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1.
An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro. 相似文献
2.
Investigation of rifampicin-induced hepatotoxicity in rat hepatocytes maintained in gel entrapment culture 总被引:1,自引:0,他引:1
Rifampicin-induced hepatotoxicity has been well recognized in animals and patients. However, it is undetectable in cultured
hepatocyte monolayers in vitro at the equivalent toxic concentration in vivo. This study investigated the rifampicin-induced
toxicity on rat hepatocytes in gel entrapment vs. in monolayer culture. Thiazolyl tetrazolium reduction and albumin secretion
were routinely detected to identify the toxic responses of rat hepatocytes to rifampicin, while reactive oxygen species (ROS)
accumulation and intracellular glutathione (GSH) content were assayed as biomarkers of oxidative stress. In addition, Nile
red staining and malondialdehyde (MDA) generation were, respectively, used as endpoints for lipid accumulation and peroxidation.
After treatment of hepatocytes for 96 h at a serum rifampicin concentration (12 μM), gel-entrapped rat hepatocytes showed
significant cellular damage indicated by alternations of all parameters indicated above, while hepatocyte monolayers did not
show severe responses. In contrast to a lack of protections by cytochrome P 450 inhibitors, the ROS scavenger (glycyrrhizic
acid) and thiol compounds (N-acetylcysteine and GSH) significantly reduced rifampicin toxicity in gel-entrapped hepatocytes. It appears that gel-entrapped
rat hepatocytes reflected significant hepatotoxicity of rifampicin in vivo, and this toxicity was most possibly associated
with oxidative stress and lipid accumulation. 相似文献
3.
Arye Lazar Henry J. Mann Rory P. Remmel Russell A. Shatford Frank B. Cerra Wei-Shou Hu 《In vitro cellular & developmental biology. Animal》1995,31(5):340-346
The potential use of porcine hepatocytes in a bioartificial liver device requires large quantities of viable and highly active
cells. To facilitate the scaling up of the system, liver specific activities of hepatocytes should be maximized. One way of
enhancing the specific activities is to cultivate hepatocytes as multicellular spheroids. Freshly isolated porcine hepatocytes
form spheroids when cultivated in suspended cultures. These spheroids exhibit higher activities for a number of liver specific
functions compared to hepatocytes cultivated as monolayers. However, these activities decreased in a few days in culture.
Entrappment of spheroids in collagen gel sustained their metabolic activities at a stable level over 21 days. Production of
albumin and urea by spheroid hepatocytes entrapped in collagen gels were 2 to 3 times higher than those by freshly isolated
single cells. P-450 activity was demonstrated by metabolism of lidocaine to its main metabolite, monoethylglycinexylidide.
Phase II drug metabolism was demonstrated by glucuronidation of 4-methylumbelliferone. This work shows that porcine hepatocyte
spheroids entrapped in collagen maintain differentiated functions for an extended time period. Such hepatocyte spheroid entrappment
system may facilitate the development of a bioartificial liver support device. 相似文献
4.
J Fantini J B Rognoni B Verrier M Lehmann M Roccabianca J Mauchamp J Marvaldi 《European journal of cell biology》1990,51(1):110-119
The clonal cell line HT29-D4 is able to differentiate by two different ways: i) by replacing glucose by galactose in the culture medium; ii) by addition of suramin (a drug known to interfere with the growth promoting activity of growth factors) in the medium. In both cases the transition in the organization of the cell monolayer occurred without cell loss. The two ways (i.e., glucose starvation or suramin addition) lead to polarized cells which generate electrically active cell monolayers (Fantini et al., Biol. Cell 65, 163-169 (1989) and this paper). Yet several important differences can be observed at the morphological or at the electrophysiological levels. 1) The suramin-treated cells (HT29-D4-S cells) organized into monolayers of high (40-50 microns) columnar cells while glucose-starved cells (HT29-D4-Gal cells) were rather cuboidal (20-25 microns). 2) HT29-D4-S cells were highly polarized; the nucleus was rejected at the basal side of the cell and lysosomes in the upper part of the cytoplasm. Numerous lipid-like droplets surrounded with glycogen were observed underneath the nucleus. HT29-D4-Gal cells never presented such a degree of organization. 3) The transepithelial resistance and the potential difference of HT29-D4-S monolayers reached values significantly higher than those for HT29-D4-Gal monolayers, reflecting a higher degree of organization. Specific proteins such as sucrase-isomaltase, alkaline phosphatase and carcinoembryonic antigen were localized exclusively on the apical membrane while human lymphocyte antigen (HLA) class I molecules were restricted to the basolateral membrane for both HT29-D4-S and HT29-D4-Gal cells. The present data demonstrate that the same cells can generate a different degree of cellular organization according to the experimental conditions of cell growth, the most elaborate state of differentiation being obtained in the presence of suramin. 相似文献
5.
《Biochemical Engineering Journal》2007,35(3):267-272
A long-term hepatocyte model in vitro is preferable for chronic hepatotoxicity research because hepatocytes in this model of culture can preserve liver-specific functions for long period. Micro-hollow fiber reactors (MHFR), composed of polysulphone (PS) hollow fibers with a molecular weight cut-off 100 kDa, were applied to test the hepatotoxicity of acetaminophen, isoniazid and rifampicin, respectively. Monolayer culture was used as a control model for hepatocyte culture. It was found that hepatocytes within MHFR were more sensitive to toxicity of acetaminophen (0.38–1.51 g/L) than those in monolayer cultures. Furthermore, significant hepatotoxicity of isoniazid (15 mg/L) and rifampicin (10 mg/L) were detected in hepatocytes cultured in MHFR but not detected in hepatocyte monolayer, which could be due to well-preserved drug metabolizing enzymes in MHFR. These results indicate that the MHFR may be an effective model for long-term hepatotoxicity research in vitro. 相似文献
6.
J Fantini B Verrier J Marvaldi J Mauchamp 《Biology of the cell / under the auspices of the European Cell Biology Organization》1989,65(2):163-169
HT 29-D4 is a clonal cell line, derived from the human colon adenocarcinoma cell line HT 29, which can be induced to differentiate into enterocyte-like cells by replacing glucose with galactose in the culture medium (Fantini et al. [1986], J. Cell Sci. 83, 235-249). Both undifferentiated and differentiated HT 29-D4 cells have been successfully grown to confluency in Costar Transwell permeable chambers. Only HT 29-D4 cells grown in glucose-free, galactose-containing medium were able to form leakproof monolayers, as demonstrated by their ability to prevent diffusion of serum proteins. These monolayers consist of highly polarized epithelial-like cells with a well organized apical brush border. Transepithelial electrical parameters have been measured under sterile conditions for both types of monolayer. Only HT 29-D4 monolayers cultured in glucose-free, galactose-containing medium were electrically active, with a transepithelial resistance R = 172 +/- 46 omega.cm2, a potential difference PD = 0.35 +/- 0.05 mV, apical negative and a short-circuit current Isc = 2.0 +/- 0.4 microA.cm-2. Apical addition of amphotericin B induced a rapid and considerable increase in Isc and PD, which was abolished by basal ouabain. In contrast, HT 29-D4 cells grown in glucose-containing medium did not generate any potential difference (PD = 0 mV) and their resistance was very low (R = 34.1 +/- 0.9 omega.cm2). It is concluded from these studies that HT 29-D4 cells grown in glucose-free, galactose-containing medium acquire functional characteristics of epithelia, compared to HT 29-D4 cells grown in glucose-containing medium.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers 总被引:4,自引:0,他引:4
Geraldine A. Hamilton Carl Westmoreland Elisabeth George 《In vitro cellular & developmental biology. Animal》2001,37(10):656-667
Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial. 相似文献
8.
Yuichi Adachi Tadashi Mio Keiichi Takigawa Ilja Striz Debra J. Romberger John R. Spurzem Stephen I. Rennard 《In vitro cellular & developmental biology. Animal》1998,34(3):203-210
Summary
In vivo, fibroblasts are distributed in a three-dimensional (3-D) connective tissue matrix. Fibronectin is a major product of fibroblasts
in routine cell culture and is thought to regulate many aspects of fibroblast biology. In this context, we sought to determine
if the interaction of fibroblasts with a 3-D matrix might affect fibronectin production. To examine this hypothesis, fibronectin
production by fibroblasts cultured in a 3-D collagen gel or on plastic dishes was measured by ELISA. Fibroblasts in 3-D gel
culture produced more fibronectin than those in monolayer culture. Fibroblasts in 3-D culture produced increasing amounts
of fibronectin when the collagen concentration of the gel was increased. The 3-D nature of the matrix appeared to be crucial
because plating the fibroblasts on the surface of a plastic dish underneath a collagen gel was not different from plating
them on a plastic dish in the absence of collagen. In addition to increased fibronectin production, the distribution of the
fibronectin produced in 3-D culture was different from that of monolayer culture. In monolayer culture, more than half of
the fibronectin was released into the culture medium. In 3-D culture, however, approximately two-thirds remained in the collagen
gel. In summary, the presence of a 3-D collagen matrix increases fibroblast fibronectin production and results in greater
retention of fibronectin in the vicinity of the producing cells. 相似文献
9.
Imran Ullah Yeongji Kim Malgum Lim Keon Bong Oh Seongsoo Hwang Yurianna Shin 《Animal cells and systems.》2017,21(5):332-340
Primary hepatocytes (PHs) are considered the ‘gold standard’ in drug screening owing to their ability to express many drug-metabolizing enzymes and transporters. Culturing hepatocytes and maintaining their fate in vitro is a major issue since last decade. The main problem with in vitro hepatocytes culture is that they rapidly lose their hepatic morphology and liver-specific functions in culture. Herein, we isolated rat PHs, and cultured them in monolayers (2-D) and spheroids (3-D). The 2-D-cultured PHs exhibited elongated morphology, whereas the 3-D-cultured PHs exhibited spheroid morphology with gradual diameter decrease until 7 days. After 7 days of in vitro culture, PHs were analyzed for the expression of hepatic (Alb, Tf, and Afp) and apoptotic markers (Bax and Bcl2), and co-expression of CYP3A1 and Abumin after 2 and 7 days. Furthermore, in both cultures, PHs were induced with 3-methylcholanthrene (3-MC, Cyp1a-specific inducer) and dexamethasone (Cyp3a-specific inducer) for 48 and 72?h, respectively. The mRNA levels of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PHs. After 7 days of in vitro culture, PHs exhibited dramatic downregulation of hepatic marker expression in both cultures. Furthermore, apoptotic marker expression was higher in the 2-D-cultured PHs than 3-D-cultured PHs. The mRNA levels of Cyp1a and Cyp3a indicated higher RNA content in the 2-D-cultured PHs after 48?h of induction. Therefore, we concluded that there was no significant difference between the culture systems, and further studies are required to identify the essential components for in vitro PH culture rather than culture systems. 相似文献
10.
Gel entrapment culture of primary mammalian cells within collagen gel is one important configuration for construction of bioartificial organ as well as in vitro model for predicting drug situation in vivo. Gel contraction in entrapment culture, resulting from cell-mediated reorganization of the extracellular matrix, was commonly used to estimate cell viability. However, the exact influence of gel contraction on cell activities has rarely been addressed. This paper investigated the gel contraction under varying culture conditions and its effect on the activities of rat hepatocyte entrapped in collagen gel within hollow fibers. The hepatocyte activities were reflected by cell viability together with liver-specific functions on urea secretion and cytochrome P450 2E1. Unexpectedly, no gel contraction occurred during gel entrapment culture of hepatocyte under a high collagen concentration, but hepatocytes still maintained cell viability and liver-specific functions at a similar level to the other cultures with normal gel contraction. It seems that cell activities are unassociated with gel contraction. Alternatively, the mass transfer resistance induced by the combined effect of collagen concentration, gel contraction and cell density could be a side effect to reduce cell activities. The findings with gel entrapment culture of hepatocytes would be also informative for the other cell culture targeting pathological studies and tissue engineering. 相似文献
11.
Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes
on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined
medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03–3%). When cultured in a
FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological
characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes
cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured
on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated
in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers
on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes
in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer
cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium,
are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development
of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
12.
Summary We established an in vitro hepatocyte primary culture system from Oreochromis niloticus, a tropical fish species of great economical importance, and evaluated its ability to express albumin, a liver-specific protein,
consistently for a period of 3 wk. Serum requirements for fish hepatocyte cultures were assessed. A one-step in situ perfusion
of tilapia liver retrogradely followed by collagenase liver dissociation and subsequent washing produced nearly 90% homogenous
viable hepatocytes, as shown by trypan blue exclusion test. Mixed primary monolayer and aggregate hepatocyte cultures achieved
by 10% fetal calf serum medium supplements expressed consistent levels of albumin. The results of light and electron microscopy
showed that the hepatocytes did not significantly proliferate (P<0.05) but remained viable for at least 3 wk. The results of this study show that in vitro cultures of mixed primary hepatocyte
monolayers and aggregates established from Nile tilapia may be useful models for studying transient cellular stress induction. 相似文献
13.
L. Agius C. Battersby K. G. M. M. Alberti 《In vitro cellular & developmental biology. Plant》1985,21(5):254-259
Summary A method is described for the attachment to and monolayer culture of adult rat hepatocytes on collagen-coated or fibronectin-coated
microbeads or both in a chemically defined serum-free medium. Protein synthesis measured by the incorporation of [3H]leucine into protein was four-fold higher in the hepatocyte microcarrier cultures than in isolated hepatocyte suspensions.
The hepatocyte microcarrier cultures showed acute responsiveness to insulin of fatty acid synthesis, glucose incorporation
into glycogen, and decarboxylation of [1-14C]pyruvate. Microcarrier-cultured hepatocytes have the combined advantages of monolayer culture and suspension systems. They
are a potential tool for the study of long-term as well as acute effects of hormones.
This work was supported by the British Diabetic Association. 相似文献
14.
Nanoparticle carriers are attractive vehicles for a variety of drug delivery applications. In order to evaluate nanoparticle formulations for biological efficacy, monolayer cell cultures are typically used as in vitro testing platforms. However, these studies sometimes poorly predict the efficacy of the drug in vivo. The poor in vitro and in vivo correlation may be attributed in part to the inability of two-dimensional cultures to reproduce extracellular barriers, and may also be due to differences in cell phenotype between cells cultured as monolayers and cells in native tissue. In order to more accurately predict in vivo results, it is desirable to test nanoparticle therapeutics in cells cultured in three-dimensional (3-D) models that mimic in vivo conditions. In this review, we discuss some 3-D culture systems that have been used to assess nanoparticle delivery and highlight several implications for nanoparticle design garnered from studies using these systems. While our focus will be on nanoparticle drug formulations, many of the systems discussed here could, or have been, used for the assessment of small molecule or peptide/protein drugs. We also offer some examples of advancements in 3-D culture that could provide even more highly predictive data for designing nanoparticle therapeutics for in vivo applications. 相似文献
15.
Kaipparettu BA Kuiatse I Tak-Yee Chan B Benny Kaipparettu M Lee AV Oesterreich S 《BioTechniques》2008,45(2):165-8, 170-1
Although three dimensional (3-D) cell culture systems have numerous advantages over traditional monolayer culture, the currently available 3-D cell culture media are cost-prohibitive for regular use by the majority of research laboratories. Here we show a simple system based on avian egg white that supports growth of cells in 3-D, at a significantly decreased cost. Specifically, we show that growth of immortalized human breast epithelial cells (MCF10A) in egg white-based medium results in formation of acini with hollow lumens, apoptotic clearance of the cells in the lumen, and apicobasal polarization comparable to what has been described using established 3-D culture media such as reconstituted basement membrane preparations (BM). There was no significant difference in MCF10A proliferation and acinar size between egg white and BM. We also cultured different established cell lines, oncogene-transformed MCF10A, and mouse mammary epithelial cells in egg white and BM, and observed similar morphology. In summary, our data convincingly argue that egg white can be used as a suitable alternative model for 3-D cell culture studies. We strongly believe that this simple and inexpensive method should allow researchers to perform 3-D cell culture experiments on a regular basis, and result in a dramatic increase of use of the 3-D cell culture in research. Thus, this finding lays the foundation for significantly increased, cost-effective use of 3-D cultures in cell biology. 相似文献
16.
Past in vitro studies of liver-cell functions have been performed on nonproliferating primary cells or serially propagated hepatic monolayers of neoplastic or fetal origin. We optimized conditions for the selective culture of adult rabbit and canine liver parenchymal cells and presently have four differentiated proliferating monolayer strains. At the 30th passage level these hepatic cultures still display the specific liver parenchymal functions of albumin and fibrinogen synthesis as well as tyrosine aminotransferase activity. Optimization of the conditions for hepatocyte culture was monitored by [3H]thymidine incorporation. Albumin and fibrinogen synthesis were measured by bioradioimmunoassay and tryosine transaminase activity by a modification of Diamondstone's assay. Albumin and fibrinogen synthesis were correlated with hepatocyte growth kinetics. 相似文献
17.
Amphibian hepatocytes have been prepared in both high yield and purity using a collagenase perfusion technique. The isolated cells attach efficiently in serum-free medium to collagen-coated culture dishes and subsequently form monolayers. These cultures can be maintained in an appropriate medium for over one week with minimal cell loss. The nuclear labelling index of cells exposed to [3H]thymidine indicates a very low level of cell growth. Twenty-four hour exposure to dexamethasone induces tyrosine aminotransferase activity throughout the culture period. Monolayers incorporate [3H]leucine linearly into acid-insoluble material with approx. 40% of all synthesis devoted to secreted protein. Polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate shows the majority of proteins present in whole serum are synthesized and secreted by the cultured hepatocytes. The absolute rate of protein secretion on the first day of culture is approx. 73 μg/day/mg cell protein which subsequently declines and plateaus at 30% of this level by the 4th–5th day of culture. However, when hepatocytes are cultured in the continued presence of insulin, the drop in protein secretion is completely inhibited.Cultures of hepatocytes isolated from female frogs and subsequently exposed to 17-B estradiol in culture, synthesize and secrete the egg-yolk protein precursor vitellogenin. The protein initially appears as a minor component in the medium 1–2 days after hormone addition. Its rate of synthesis, relative to other secreted proteins, increases with time so that it ultimately constitutes the majority of protein being exported after 6 days of treatment. Parallel with vitellogenin induction is an increase in rate of total protein secretion reaching a 2-fold increase at maximal stimulation.The results show that viable, monolayer cultures of amphibian hepatocytes can be prepared which retain the ability to respond directly to added estrogen by synthesizing vitellogenin. 相似文献
18.
Suramin is an anti-cancer drug which induces the differentiation of the human colon cancer clone HT29-D4. Yet chronic suramin treatment of these cells eventually leads to a marked disturbance of the lysosomal system, which consists in an accumulation of hypertrophied autophagic vacuoles and the occurrence of lamellated inclusion bodies. We report here the effect of a prime treatment of HT29-D4 cells with suramin during various periods of time, followed by the removal of the drug and a subsequent culture in suramin-free medium. A prime treatment of cells in the presence of the drug for 2 days or 4 days was found ineffective to induce the organization of cells into polarized monolayers. On the contrary, a prime treatment of cells for 5 days is sufficient to allow the cellular organization to proceed normally toward a fully polarized monolayer, without any lysosomal damage. The cells did not require the continuous presence of suramin to develop an electrical resistance and a transepithelial potential difference. Moreover the basolateral localization of HLA class I molecules was achieved 9 days after the removal of the drug from the culture medium. Finally prime treatment of cells in the presence of suramin for times longer than 5 days induced the morphological, biochemical, and electrophysiological differentiation of HT29-D4 cells. However, in this case, severe lysosomal disturbances were constantly observed. These data demonstrate that the impaired lysosomal system is a post-differentiation event due to prolonged exposure of the cells to suramin. A metabolic analysis of HT29-D4 cells primed for various times with the drug showed that differentiated cells have a reduced glycolytic activity and this suggests an action of suramin at the level of autocrine growth factors which are known to regulate glucose uptake and degradation. 相似文献
19.
Albert P. Li George Barker Dale Beck Susan Colburn Roger Monsell Carol Pellegrin 《In vitro cellular & developmental biology. Animal》1993,29(3):249-254
Summary Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell
contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell
contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes
with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor
and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently
entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen
tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in
culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for
15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver
organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including
the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes.
We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial
applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also
be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with
hepatic failure. 相似文献
20.
Kazuyoshi Takeshita William C. Bowen George K. Michalopoulos 《In vitro cellular & developmental biology. Animal》1998,34(6):482-485
Summary Rat hepatocytes were maintained on three-dimensional cultures on sponge discs kept in Spinner Baskets (New Brunswick Scientific
Co., New Brunswick, NJ, USA) with continuously circulating serum-free hepatocyte growth medium (HGM) containing hepatocyte
growth factor (HGF) and epidermal growth factor (EGF). Hepatocytes were embedded in polyester sponge discs with a collagen
gel at the concentration of 5 million cells/ml. Atmospheric gas containing 7% CO2 was directly bubbled into the medium. Agitation by the impeller created a continuous medium-flow through the packed hepatocytes.
Comparison between identically prepared perfused and stationery cultures showed that hepatocytes in the perfused cultures
maintain higher levels of DNA synthesis. These results demonstrate the value of perfusion systems and also show that hepatocytes
can proliferate and maintain differentiation in three-dimensional culture environments. 相似文献