Mesenchymal stromal cell delivery via an ex vivo bioreactor preclinical test system attenuates clot formation for intravascular application.

While mesenchymal stromal cells are an appealing therapeutic option for a range of clinical applications, their potential to induce clotting when used systemically remains a safety concern, particularly in hypercoagulable conditions, such as in patients with severe COVID-19, trauma, or cancers. Here, we tested a novel preclinical approach aimed at improving the safety of mesenchymal stromal cell (MSC) systemic administration by use of a bioreactor. In this system, MSCs are seeded on the exterior of a hollow-fiber filter, sequestering them behind a hemocompatible semipermeable membrane with defined pore-size and permeability to allow for a molecularly defined cross talk between the therapeutic cells and the whole blood environment, including blood cells and signaling molecules. The potential for these bioreactor MSCs to induce clots in coagulable plasma was compared against directly injected "free" MSCs, a model of systemic administration. Our results showed that restricting MSCs exposure to plasma via a bioreactor extends the time necessary for clot formation to occur when compared with "free" MSCs. Measurement of cell surface data indicates the presence of known clot inducing factors, namely tissue factor and phosphatidylserine. Results also showed that recovering cells and flushing the bioreactor prior to use further prolonged clot formation time. Furthermore, application of this technology in two in vivo models did not require additional heparin in fully anticoagulated experimental animals to maintain target activated clotting time levels relative to heparin anticoagulated controls. Taken together the clinical use of bioreactor housed MSCs could offer a novel method to control systemic MSC exposure and prolong clot formation time.

Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells.

Bone marrow mesenchymal stromal cells (MSCs) have been studied for decades as potent immunomodulators. Clinically, they have shown some promise but with limited success. Here, we report the ability of a scalable hollow fiber bioreactor to effectively maintain ideal MSC function as a single population while also being able to impart an immunoregulatory effect when cultured in tandem with an inflamed lymphocyte population. MSCs were seeded on the extraluminal side of hollow fibers within a bioreactor where they indirectly interact with immune cells flowing within the lumen of the fibers. MSCs showed a stable and predictable metabolite and secreted factor profile during several days of perfusion culture. Exposure of bioreactor-seeded MSCs to inflammatory stimuli reproducibly switched MSC secreted factor profiles and altered microvesicle composition. Furthermore, circulating, activated human peripheral blood mononuclear cells (PBMCs) were suppressed by MSC bioreactor culture confirmed by a durable change in their immunophenotype and function. This platform was useful to study a model of immobilized MSCs and circulating immune cells and showed that monocytes play an important role in MSC driven immunomodulation. This coculture technology can have broad implications for use in studying MSC-immune interactions under flow conditions as well as in the generation of ex vivo derived immune cellular therapeutics.

Extracorporeal Stromal Cell Therapy for Subjects With Dialysis-Dependent Acute Kidney Injury.

INTRODUCTION: The pathophysiology of acute kidney injury (AKI) involves damage to renal epithelial cells, podocytes, and vascular beds that manifests into a deranged, self-perpetuating immune response and peripheral organ dysfunction. Such an injury pattern requires a multifaceted therapeutic to alter the wound healing response systemically. Mesenchymal stromal cells (MSCs) are a unique source of secreted factors that can modulate an inflammatory response to acute organ injury and enhance the repair of injured tissue at the parenchymal and endothelial levels. This phase Ib/IIa clinical trial evaluates SBI-101, a combination product that administers MSCs extracorporeally to overcome pharmacokinetic barriers of MSC transplantation. SBI-101 contains allogeneic human MSCs inoculated into a hollow-fiber hemofilter for the treatment of patients with severe AKI who are receiving continuous renal replacement therapy (CRRT). SBI-101 therapy is designed to reprogram the molecular and cellular components of blood in patients with severe organ injury. METHODS: This study is a prospective, multicenter, randomized, double-blind, sham-controlled, study of subjects with a clinical diagnosis of AKI who are receiving CRRT. Up to 32 subjects may be enrolled to provide 24 evaluable subjects (as a per protocol population). Subjects will receive CRRT in tandem with a sham control (0 MSCs), or the low- (250 × 106 MSCs) or high-dose (750 × 106 MSCs) SBI-101 therapeutic. RESULTS: The study will measure dose-dependent safety, renal efficacy, and exploratory biomarkers to characterize the pharmacokinetics and pharmacodynamics of SBI-101 in treated subjects. CONCLUSION: This first-in-human clinical trial will evaluate the safety and tolerability of SBI-101 in patients with AKI who require CRRT.

Suppressive effects of interleukin-18 on liver function in rat liver allografts.

BACKGROUND: Interleukin-18 (IL-18) is a potent proinflammatory cytokine that augments both innate and acquired immune responses. It is also a crucial regulator of lymphocyte production of interferon-γ (IFN-γ), which can promote acute cellular rejection of transplanted solid organs. METHODS: To evaluate the role of IL-18 in liver transplantation, we constructed an adenoviral vector encoding IL-18 binding protein (Adex-IL18bp), which specifically suppressed the biologic activity of IL-18, and examined the effect of this suppression on liver allografts by using a high-responder rat model (ACI to Lewis) of orthotopic liver transplantation (OLTx). Donor rats were given one intravenous injection of Adex-IL18bp or Adex-LacZ (control vector) 2 d before OLTx. RESULTS: Seven days after OLTx, overexpression of IL-18bp resulting from the adenovirus gene transfer was associated with significantly decreased serum alanine aminotransferase levels and less histologic hepatic injury in recipient rats with Adex-IL18bp-pretreated donors compared with Adex-LacZ controls. Adex-IL18bp pretreatment also significantly prolonged rat/allograft survival, inhibited expression of IFN-γ, and reduced levels (versus control values) of both CXCL10 and CX3CL1, which can be induced by IFN-γ. CONCLUSION: These results suggest that IL-18 has an important role in liver allograft rejection through IFN-γ and chemokines and that specific suppression of IL-18 may improve liver function early after transplantation.

Phenotypic and functional characterization of human bone marrow stromal cells in hollow-fibre bioreactors.

The transplantation of human bone marrow stromal cells (BMSCs) is a novel immunotherapeutic approach that is currently being explored in many clinical settings. Evidence suggests that the efficacy of cell transplantation is directly associated with soluble factors released by human BMSCs. In order to harness these secreted factors, we integrated BMSCs into large-scale hollow-fibre bioreactor devices in which the cells, separated by a semipermeable polyethersulphone (PES) membrane, can directly and continuously release therapeutic factors into the blood stream. BMSCs were found to be rapidly adherent and exhibited long-term viability on PES fibres. The cells also preserved their immunophenotype under physiological fluid flow rates in the bioreactor, and exhibited no signs of differentiation during device operation, but still retained the capacity to differentiate into osteoblastic lineages. BMSC devices released growth factors and cytokines at comparable levels on a per-cell basis to conventional cell culture platforms. Finally, we utilized a potency assay to demonstrate the therapeutic potential of the collected secreted factors from the BMSC devices. In summary, we have shown that culturing BMSCs in a large-scale hollow-fibre bioreactor is feasible without deleterious effects on phenotype, thus providing a platform for collecting and delivering the paracrine secretions of these cells.

Secreted factors from bone marrow stromal cells upregulate IL-10 and reverse acute kidney injury.

Acute kidney injury is a devastating syndrome that afflicts over 2,000,000 people in the US per year, with an associated mortality of greater than 70% in severe cases. Unfortunately, standard-of-care treatments are not sufficient for modifying the course of disease. Many groups have explored the use of bone marrow stromal cells (BMSCs) for the treatment of AKI because BMSCs have been shown to possess unique anti-inflammatory, cytoprotective, and regenerative properties in vitro and in vivo. It is yet unresolved whether the primary mechanisms controlling BMSC therapy in AKI depend on direct cell infusion, or whether BMSC-secreted factors alone are sufficient for mitigating the injury. Here we show that BMSC-secreted factors are capable of providing a survival benefit to rats subjected to cisplatin-induced AKI. We observed that when BMSC-conditioned medium (BMSC-CM) is administered intravenously, it prevents tubular apoptosis and necrosis and ameliorates AKI. In addition, we observed that BMSC-CM causes IL-10 upregulation in treated animals, which is important to animal survival and protection of the kidney. In all, these results demonstrate that BMSC-secreted factors are capable of providing support without cell transplantation, and the IL-10 increase seen in BMSC-CM-treated animals correlates with attenuation of severe AKI.

The inflammatory response to double stranded DNA in endothelial cells is mediated by NFκB and TNFα.

Endothelial cells represent an important barrier between the intravascular compartment and extravascular tissues, and therefore serve as key sensors, communicators, and amplifiers of danger signals in innate immunity and inflammation. Double stranded DNA (dsDNA) released from damaged host cells during injury or introduced by pathogens during infection, has emerged as a potent danger signal. While the dsDNA-mediated immune response has been extensively studied in immune cells, little is known about the direct and indirect effects of dsDNA on the vascular endothelium. In this study we show that direct dsDNA stimulation of endothelial cells induces a potent proinflammatory response as demonstrated by increased expression of ICAM1, E-selectin and VCAM1, and enhanced leukocyte adhesion. This response was dependent on the stress kinases JNK and p38 MAPK, required the activation of proinflammatory transcription factors NFκB and IRF3, and triggered the robust secretion of TNFα for sustained secondary activation of the endothelium. DNA-induced TNFα secretion proved to be essential in vivo, as mice deficient in the TNF receptor were unable to mount an acute inflammatory response to dsDNA. Our findings suggest that the endothelium plays an active role in mediating dsDNA-induced inflammatory responses, and implicate its importance in establishing an acute inflammatory response to sterile injury or systemic infection, where host or pathogen derived dsDNA may serve as a danger signal.

Reactive bone marrow stromal cells attenuate systemic inflammation via sTNFR1.

Excessive systemic inflammation following trauma, sepsis, or burn could lead to distant organ damage. The transplantation of bone marrow stromal cells or mesenchymal stem cells (MSCs) has been reported to be an effective treatment for several immune disorders by modulating the inflammatory response to injury. We hypothesized that MSCs can dynamically secrete systemic factors that can neutralize the activity of inflammatory cytokines. In this study, we showed that cocultured MSCs are able to decrease nuclear factor κ-B (NFκB) activation in target epithelial cells incubated in inflammatory serum conditions. Proteomic screening revealed a responsive secretion of soluble tumor necrosis factor (TNF) receptor 1 (sTNFR1) when MSCs were exposed to lipopolysaccharide (LPS)-stimulated rat serum. The responsive effect was eliminated when NFκB activation was blocked in MSCs. Intramuscular transplantation of MSCs in LPS-endotoxic rats decreased a panel of inflammatory cytokines and inflammatory infiltration of macrophages and neutrophils in lung, kidney, and liver when compared to controls. These results suggest that improvements of inflammatory responses in animal models after local transplantation of MSCs are at least, in part, explained by the NFκB-dependent secretion of sTNFR1 by MSCs.

Bone marrow mesenchymal stromal cells attenuate organ injury induced by LPS and burn.

Bone marrow mesenchymal stromal cells (MSCs) suppress immune cell responses and have beneficial effects in various inflammatory-related immune disorders. A therapeutic modality for systemic inflammation and its consequences is not available yet. Thus, this work investigates the therapeutic effects of MSCs in injury models induced by lipopolysaccharide (LPS) or burn. Gene expression was analyzed in MSCs when exposed to inflammatory serum from injured animals and it showed remarkable alterations compared to normal culture. In addition, injured animals were transplanted intramuscularly with MSCs. Forty-eight hours after cell transplantation, kidney, lung, and liver were analyzed for infiltration of inflammatory cells and TUNEL-expressing cells. Results showed that MSCs attenuate injury by reducing the infiltration of inflammatory cells in various target organs and by reducing cell death. These data suggest that MSCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for systemic inflammatory-based disorders.

Long-term superior performance of a stem cell/hepatocyte device for the treatment of acute liver failure.

Cell-based technologies to support/restore organ function represent one of the most promising avenues in the treatment of acute liver failure (ALF). Recently, mesenchymal stem cells (MSCs) have been reported as a new therapeutic for inflammatory conditions. Here, we demonstrate the efficacy of MSCs, when cocultured with hepatocytes, to provide combination hepatic and antiinflammatory therapy in the setting of ALF. MSCs were shown to have multiple beneficial effects in vitro that were relevant in a therapeutic context, including (1) hepatocellular functional support, (2) secretion of molecules that inhibit hepatocyte apoptosis, and (3) modulation of an acute phase response by hepatocytes cultured in ALF-induced serum. In addition, we show that the MSC secretome is dynamically changed in response to serum exposure from ALF rats. We then conducted a therapeutic trial of liver assist devices (LADs). LADs containing cocultures of MSCs and hepatocytes provided a greater survival benefit compared to other coculture and monocellular control LADs. Treatment with MSC-hepatocyte devices was associated with specific improvements in hepatic functional and histological parameters as well as decreasing inflammatory serum cytokine levels, validating a combined therapeutic effect. Moreover, MSC coculture reduced the overall cell mass of the device by an order of magnitude. These findings demonstrate the importance of nonparenchymal cells in the cellular composition of LADs, and strongly support the integration of MSCs into hepatocyte-coculture-based LADs as a potential destination therapy for ALF.

Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo.

UNLABELLED: Orthotopic liver transplantation is the only proven effective treatment for fulminant hepatic failure (FHF), but its use is limited because of organ donor shortage, associated high costs, and the requirement for lifelong immunosuppression. FHF is usually accompanied by massive hepatocellular death with compensatory liver regeneration that fails to meet the cellular losses. Therefore, therapy aimed at inhibiting cell death and stimulating endogenous repair pathways could offer major benefits in the treatment of FHF. Recent studies have demonstrated that mesenchymal stem cell (MSC) therapy can prevent parenchymal cell loss and promote tissue repair in models of myocardial infarction, acute kidney failure, and stroke through the action of trophic secreted molecules. In this study, we investigated whether MSC therapy can protect the acutely injured liver and stimulate regeneration. In a D-galactosamine-induced rat model of acute liver injury, we show that systemic infusion of MSC-conditioned medium (MSC-CM) provides a significant survival benefit and prevents the release of liver injury biomarkers. Furthermore, MSC-CM therapy resulted in a 90% reduction of apoptotic hepatocellular death and a three-fold increment in the number of proliferating hepatocytes. This was accompanied by a dramatic increase in the expression levels of 10 genes known to be up-regulated during hepatocyte replication. Direct antiapoptotic and promitotic effects of MSC-CM on hepatocytes were demonstrated using in vitro assays. CONCLUSION: These data provide the first clear evidence that MSC-CM therapy provides trophic support to the injured liver by inhibiting hepatocellular death and stimulating regeneration, potentially creating new avenues for the treatment of FHF.

Bone marrow-derived mesenchymal stem cells ameliorate autoimmune enteropathy independently of regulatory T cells.

Cell-based tolerogenic therapy is a relatively new approach for the treatment of autoimmune diseases. Mesenchymal stem cells (MSCs) have been shown to be potent immunomodulatory agents in a number of experimental and clinical scenarios; however, their use in various autoimmune diseases is undefined. Herein, we report the efficacy of MSC transplantation in a multiorgan autoimmunity model. Mice with defective peripheral tolerance caused by a deficiency in regulatory T cells were used as a testbed for therapy. After screening multiple target tissues of autoimmune attack, we observed an MSC-specific improvement in the histopathology of the distal ileum of treated mice. We then showed that MSCs can reduce mesenteric lymph node (MLN) cellularity in autoimmune mice during active disease and decrease activated T-cell populations in the MLN. Trafficking studies using enhanced green fluorescent protein (eGFP)-reporter MSCs revealed no appreciable engraftment in the intestine, but it did reveal the presence of eGFP+ cells organized in clusters within the MLN, as well as ancillary nodes. Semiquantitative analysis showed no difference in the number of clusters; however, eGFP+ cells in MLNs compared with ancillary nodes had distinct fibroblastoid morphology and formed a network with neighboring eGFP+ cells. Finally, we show evidence that transplantation of MSCs caused global immunosuppression, as measured by increased CD4+ CD8+ thymocyte production and serum interleukin-10 and decreased serum interferon-gamma. These data implicate the intestine as a new site of MSC tolerance induction and should motivate additional studies evaluating the use of MSCs as a treatment for autoimmune enteropathies.

Activin alters the kinetics of endoderm induction in embryonic stem cells cultured on collagen gels.

Embryonic stem cell-derived endoderm is critical for the development of cellular therapies for the treatment of disease such as diabetes, liver cirrhosis, or pulmonary emphysema. Here, we describe a novel approach to induce endoderm from mouse embryonic stem (mES) cells using fibronectin-coated collagen gels. This technique results in a homogeneous endoderm-like cell population, demonstrating endoderm-specific gene and protein expression, which remains committed following in vivo transplantation. In this system, activin, normally an endoderm inducer, caused an 80% decrease in the Foxa2-positive endoderm fraction, whereas follistatin increased the Foxa2-positive endoderm fraction to 78%. Our work suggests that activin delays the induction of endoderm through its transient precursors, the epiblast and mesendoderm. Long-term differentiation displays a twofold reduction in hepatic gene expression and threefold reduction in hepatic protein expression of activin-treated cells compared with follistatin-treated cells. Moreover, subcutaneous transplantation of activin-treated cells in a syngeneic mouse generated a heterogeneous teratoma-like mass, suggesting that these were a more primitive population. In contrast, follistatin-treated cells resulted in an encapsulated epithelial-like mass, suggesting that these cells remained committed to the endoderm lineage. In conclusion, we demonstrate a novel technique to induce the direct differentiation of endoderm from mES cells without cell sorting. In addition, our work suggests a new role for activin in induction of the precursors to endoderm and a new endoderm-enrichment technique using follistatin.

Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-alpha abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.

Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure.

Modulation of the immune system may be a viable alternative in the treatment of fulminant hepatic failure (FHF) and can potentially eliminate the need for donor hepatocytes for cellular therapies. Multipotent bone marrow-derived mesenchymal stem cells (MSCs) have been shown to inhibit the function of various immune cells by undefined paracrine mediators in vitro. Yet, the therapeutic potential of MSC-derived molecules has not been tested in immunological conditions in vivo. Herein, we report that the administration of MSC-derived molecules in two clinically relevant forms-intravenous bolus of conditioned medium (MSC-CM) or extracorporeal perfusion with a bioreactor containing MSCs (MSC-EB)-can provide a significant survival benefit in rats undergoing FHF. We observed a cell mass-dependent reduction in mortality that was abolished at high cell numbers indicating a therapeutic window. Histopathological analysis of liver tissue after MSC-CM treatment showed dramatic reduction of panlobular leukocytic infiltrates, hepatocellular death and bile duct duplication. Furthermore, we demonstrate using computed tomography of adoptively transferred leukocytes that MSC-CM functionally diverts immune cells from the injured organ indicating that altered leukocyte migration by MSC-CM therapy may account for the absence of immune cells in liver tissue. Preliminary analysis of the MSC secretome using a protein array screen revealed a large fraction of chemotactic cytokines, or chemokines. When MSC-CM was fractionated based on heparin binding affinity, a known ligand for all chemokines, only the heparin-bound eluent reversed FHF indicating that the active components of MSC-CM reside in this fraction. These data provide the first experimental evidence of the medicinal use of MSC-derived molecules in the treatment of an inflammatory condition and support the role of chemokines and altered leukocyte migration as a novel therapeutic modality for FHF.

A bioartificial liver device secreting interleukin-1 receptor antagonist for the treatment of hepatic failure in rats.

BACKGROUND: Liver transplantation is the treatment of choice for many patients with fulminant hepatic failure (FHF). A major limitation of this treatment is the lack of available donors. An optimally functioning bio-artificial liver (BAL) device has the potential to provide critical hepatic support to patients with FHF. In this study, we examined the efficacy of combining interleukin-1 (IL-1) receptor blockade with the synthetic function of hepatocytes in a BAL device for the treatment of FHF. MATERIALS AND METHODS: We injected an adenoviral vector encoding human IL-1 receptor antagonist (AdIL-1Ra) into the liver of D-galactosamine (GalN) intoxicated rats via the portal vein. We also transfected primary rat hepatocytes and reversibly immortalized human hepatocytes (TTNT cells) with AdIL-1Ra, and incorporated these transfected hepatocytes into our flat-plate BAL device and evaluated their efficacy in our GalN-induced FHF rat model after 10 h of extracorporeal perfusion. RESULTS: Rats injected with AdIL-1Ra showed significant reductions in the plasma levels of hepatic enzymes. Primary rat hepatocytes transfected with AdIL-1Ra secreted IL-1Ra without losing their original synthetic function. Incorporating these cells into the BAL device and testing in a GalN-induced FHF rat model resulted in significant reductions in plasma IL-6 levels and significantly improved animal survival. Incorporating the AdIL-1Ra transfected TTNT cells in the BAL device and testing in the GalN-induced FHF rat model resulted in significantly reduced plasma IL-6 levels, and a trend toward improved survival was seen. CONCLUSION: Hepatocytes producing IL-1Ra are a promising cell source for BAL devices in the treatment of GalN-induced FHF.

Treatment of fulminant hepatic failure in rats using a bioartificial liver device containing porcine hepatocytes producing interleukin-1 receptor antagonist.

Fulminant hepatic failure (FHF) is a serious clinical condition that is associated with high mortality. There is evidence that FHF is an inflammatory disease, which is supported clinically by elevated serum levels of cytokines. In an effort to develop hepatocytes with additional functions for use in our bioartificial liver (BAL) device, we focused on interleukin-1 (IL-1) blockade as a therapeutic modality. Primary porcine hepatocytes were isolated from the livers of miniature swine and then transfected with an adenoviral vector encoding human interleukin-1 receptor antagonist (AdIL-1Ra). The transfected hepatocytes secreted human IL-1Ra. These transfected hepatocytes were incorporated into a flat-plate BAL device to evaluate their efficacy in treating D-galactosamine (GalN)- induced FHF in a rat model. After extracorporeal perfusion with the BAL device containing the transfected hepatocytes, there were significant reductions in the plasma levels of hepatic enzymes (aspartate aminotransferase and alanine aminotransferase) and cytokines (IL-1 and IL-6), indicating a beneficial effect. Animal survival was significantly improved in the treated group compared to the control group. These experiments demonstrate that combining inflammatory cytokine blockade with a functional BAL device may be an effective therapeutic option in the treatment of FHF.

Designing a hepatocellular microenvironment with protein microarraying and poly(ethylene glycol) photolithography.

In this study, robotic protein printing was employed as a method for designing a cellular microenvironment. Protein printing proved to be an effective strategy for creating micropatterned co-cultures of primary rat hepatocytes and 3T3 fibroblasts. Collagen spots (ca. 170 microm in diameter) were printed onto amino-silane- and glutaraldehyde-modified glass slides. Groups of 15-20 hepatocytes attached to collagen regions in a highly selective manner forming cell clusters corresponding in size to the printed collagen domains. Fibroblasts, seeded onto the same surface, adhered and spread around arrays of hepatocyte islands creating a heterotypic environment. The co-cultured hepatocytes produced and maintained high levels of liver-specific biomarkers, albumin and urea, over the course of 2 weeks. In addition, protein printing was combined with poly(ethylene glycol) photolithography to define intercellular contacts within the clusters of hepatocytes residing on individual collagen islands. Glass slides, treated with 3-acryloxypropyl trichlorosilane and imprinted with 170 m diameter collagen spots, were micropatterned with a high-density array of 30 microm x 30 microm poly(ethylene glycol) (PEG) wells. As a result, discrete groups of ca. 9 PEG microwells became functionalized with the cell-adhesive ligand. When exposed to micropatterned surfaces, hepatocytes interacted exclusively with collagen-modified regions, attaching and becoming confined at a single-cell level within the hydrogel wells. Micropatterning strategies proposed here will lead to greater insights into hepatocellular behavior and will benefit the fields of hepatic tissue engineering and liver biology.

Bioengineering of liver assist devices.

Over 30 000 patients die annually in the United States from liver failure. In fulminant hepatic failure, a clinical syndrome associated with high mortality, orthotopic liver transplantation is the primary therapeutic option for patients not responding to supportive therapy. However, the persistent scarcity of donor organs has limited this therapeutic modality, resulting in a continued increase in the number of patients who die waiting for a donor liver. An extracorporeal bioartificial liver device could provide vital support to a liver failure patient until a donor liver was available or until the patient's own liver regenerated. Although it is unclear which liver-specific functions must be provided by such a device to be effective, a constant challenge has been to obtain stable, well-differentiated, and normally functioning hepatocytes that can be cultured at high cell densities. Many of the devices currently undergoing clinical trials are limited by designs which are prone to substrate limitations, resulting in compromised hepatocyte function. In devices that avoid substrate limitations, hepatocyte functions can be optimized, thereby leading to increased device efficiency. In this overview, the authors describe the critical issues involved in bioartificial liver development and discuss their experiences in hepatocyte culture optimization within the context of a microchannel, flat-plate bioartificial liver device with an internal membrane oxygenator.