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A schematic diagram of the extracorporeal circulation system containing the PUF-HAL module. Rats with a partial hepatectomy were treated with a PUF-HAL module with immobilized differentiating mouse ES cells (ES-HAL, n = 3) or primary mouse hepatocytes (Hep-HAL, n = 3). The same system without cells in the PUF-HAL module was used as a control (Control, n = 4).
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Hybrid artificial liver (HAL) is an extracorporeal circulation system comprised of a bioreactor containing immobilized functional liver cells. It is expected to not only serve as a temporary liver function support system, but also to accelerate liver regeneration in recovery from hepatic failure. One of the most difficult problems in developing a h...
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Aim:
There are no beneficial therapies except for emergency liver transplantation for acute liver failure (ALF). However, in Japan where a serious problem of a shortage of donor livers still remains, therapies other than transplantation must be further investigated for patients with ALF. Pro-inflammatory cytokines promoting tissue destruction are...
Citations
... In one study, mouse ESCs (mESCs) were used in ALF-model rats. Overall, the authors showed that, even if cells were not fully differentiated in hepatocytes-like cells, their bioreactor containing mESCs-derived hepatocytes had a good potential to support liver function 115 . In another study, the authors used mESCs in a hollow-fibre based BAL. ...
For all patients suffering of acute liver failure, there is an urgent need of alternatives to liver transplantation. Due to a variety of factors, those patients do not always have easy access to an available organ and die waiting for a transplant. Therefore, it is imperative to find viable alternatives to liver transplantation. Among the various alternatives that emerged in recent years, our group has mainly investigated the concept of bioartificial liver. It is an extracorporeal circulation device equipped with artificial elements (activated charcoal and ionic resin) and a biological element (hepatic biomass) capable of supporting the failing organ. This device has the role of providing hepatic functions, lost due to the disease, helping the patient to remain alive until an organ is available or, otherwise, capable of promoting natural liver regeneration. This thesis retraces the evolution of these devices over time, describing their principle of operation and the main results proposed by the literature. The focus is then moved on the development of our liver supply device and the scientific path that allowed optimizing the hepatic biomass at best. This thesis work led to the development of a ready-to-use BAL device on animal models of acute liver failure.
... Hiroshi Mizumoto used a polyurethane foam (PUF)/spheroid culture to differentiate embryonic stem (ES) cells of hepatic lineage, and cells spontaneously formed spherical multicellular aggregates (spheroids) in the pores of the PUF. However, cell number decreased after 15 days of culture [10]. We worked on spacing tissue elements with the inclusion of poly (l, l-lactide) (PLLA) fibers in cellular aggregates, which partly improved local mass transfer for cultured cells, but was still limited by aggregate disintegration during a prolonged period of perfusion culture [11]. ...
The aim of the present study was to design and fabricate polyglycolic acid (PGA) modules on the basis of the Raschig ring as a tissue element for bottom–top tissue engineering to increase the feasibility of cellular-assembly technology. Three types of modules, namely, cylindrical, Raschig ring, and transverse-pore modules, with different numbers and orientations of canals, were designed and fabricated by modified selective-laser-sintering (SLS) technology. These modules maintained their structure in a flowing culture environment, and degradation did not create an acidic environment, hence promoting their ability to scale up to highly functional tissue. The modules were seeded with human hepatoma Hep G2 cells and cultured for 10 days. The transverse-pore modules were found to have the highest glucose consumption, albumin production, and cell viability among the three tested modules. Our study showed that the proposed module design provided better mass transfer and possessed the required mechanical strength to enable use in the construction of large tissue.
... Stem cells are other alternative cell source candidates. Embryonic stem (ES) cells (Martin 1981;Evans and Kaufman 1981) and induced pluripotent stem cells (Takahashi and Yamanaka 2006;Takahashi et al. 2007) have recently gained attention as new cell sources due to their infinite proliferation capacity and ability to differentiate into hepatocytes (Mizumoto et al. 2012(Mizumoto et al. , 2018Sakiyama et al. 2017). On the other hand, before employing pluripotent stem cells in BAL devices, problems such as teratoma formation risk and low efficiency of differentiation into hepatocytes must be resolved. ...
The bioartificial liver (BAL) device is an extracorporeal liver support system incorporating living hepatocytes. A major problem in BAL device development is to obtain a high number of functional cells. In this study, we focused on a genetically engineered mouse hepatoma cell line, Hepa/8F5, in which elevated liver functions are induced via overexpression of liver-enriched transcription factors activated by doxycycline (Dox) addition. We applied a three-dimensional culture technique using hollow fibers (HFs) to Hepa/8F5 cells. Hepa/8F5 cells responded to Dox addition by reducing their proliferative activity and performing liver-specific functions of ammonia removal and albumin secretion. The functional activities of cells depended on the timing of Dox addition. We also found that Hepa/8F5 cells in the HF culture were highly functional in a low rather than high cell density environment. We further fabricated an HF-type bioreactor with immobilized Hepa/8F5 cells as a BAL device. Although ammonia removal activity of this BAL device was lower than that of the small-scale HF bundle, albumin secretion activity was slightly higher. These results indicated that the BAL device with immobilized Hepa/8F5 cells was highly functional with potential to show curative effects in liver failure treatment.
... Even though fully matured primary human hepatocytes (PHH) exhibit all the specific liver functions, their limited availability and loss of liver specific functions during culturing in vitro are still the major limitations for their application in a bioartificial liver (BAL) (Ordovás et al., 2013, Mizumoto et al., 2012. Therefore, porcine primary hepatocytes and carcinoma cell lines (HepG2, HepG2/C3A and HepaRG) have been widely employed in liver engineer- ing (Nibourg et al., 2012, Palakkan et al., 2013, Schwartz et al., 2014). ...
Hepatic differentiation of hiPSCs under flow conditions in a 3D scaffold is expected to be a major step forward for construction of bioartificial livers. The aims of this study were to induce hepatic differentiation of hiPSCs under perfusion conditions and to perform functional comparisons with fresh human precision cut liver slices (hPCLS), an excellent benchmark for the human liver in vivo. The majority of the mRNA expression of CYP isoenzymes and transporters and the tested CYP activities, phase II metabolism, and albumin, urea and bile acid synthesis in the hiPSC derived cells reached values that overlap those of hPCLS, which indicates a higher degree of hepatic differentiation than observed until now. Differentiation under flow compared to static conditions had a strong inducing effect on phase II metabolism, suppressed AFP expression but resulted in slightly lower activity of some of the phase I metabolism enzymes. Gene expression data indicates that hiPSCs differentiated into both hepatic and biliary directions. In conclusion, the hiPSC differentiated under flow conditions towards hepatocytes express a wide spectrum of liver functions at levels comparable to hPCLS indicating excellent future perspectives for the development of a bioartificial liver system for toxicity testing or as liver support device for patients.
... These include the spatial organization of heterogeneous tissue-specific cells, cell-cell, and cell-matrix interactions, and certain physiological functions of tissue-specific cells [9]. Liver organoids can be derived directly by liver tissue isolation, and can also be independently established by Yimlamai D 2014 [78] FLCs (1) Gene expression: Afp, Alb, α1-antitrypsin, glucose-6 phosphatase, Ck19, Bgp; (2) Suspended/embedded (collagen Type I) Gabriel E 2012 [82] (1) Ammonia removal activity; (2) albumin concentration (ELISA); (3) ex vivo experiment Scaffold Mizumoto H 2012 [83] (1) Glucose levels; (2) albumin concentration (ELISA); (3) activity of ammonia detoxification; (4) urea synthesis ability Scaffold: transferrin, hydrocortisone-21-hemisucci-nate, ascorbic acid, insulin, niacinamide, dexamethasone Teratani T 2005 [84] (1) Gene expression: Sox17, Hnf3β, Afp, Alb, Aat, Glut2, Gys2, Tat, Lst-1, CK8, CK18, CK19, CD34, etc.; (2) protein expression: AAT, AFP, ALB, CK18, CD34, c-KIT, THY-1, DLK; (3) differentiation potential; (4) PAS staining ...
... Microfluidic devices have been utilized in cell culture [16][17][18], cell differentiation [19][20][21][22], dynamic gene expression [23,24], and test of cellular response to chemical gradients [25][26][27]. Studies of hepatic differentiation from MSCs using microfluidic devices [20] and bioreactors [28][29][30][31][32][33][34][35][36][37][38][39][40], together with 3D scaffolds [41], have been reported. However, there are shortcomings when using conventional microfluidic devices to promote hepatic differentiation. ...
Background
Mesenchymal stromal cells (MSCs) are multipotent and have great potential in cell therapy. Previously we reported the differentiation potential of human MSCs into hepatocytes in vitro and that these cells can rescue fulminant hepatic failure. However, the conventional static culture method neither maintains growth factors at an optimal level constantly nor removes cellular waste efficiently. In addition, not only is the duration of differentiating hepatocyte lineage cells from MSCs required to improve, but also the need for a large number of hepatocytes for cell therapy has not to date been addressed fully. The purpose of this study is to design and develop an innovative microfluidic device to overcome these shortcomings. Methods
We designed and fabricated a microfluidic device and a culture system for hepatic differentiation of MSCs using our protocol reported previously. The microfluidic device contains a large culture chamber with a stable uniform flow to allow homogeneous distribution and expansion as well as efficient induction of hepatic differentiation for MSCs. ResultsThe device enables real-time observation under light microscopy and exhibits a better differentiation efficiency for MSCs compared with conventional static culture. MSCs grown in the microfluidic device showed a higher level of hepatocyte marker gene expression under hepatic induction. Functional analysis of hepatic differentiation demonstrated significantly higher urea production in the microfluidic device after 21 days of hepatic differentiation. Conclusions
The microfluidic device allows the generation of a large number of MSCs and induces hepatic differentiation of MSCs efficiently. The device can be adapted for scale-up production of hepatic cells from MSCs for cellular therapy.
... The liver-specific functions of the incubated cells on the galactose-carrying polymer, including ammonia removal and albumin secretion, have been found to be enhanced over the long term. 10,11 Moreover, a chimeric protein, E-cadherin-Fc (EFC), composed of the E-cadherin extracellular domain and the immunoglobulin G (IgG) Fc domain, is capable of enhancing the cellular viability of endodermal cells. 12 The protein-coated surface could further preserve the hepatocellular functions and metabolism of hepatic cell lineages. ...
Cell culture systems have proven to be crucial for the in vitro maintenance of primary hepatocytes and the preservation of hepatic functional expression at a high level. A poly-(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide) matrix can recognize cells and promote liver function in a spheroid structure because of a specific galactose–asialoglycoprotein receptor interaction. Meanwhile, a fusion protein, E-cadherin-Fc, when incubated with various cells, has shown an enhancing effect on cellular viability and metabolism. Therefore, a hybrid substratum was developed for biomedical applications by using both of these materials to combine their advantages for primary hepatocyte cultures. The isolated cells showed a monolayer aggregate morphology on the coimmobilized surface and displayed higher functional expression than cells on traditional matrices. Furthermore, the hybrid system, in which the highest levels of cell adhesion and hepatocellular metabolism were achieved with the addition of 1% fetal bovine serum, showed a lower serum dependency than the collagen/gelatin-coated surface. Accordingly, this substrate may attenuate the negative effects of serum and further contribute to establishing a defined culture system for primary hepatocytes.
... The rotating bioreactor of the RCCS creates a simulated microgravity, allowing the ES cells to grow and differentiate in three-dimensional (3D) culture [15]. Hepatocytes in 3D multicellular aggregates or spheroids have been shown to maintain their morphology and ultrastructural characteristics, resulting in higher degrees of liver-specific functions [16,17]. Previous reports have also revealed that mouse ES (mES) cells differentiate into hepatocytes via embryoid body (EB) formation in vitro [18,19]. ...
Embryonic 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.
In 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.
During the rotating culture, most of the EB-derived cells gradually showed the histological 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. Upon further growing the EBs on tissue-culture plates, most of the EB-derived cells were found to display the morphological 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 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.
Large 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.
... 3D constructs remained viable, undergoing further differentiation and maturation to hepatic-like cells in vivo. In other studies, polyurethane foam was used to capture and differentiate mESC to hepatic cells in a hybrid bioartificial liver system (Matsumoto et al., 2008;Mizumoto et al., 2012). Polyurethane foam spheroid culture allowed spontaneous formation of aggregates inside the scaffold and large-scale differentiation of stem cells. ...
