3D Bioprinted Liver-on-a-Chip for Drug Cytotoxicity Screening.

Tissues on a chip are sophisticated three-dimensional (3D) in vitro microphysiological systems designed to replicate human tissue conditions within dynamic physicochemical environments. However, the current fabrication methods for tissue spheroids on a chip require multiple parts and manual processing steps, including the deposition of spheroids onto prefabricated "chips." These challenges also lead to limitations regarding scalability and reproducibility. To overcome these challenges, we employed 3D printing techniques to automate the fabrication process of tissue spheroids on a chip. This allowed the simultaneous high-throughput printing of human liver spheroids and their surrounding polymeric flow chamber "chips" containing inner channels in a single step. The fabricated liver tissue spheroids on a liver-on-a-chip (LOC) were subsequently subjected to dynamic culturing by a peristaltic pump, enabling assessment of cell viability and metabolic activities. The 3D printed liver spheroids within the printed chips demonstrated high cell viability (>80%), increased spheroid size, and consistent adenosine triphosphate (ATP) activity and albumin production for up to 14 days. Furthermore, we conducted a study on the effects of acetaminophen (APAP), a nonsteroidal anti-inflammatory drug, on the LOC. Comparative analysis revealed a substantial decline in cell viability (<40%), diminished ATP activity, and reduced spheroid size after 7 days of culture within the APAP-treated LOC group, compared to the nontreated groups. These results underscore the potential of 3D bioprinted tissue chips as an advanced in vitro model that holds promise for accurately studying in vivo biological processes, including the assessment of tissue response to administered drugs, in a high-throughput manner.

Bioreactor design and validation for manufacturing strategies in tissue engineering.

The fields of regenerative medicine and tissue engineering offer new therapeutic options to restore, maintain or improve tissue function following disease or injury. To maximize the biological function of a tissue-engineered clinical product, specific conditions must be maintained within a bioreactor to allow the maturation of the product in preparation for implantation. Specifically, the bioreactor should be designed to mimic the mechanical, electrochemical and biochemical environment that the product will be exposed to in vivo. Real-time monitoring of the functional capacity of tissue-engineered products during manufacturing is a critical component of the quality management process. The present review provides a brief overview of bioreactor engineering considerations. In addition, strategies for bioreactor automation, in-line product monitoring and quality assurance are discussed.

Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half-life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis-related genes in primary human stellate cells. RNA sequencing analysis of TGF-ß1-activated LX-2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS-induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. In mice expressing human RXFP1 gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α-smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.-Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Urothelium with barrier function differentiated from human urine-derived stem cells for potential use in urinary tract reconstruction.

BACKGROUND: Autologous urothelial cells are often obtained via bladder biopsy to generate the bio-engineered urethra or bladder, while urine-derived stem cells (USC) can be obtained by a non-invasive approach. The objective of this study is to develop an optimal strategy for urothelium with permeability barrier properties using human USC which could be used for tissue repair in the urinary tract system. METHODS: USC were harvested from six healthy adult individuals. To optimize urothelial differentiation, five different differentiation methods were studied. The induced cells were assessed for gene and protein expression markers of urothelial cells via RT-PCR, Western blotting, and immunofluorescent staining. Barrier function and ultrastructure of the tight junction were assessed with permeability assays and transmission electron microscopy (TEM). Induced cells were both cultured on trans-well membranes and small intestinal submucosa, then investigated under histology analysis. RESULTS: Differentiated USC expressed significantly higher levels of urothelial-specific transcripts and proteins (Uroplakin III and Ia), epithelial cell markers (CK20 and AE1/AE3), and tight junction markers (ZO-1, ZO-2, E-cadherin, and Cingulin) in a time-dependent manner, compared to non-induced USC. In vitro assays using fluorescent dye demonstrated a significant reduction in permeability of differentiated USC. In addition, transmission electron microscopy confirmed appropriate ultrastructure of urothelium differentiated from USC, including tight junction formation between neighboring cells, which was similar to positive controls. Furthermore, multilayered urothelial tissues formed 2 weeks after USC were differentiated on intestine submucosal matrix. CONCLUSION: The present study illustrates an optimal strategy for the generation of differentiated urothelium from stem cells isolated from the urine. The induced urothelium is phenotypically and functionally like native urothelium and has proposed uses in in vivo urological tissue repair or in vitro urethra or bladder modeling.

Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform.

Many drugs have progressed through preclinical and clinical trials and have been available - for years in some cases - before being recalled by the FDA for unanticipated toxicity in humans. One reason for such poor translation from drug candidate to successful use is a lack of model systems that accurately recapitulate normal tissue function of human organs and their response to drug compounds. Moreover, tissues in the body do not exist in isolation, but reside in a highly integrated and dynamically interactive environment, in which actions in one tissue can affect other downstream tissues. Few engineered model systems, including the growing variety of organoid and organ-on-a-chip platforms, have so far reflected the interactive nature of the human body. To address this challenge, we have developed an assortment of bioengineered tissue organoids and tissue constructs that are integrated in a closed circulatory perfusion system, facilitating inter-organ responses. We describe a three-tissue organ-on-a-chip system, comprised of liver, heart, and lung, and highlight examples of inter-organ responses to drug administration. We observe drug responses that depend on inter-tissue interaction, illustrating the value of multiple tissue integration for in vitro study of both the efficacy of and side effects associated with candidate drugs.

Optical Tracking and Digital Quantification of Beating Behavior in Bioengineered Human Cardiac Organoids.

Organoid and organ-on-a-chip technologies are rapidly advancing towards deployment for drug and toxicology screening applications. Liver and cardiac toxicities account for the majority of drug candidate failures in human trials. Liver toxicity generally produces liver cell death, while cardiac toxicity causes adverse changes in heart beat kinetics. In traditional 2D cultures, beating kinetics can be measured by electrode arrays, but in some 3D constructs, quantifying beating kinetics can be more challenging. For example, real time measurements of calcium flux or contractile forces are possible, yet rather complex. In this communication article, we demonstrate a simple sensing system based on software code that optically analyzes video capture files of beating cardiac organoids, translates these files in representations of moving pixels, and quantifies pixel movement activity over time to generate beat kinetic plots. We demonstrate this system using bioengineered cardiac organoids under baseline and drug conditions. This technology offers a non-invasive, low-cost, and incredibly simple method for tracking and quantifying beating behavior in cardiac organoids and organ-on-a-chip systems for drug and toxicology screening.

A liver-on-a-chip platform with bioprinted hepatic spheroids.

The inadequacy of animal models in correctly predicting drug and biothreat agent toxicity in humans has resulted in a pressing need for in vitro models that can recreate the in vivo scenario. One of the most important organs in the assessment of drug toxicity is liver. Here, we report the development of a liver-on-a-chip platform for long-term culture of three-dimensional (3D) human HepG2/C3A spheroids for drug toxicity assessment. The bioreactor design allowed for in situ monitoring of the culture environment by enabling direct access to the hepatic construct during the experiment without compromising the platform operation. The engineered bioreactor could be interfaced with a bioprinter to fabricate 3D hepatic constructs of spheroids encapsulated within photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. The engineered hepatic construct remained functional during the 30 days culture period as assessed by monitoring the secretion rates of albumin, alpha-1 antitrypsin, transferrin, and ceruloplasmin, as well as immunostaining for the hepatocyte markers, cytokeratin 18, MRP2 bile canalicular protein and tight junction protein ZO-1. Treatment with 15 mM acetaminophen induced a toxic response in the hepatic construct that was similar to published studies on animal and other in vitro models, thus providing a proof-of-concept demonstration of the utility of this liver-on-a-chip platform for toxicity assessment.

Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.

Tissue engineering and cell based liver therapies have utilized primary hepatocytes with limited success due to the failure of hepatocytes to maintain their phenotype in vitro. In order to overcome this challenge, hyaluronic acid (HA) cell culture substrates were formulated to closely mimic the composition and stiffness of the normal liver cellular microenvironment. The stiffness of the substrate was modulated by adjusting HA hydrogel crosslinking. Additionally, the repertoire of bioactive molecules within the HA substrate was bolstered by supplementation with normal liver extracellular matrix (ECM). Primary human hepatocyte viability and phenotype were determined over a narrow physiologically relevant range of substrate stiffnesses from 600 to 4600Pa in both the presence and absence of liver ECM. Cell attachment, viability, and organization of the actin cytoskeleton improved with increased stiffness up to 4600Pa. These differences were not evident in earlier time points or substrates containing only HA. However, gene expression for the hepatocyte markers hepatocyte nuclear factor 4 alpha (HNF4α) and albumin significantly decreased on the 4600Pa stiffness at day 7 indicating that cells may not have maintained their phenotype long-term at this stiffness. Function, as measured by albumin secretion, varied with both stiffness and time in culture and peaked at day 7 at the 1200Pa stiffness, slightly below the stiffness of normal liver ECM at 3000Pa. Overall, gel stiffness affected primary human hepatocyte cell adhesion, functional marker expression, and morphological characteristics dependent on both the presence of liver ECM in gel substrates and time in culture.

Bioengineered transplantable porcine livers with re-endothelialized vasculature.

Donor shortage remains a continued challenge in liver transplantation. Recent advances in tissue engineering have provided the possibility of creating functional liver tissues as an alternative to donor organ transplantation. Small bioengineered liver constructs have been developed, however a major challenge in achieving functional bioengineered liver in vivo is the establishment of a functional vasculature within the scaffolds. Our overall goal is to bioengineer intact livers, suitable for transplantation, using acellular porcine liver scaffolds. We developed an effective method for reestablishing the vascular network within decellularized liver scaffolds by conjugating anti-endothelial cell antibodies to maximize coverage of the vessel walls with endothelial cells. This procedure resulted in uniform endothelial attachment throughout the liver vasculature extending to the capillary bed of the liver scaffold and greatly reduced platelet adhesion upon blood perfusion in vitro. The re-endothelialized livers, when transplanted to recipient pigs, were able to withstand physiological blood flow and maintained for up to 24 h. This study demonstrates, for the first time, that vascularized bioengineered livers, of clinically relevant size, can be transplanted and maintained in vivo, and represents the first step towards generating engineered livers for transplantation to patients with end-stage liver failure.

Immunogenicity of decellularized porcine liver for bioengineered hepatic tissue.

Liver disease affects millions of patients each year. The field of regenerative medicine promises alternative therapeutic approaches, including the potential to bioengineer replacement hepatic tissue. One approach combines cells with acellular scaffolds derived from animal tissue. The goal of this study was to scale up our rodent liver decellularization method to livers of a clinically relevant size. Porcine livers were cannulated via the hepatic artery, then perfused with PBS, followed by successive Triton X-100 and SDS solutions in saline buffer. After several days of rinsing, decellularized liver samples were histologically analyzed. In addition, biopsy specimens of decellularized scaffolds were seeded with hepatoblastoma cells for cytotoxicity testing or implanted s.c. into rodents to investigate scaffold immunogenicity. Histological staining confirmed cellular clearance from pig livers, with removal of nuclei and cytoskeletal components and widespread preservation of structural extracellular molecules. Scanning electron microscopy confirmed preservation of an intact liver capsule, a porous acellular lattice structure with intact vessels and striated basement membrane. Liver scaffolds supported cells over 21 days, and no increased immune response was seen with either allogeneic (rat-into-rat) or xenogeneic (pig-into-rat) transplants over 28 days, compared with sham-operated on controls. These studies demonstrate that successful decellularization of the porcine liver could be achieved with protocols developed for rat livers, yielding nonimmunogenic scaffolds for future hepatic bioengineering studies.

Inhibition of Notch signaling affects hepatic oval cell response in rat model of 2AAF-PH.

BACKGROUND AND AIMS: Activation of the oval cell compartment occurs in the liver when hepatocytes are functionally compromised and/or unable to divide. Our goal was to investigate the systemic signals responsible for determining the efficiency of oval cell-mediated liver regeneration, focusing on the Notch signaling cascade. METHODS: The established oval cell induction protocol of 2-acetylaminofluorine (2-AAF) implantation followed by 70% surgical resection of the liver (partial hepatectomy, PH) was employed in a rat model. This oval cell induction model was further combined with injections of a γ-secretase inhibitor (GSI XX) to examine the effects of Notch inhibition on oval cell-aided regeneration of the liver. RESULTS: Notch signaling was found to be upregulated at the peak of oval cell induction during 2AAF-PH alone. Treatment with GSI XX led to interruption of the Notch signal, as shown by a decrease in expression of Hes1. While there was a robust oval cell response seen at day 11 post-PH, there was a measurable delay in differentiation when Notch was inhibited. This was confirmed morphologically as well as by immunohistochemistry for the oval cell markers, α-fetoprotein, OV-6, and CK19. The hepatocytes seen at day 22 demonstrated an enhanced hepatocellular mitoinhibition index (p21(Waf1)/Ki67), suggestive of dysregulated proliferation and cell cycle progression. Moreover, these hepatocytes exhibited decreased expression of hepatocyte functional markers, such as cytochrome P450 and glucose-6-phosphatase-α. CONCLUSIONS: Taken together, these results identify the Notch signaling pathway as a potent regulator of differentiation and proliferation in oval cells, which is necessary for functional for repair of the liver by oval cells.

Hepatic stellate cells' involvement in progenitor-mediated liver regeneration.

Earlier studies conducted by our laboratory have shown that suppression of transforming growth factor-beta (TGFbeta)-mediated upregulation of connective tissue growth factor (CTGF) by iloprost resulted in a greatly diminished oval cell response to 2-acetylaminofluorene/partial hepatectomy (2AAF/PH) in rats. We hypothesized that this effect is due to decreased activation of hepatic stellate cells. To test this hypothesis, we maintained rats on a diet supplemented with 2% L-cysteine as a means of inhibiting stellate cell activation during the oval cell response to 2AAF/PH. In vitro experiments show that L-cysteine did, indeed, prevent the activation of stellate cells while exerting no direct effect on oval cells. Desmin immunostaining of liver sections from 2AAF/PH animals indicated that maintenance on the L-cysteine diet resulted in an 11.1-fold decrease in the number of activated stellate cells within the periportal zones. The total number of cells proliferating in the periportal zones of livers from animals treated with L-cysteine was drastically reduced. Further analyses showed a greater than fourfold decrease in the magnitude of the oval cell response in animals maintained on the L-cysteine diet as determined by immunostaining for both OV6 and alpha-fetoprotein (AFP). Global liver expression of AFP as measured by real-time PCR was shown to be decreased 4.7-fold in the L-cysteine-treated animals. These data indicate that the activation of hepatic stellate cells is required for an appropriate oval cell response to 2AAF/PH.

A detailed characterization of the adult mouse model of glycogen storage disease Ia.

Glycogen storage disease type Ia (GSDIa) is caused by a genetic defect in the hepatic enzyme glucose-6-phosphatase (G6Pase-alpha), which manifests as life-threatening hypoglycemia with related metabolic complications. A G6Pase-alpha knockout (KO) mouse model was generated to study potential therapies for correcting this disorder. Since then, gene therapy studies have produced promising results, showing long-term improvement in liver histology and glycogen metabolism. Under existing protocols, however, untreated KO pups seldom survived weaning. Here, we present a thorough characterization of the G6Pase-alpha KO mouse, as well as the husbandry protocol for rearing this strain to adulthood. These mice were raised with only palliative care, and characterized from birth through 6 months of age. Once KO mice have survived the very frail weaning period, their size, agility, serum lipids and glycemic control improve dramatically, reaching levels approaching their wild-type littermates. In addition, our data reveal that adult mice lacking G6Pase-alpha are able to mate and produce viable offspring. However, liver histology and glycogen accumulation do not improve with age. Overall, the reliable production of mature KO mice could provide a critical tool for advancing the GSDIa field, as the availability of a robust enzyme-deficient adult offers a new spectrum of treatment avenues that would not be tolerated by the frail pups. Most importantly, our detailed characterization of the adult KO mouse provides a crucial baseline for accurately gauging the efficacy of experimental therapies in this important model.

Establishment of cancer cell lines from rat hepatocholangiocarcinoma and assessment of the role of granulocyte-colony stimulating factor and hepatocyte growth factor in their growth, motility and survival.

BACKGROUND/AIMS: Oval cells (OCs), putative hepatic stem cells, may give rise to liver cancers. We developed a carcinogenesis regimen, based upon induction of OC proliferation prior to carcinogen exposure. In our model, rats subjected to 2-acetylaminofluorene/ partial-hepatectomy followed by aflatoxin injection (APA regimen) developed well-differentiated hepatocholangiocarcinomas. The aim of this study was to establish and characterize cancer cell lines from this animal model. METHODS: Cancer cells were cultured from animals sacrificed eight months after treatment, and single clones were selected. The established cell lines, named LCSCs, were characterized, and their tumorigenicity was assessed in vivo. The roles of granulocyte-colony stimulating factor (G-CSF) and hepatocyte growth factor (HGF) in LCSC growth, survival and motility were also investigated. RESULTS: From primary tumors, six cell lines were developed. LCSCs shared with the primary tumors the expression of various OC-associated markers, including cMet and G-CSF receptor. In vitro, HGF conferred protection from death by serum withdrawal. Stimulation with G-CSF increased LCSC growth and motility, while the blockage of its receptor inhibited LCSC proliferation and migration. CONCLUSIONS: Six cancer cell lines were established from our model of hepatocholangiocarcinoma. HGF modulated LCSC resistance to apoptosis, while G-CSF acted on LCSCs as a proliferative and chemotactic agent.

Isolation and characterization of hepatic stem cells, or "oval cells," from rat livers.

The pace of research on the potential therapeutic uses of liver stem cells or "oval cells" has accelerated significantly in recent years. Concurrent advancements in techniques for the isolation and characterization of these cells have helped fuel this research. Several models now exist for the induction of oval cell proliferation in rodents. Protocols for the isolation and culture of these cells have evolved to the point that they may be set up in any laboratory equipped for cell culture. The advent of magnetic cell sorting has eliminated reliance on expensive flow cytometric sorting equipment to generate highly enriched populations of oval cells. Our laboratory has had much success in using the oval cell surface marker Thy-1 in combination with magnetic sorting to produce material suitable for testing the influence of a myriad of chemical signaling molecules on the oval cell phenotype. This chapter will describe our basic strategy for oval cell induction and isolation. Additionally, two in vitro procedures are described which the reader may find useful in the early stages of developing an oval cell research project.

Granulocyte-colony stimulating factor promotes liver repair and induces oval cell migration and proliferation in rats.

BACKGROUND AND AIMS: Hepatic regeneration is a heterogeneous phenomenon involving several cell populations. Oval cells are considered liver stem cells, a portion of which derive from bone marrow (BM). Recent studies have shown that granulocyte-colony stimulating factor (G-CSF) may be effective in facilitating liver repair. However, it remains unclear if G-CSF acts by mobilizing BM cells, or if it acts locally within the liver microenvironment to facilitate the endogenous restoration program. In the present study, we assessed the involvement of G-CSF during oval cell activation. METHODS: Dipeptidyl-peptidase-IV-deficient female rats received BM transplants from wild-type male donors. Four weeks later, rats were subjected to the 2-acetylaminofluorene/partial hepatectomy model of oval cell-mediated liver regeneration, followed by administration of either nonpegylated G-CSF or pegylated G-CSF. Control animals did not receive further treatments after surgery. The magnitude of oval cell reaction, the entity of BM contribution to liver repopulation, as well as the G-CSF/G-CSF-receptor expression levels were evaluated. In addition, in vitro proliferation and migration assays were performed on freshly isolated oval cells. RESULTS: Oval cells were found to express G-CSF receptor and G-CSF was produced within the regenerating liver. G-CSF administration significantly increased both the magnitude of the oval cell reaction, and the contribution of BM to liver repair. Finally, G-CSF acted as a chemoattractant and a mitogen for oval cells in vitro. CONCLUSIONS: We have shown that G-CSF facilitates hepatic regeneration by increasing the migration of BM-derived progenitors to the liver, as well as enhancing the endogenous oval cell reaction.

A potential role of somatostatin and its receptor SSTR4 in the migration of hepatic oval cells.

Somatostatin (SST) is a regulatory peptide that activates G protein-coupled receptors comprised of five members (somatostatin receptors (SSTRs) 1-5). Despite the broad use of SST and its analogs in clinical practice, the spectrum of SST activities has been incompletely defined. Recently, it has been demonstrated that SST can be a chemoattractant for hematopoietic precursor cells. Since hepatic oval cells (HOCs) share common characteristics with hematopoietic stem cells, we hypothesized that SST could act as a chemoattractant for HOCs by stimulating SSTRs. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot assay revealed an increased expression of SST in the 2-acetyl-aminofluorene (2AAF)/partial hepatectomy (PHx) HOC induction model. Immunohistochemical staining showed the expression of SST in 2AAF/PHx-treated rat liver, as compared to normal liver. Proliferation and migration assays demonstrated that the increase of SST was related to migration of HOCs, but not their proliferation. RT-PCR and quantitative real-time PCR showed that SSTR4 was preferentially expressed by HOCs. Western blot assay and immunohistochemical staining confirmed the expression of SSTR4 by HOCs. In addition, pretreatment with anti-SSTR4 antibody cultures resulted in a dramatic reduction of cell migration as compared to that of control. Lastly, SST stimulated the rearrangement of actin filaments in HOCs, while HOCs treated with anti-SSTR4 antibody failed to do so. These results suggest a positive role for SST in the migration of HOCs, and that this effect is mediated through SSTR4.