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1.
Enhanced maintenance and functions of rat hepatocytes induced by combination of on-site oxygenation and coculture with fibroblasts 总被引:4,自引:0,他引:4
Nishikawa M Kojima N Komori K Yamamoto T Fujii T Sakai Y 《Journal of biotechnology》2008,133(2):253-260
In in vivo liver tissue, each hepatocyte has intimate interactions not only with adjacent hepatocytes but also with nonparenchymal cells in a three-dimensional (3D) manner. We recently reported that hepatic function is highly maintained on collagen covalently immobilized poly-dimethylsiloxane (PDMS) membranes through which oxygen is supplied directly to the cells. In this study, to further enhance performances of hepatocytes culture, we investigated cocultivation of rat hepatocytes with a mouse fibroblast, NIH/3T3 (3T3) in the same PDMS membranes. Various functions of hepatocytes were better maintained on the membrane at remarkably higher levels, particularly albumin secretion on such coculture was about 20 times higher than that in conventional coculture on tissue-culture-treated polystyrene (TCPS) surfaces. The remarkable functional enhancements are likely to be explained by the net growth of hepatocytes (from 1.2- to 1.4-fold inoculated number) and very intimate contact between hepatocytes and 3T3 cells in almost continuous double-layered structures under the adequate oxygen supply. The results demonstrate that simultaneous realization of different requirements toward mimicking in vivo liver tissue microstructure is effective in improving performance of hepatocytes culture system. 相似文献
2.
Microfluidic PDMS (polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes 总被引:5,自引:0,他引:5
Microfluidics could provide suitable environments for cell culture because of the larger surface-to-volume ratio and fluidic behavior similar to the environments in vivo. Such microfluidic environments are now used to investigate cell-to-cell interactions and behaviors in vitro, emulating situations observed in vivo, for example, microscale blood vessels modeled by microfluidic channels. These emulated situations cannot be realized by conventional technologies. In our previous works, microfluidic channels composed of two PDMS (poly(dimethylsiloxane)) layers were successfully used for Hep G2 cell culture. To achieve physiologically meaningful functions in vitro, a culture with a larger number of cells and higher density must be performed. This will require bioreactors with larger surface areas for cell attachment and sufficient amounts of oxygen and nutrition supply. For those purposes, we fabricated a bioreactor by stacking 10 PDMS layers together, i.e., four cell culture chambers, and a chamber dedicated to the oxygen supply inserted in the middle of the 10-stacked layers. The oxygen supply chamber is separated from the microfluidic channels for the culture medium perfusion by thin 300-microm PDMS walls. The high gas permeability of PDMS allows oxygen supply to the microfluidic channels through the thin walls. On the basis of the measurement of glucose consumption and albumin production, it is shown that cellular activity exhibits a gradual increase and saturation throughout the culture. We clearly observed that in the case of the microfluidic bioreactor for large-scale cultures, the oxygen chamber is indispensable to achieve longer and healthy cultures. In the present bioreactor, the cell density was found to be about 3-4 x 10(7) cells/cm(3), which is in the same order of magnitude as the conventional macroscale bioreactors. Consequently, by stacking single culture chambers and oxygen chambers in between, we could have a scalable method to realize the microfluidic bioreactor for large-scale cultures. 相似文献
3.
《Biochemical Engineering Journal》2009,46(3):226-231
Co-culture of primary rat hepatocytes with hepatic non-parenchymal cells or sinusoidal endothelial cells for albumin production activity as an index of liver-specific function was studied. The co-cultures were effective for the expression and maintenance of albumin production activity. However, the co-culture effect was not observed when we used a suitable culture medium, which had already been reported to be sufficient for albumin production activity. Albumin production of dispersed cells in collagen gel culture was higher than that of spheroid culture. In addition, albumin production of spheroids in collagen gel culture was higher than that of spheroid culture and dispersed cell collagen gel culture with a suitable culture medium. We found that culture medium composition was more important than co-culture for expression and maintenance of albumin production. Furthermore, we found that cell–cell interaction was effective for the expression of albumin production, but heterotypic cell–cell interaction was not necessary. 相似文献
4.
Perfusion enhanced polydimethylsiloxane based scaffold cell culturing system for multi‐well drug screening platform
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Marshella Tania Myat Noe Hsu Si Ning Png Hwa Liang Leo Guoyang William Toh Erik Birgersson 《Biotechnology progress》2014,30(2):418-428
Conventional two‐dimensional cultures in monolayer and sandwich configuration have been used as a model for in vitro drug testing. However, these culture configurations do not present the actual in vivo liver cytoarchitecture for the hepatocytes cultures and thus they may compromise the cells liver‐specific functions and their cuboidal morphology over longer term culture. In this study, we present a three‐dimensional polydimethylsiloxane (PDMS) scaffold with interconnected spherical macropores for the culturing of rat liver cells (hepatocytes). The scaffolds were integrated into our perfusion enhanced bioreactor to improve the nutrients and gas supply for cell cultures. The liver‐specific functions of the cell culture were assessed by their albumin and urea production, and the changes in the cell morphology were tracked by immunofluorescence staining over 9 days of culture period. N‐Acetyl‐Para‐Amino‐Phenol (acetaminophen) was used as drug model to investigate the response of cells to drug in our scaffold‐bioreactor system. Our experimental results revealed that the perfusion enhanced PDMS‐based scaffold system provides a more conducive microenvironment with better cell‐to‐cell contacts among the hepatocytes that maintains the culture specific enzymatic functions and their cuboidal morphology during the culturing period. The numerical simulation results further showed improved oxygen distribution within the culturing chamber with the scaffold providing an additional function of shielding the cell cultures from the potentially detrimental fluid induced shear stresses. In conclusion, this study could serve a crucial role as a platform for future preclinical hepatotoxicity testing. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:418–428, 2014 相似文献
5.
Various types of bioreactors composed of microstructured PDMS (Polydimethylsiloxane) layers have recently been fabricated for perfusion culture of mammalian cells such as adult rat hepatocytes. As a new feature of those bioreactors, in this study, cultivation of fetal human hepatocytes (FHHs) was attempted, because they have high possibility to mature in vitro with preserving their normality, which is suitable for inplantation of liver tissue equivalents reconstituted in vitro. During the perfusion culture in the PDMS bioreactors for 1 week, cells showed good attachment, spreading and reached their confluence over the channels. In addition, their albumin production was significantly enhanced in the perfusion culture using the PDMS bioreactors up to about four times during the FHH perfusion culture when compared in dish-level static culture. Hep G2 cell cultures were also performed and have also shown under perfusion conditions an enhanced cell activity multiplied by 2 compared to static conditions. Although, the cellular activities of FHH cells are still low even compared to those of the Hep G2 cells, the conclusions of this work is encouraging toward future liver tissue engineering based on in vitro propagation and maturation of hepatocyte progenitors combined with microfabrication technologies. 相似文献
6.
Primary cultured rat hepatocytes in a membrane-supported collagen sandwich maintained their normal cell morphology and high level of albumin secretion for over 56 days. It was found that the existence of an upper layer of collagen gel is crucial for long-term culture and that the transference of cellular nutrients between the culture media and hepatocytes from both the upper and the lower sides of gel layers promotes albumin secretion. These facts suggest that the membrane-supported collagen sandwich mimics well thein vivo environment of hepatocytes. This method has great potential for the long-term culture of primary cells. 相似文献
7.
8.
Jian Zhong Tong Pascal De Lagausie Valerie Furlan Thierry Cresteil Olivier Bernard Fernando Alvarez 《Experimental cell research》1992,200(2):326-332
Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system. 相似文献
9.
Fanny Evenou Morgan Hamon Teruo Fujii Shoji Takeuchi Yasuyuki Sakai 《Biotechnology progress》2011,27(4):1146-1153
To engineer reliable in vitro liver tissue equivalents expressing differentiated hepatic functions at a high level and over a long period of time, it appears necessary to have liver cells organized into a three‐dimensional (3D) multicellular structure closely resembling in vivo liver cytoarchitecture and promoting both homotypic and heterotypic cell–cell contacts. In addition, such high density 3D hepatocyte cultures should be adequately supplied with nutrients and particularly with oxygen since it is one of the most limiting nutrients in hepatocyte cultures. Here we propose a novel but simple hepatocyte culture system in a microplate‐based format, enabling high density hepatocyte culture as a stable 3D‐multilayer. Multilayered co‐cultures of hepatocytes and 3T3 fibroblasts were engineered on collagen‐conjugated thin polydimethylsiloxane (PDMS) membranes which were assembled on bottomless frames to enable oxygen diffusion through the membrane. To achieve high density multilayered co‐cultures, primary rat hepatocytes were seeded in large excess what was rendered possible due to the removal of oxygen shortage generally encountered in microplate‐based hepatocyte cultures. Hepatocyte/3T3 fibroblasts multilayered co‐cultures were maintained for at least 1 week; the so‐cultured cells were normoxic and sustained differentiated metabolic functions like albumin and urea synthesis at higher levels than hepatocytes monocultures. Such a microplate‐based cell culture system appears suitable for engineering in vitro miniature liver tissues for implantation, bioartificial liver (BAL) development, or chemical/drug screening. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011. 相似文献
10.
The albumin receptor effect may be due to a surface-induced conformational change in albumin 总被引:5,自引:0,他引:5
To determine whether equilibrium binding between albumin and hepatocytes involves a cell surface receptor for albumin, we incubated freshly isolated rat hepatocytes with 125I-albumin and determined the amount of albumin associated with the cells as a function of the total albumin concentration. The resulting two-phase binding curve showed the rat albumin-hepatocyte interaction to consist of a saturable binding interaction with a dissociation constant of 1.1 microM and 2 X 10(6) sites/cell in addition to a weak, nonsaturable binding interaction. However, the saturable binding of albumin to hepatocytes did not appear to result from the presence of an albumin receptor on the cell surface; the interaction was the same for different species of albumin, for chemically modified albumins, and for fragments of albumin representing mutually exclusive domains of the molecule. The saturable binding was, instead, found to involve a subpopulation of albumin with an enhanced affinity for the cell surface. We show that this subpopulation of albumin is generated upon contact with either solid surfaces or cell surfaces and can be transferred from one surface to another. We propose that the two-phase Scatchard binding curve and the "albumin receptor effect" reflect two populations of albumin that bind to the cell surface with different affinities rather than one population of albumin that binds to two classes of binding sites. 相似文献
11.
Oxygen uptake rates in cultured rat hepatocytes 总被引:5,自引:0,他引:5
One potential treatment of acute liver failure involves the use of an extracorporeal device composed of functional hepatocytes. A major issue in the design of such a large-scale device is providing the hepatocytes with a sufficient supply of oxygen and other nutrients. In this study, we have designed and characterized a simple perfusion system hepatocytes using this system. The OUR of hepatocytes was determined during the first day after seeding on a single collagen gel and during the long-term stable culture after the addition of a top layer of collagen. The OUR increased to 20.7 +/- 0.57 pmol/sec/mug DNA during the first 13 hours of culture on a single collagen gel, while during the next 11 hours, the OUR declined to 10.6 +/- 1.5 pmol/sec/mug DNA. In parallel with the increase in OUR during the first 10 hours, we observed significant cell spreading, suggesting that the oxygen supply to the cells may be critical for the spreading and adaptation of the anchorage-dependent hepatocytes following isolation. Addition of a top layer of collagen to hepatocyte cultures for 24 hours of culture on a single collagen layer resulted in a stable OUR for 15 days. These results indicate that OUR of hepatocytes in culture may vary depending on the phase of culture (i.e., early vs. late) and on the extracellular environment. (c) 1992 John Wiley & Sons, Inc. 相似文献
12.
Beken S De Smet K Depreter M Roels F Vercruysse A Rogiers V 《Alternatives to laboratory animals : ATLA》2001,29(1):35-53
L-Proline supplementation of the medium for collagen gel cultures of hepatocytes has been shown to improve albumin secretion. A study was made as to whether L-proline is also essential for the maintenance of xenobiotic biotransformation capacities in collagen gel sandwich and immobilisation cultures of rat and human hepatocytes. Key phase I (cytochrome P450-dependent monooxygenase [CYP)] and microsomal epoxide hydrase [mEH]) and phase II (glutathione S-transferase [GST]) biotransformation enzyme activities and the secretion of albumin in the culture medium were assessed in the absence and presence of L-proline. CYP and mEH activities were not affected by the addition of L-proline, whereas phase II alpha-Class GST activity of rat hepatocytes in collagen cultures was decreased. Species differences were demonstrated, as human hepatocytes showed a better maintenance of GST activities than their rat counterparts in the presence of L-proline. Albumin secretion, often considered to be a marker for differentiated cell function, does not parallel the biotransformation capacities of the hepatocytes in culture. Additional results demonstrated an L-proline-mediated enhancement of the proliferation rate of contaminating stellate cells in conventional monolayer culture. Transdifferentiation of stellate cells to proliferating myofibroblasts, along with an increased albumin secretion and collagen synthesis, are characteristic of fibrotic liver. Since the last two phenomena have been observed in L-proline-supplemented collagen gel cultures, it can be concluded that when stable collagen gel cultures of rat hepatocytes are needed for long-term pharmacotoxicological studies, it is preferable to use an L-proline-free culture medium. Further studies on medium optimisation are required for hepatocytes from species other than rat. 相似文献
13.
William J. McCarty Ljupcho Prodanov Shyam Sundhar Bale Abhinav Bhushan Rohit Jindal Martin L. Yarmush O. Berk Usta 《Journal of visualized experiments : JoVE》2015,(103)
Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices. 相似文献
14.
《Biochemical Engineering Journal》2011,53(2-3):255-262
In this study, we established rat primary hepatocyte sandwich cultures on oxygen-permeable membranes and investigated the change in their repolarization. Functional bile canaliculi in sandwich-cultured hepatocytes on oxygen-permeable polydimethylsiloxane (PDMS) membranes were re-established more quickly than those in a conventional sandwich culture on polystyrene (PS). This enhanced biliary excretory activity was also observed in hepatocytes on another oxygen-permeable membrane plate but not on a PDMS surface whose oxygen permeability is blocked. An apical efflux transporter protein, Mrp2, was more rapidly distributed in hepatocytes cultured on PDMS membranes than in hepatocytes cultured on conventional PS plates. Moreover, the area of distribution of the Mrp2 in polarized hepatocytes cultured on PDMS membranes was more widespread than that for the hepatocytes grown on sandwich-cultured PS plates. The observation of ultrastructure in transmission electron microscopy clearly confirmed the presence of bile canalicular lumens possessing microvilli and tight junctions. Additionally, we demonstrated that the 7-ethoxyresorufin-O-deethylation activity of hepatocytes on PDMS membranes was also improved as compared to those on a PS surface. Therefore, sandwich-cultured hepatocytes on oxygen-permeable substrates can provide a simple tool for predicting the hepatic metabolism and toxicity of xenobiotics in vivo with short span and low cost in the course of drug discovery and evaluation. 相似文献
15.
The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas‐permeable membranes
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Wenjin Xiao Marie Shinohara Kikuo Komori Yasuyuki Sakai Hitoshi Matsui Tomoharu Osada 《Biotechnology progress》2014,30(6):1401-1410
Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%‐O2 (+)] or physiological oxygen concentrations [10%‐O2 (+), 5%‐O2 (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas‐impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS‐O2 (?)]. The results indicated that the hepatocytes under 10%‐O2 (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS‐O2 (?) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug‐metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long‐term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen‐permeable membrane system to provide a simple method for in vitro studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1401–1410, 2014 相似文献
16.
Postthaw viability of precultured hepatocytes 总被引:8,自引:0,他引:8
Hepatocytes are being studied for a wide variety of applications, including drug metabolism studies, gene therapy, and use in liver-assist devices for temporary liver support. The ability to cryopreserve isolated hepatocytes would permit the pooling of cells to reach the required therapeutic coordination of the cell supply with patient care regimes and the completion of safety and quality-control testing. The objective of this investigation was to develop a method of cryopreserving isolated hepatocytes that will retain high levels of function and facilitate the use of the cells in different applications. Freshly isolated hepatocytes were cultured in a spinner flask for different periods of time, up to 48 h. The cells were cryopreserved by use of a range of solution concentrations and cooling rates. For fresh, nonfrozen hepatocytes precultured for 24 h prior to being plated on collagen, the albumin secretion rate was 0.88 +/- 0.62 mg/ml/h. When the cells were precultured for 24 h, frozen in a solution containing 10% Me2SO with a cooling rate of 1 degrees C/min, thawed, plated on collagen, and cultured, the albumin secretion rate was 0.21 +/- 0.24 microg/ml/h. In contrast, freshly isolated hepatocytes cryopreserved without preculture and cultured on collagen had an albumin secretion rate of 0.07 +/- 0.08 mg/ml/h. The influences of different solution compositions and cooling rates on postthaw function of precultured hepatocytes were also determined. These results indicate that the use of a preliminary culture step prior to cryopreservation can enhance the postthaw function of hepatocytes. 相似文献
17.
Glycosaminoglycans partially substitute for proteoglycans in spheroid formation of adult rat hepatocytes in primary culture 总被引:3,自引:0,他引:3
Adult rat hepatocytes seeded in a noncoated plastic dish containing serum-free medium formed a monolayer within 24 h of culture. Those seeded in a dish coated with a proteoglycan fraction isolated from rat liver reticulin fibers attached to the dish but did not spread within 4 h, and then gradually assembled to form floating spherical aggregates (spheroids) with a diameter of 120 +/- 40 micron, within 72 h. The proteoglycan fraction appeared to contain dermatan sulfate, heparan sulfate and an unidentified glycosaminoglycan in its glycan moieties by glycosaminoglycan analysis after pronase digestion and high molecular weight proteoglycan molecules (mw: over 300,000 and about 200,000) by SDS-PAGE analysis. Cells seeded in dishes coated with these defined glycosaminoglycans and heparin assembled to form hemispheroids and multilayer islands, but not floating spheroids, within 72 h of culture. Dermatan sulfate had a stronger ability to induce hemispheroids than heparan sulfate or heparin. As the hemispheroid and multilayer islands were the intermediate form between monolayer and floating spheroids, the glycosaminoglycan moieties of the proteoglycan fraction were thought to participate in the formation of spheroid. 相似文献
18.
Yumi Kono Suyun Yang Eve A. Roberts 《In vitro cellular & developmental biology. Animal》1997,33(6):467-472
Summary To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of
collagen gel, called a “collagen gel sandwich.” Maintenance of hepatocellular functions in this system was compared with that
of identical hepatocyte preparations cultured on dry-collagen coated dishes or co-cultured with rat liver epithelial cells.
Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period
of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin
secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion
could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer
of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture.
These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes
in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves
differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system
is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro. 相似文献
19.
20.
Nazaré Pereira-Rodrigues Paul-Emile Poleni Denis Guimard Yasuhiko Arakawa Yasuyuki Sakai Teruo Fujii 《PloS one》2010,5(3)