Development Of Bioartificial Liver Research Articles

Utilizing an automation-on-demand analytical tool for cancer immunotherapy

Richard Parker-Manuel, PhD, is a Group Leader for Plasmid Engineering and Production at OXGENE, A WuXi Advanced Therapies Company. He has twenty years’ experience in molecular biology with nearly seven years in the cell and gene therapy space. He oversees the design and engineering of adeno associated virus and lentivirus vector plasmids for a variety of clients. He is also involved in several R&D initiatives with the aim of making OXGENE’s excellent vectors even better.Qian Liu, PhD, joined OXGENE in July 2017 as a Cell Line Engineering Scientist. She has a background in cell and molecular biology, She was then gradually promoted to lead all biomanufacturing services OXGENE provide. Prior to joining OXGENE, Qian was a postdoctoral researcher in the field of Regenerative Medicine, involved in bioartificial liver development and stem cell differentiation mechanism investigation in Loughborough University and the University of Nottingham, respectively. Qian obtained her PhD in Molecular Nutrition from the University of Nottingham.

Function of hepatocyte spheroids in bioactive microcapsules is enhanced by endogenous and exogenous hepatocyte growth factor

The ability to maintain functional hepatocytes has important implications for bioartificial liver development, cell-based therapies, drug screening, and tissue engineering. Several approaches can be used to restore hepatocyte function in vitro, including coating a culture substrate with extracellular matrix (ECM), encapsulating cells within biomimetic gels (Collagen- or Matrigel-based), or co-cultivation with other cells. This paper describes the use of bioactive heparin-based core-shell microcapsules to form and cultivate hepatocyte spheroids. These microcapsules are comprised of an aqueous core that facilitates hepatocyte aggregation into spheroids and a heparin hydrogel shell that binds and releases growth factors. We demonstrate that bioactive microcapsules retain and release endogenous signals thus enhancing the function of encapsulated hepatocytes. We also demonstrate that hepatic function may be further enhanced by loading exogenous hepatocyte growth factor (HGF) into microcapsules and inhibiting transforming growth factor (TGF)-β1 signaling. Overall, bioactive microcapsules described here represent a promising new strategy for the encapsulation and maintenance of primary hepatocytes and will be beneficial for liver tissue engineering, regenerative medicine, and drug testing applications.

Bioartificial Liver Manufacturing Methodologies in Comparison to Hepatogenesis

End-stage organ failure is a major global issue, with the liver being the second-highest transplanted organ due to lifestyle choices or other conditions. In the field of biomedical engineering, artificial organ manufacturing has been a possible alternative to organ transplants by aiming to achieve less immune rejection, more efficient production, and higher accessibility. Of the biological manufacturing methods today, two up-and-coming technologies for the liver include 3D bioprinting and decellularized organ regeneration. By analyzing general stages of 3D bioprinting and decellularization for liver development and comparing it to the cellular and genetic stages of hepatic development in the embryo, dilemmas and successes in bioartificial manufacturing can be identified. Overall, neither artificial methodology can replicate the genetic influence or non-liver based influence of hepatogenesis. 3D bioprinting is similar to hepatogenesis in cell development and construction, but contains shortcomings in vascularization, which can be addressed using the vasculogenesis and angiogenesis processes in hepatic development. Decellularization allows for cell differentiation, although it is unable to instill natural cellular distribution and gradual ECM development. This may cause blockages for clinical use in the future. Both methods have potential for bioartificial liver development and clinical application, but 3D bioprinting technology is more aligned with the stages of hepatic development, which has proven to induce fewer errors in the physiological nature of the manufactured liver. In the future, modification and research must be conducted on the specific stages of these methodologies for either to create an operational bioartificial liver for clinical use.

Hepatic organoids: What are the challenges?

The study and understanding of liver organogenesis have allowed the development of protocols for pluripotent stem cells differentiation to overcome the lack of primary cells, providing an almost unlimited source of liver cells. However, as their differentiation in conventional 2D culture systems has shown serious limits, hepatic organoids derived from human pluripotent stem cells represent a promising alternative. These complex and organized structures, containing one or more cell types, make it possible to recapitulate in vitro some of the organ functions, thus enabling numerous applications such as the study of the liver development, the mass production of functional liver cells for transplantation or the development of bioartificial livers, as well as the in vitro modeling of hepatic pathologies allowing high throughput applications in drug screening or toxicity studies. Economic and ethical issues must also be taken into account before using these organoids in therapeutic applications.

Polyethersulfone-carbon nanotubes composite hollow fiber membranes with improved biocompatibility for bioartificial liver

Carbon nanotubes (CNTs) blended hollow fiber membranes (HFMs) are a promising new material in the area of biomedical engineering because they simultaneously provide tunable hydrophilicity along with selective permeability. In the present study, composite polyethersulfone (P) HFMs were fabricated using d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS or T) as compatibilizer, and carboxylated multiwalled CNTs (MWCNTs or C) as filler. The amount of MWCNTs was optimized for the improved hemocompatibility, cell viability, and cellular functionality. An optimum was found with the composte HFMs (PTC-2), where MWCNTs were used at concentration of 0.030 wt.%, as it exhibited improved compatibility with human blood. Further, these PTC-2 HFMs showed enhanced liver (HepG2) cells growth with the enhanced cell functional activities, mainly albumin secretion and glucose consumption. These developed composite membrane can act as a membrane material for liver cell bioreactor and bioartificial liver development because of their 3D scaffold like characteristic which enables cell growth, and selective permeability which helps in immunoisolation.

Bioinspired Engineering for Liver Tissue Regeneration and Development of Bioartificial Liver: A Review.

Patients with advanced liver disease have very high mortality due to associated complications such as hepatic encephalopathy, hepatorenal syndrome, and multiorgan failure. Liver transplant is the only therapeutic option for such patients; however, problems linked to availability, cost, complications, and side effects limit its practical application. The strong potential for hepatic regeneration and recovery has been documented in liver disease, without advance decomposition of the liver. Recently, hepatocyte transplantation was used as an alternative to liver transplantation, but insufficient numbers of functional hepatocytes for therapeutic efficacy limits its use. In addition, extracorporeal liver support systems are a modality for patient management or they can be used as a bridge to a possible liver transplant. But such systems lack clear-cut survival benefits, specifically in advanced liver disease. Due to the limited amount of organ donations and living donor liver transplantation across the globe, novel technologies have been proposed for development of three-dimensional (3D) composite constructs, 3D perfused bioreactors for spheroid culture, liver-on-chip platforms, and bioprinted liver to produce an implantable in vitro liver that can reliably predict in vivo-like tissue responses and suitability for drug toxicity testing. These research fields stand to be game changers in regeneration and repair of liver tissues in patients. This review focuses on novel technologies that are currently used for liver regeneration and production of transplantable liver.

Enhanced intrinsic CYP3A4 activity in human hepatic C3A cells with optically controlled CRISPR/dCas9 activator complex.

Human hepatic C3A cells have been applied in bioartificial liver development, although these cells display low intrinsic cytochrome P450 3A4 (CYP3A4) enzyme activity. We aimed to enhance CYP3A4 enzyme activity of C3A cells utilizing CRISPR gene editing technology. We designed two CYP3A4 expression enhanced systems applying clustered regularly interspaced short palindromic repeats (CRISPR) gene technology: a CRISPR-on activation system including dCas9-VP64-GFP and two U6-sgRNA-mCherry elements, and a light-controlled CRISPR-on activation system combining our CRISPR-on activation system with an optical control system to facilitate regulation of CYP3A4 expression for various applications. Results of enzymatic activity assays displayed increased CYP3A4 activity in C3A cells expressing the CRISPR-on activation system compared with C3A cells. In addition, CYP3A4 activity increased in C3A cells expressing the light-controlled CRISPR-on activation system under blue light radiation compared with C3A cells. Notably, there was no statistical difference in the increase of CYP3A4 protein amounts induced by these two methods. After expansion in culture, C3A cells with the light-controlled CRISPR-on activation system exhibited no statistical difference in CYP3A4 mRNA levels between generations. Our findings provide a method to stably enhance functional gene expression in bioartificial liver cells with the potential for large-scale cell expansion.

The development and status of bioartificial liver

Liver failure is a serious stage during liver disease development of which mobidity is high. There is no effective treatment at present.Artificial liver support system is one of the important methods to treat liver failure which includes non-biological artificial liver, biological artificial liver and hybrid artificial liver. Among the artificial liver devices. The bioartificial liver is the most similar artifical liver device to human liver in terms of detoxification, synthesis and metabolism currently.The complexity of human liver function makes the biological artificial liver facing great challenges in selection of liver seed cells, construction of bioreactor and the best combination with auxiliary device, which leads to the slow development of bioartificial liver. In order to provide theoretical support for the study of bioartificial liver, the current status and its development are reviewed in the following aspects, the source of seed cells, the construction of bioreactor, the combination of auxiliary devices and the clinical application of bioartificial liver in this article.

Nanofibers promote HepG2 aggregate formation and cellular function.

Formation of hepatocyte spheroids is a necessary strategy for increasing liver-specific function in vitro. In this study, HepG2 cells showed good viability when grown on a polylactic acid-chitosan (PLA-CS) nanofiber and aggregated to form multicellular spheroids on the PLA-CS nanofibers with a diameter of approximately 100-200 mm in 5 days of culture, whereas no such aggregation was observed in cells cultured on 24-well plates. Hepatocyte spheroids formed on the PLA-CS nanofibers displayed excellent hepatic-related protein expression, such as albumin and urea, compared to HepG2 cells cultured on the 24-well plates. These results indicated that formation of the hepatocyte spheroids in nanofibers can increase and maintain hepatocyte functions for a longer time, supporting a new strategy for bioartificial liver development.

Decellularization and Recellularization of Rat Livers With Hepatocytes and Endothelial Progenitor Cells.

Whole-organ decellularization has been identified as a promising choice for tissue engineering. The aim of the present study was to engineer intact whole rat liver scaffolds and repopulate them with hepatocytes and endothelial progenitor cells (EPCs) in a bioreactor. Decellularized liver scaffolds were obtained by perfusing Triton X-100 with ammonium hydroxide. The architecture and composition of the original extracellular matrix were preserved, as confirmed by morphologic, histological, and immunolabeling methods. To determine biocompatibility, the scaffold was embedded in the subcutaneous adipose layer of the back of a heterologous animal to observe the infiltration of inflammatory cells. Hepatocytes were reseeded using a parenchymal injection method and cultured by continuous perfusion. EPCs were reseeded using a portal vein infusion method. Morphologic and functional examination showed that the hepatocytes and EPCs grew well in the scaffold. The present study describes an effective method of decellularization and recellularization of rat livers, providing the foundation for liver engineering and the development of bioartificial livers.

Effect of Dimethyl Sulfoxide and Melatonin on the Isolation of Human Primary Hepatocytes

The availability of fully functional human hepatocytes is critical for progress in human hepatocyte transplantation and the development of bioartificial livers and in vitro liver systems. However, the cell isolation process impairs the hepatocyte status and determines the number of viable cells that can be obtained. This study aimed to evaluate the effects of using dimethyl sulfoxide (DMSO) and melatonin in the human hepatocyte isolation protocol. Human hepatocytes were isolated from liver pieces resected from 10 patients undergoing partial hepatectomy. Each piece was dissected into 2 equally sized pieces and randomized, in 5 of 10 isolations, to perfusion with 1% DMSO-containing perfusion buffer or buffer also containing 5 m<smlcap>M</smlcap> melatonin using the 2-step collagenase perfusion technique (experiment 1), and in the other 5 isolations to standard perfusion or perfusion including 1% DMSO (experiment 2). Tissues perfused with DMSO yielded 70.6% more viable hepatocytes per gram of tissue (p = 0.076), with a 26.1% greater albumin production (p < 0.05) than those perfused with control buffer. Melatonin did not significantly affect (p > 0.05) any of the studied parameters, but cell viability, dehydrogenase activity, albumin production, urea secretion, and 7-ethoxycoumarin O-deethylase activity were slightly higher in cells isolated with melatonin-containing perfusion buffer compared to those isolated with DMSO. In conclusion, addition of 1% DMSO to the hepatocyte isolation protocol could improve the availability and functionality of hepatocytes for transplantation, but further studies are needed to clarify the mechanisms involved.

Blood-Compatible Polymer for Hepatocyte Culture with High Hepatocyte-Specific Functions toward Bioartificial Liver Development.

The development of bioartificial liver (BAL) is expected because of the shortage of donor liver for transplantation. The substrates for BAL require the following criteria: (a) blood compatibility, (b) hepatocyte adhesiveness, and (c) the ability to maintain hepatocyte-specific functions. Here, we examined blood-compatible poly(2-methoxyethyl acrylate) (PMEA) and poly(tetrahydrofurfuryl acrylate) (PTHFA) (PTHFA) as the substrates for BAL. HepG2, a human hepatocyte model, could adhere on PMEA and PTHFA substrates. The spreading of HepG2 cells was suppressed on PMEA substrates because integrin contribution to cell adhesion on PMEA substrate was low and integrin signaling was not sufficiently activated. Hepatocyte-specific gene expression in HepG2 cells increased on PMEA substrate, whereas the expression decreased on PTHFA substrates due to the nuclear localization of Yes-associated protein (YAP). These results indicate that blood-compatible PMEA is suitable for BAL substrate. Also, PMEA is expected to be used to regulate cell functions for blood-contacting tissue engineering.

Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation.

Liver synthetic and metabolic function can only be optimised by the growth of cells within a supportive liver matrix. This can be achieved by the utilisation of decellularised human liver tissue. Here we demonstrate complete decellularization of whole human liver and lobes to form an extracellular matrix scaffold with a preserved architecture. Decellularized human liver cubic scaffolds were repopulated for up to 21 days using human cell lines hepatic stellate cells (LX2), hepatocellular carcinoma (Sk-Hep-1) and hepatoblastoma (HepG2), with excellent viability, motility and proliferation and remodelling of the extracellular matrix. Biocompatibility was demonstrated by either omental or subcutaneous xenotransplantation of liver scaffold cubes (5 × 5 × 5 mm) into immune competent mice resulting in absent foreign body responses. We demonstrate decellularization of human liver and repopulation with derived human liver cells. This is a key advance in bioartificial liver development.

Rapid cell-fate conversion of mouse fibroblasts into hepatocyte-like cells

Recent progress in studies on direct cell-fate conversion of differentiated somatic cells into other cell types, which is known as “direct reprogramming”, is expected to lead to innovations in health care. In our previous study, we found that three specific combinations of two transcription factors, comprising Hnf4α plus Foxa1, Foxa2, or Foxa3, were able to induce conversion of mouse fibroblasts into functional hepatocyte-like cells. These induced hepatocyte-like (iHep) cells will be useful for developing regenerative therapies for liver diseases and examining the pharmacological effects of drugs. However, to evaluate the potential utility of iHep cells, the phenomena involved in the direct conversion of fibroblasts into iHep cells should be examined in detail. Thus, in this study, we sequentially analyzed the early stage of fibroblast conversion into iHep cells after infection with retroviruses expressing Hnf4α and Foxa3. Our data demonstrated that the conversion into iHep cells began within 2 days after introduction of the transgenes into fibroblasts, and the number of iHep cells increased gradually as the culture progressed. The rapid cell-fate conversion of fibroblasts into iHep cells and stable expansion of iHep cells are two pieces of evidence suggesting the utility of iHep cells for cell transplantation therapy, bioartificial liver development, and screening of drugs for patients with liver diseases, which require many hepatocytes within a short period of time.

Efficient large-scale generation of functional hepatocytes from mouse embryonic stem cells grown in a rotating bioreactor with exogenous growth factors and hormones

IntroductionEmbryonic stem (ES) cells are considered a potentially advantageous source of hepatocytes for both transplantation and the development of bioartificial livers. However, the efficient large-scale generation of functional hepatocytes from ES cells remains a major challenge, especially for those methods compatible with clinical applications.MethodsIn this study, we investigated whether a large number of functional hepatocytes can be differentiated from mouse ES (mES) cells using a simulated microgravity bioreactor. mES cells were cultured in a rotating bioreactor in the presence of exogenous growth factors and hormones to form embryoid bodies (EBs), which then differentiated into hepatocytes.ResultsDuring the rotating culture, most of the EB-derived cells gradually showed the histologic characteristics of normal hepatocytes. More specifically, the expression of hepatic genes and proteins was detected at a higher level in the differentiated cells from the bioreactor culture than in cells from a static culture. On further growing, the EBs on tissue-culture plates, most of the EB-derived cells were found to display the morphologic features of hepatocytes, as well as albumin synthesis. In addition, the EB-derived cells grown in the rotating bioreactor exhibited higher levels of liver-specific functions, such as glycogen storage, cytochrome P450 activity, low-density lipoprotein, and indocyanine green uptake, than did differentiated cells grown in static culture. When the EB-derived cells from day-14 EBs and the cells’ culture supernatant were injected into nude mice, the transplanted cells were engrafted into the recipient livers.ConclusionsLarge quantities of high-quality hepatocytes can be generated from mES cells in a rotating bioreactor via EB formation. This system may be useful in the large-scale generation of hepatocytes for both cell transplantation and the development of bioartificial livers.

The effects of human umbilical cord perivascular cells on rat hepatocyte structure and functional polarity

Hepatocyte culture is a useful tool for the study of their biology and the development of bioartificial livers. However, many challenges have to be overcome since hepatocytes rapidly lose their normal phenotype in vitro. We have recently demonstrated that human umbilical cord perivascular cells (HUCPVCs) are able to provide support to hepatocytes. In the present study we go further into exploring the effects that HUCPVCs have in the functional polarization, and both the internal and external organization, of hepatocytes. Also, we investigate HUCPVC-hepatocyte crosstalk by tracking both the effects of HUCPVCs on hepatocyte transcription factors and those of hepatocytes on the expression of hepatotrophic factors in HUCPVCs. Our results show that HUCPVCs maintain the functional polarity of hepatocytes ex vivo, as judged by the secretion of fluorescein into bile canaliculi, for at least 40 days. Transmission electron microscopy revealed that hepatocytes in coculture organize in an organoid-like structure embedded in extracellular matrix surrounded by HUCPVCs. In coculture, hepatocytes displayed a higher expression of C/EBPα, implicated in maintenance of the mature hepatocyte phenotype, and HUCPVCs upregulated hepatocyte growth factor and Jagged1 indicating that these genes may play important roles in HUCPVC-hepatocyte interactions.

Effect of Fulminant Hepatic Failure Porcine Plasma Supplemented with Essential Components on Encapsulated Rat Hepatocyte Spheroids

The development of bioartificial liver (BAL) systems has required detailed information about the functional capabilities of cultured hepatocytes during blood or plasma passage. In this study we investigated the effects of porcine plasma and various supplements on the viability and function of adult rat hepatocytes in vitro. Primary rat hepatocytes cultured in porcine plasma supplemented with various substances showed albumin synthesis rates and viability equal to or higher than those of controls. Supplementation with calcium chloride, magnesium sulfate, trace elements, amino acids, insulin, and epidermal growth factor were essential to maintain viability and high albumin synthesis. Especially, trace elements showed significantly higher and longer albumin secretion.Isolated rat hepatocytes were cultured in Spinner flasks for 24 hours to form spheroids that were harvested and encapsulated with chitosan-alginate solution before transfer to the bioreactor in the BAL system. Encapsulated rat hepatocyte spheroids cultured with porcine plasma including trace elements showed higher viability (57%) than controls (40%) after 24 hours, with ammonia removal values of 30.92 μg/106 cells versus the control 9.04 μg/106 cells. After 24 hours of operation the urea secretion value of encapsulated rat hepatocyte spheroids cultured in porcine plasma in the presence versus absence of trace elements was 76.73 μg/106 cells and 18.80 μg/106 cells, respectively. We concluded that encapsulated hepatocyte spheroids in a packed-bed bioreactor operated with human plasma including trace elements enhanced cell viability and liver function as a bases for an in vivo clinical trial of the BAL system.

Current development of bioreactors for extracorporeal bioartificial liver (Review)

The research and development of extracorporeal bioartificial liver is gaining pace in recent years with the introduction of a myriad of optimally designed bioreactors with the ability to maintain long-term viability and liver-specific functions of hepatocytes. The design considerations for bioartificial liver are not trivial; it needs to consider factors such as the types of cell to be cultured in the bioreactor, the bioreactor configuration, the magnitude of fluid-induced shear stress, nutrients' supply, and wastes' removal, and other relevant issues before the bioreactor is ready for testing. This review discusses the exciting development of bioartificial liver devices, particularly the various types of cell used in current reactor designs, the state-of-the-art culturing and cryopreservation techniques, and the comparison among many today's bioreactor configurations. This review will also discuss in depth the importance of maintaining optimal mass transfer of nutrients and oxygen partial pressure in the bioreactor system. Finally, this review will discuss the commercially available bioreactors that are currently undergoing preclinical and clinical trials.

Three-Dimensional Primary Hepatocyte Culture in Synthetic Self-Assembling Peptide Hydrogel

Drug metabolism studies and liver tissue engineering necessitate stable hepatocyte cultures that express liver functions for a minimum of 4 days to 3 weeks. Current techniques, using different biomaterials and geometries, that maintain hepatocellular function in vitro exhibit a low cell density and functional capacity per unit volume. Herein we investigated a well-defined synthetic peptide that can self-assemble into three-dimensional interweaving nanofiber scaffolds to form a hydrogel, PuraMatrix, as a substrate for hepatocyte culture. Freshly isolated primary rat hepatocytes attached, migrated, and formed spheroids within 3 days after seeding on PuraMatrix. Hepatocytes expressed the apical membrane marker dipeptidyl peptidase IV at cell-cell contacts. Compared to the collagen sandwich, albumin and urea secretion on PuraMatrix were higher for the first week, and cytochrome P450IA1 activity was higher throughout the culture period. Mitochondrial membrane potential 1 day after seeding was higher on PuraMatrix than in the collagen sandwich, suggesting better preservation of the metabolic machinery. PuraMatrix and Matrigel showed similar albumin and urea production. PuraMatrix is an attractive system for generating hepatocyte spheroids that quickly restore liver functions after seeding. This system is also amenable to scale-up, which makes it suitable for in vitro toxicity, hepatocyte transplantation, and bioartificial liver development studies.

Induction and High Density Culture of Human Hepatoblasts from Fetal Hepatocytes with Suppressing Transformation

It is well known that it would be important to cultivate human hepatocytes of about 10(10) cells at a high cell density, about 1 x 10(7) cells/cm(3), in the bioreactor for the development of bioartificial liver. However, since primary human hepatocytes lack an ability to proliferate in vitro, it is essential to establish a culture method for the proliferation of normal human hepatic stem cells as a cell source. In this study, it was found that human hepatoblasts, a kind of hepatic stem cells, were induced from human fetal hepatocytes while keeping the ability of proliferation by the treatment of 1mM sodium butyrate (SB) for 12 d of culture. The transformation of hepatoblasts was evaluated by abnormal prothrombin (PIVKA-II) assay, which is a clinical marker for hepatocellular carcinoma. The PIVKA-II production rate of the cells was suppressed to the normal level under 1 mM SB. The cells including hepatoblasts under 1 mM SB attached to the porous hydroxyapatite carriers and proliferated to a high cell density of about 1 x 10(7) cells/cm(3) in the carriers. The liver-specific function, cytochrome P450 3A4 activity (4.2 pmol/mg protein/min) of the cells in the carriers under 1 mM SB was comparable to that of primary human hepatocytes. Ammonia metabolizing activity (0.21 micromol/10(6) cells/h) of the cells was also comparable to that of porcine hepatocytes used in the bioartificial liver. The PIVKA-II production rate of the cells in the carrier was suppressed to the normal level. These results suggested that induction of human hepatoblasts from fetal hepatocytes by the treatment of 1mM SB and proliferation of the cells at a high cell density using hydroxyapatite carriers should be one of the more promising culture methods for bioartificial liver developments.