhy successful correction of metabolic diseases by transplantation hasbeen transient, rarely lasting more than a year or two. Through the de-velopment of improved animal models, pretreatment strategies, and en-gineered delivery platforms, ongoing efforts in the field aim to build onthese preliminary findings in patients toward an improvement in trans-plantation efficiency and clinical efficacy.

    Mature hepatocytes as sources for cellular therapiesDespite the lack of correlation between some animal model experimentsand the minimal success of hepatocyte transfer to patients, there is suf-ficient support for their clinical potential to prompt the field to discoverstrategies to obtain, maintain, and expand human hepatocytes. Accessto sufficient functional cell numbers is a major hurdle despite evidencefrom serial transplantation using genetically marked, mature donor he-patocytes showing that such cells have a remarkable ability to replicatein vivo, many times more than that seen after partial hepatectomy (36).Thus, unlike many other fully differentiated cell types, hepatocytes havethe intrinsic capacity for robust replication, at least under a set of poorlydefined circumstances. Still, despite decades of research and partial suc-cesses with culture conditions, hepatocytes in vitro rapidly lose their dif-ferentiated characteristics and proliferate poorly (37, 38). It is currentlynot feasible to routinely obtain hepatocytes from healthy human donorlivers, amplify the cell populations in vitro while maintaining their func-tional capacities, and obtain sufficient cells for transplantation or use inextracorporeal devices. This situation will be helped by further researchon the hepatocyte microenvironment including the roles of extracellularsignaling, matrix, cell-cell interactions, physical forces, and soluble

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    factors (39, 40), to explore diverse and potentially unexpected ways togenerate a replicative environment for the hepatocyte.

    A different approach to controlling hepatocyte replication is to ma-nipulate the intrinsic cellular mechanisms that either keep hepatocytesquiescent in the liver or allow the cells to enter the cell cycle after lossof tissue mass (41). In the past decade, substantial inroads have beenmade in identifying how different cellular networks converge on theproteins and complexes that govern the hepatocyte cell cycle duringhepatic regeneration (42). The FoxM1b transcription factor (43) acti-vates Cdc25B, which in turn is needed for Cdk1 activation and entryinto mitosis after partial hepatectomy. The anaphase-promoting com-plex restrains hepatocytes in vivo from entering the cell cycle (44), andthe Hippo/YAP signaling pathway promotes cell cycle reentry (45). Re-cently, the kinase mitogen-activated protein kinase kinase 4 (MKK4)was found to restrain hepatocyte proliferation, such that experimentalinactivation of MKK4 stimulated hepatocyte regeneration in mousemodels of acute and chronic liver failure (46). These disparate findingsand others could be brought to bear in a united fashion to better un-derstand how the hepatocyte cell cycle can be controlled.

    Extracorporeal bioartificial liver devicesWith improved understanding of the signals needed to expand and/ormaintain long-term function of isolated hepatocytes, it may be possi-ble to provide appropriate architecture- and soluble-based signals topopulate extracorporeal liver devices. Extracorporeal liver devices areprincipally aimed at providing temporary support to patients with liverfailure. Early attempts at developing extracorporeal support technologieswere based on nonbiological mechanisms such as hemodialysis, hemo-perfusion, hemodiabsorption, plasmapheresis, and plasma exchange(47), in part due to the lack of available hepatocytes needed to populatesuch devices. More recent configurations of artificial, nonbiological sup-port systems have focused on the elimination of albumin-bound toxins,using a method termed albumin dialysis. These devices, such as theMolecular Adsorbent Recirculating System (MARS; Gambro) andthe Prometheus (Fresenius Medical Care) platform, have been shownto be effective for the reduction of plasma bilirubin, bile acids, and otheralbumin-bound molecules (48, 49). Although some reports point tothe potential utility of these approaches as bridge treatments beforeorgan transplantation (50–52), improvements in patient outcome havenot been fully demonstrated (53, 54), suggesting that additional trialsare required to fully evaluate their effectiveness. In particular, initialclinical studies have illustrated beneficial effects on organ function with-out an associated improvement in transplant-free survival (54, 55), whichfurther underscores the complexity of liver failure events and the need forcontrolled clinical trials. In light of the broad range of vital synthetic func-tions carried out by the liver and the lack of a liver “biomarker” aroundwhich to design these devices, much of the field has gravitated towardcellular rather than purely device-based solutions.

    Accordingly, to provide a more complete array of synthetic and bio-chemical functions, which is lacking in strictly artificial support systems,considerable efforts have been placed in the development of bioartificialliver (BAL) devices containing hepatic cells. Following early work in-cluding the study by Matsumura et al. in 1987 (56), a broad range ofBAL device designs have been reported, and have highlighted the impor-tance of several criteria in the development of an effective device (Fig. 4).These include issues of cell sourcing, maintenance of cell viability andhepatic functions, sufficient bidirectional mass transport, and scalabilityto therapeutic levels. As discussed earlier, the sourcing of hepatic cells

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    is a fundamental challenge for all liver cell–based therapies. As a result,xenogeneic sources (primarily porcine) or transformed/immortalizedhuman hepatocyte cell lines have formed the basis for the majority ofBAL devices (which typically include about 1 × 1010 cells) tested in theclinic to date (57–60). However, lack of hepatocyte function in trans-formed cell lines (61) and cryopreserved cells and the potential risk forporcine endogenous retroviruses have hampered attempts to demon-strate efficacy. More recently, studies have begun to incorporate pri-mary liver cells by exploiting advances in cryopreservation, fetal cellprocurement, and stem cell differentiation (59, 62–65). Magnifying thecell sourcing challenge are the scalability requirements for translation.

    Furthermore, the design of an effective BAL device is dependent onthe incorporation of the appropriate environmental and organization-al cues that enable maximal survival and function of the hepatocellularcomponent. Hollow fiber devices are the most common BAL designand contain hepatic cells within cartridge units (66), with the hollowfiber membranes serving as a scaffold for cell attachment and com-partmentalization (Fig. 4). A range of modifications aimed at optimiz-ing cellular performance have been explored. In particular, due to theenhanced function of hepatocyte aggregates relative to single-cell sus-pensions, many device configurations contain either attached orencapsulated hepatocellular spheroids (66–70). In the modular extra-corporeal liver support (MELS) system (Charite), hepatocytes areaggregated in coculture with liver nonparenchymal cells, resulting inthe formation of tissue-like organoid structures (71). Furthermore, ex-posure of hepatocytes to plasma of a sick patient may necessitatespecific alterations in hepatocyte culture conditions. For example, thesupplementation of plasma with amino acids has been shown to in-crease albumin and urea synthesis (72), and preconditioning withphysiological levels of insulin (lower than standard culture medium)has been demonstrated to prevent abnormal lipid accumulation inhepatocytes (73). Overall, environmental conditions within a BAL de-vice, such as oxygen tension and fluid shear forces, can significantlyaffect hepatocyte functions (74). In addition, both the convective anddiffusive properties of the systems must be optimized to provide vitalnutrients to the cells while simultaneously allowing export of thera-peutic cellular products. Currently, although clinical efficacy of BALdevices remains limited, improvements in device and trial design con-tinue to be implemented. It is anticipated that parallel progress towardthe development of highly functional in vitro platforms will provide areciprocal benefit for the advancement of BAL approaches. For ex-ample, small-scale in vitro bioreactor systems have been used to sys-tematically examine the effects of shear stress and oxygen tension onhepatocyte function (75, 76). BAL device development is in desperateneed of predictive biomarkers that point to the degree of hepaticfunction that is represented by a device. For example, the ability tomonitor a single protein or metabolite in the perfusate as a broaderindicator of reduced accumulation of systemic toxic metabolites, andhence the neuroprotective effects of a device, would be invaluable. Ap-plying “omics” methods in an effort to evaluate the state of these de-vices might provide clues toward the development of appropriatebiomarker readouts.

    In vitro strategies for improving hepatocyte viabilityand functionA major research area is the development of improved in vitro culturemodels, which would improve BAL design as well as provide platformsfor the study of human hepatic function. Although systems using single

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    Fig. 4. Extracorporeal BAL devices. Extracorporeal BAL devices incorpo-rate liver cells and aim to provide an array of important liver functions

    (detoxification, metabolic, and synthetic) for a patient by processing thepatient’s blood/plasma outside of the body. This approach could serve asa temporary bridge until a liver becomes available for transplantation. Cur-rently, such devices are either in clinical trials or in the exploratory researchstage. Liver cell–based bioreactor designs primarily fall into four generalcategories based on device configurations. These include hollow fiber de-vices, packed beds, flat plate systems, and encapsulation-based reactors.The majority of current clinical trials use a hollow fiber design in whichcells are positioned outside the fibers and the patient’s blood/plasma isperfused through the fiber lumen. Cell sources include three categories:tumor cell lines, porcine and human primary hepatocytes, or hepatic cellsderived from embryonic stem cells (ESCs) or induced pluripotent stem cells(iPSCs) or reprogrammed from other cell types. Because of their increasedavailability compared to primary human hepatocytes, primary porcinehepatocytes are the most common cellular component of current BAL de-vices. Device design characteristics have been shown to affect the func-tional stability of the cellular components. Many design challenges existincluding the balanced delivery of oxygen and nutrients to the cells,preventing mechanical shear forces from damaging the cells, and clini-cally relevant scale-up.

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    enzymes, liver slices, or liver cell lines have found utility for addressingfocused questions, each has limitations. For example, liver slices havelimited viability and are not amenable to high-throughput screening;cell-free microsomes lack the dynamic gene expression and intactcellular machinery required for cellular responses (that is, cytotoxicity);and carcinoma-derived cell lines and immortalized hepatocytes displayan abnormal repertoire of liver proteins and limited liver-specificfunctions (5). For these reasons, isolated primary hepatocytes areconsidered to be the most suitable platform for a range of in vitro ap-plications (3, 6). As a result, extensive research over several decades hasbeen focused on identifying specific culture configurations and molec-ular stimuli that can maintain the phenotypic functions of hepatocytes(Fig. 5). In general, the phenotype of an isolated hepatocyte is quiteplastic and is exquisitely sensitive to its microenvironment. Many dif-ferent manipulations regulate aspects of the differentiation program, al-though not necessarily in equivalent ways (the plethora of hepatocytefunctions and the absence of a clinical biomarker of rescue means thatmany parameters must be measured). Such manipulations include cul-ture medium, extracellular matrix, and interactions with nonparenchy-mal cells. Engineers and biologists have also tried manipulations that arenot explicitly “biomimetic” or obviously physiological but still manageto influence hepatocyte fate and function, presumably through some ofthe existing adult or developmental signaling pathways. For example,additives such as hormones, amino acids, corticosteroids, and growthfactors, as well as nonphysiological small molecules, have been demon-strated to affect hepatocyte functions (77–80). Additionally, both extra-cellular matrix composition and topology can modulate hepatocytemorphology and phenotype. In the classic “double gel” culture format,hepatocytes are sandwiched between two layers of collagen gel (81). Inthis configuration, hepatocytes exhibit improved morphologicalfeatures, such as polarized bile canaliculi, and stabilized functions, in-

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    cluding albumin and transferrin secretion, for weeks. However, key de-toxification pathways such as oxidation and conjugation reactions havebeen shown to become imbalanced over time in this system (82, 83).Studies investigating the potential added benefits of complex mixturesof extracellular matrix components on hepatocyte function have usedstrategies including extracellular matrix preparations from native livertissue (84, 85) or, conversely, screening methods to systematically inves-tigate defined extracellular matrix combinations (39, 86). Synthetic sur-face modifications, such as polyelectrolyte chemistries, also influencehepatocyte function in vitro (87, 88), and a polyurethane matrix identi-fied by polymer library screening was shown to support hepatocellulardifferentiation and function (89). In addition to strictly two-dimensional(2D) systems, hepatocyte culture under conditions that promote aggre-gation into 3D spheroids can affect functionality, with spheroid config-urations displaying superior hepatocyte function compared to standardcollagen monolayer cultures (90, 91). Potential mechanisms underlyingthese effects include the increased number of homotypic cell-cellcontacts between hepatocytes, the retention of a 3D cytoarchitecture,and the asymmetric presentation of extracellular matrix and othersignals surrounding the spheroids (92). Numerous technologies includ-ing arrays, rotational cultures, and encapsulation methods have beendeveloped for the optimization and scale-up of hepatocyte spheroidculture (93–95).

    In both 2D and 3D formats, the addition of secondary supportivecells for heterotypic interactions is one of the most robust approachesfor preserving the viability, morphology, and function of cultured he-patocytes. In addition to modulating cell fate through cell-cell sensingpathways such as cadherins, stromal cells additionally modify the ex-tracellular matrix and the paracrine signals in the microenvironment.Specifically, beginning with the initial experiments by Guguen-Guillouzoand colleagues (96), substantial research efforts have demonstrated

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    Fig. 5. In vitro culture systems for hepatocytes. Improved in vitro sys-tems have been developed for culturing primary human hepatocytes.

    technologies to in vitro hepatic tissue engineering has facilitated the de-velopment of microscale culture platforms (middle). Natural and synthetic

    Elucidating the roles of microenvironmental signals in governing hepato-cellular processes has enabled optimization of in vitro culture systems.These optimized systems include cocultures to provide specific cell-cell in-teractions as well as defined concentrations of soluble factors andextracellular matrix molecules (left). The application of microfabrication

    biomaterial systems have been applied to optimize 3D in vitro culture plat-forms (right). Collectively, these engineering approaches have further ad-vanced the understanding of how combinations of microenvironmentalcues influence cell processes, and have provided important insights intothe temporal and spatial dynamics of hepatic cell and tissue function.

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    that both liver-derived cell types, including liver biliary epithelial cellsand nonparenchymal cells (for example, stellate cells and sinusoidalendothelial cells), and numerous non–liver-derived cells (for example,embryonic fibroblasts) are capable of supporting hepatocyte functionin coculture contexts (97). Although varied culture conditions have beenexplored, typically, primary hepatocyte functions can be rescued if cocul-tures are initiated within 3 to 7 days after isolation. Transcriptional andposttranscriptional mechanisms have been implicated in the coculturestabilization of hepatocyte-specific genes including albumin, transferrin,pyruvate kinase, and glutathione S-transferase (98, 99). Furthermore,cytochrome P450 (CYP) detoxification enzymes are elevated in cocul-tures compared to hepatocytes cultured alone (100).

    Coculture systems have formed the basis for investigations into abroad range of hepatocellular processes such as the acute phase response,oxidative stress, mutagenesis, lipid and drug metabolism, and xenobiotictoxicity (97). For example, cocultures of hepatocytes and Kupffer cells(the liver’s resident macrophages) have been used to examine mechan-isms of hepatocellular damage (101, 102). Meanwhile cocultures with liversinusoidal endothelial cells have highlighted the importance of hepato-cyte–endothelial cell interactions in the bidirectional stabilization of thesecell types (103–108). Research efforts continue to use coculture platformsas in vitro models aimed at dissecting physiological cell-cell interactions inthe liver, and as a tool for optimizing engineered liver microenviron-ments. In particular, for certain tissue engineering applications, replacingsupportive cell types with acellular components that do not consume nu-trients and occupy limited space may be advantageous. Studies focusedon the underlying mechanisms of hepatocyte stabilization in coculturehave identified several cell surface and secreted factors that play a role,including T-cadherin, E-cadherin, decorin, transforming growth factor–b1,and liver-regulating protein (109–115). Although such factors are cur-

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    rently incapable of supporting hepatocytefunction at a level comparable to support-ive stromal cell types, they represent aproof of concept for highly functionalhepatocyte-only culture platforms.

    Collectively, a set of in vitro strategiesis emerging that may aid in BAL designas well as inform the development of invitro liver models for discovery. Becausethe hepatocyte phenotype is quite plasticonce the cell is isolated from its nativeenvironment, much of the focus has beenon rescuing the hepatocyte phenotypeex vivo. There seems to be a shift in thefield from strategies that are solely bio-mimetic (for example, culturing hepato-cytes in sandwich extracellular matrix,or under gradients of oxygen tension, orin coculture with nonparenchymal he-patic cells) to culture models that do notnecessarily mimic the native hepatic mi-croenvironment (for example, spheroidsand cocultures with embryonic fibroblasts).Strategies that optimize hepatic function,duration of rescue, accessibility to per-fusate, and amenability to scale-up mayor may not ultimately resemble nativeliver architecture. A systems-level picture

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    of molecular signals that influence phenotypic stability is emergingand will aid these efforts (116).

    Stem cells as sources of hepatocytes for cellular therapiesAn independent approach to generating hepatocytes for therapeuticsis to use stem and/or progenitor cells, which by definition have a highcapacity for expansion (Fig. 6), and may be sourced from a variety of tis-sues. Theoretically, such cells could be amplified, induced to differentiate,and used in diverse applications. Whereas progress has been made ingenetically or immunologically identifying tissue-resident stem cell–likepopulations that arise in chemically damaged livers (41, 117–121), fur-ther work is needed to understand the role of these cells in normal liverphysiology and repair (122) and to assess whether these populationsrepresent a clinically relevant source of hepatocytes. Other cell sources,including mesenchymal stem cells (MSCs), have been discussed re-cently in this context (123). Another current interest is on the potentialof pluripotent stem cells, including human ESC (hESC) and inducedpluripotent stem cell (iPSC) lines, which have a high proliferative capac-ity and can differentiate into diverse lineages in vitro and in vivo (124).Various directed differentiation strategies have been applied to hESCand iPSC cultures, and have yielded populations that exhibit manyphenotypic and some functional traits of mature hepatocytes, earningthe derived cells the label of “hepatocyte-like cells” (11, 125–135). Yet,hepatocyte-like cells exhibit many characteristics, such as distinctiveCYP activities as well as expression of a-fetoprotein, that more closelyresemble fetal-stage hepatocytes rather than mature adult cells (136). Tofurther characterize these cells, some studies have tested the maturationand repopulation potential of both mouse and hESC-derived hepaticcells in rodent transplantation models, such as immunodeficient re-cipient mice that harbor genetic defects in resident hepatocytes

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    Fig. 6. Sources of hepatocytes. Obtaining appropriate sources of hepatocytes is a major limitation fordeveloping cell-based therapies for treating liver disease. Many different approaches are under investiga-

    tion including methods for improving the expansion of primary human hepatocytes in vitro, the directeddifferentiation of pluripotent stem cells (both ESCs and iPSCs), and the differentiation of either intrahepaticor extrahepatic adult progenitor cells, as well as new methods for the direct reprogramming of adultsomatic cells to hepatic cells.

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    (13, 137, 138). This form of inquiry helps to support the conclusionthat hepatocyte-like cell populations can support at least some liverfunction in a replacement setting, but the best choice of animal modelsfor these studies is still under debate (139). Attempts to identify smallmolecules that can induce the maturation of hepatocyte-like cells havesuggested that ex vivo maturation may be possible (40).

    One study used partially differentiated human iPSCs and admixedsupportive stromal and endothelial cells to mimic aspects of early liverdevelopment. The resulting liver “buds” were able to vascularize uponectopic transplantation and provide functional rescue of mouse mod-els of liver injury (140). Furthermore, recent progress suggests thathepatocyte-like cells derived by directed differentiation of fibroblastsalso exhibit more adult hepatic traits and, like iPSCs, might providean autologous stem cell source that would bypass a need for clinical im-mune suppression (141–145). In light of this progress, the strategy of usingpluripotent cells as a source material for generating hepatocytes—eitherfor clinical transplantation, as a cellular component in BAL devices, orin various model platforms to study disease and drug development—holds promise. Considerable experimental energy continues to be ap-plied to find methods that improve the efficiency and extent of differ-entiation of these expansion-ready precursor cells (Fig. 6). More needsto be done to drive expansion of human stem cell–derived hepatocytesimplanted into mouse livers and to evaluate their performance and safe-ty. Regardless, the capacity to populate mouse model livers with humanhepatocyte-like cells both from normal individuals and patients offersan experimental system of “humanized” mouse livers for use in toxico-logical studies on human hepatocytes (140, 146–149).

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    TRANSLATING EXISTING TECHNOLOGY INTOPRECLINICAL AND THERAPEUTIC APPLICATIONS

    Implantable therapeutic constructs for preclinical testingDelivery of hepatocytes by cell transplantation requires cells to hometo the liver from the portal blood stream and extravasate across thespace of Disse into the hepatic lobular compartment. Early “tissue en-gineers” sought to support these adhesion-dependent cells with bioma-terials that could alleviate the need for homing and attachment. Theseseminal studies used available biomaterials [such as poly-lactic co-glycolicacid (PLGA)] and demonstrated an improvement in the survival of thetransplanted cells. Nonetheless, animal studies suggested that the per-sistence of hepatocyte phenotype and survival were dependent on thesite of implantation, at least in this delivery context. For example, somestudies demonstrated an improvement in the function of transplantedhepatocytes when bathed in the so-called hepatotropic factors drainingfrom the gut to the portal vein (150). Because the portal vein is an un-attractive site for transplantation in chronic liver failure patients withelevated portal pressures, investigators have begun to explore ectopicsites (spleen, subcutaneous, renal capsule, intraperitoneal) (63, 151, 152).In these ectopic sites, manipulating the cellular microenvironment usingbiomaterials, peptides, or even other cells has led to a diminished depen-dence on the portal circulation, setting the stage for ectopic transplan-tation of engineered hepatic tissues (as is currently the practice for renaltransplantation). Under strong regenerative stimulation such as thatfound in genetic mouse models of tyrosinemia, the transplanted cellscan even repopulate host lymph nodes ectopically, suggesting that eitherthe cells in these sites receive some hepatotropic stimuli or the relianceon portal hepatotropic factors may be mitigated in some regenerative

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    contexts (26, 153). These findings are consistent with the classic para-biosis experiments of Bucher and colleagues (154), suggesting that re-generating liver produces blood-borne factors that stimulate hepatocyteexpansion in a conjoined animal.

    Since the early experiments with PLGA, a synthetic degradablepolyester found in suture material, the community has explored manyporous scaffold materials ranging from both natural (for example, col-lagen and alginate) and synthetic [for example, PLGA and PLLA(poly-L-lactic acid)] sources. Scaffold traits including porosity, materialand chemical characteristics, and 3D architecture are among the pa-rameters that are customizable in 3D platforms, and these propertiesplay important roles in dictating cellular function and facilitating thetransport of nutrients and secreted therapeutic factors. In addition, syn-thetic hydrogel systems based on poly(ethylene glycol) (PEG), whichhave been extensively used for tissue engineering studies (155), havefound recent utility in 3D liver platforms (156–158). Hydrogel platformsoffer the advantage of polymerization in the presence of cells, therebyenabling the fabrication of 3D networks with uniform cellular dis-tribution without the need for cell migration or expansion in situ.Modifications in polymer chain length and the conjugation of bioactivefactors such as adhesive peptides improve the survival and function ofPEG hydrogel–encapsulated hepatic cells (156, 157, 159). Moreover, togenerate scaffolds with a highly defined architecture that provide bettercontrol over the 3D environment at the microscale, a range of rapidprototyping and patterning strategies have been developed and testedfor liver applications (160–163). For hydrogel systems, these includephotolithography-based techniques for dictating the size and shape ofconstructs and for building multilayer geometries (156, 158, 164–166),and dielectrophoresis methods for controlling cellular positioning (167).Notably, cell-cell interactions (both homotypic and heterotypic) havebeen suggested to substantially affect hepatocellular survival and functionin 3D biomaterial scaffolds (157, 166, 168, 169). As a result, similar to 2Dculture models, the controlled integration of additional cell types or acel-lular factors that mimic key cell-cell interactions will be critical for theadvancement of 3D liver platforms.

    Early studies also uncovered a scale-up issue that paralleled thatfound with BAL devices: Transplanting large numbers of cells in bio-materials without a sufficient nutrient supply rapidly leads to necrosisof the engineered tissue. Hepatocytes are highly metabolic and are nor-mally in close contact with an extensive sinusoidal vasculature. As a re-sult, significant efforts must be made in the design of implantable liversystems to avoid transport limitations that can greatly diminish tissuefunction. In particular, the site of implantation is a critical parameter inliver tissue engineering studies, with more highly vascularized sites suchas the peritoneum or renal capsule generally promoting improved en-graftment and function (157, 170, 171). Thus, another area of focus isthe establishment of a vascular network that supports a large number ofhepatocytes. Priming of the implantation site through prevasculariza-tion is a particularly effective strategy owing to the minimization of earlytransport restrictions that occur before the establishment of functionalvasculature. Microfabrication approaches, such as polymer moldingwith etched silicon, dissolvable sugar lattices, and microtissue moldingof aligned endothelial cords, have been used as strategies to preformcapillary-sized channel networks (162, 172, 173). Additionally, the in-tegration of angiogenic growth factors into the implantable scaffoldshas been shown to promote the recruitment of host vessels (174–177).Alternative strategies to achieve this goal include multilayer microfluidicnetworks, prevascularization through release of angiogenic growth

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    factors, and inclusion of nonparenchymal cells that promote angioge-nesis and vascular stabilization with pericytes. For instance, precedinghepatocyte delivery with the implantation of scaffolds that release angi-ogenic VEGF (vascular endothelial growth factor) enhanced capillarydensity and improved engraftment in rat liver lobules (178). Similarly,FGF2 (fibroblast growth factor 2)–coated scaffolds served as a sup-portive environment for mouse ESC–derived hepatocyte inoculationin an in vivo hepatic failure mouse model (63). Furthermore, severalrecent studies aimed at engineering skeletal or cardiac muscle tissuehave illustrated that significant improvements in survival and host vas-culature connections can be achieved after in vitro formation of vesselstructures within optimized tri-culture systems (muscle, endothelial, andmesenchymal cells) (172, 179–181).

    In addition to vascular integration, an improved understanding ofmulticellular organization and morphogenesis in the liver could alsoaid in the formation of functional biliary transport systems. Various invitro models have been developed that exhibit organized bile canaliculi(182–184) or artificial duct structures (185), but their incorporationinto implantable systems has yet to be fully explored. Although earlywork demonstrated engrafted bile ducts in ectopic sites (186), the de-gree to which the biliary tree must be reconstructed has not yet beenestablished; in ectopic cell transplantation experiments, the hepato-cytes do not appear cholestatic and biliary products do appear to findtheir way to the digestive tract. One hypothesis is that the biliaryproducts are redirected or “leak” into the bloodstream where they cir-culate and are processed by the remnant liver into bile. This scenariowould argue against removal of the diseased liver in the setting of trans-plantable tissue-engineered constructs, and is consistent with the function-al outcome achieved in peritoneal transplantation of mature hepatocytesand hepatocyte-like cells that lacked biliary networks (26, 140). Theneed for reconstruction of the nervous and lymphatic systems is likewiseeven less well understood, although, if clinical transplantation is a guide,these are likely to be less critical.

    Similar to whole organ transplantation, the host immune responsefollowing the introduction of tissue-engineered constructs is a criticaldeterminant of successful engraftment. Accordingly, the immune iso-lation capability of polymer scaffolds is a highly active research area(187) and represents the foundation of numerous tissue engineeringapproaches such as pancreatic islet transplantation (188–190). Recentliver tissue engineering studies demonstrate that hydrogel encapsula-tion enabled detectable human hepatic function in transplanted immu-nocompetent mouse strains for more than 1 week (157), suggesting thataspects of immune isolation may find utility in various liver applica-tions. In general, approaches to tune the degree of inflammatory cellrecruitment to engineered implants could substantially aid in preventingforeign body responses that severely impair engraftment while poten-tially maintaining the positive remodeling effects that inflammatory cellsprovide in numerous normal tissue regeneration contexts (191). For ex-ample, liver regeneration proceeds with a sequence of remodeling pro-cesses including protease expression and extracellular matrix deposition(192–194). The incorporation of protease-sensitive domains into hepatichydrogel systems could allow for the degradation of the constructs afterimplantation and subsequent changes in ligand presentation and gelmechanics (195). Tailoring the degradation properties of constructsbased on the kinetics of cell proliferation, inflammation, and angiogen-esis could provide a means for efficient integration. Overall, the devel-opment of new biomaterial platforms that can collectively modulatehost cell recruitment and material-resident cell function, as well as en-

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    graftment and release of implanted cell types (196), is a major focus andshould advance liver tissue engineering approaches.

    Animal models for preclinical testing of candidateinterventions and barriers to overcome inclinical translationTransplantation of liver cells has been shown to improve the survivalof animals with chemically and surgically induced acute liver failure(197–206) and end-stage liver failure secondary to cirrhosis (207), andto correct metabolic deficiencies and prolong survival in numerousmodels of liver-based metabolic diseases (198, 208). Whereas animalmodels have provided a foundation for clinical translation, the resultsof clinical trials using liver cell transplantation have been disappoint-ing. The most promising results have come from the treatment of chil-dren with liver-based metabolic diseases, where evidence of functionalreplacement of the deficient enzyme has been documented and post-transplant biopsies have shown engrafted cells expressing a correctedform of the deficient enzyme (10).

    Most disappointing has been the failure to successfully reverseacute hepatic failure in patients by hepatocyte transplantation. Be-cause the normal hepatic architecture remains intact in most cases ofacute liver failure, transplanted hepatocytes, which in animals are in-fused through the portal vein or directly into the spleen, would be ex-pected to translocate to the liver, engraft, and provide life-saving metabolicsupport while residual host hepatocytes regenerate. Yet, the field lacksan animal model that adequately recapitulates clinical hepatic failuresuch that the potential clinical effectiveness of cell transplantation or aliver assist device may be predicted. In the most used chemical andsurgically induced models of acute liver failure, animals develop severehistologic injury to the liver and elevated serum levels of ALT, AST,and bilirubin; however, the majority of animals do not die as a resultof intracranial hypertension, which is the most common cause of deathin acute liver failure. In addition, if the animals can be kept alive for aslittle as 72 hours after the injury, regeneration is rapid enough to com-pletely correct the liver injury and animal survival approaches 100%.In contrast, experience with auxiliary liver transplant in patients withacute liver failure indicates that native liver recovery could take weeksto months (209–211). Thus, the marked results obtained in animalstudies may result from the short-term metabolic support providedby the transplanted hepatocytes rather than from stable replace-ment of hepatic function. Finally, it has not been possible to determinehow many of each of the immediately available sources of function-ing hepatocytes, which include cryopreserved cells, xenografts, or stemcell–derived hepatocytes, need to be transplanted to reverse clinicalliver failure.

    In addition to acute hepatic failure, intrasplenic hepatocyte trans-plantation to treat mouse models of chronic hepatic failure secondaryto cirrhosis has been effective but transient, and the clinical experiencehas produced only anecdotal reports of improvement in some aspectsof hepatic function. This inability to successfully reverse human liverfailure associated with cirrhosis is not unexpected, as the cause of he-patic failure in cirrhosis is not completely understood (18, 194, 212).Furthermore, changes in liver architecture inhibit entry of transplantedhepatocytes into the abnormal cirrhotic environment, thus supportingthe exploration of ectopic transplantation sites (194, 213). Cirrhosisrepresents a final phase of liver disease characterized by advancedliver fibrosis and nodular architecture. The two most commonly usedanimal models of experimental fibrosis are toxic damage [using the

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    chemical carbon tetrachloride (CCl4)] and bile duct ligation (18). Othermouse models that mimic specific liver diseases, including nonalcoholicsteatohepatitis, may require special diets to induce injury (214), or theyare genetic knockout models, such as mdr2-null mice, that spontane-ously develop fibrosis (215). Notably, the degree of reversibility of liverfibrosis in rodents after the discontinuation of the toxic agent variesbetween experimental model systems (207, 216). In addition, changesin hepatic function may result from acute effects of the intoxicatingagent rather than from chronic injury to the liver. Reversal of humancirrhosis and secondary hepatic failure is not so easily accomplished(18, 217, 218). In rats, treatment with CCl4 for 28 to 32 weeks canreliably recapitulate many physiologic abnormalities found in patientswith advanced cirrhosis and liver failure (216). In this model, hepato-cytes transplanted into the spleen, to bypass vascular changes in the liverincluding portal hypertension, minimized hepatic encephalopathy andsupported survival for a period of months (207, 219, 220). Alternatively,rodents that receive portacaval shunts and ammonium chloride treatmentsexhibit neurobehavioral changes similar to those associated with cirrhosis,without other hallmarks of hepatic failure. In this setting, hepatocytetransplantation into the spleen markedly improves hyperammonemia-induced hepatic encephalopathy and amino acid imbalances and pre-vents the development of hepatic coma (221, 222).

    The failure of isolated hepatocyte transplantation to affect liverfunction and survival in patients with cirrhosis may be attributed totheir infusion through the splenic artery, rather than by direct injectionthrough the splenic capsule, which is the route of engraftment success-fully used in rodents (223). Given that it appears that cells need to engraftin an extrahepatic site, treatment of chronic liver failure might also ben-efit from work in tissue engineering and whole organ liver bioengineer-ing. Decellularized human or animal livers could serve as a biologicalscaffold for transplanted cells forming engineered internal auxiliary livergrafts (63, 224–227).

    Transplantation of MSCs derived from bone marrow, adipose tis-sue, amniotic fluid, and other tissues has been associated in clinicaltrials with correction of portal hypertension and decompensated he-patic function. Evidence that this effect is mediated by replacement ofdiseased hepatocytes by MSC-derived cells, however, is lacking. [Thecomplex role of MSCs in the treatment of liver disease is reviewed in(228, 229).] Of course, any therapy that allows the native cirrhotic liverto remain in place will leave unresolved the management of coexistingportal hypertension and the risk of developing hepatocellular carcino-ma unless the rescue is sufficient to ultimately allow explant of thecirrhotic liver.

    Finally, beneficial improvement in liver function has been reportedafter hepatocyte transplantation in several models of life-threateningliver-based metabolic disease. Multiple animal models faithfully repre-sent this class of diseases in man. These include the Gunn rat, a modelof Crigler-Najjar syndrome type 1 (208); the fumaryl acetoacetate hy-drolase (FAH)–deficient mouse, a model of tyrosinemia type I (230);the Long-Evans cinnamon rat, a model of Wilson’s disease (231); themdr2-null mouse, a model of progressive familial intrahepatic chole-stasis type 3 (215); an arginosuccinate synthetase–deficient mouse (232);the spf-ash mouse, a model of ornithine transcarbamylase deficiency(233, 234); a liver arginase null mouse (235); the Agxt−/− mouse, a mod-el for primary hyperoxaluria-1 (236); the Watanabe heritable hyperlipi-demic (WHHL) rabbit, a model of familial hypercholesterolemia (237);and the hyperuricemic Dalmatian dog (238) and the PiZ transgenicmouse, models of a1-antitrypsin deficiency (239). Unfortunately, trans-

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    plantation of liver cells has resulted in only partial correction of the ge-netic abnormality in most of these animal models, and the experiencein humans has mirrored these results. In most cases of liver-basedinherited diseases, the life span and regenerative capacity of the hosthepatocytes are normal, and transplanted hepatocytes do not competesuccessfully for survival in the host liver.

    Transplantation studies in animal models of liver-based metabolicdisease have also shown that it is not possible to engraft enough he-patocytes in a 24- to 48-hour period to completely correct an enzymedeficiency even with multiple cell infusions. The number of donor cellsthat can be safely transplanted into the liver at any one time via theportal vein is usually less than 5% of the liver mass, or about 2 × 108

    cells/kg, as transplantation of greater numbers leads to either portal hy-pertension or translocation of cells out of the liver into the systemiccirculation and embolization of cells in the lungs. Whereas replacementof 5% enzyme activity should be adequate to correct most metabolicliver diseases, it is thought that only 10 to 20% of transplanted cellsengraft. Thus, improving engraftment rates with biomaterials, use ofectopic sites that do not harbor ongoing injurious stimuli, and the needfor expansion of engrafted cells in response to regenerative cues are allstrategies worth exploring further.

    Experience in a selected group of animal models of human meta-bolic disease, however, has highlighted a strategy that could improve theoutcome of hepatocyte transplantation in patients. In the FAH-deficientmouse model of hereditary tyrosinemia (240), the albumin-uPA trans-genic mouse (241–243), and the transgenic mouse model of human a1-antitrypsin deficiency (239), host hepatocytes exhibit markedly reducedsurvival due to the inherited metabolic defect. In these cases, trans-planted wild-type hepatocytes spontaneously replace the host cells overtime, leading to near-complete replacement of host liver cells by donorhepatocytes. Recent advances in genome editing techniques suggestthat in situ gene correction may even be beneficial in this model. Inaddition, another chimeric mouse model has been recently developed,which is based on thymidine kinase transgene expression in the liverof an immunodeficient mouse strain (TK-NOG) (244). In this model,brief exposure to ganciclovir causes a time-limited toxicity to host livercells, which opens a window for chimerism in the absence of contin-uous injury during the repopulation phase. Partial repopulation by en-grafted hepatocytes also occurs in the LEC rat model of Wilson’sdisease and in the mdr2-deficient mouse, where the hepatocellular in-jury to native liver cells is more modest. Strategies have been developedto try to recapitulate this effect by exogenous means. For example, aselective growth advantage can be given to transplanted hepatocytesby preconditioning the liver with drugs that impair native liver regen-eration or that damage hepatocytes or sinusoidal endothelial cells, fol-lowed by a proliferation stimulus for the transplanted cells. Plant alkaloids,such as retrorsine, prevent hepatocellular proliferation (245), as doespreparative irradiation of the liver; partial hepatectomy can provide aproliferative stimulus to the engrafted cells (246). Irradiating as little as35% of the liver mass before allogeneic hepatocyte transplantation re-sults in complete correction of hyperbilirubinemia in the Gunn rat modelof Crigler-Najjar syndrome (247). Whereas this approach may resolvethe problem concerning the adequacy of engraftment to treat metabol-ic liver disease, an additional barrier must be resolved, and that is theinability to directly monitor the status of the graft. As a result, rejectionor other potential sources of graft loss cannot be determined, and inap-propriate immune suppression can lead to rejection of the graft. Newapproaches for monitoring disease progression in the liver may offer a

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    path to the identification of biomarkers that improve clinical decision-making (248).

    Because of the proliferative advantage of donor hepatocytes in FAH-deficient, albumin-uPA transgenic mice and in thymidine kinase transgenicmice during ganciclovir treatment, these animals, when crossed ontoan immune-deficient background, have become the most frequentlyused animal models to study engraftment and expansion of primaryhuman hepatocytes (249). Nearly complete replacement of the nativeliver can be achieved when primary human hepatocytes are transplantedinto these models. In addition, in immune deficient FAH-deficient mice,serial transplantation of human hepatocytes produces several genera-tions of animals with humanized livers (240). Serum concentrationsof human albumin or a1-antitrypsin correlate with the degree of en-graftment in these animals, as determined by immunohistochemistryof tissue sections. Use of these serummeasurements is necessary to con-firm the extent of human cell repopulation because autofluorescence inthe liver can lead to misinterpretation of immunofluorescence data.Thus far, stem cell–derived hepatocyte-like cells have been shown to en-graft and expand to a small degree in albumin-uPA severe combinedimmunodeficient (SCID) mice (13, 250) and in PiZ SCID mice (251).Transplanted human iPSC–derived hepatocytes have also been shownto engraft and expand in the livers of Gunn rats (252). Transplantationleads to partial correction of hyperbilirubinemia when part of the hostwas irradiated; hepatocyte growth factor was provided to stimulate ex-pansion of transplanted cells and tacrolimus was used for immune sup-pression. Similar results have been obtained in the mouse model ofoxalosis (253). As mentioned above, recent studies indicate growing suc-cess in not only differentiating hESCs and iPSCs into hepatocytes butalso the direct reprogramming of human fibroblasts into hepatocytes(143–145). Collectively, these results indicate that it may be possible toachieve an expandable, patient-specific source of transplantable humanhepatocytes and also bypass the need to generate iPSCs, which carry ameasure of clinical risk of teratoma. No matter which animal model isused, it is important to apply more than one assay to monitor engraft-ment because models of liver disease and techniques for cell delivery andtransplantation are not yet standardized across most laboratories andcan generate results that have been confusing to investigators.

    Clinical translation and scale-upClinical translation of candidate cell-based therapeutics that exhibitpromise in faithful, robust preclinical models will require solutionsto a range of practical challenges such as appropriate patient diagnosisand selection, detailed trial design, overcoming potential immune bar-riers, accounting for the impact of immune suppression regimens, aswell as establishing “Good Manufacturing Practices” or GMP condi-tions for the generation of any allogeneic cellular materials. The successof cell-based therapy will depend on the ability to scale the approach toa level that provides clinical effectiveness. BAL devices tested clinicallyhave used between 0.5 × 109 and 1 × 1011 hepatoblastoma cells or por-cine hepatocytes (68). The target capacity of most BAL designs is about1 × 1010 hepatocytes corresponding to about 10% of total liver weight,the minimal mass estimated to be required to support vital metabolicfunctions such as gluconeogenesis. Hepatocyte transplantation experi-ments in rodent models and human subjects have demonstrated im-provements in blood parameters after transplantation of cell numbersthat are 1 to 10% of total liver mass (23, 254, 255). Although distinctcategories of liver diseases (acute liver failure, end-stage cirrhosis, andinherited metabolic disorders) will have different scale requirements, it

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    is expected that scale-up will represent a significant translational chal-lenge for all cell-based liver therapies. To achieve clinical efficacy willrequire (i) efficient nutrient transport within the scaled-up systems and(ii) an expandable cell source. To address nutrient limitations in large-scale engineered tissues, efforts have focused on improving integrationwith the host vasculature as well as microfabrication approaches todevelop constructs with 3D architectures and improved diffusion char-acteristics (172, 173). For extracorporeal approaches, increasing the num-ber of cartridges (256) and increasing the fiber cartridge size (257) havebeen explored to enhance the capacity of hollow fiber–based devices.Expanded configurations of flat plate or perfusion BAL device designshave been suggested, but these modifications may introduce heteroge-neous flow distributions or large fluid volumes, respectively (258). Effortsexploring the utility of proliferative stem cell sources, as well as parallelmethods for inducing substantial primary hepatocyte proliferation invitro, are aimed at addressing the cell sourcing challenge (40). Notably,donor hepatocytes exhibit a proliferative advantage in the liver within var-ious liver injury models (discussed above), which can result in near-complete replacement of the native hepatocytes (243).

    Modeling the healthy human liverProgress has been made toward clinical application of cell-based ther-apeutic models, but it should not be overlooked that both animalmodels and in vitro liver platforms offer the potential to study normalliver function and biology (Fig. 7). Insights gleaned about normal liverbiology may be applied to tissue engineering and repair efforts, andmay assist in assessing the pharmacokinetics (for example, clearance),metabolism, and potential toxicity of new drugs. In vivo, the healthyliver is perfused and the hepatocyte phenotype is stabilized in its na-tive microenvironment, leading to two classes of models: (i) perfusedhepatic cultures in bioreactors and (ii) static monolayer cultures (2D)or aggregates such as spheroids (3D). Both types of model systems willbe useful for understanding normal liver physiology and susceptibilityto insult. Many of these studies were initially performed in rodents.Whereas establishing platforms using rodent hepatocytes can aid inthe interpretation of preclinical in vivo rodent studies and serve as atest bed for optimizing formats for human cells, to achieve meaningfulpredictions of clinical outcomes it will be essential to incorporate hu-man hepatocytes into these model systems (259, 260).

    With perfused systems, a wide range of in vitro hepatocyte-basedbioreactor platforms have been developed. These platforms offer the po-tential to examine flow-dependent phenomena such as the clearance ofxenobiotics from the circulation over time or the bioactivation of a drugto a toxic metabolite that can cause damage downstream (261–264).Perfusion systems may contain hepatocellular aggregates to stabilizeliver-specific functions (265–267) and recently have been used to exam-ine the hepatic differentiation of hESCs (268, 269). The incorporationof multiple compartments in parallel may be valuable for increasingperfusion and throughput (270, 271), and integration of multiple re-actors in series could be useful for investigating organ-organ interac-tions (272, 273). There has been much interest in building a “humanon a chip” to develop predictive models of human physiology and tox-icology (274), and the liver module will be a vital part of this effort. Topromote oxygen delivery while protecting hepatocytes from the delete-rious effects of shear stress, gas-permeable membranes have beenintegrated into several bioreactor configurations (275, 276). Perfusedsystems also allow the capture of some of the physiological heterogene-ity of hepatocyte gene expression along the liver sinusoids. This “zonal”

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    distribution is thought to arise from gradients in oxygen, hormones,and extracellular matrix molecules. A parallel-plate bioreactor revealedthat a steady-state oxygen gradient contributed to the heterogeneousexpression of drug-metabolizing enzymes, which mimicked the expres-sion gradients present in the liver as well as the regional susceptibility toacetaminophen in regions of low oxygen and high CYP activity (277).

    Static systems that stabilize the hepatocyte phenotype in the absenceof flow have provided insights into drug metabolism and drug-inducedliver injury. These model systems have manipulated the compositionand geometry of the extracellular matrix and perturbed the cytokineenvironment. They also include 3D aggregates formed on engineeredsurfaces or in droplets, or coculture. Overall, coculture of hepatocyteswith nonparenchymal cell types appears to be most effective in pro-viding maintenance of long-term function in vitro, likely due to theconcordant impact on the local extracellular matrix and soluble micro-environment as well as cell-cell interactions (97). The utility of theseplatforms for drug development depends critically on their reprodu-cibility and potential for miniaturization to minimize the amount ofcandidate drug needed. The vast number of species-specific metabolicproperties of hepatocytes (278) underscores the importance of incor-porating human hepatocytes in an effective and affordable manner.

    Microtechnology offers the potential to readily miniaturize some ofthese culture environments. For instance, micropatterned coculturesof hepatocytes and fibroblasts, fabricated with either photolithographyor soft lithography techniques, have been used to precisely regulatehomotypic and heterotypic interactions and have been optimized tosupport hepatocyte phenotypic functions for several weeks, including

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    the maintenance of gene expression pro-files, canalicular transport, phase 1/2 me-tabolism, and the secretion of liver-specificproducts (97, 279). Furthermore, drug-mediated modulation of CYP expressionand activity was observed, illustrating theutility of the platform for ADME/Tox (ab-sorption, distribution, metabolism, excre-tion, and toxicity) applications. Consistentwith clinical studies and our current mech-anistic understanding of drug-drug interac-tions, acetaminophen-induced hepatocytetoxicity was increased by a chemical in-ducer of CYP expression (phenobarbital)or an inhibitor of glucuronidation (proben-ecid), and species-specific differences inCYP1A induction (280) also were demon-strated. Subsequent studies have shown im-proved potential to predict generation ofhuman metabolites and human hepatotox-icity compared to unpatterned or mono-culture models (83, 281).

    Microcontact printing and robotic spot-ting techniques have been used to fabricatemicroarrays of hepatic cells and investi-gate functional stabilization, differentia-tion, and injury responses (39, 282–284).Finally, microtechnology tools have alsobeen applied to the analysis of dynamicprocesses. These tools include microfluidicdevices for the study of drug clearance, tox-

    icity, and inflammation-mediated gene expression changes (262, 285, 286),as well as mechanically actuated microfabricated substrates for deconstruct-ing the role of contact and short-range paracrine signals in hepatocyte–stromal cell interactions (287). Overall, the fine spatial and temporalcontrol of molecular signals provided by microtechnology approachescontinues to reveal important mechanisms in liver biology and accel-erate the development of therapeutic strategies.

    In addition to in vitro approaches, humanized mouse models that ex-hibit human chimerism in the liver (discussed above) have been increasing-ly applied toward studies of human liver function and disease. In particular,for drug development, humanized mice could aid in investigations of drug-drug interactions and chronic toxicity within a framework of relevantin vivo pharmacokinetic profiles, and potentially also could detect risksthat arise when a human-metabolized drug generates a toxic responsein another organ. Recently, tissue engineering has been used to generatea humanized mouse rapidly with high yield and in an immunocompetentnon–liver injury context (157), thereby circumventing the limitations ofcurrent transgenic and transplantation approaches. Here, human hepato-cytes were transplanted into an ectopic (intraperitoneal) site within an opti-mized hydrogel scaffold, which served as a supportive microenvironmentand barrier that delayed immune rejection. The transplanted constructssynthesized human liver proteins and exhibited human drug metabolism,drug-drug interactions, and drug-induced hepatocellular injury (157).

    Modeling human disease processesEngineered in vitro liver models facilitate studies into the behavior ofpathogens that target human hepatocytes, including hepatitis C virus

    Modeling human liver functions and disease

    Development of cell-based therapies

    Optimized in vitro culture platforms Disease models

    Engineered implantable constructs Pre-clinical testing

    Co-cultivation Soluble factors

    Extracellular matrix

    3D biomaterial scaffolds

    Fig. 7. Liver cell and tissue engineering. Progress in the field of liver cell and tissue engineeringserves a bidirectional role as a means for establishing robust model systems f