• Biotechnology and Bioprocess Engineering:BBE
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• 제6권4호
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• pp.264-268
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• 2001

Aggregates of specific cells are often regarded as better from in artificial organs and mammalian cell bioreactors in terms of cell-specific functionality. In this study, the morphology and liver specific functions of freshly harvested primary rat hepatocytes, which were cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined and compared to a control (hepatocytes in single cell form). A copolymer of N-isopropylacrylamide(98 mole % in feed) and acrylic acid (poly (NiPAAm-co-AAC)), a thermo- reversible copolymer gel ma- trix, was used to entrap hepatocytes either in spheroids or single cells. During a 7-day culture pe-riod, the spheroids maintained higher viability and produced albumin and urea at a relatively con-stant rate, while, the single cell culture showed a slight increase in cell numbers and a reduction in albumin secretion Hepatocytes cultrured as spheroids present a potentially useful three-dimensional cell culture system for application in bioartificial liver device.

• KSBB Journal
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• 제7권4호
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• pp.271-277
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• 1992

쥐 간세포를collagenase perfusion method에 의해 분리한 뒤 collagen coated dish와floating collagen membrane에서 일차배양하여 간세포의 분화기능을 조사하였다. 두 경우 모두 간세포의 생존율은 5일 이후부터 점차 감소하였거나 간세포에 의한 암모니아 처리기능과 albumin생성기능은 약 7일간 유지되었다. 또 이러한 분화기능에 유지는 ollagen coated dish보다floating collagen membrane이 유리한 것으로 나타났다.

• 한국가축번식학회지
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• 제20권3호
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• pp.259-269
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• 1996

To establish whether or not androgens is responsible for the induction of vitellogenin(Vg) synthesis and secretion, primary hepatocytes prepared from immature eels were used. The results are follows: 1. Eel hepatocytes were prepared using a collagenase perfusion technique. The isolated cells attached efficiently to fibronectin-coated dishes and subsequently formed monolayers in serum-free medium. These cultures maintained in medium for 10 days with minimal cell loss. 2. Estradiol-17$\beta$(E2) alone was insufficient to induce Vg synthesis. The combination of E2 with methyltestosterone(MT) markedly stimulated Vg synthesis. High vg production occurred in MT concentration from 10-6~10-5M in the presence of E2 (10-6M). Testosterone and androsterone were also effective, but progesterone was not effective in inducing Vg synthesis. Neither MT alone nor testosterone and androsterone alone had any effect on Vg synthesis. 3. E2-primed hepatocytes showed Vg synthesis in both media with and without hormones 1 day after culture. In the cultures with the vehicle, MT, or progesterone, the rate of synthesis seemed to decrease with time. But the combination of E2 and MT showed an intense increase in Vg synthesis. Hepatocytes isolated from E2-primed eels also required androgens for continuating of Vg synthesis. 4. These results demonstrate that androgens act together with E2 in synthesis and secretion of eel Vg.

• Fisheries and Aquatic Sciences
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• 제1권2호
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• pp.153-158
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• 1998

Effects of cyclic (c) AMP and G-protein related reagents (3-isobutyl-l-methyxanthine (IBMX), Forskolin (FSK), cholera toxin (CTX), and pertussis toxin (PTX≫ on estradiol-17$\beta$ induced vitellogenin (VTG) induction were examined in primary hepatocyte cultures in rainbow trout Oncorhynchus mykiss. The addition of IBMX, FSK, or CTX to the incubation medium markedly increased VTG production, while PTX was not effective in stimulating the production. It is well known that cAMP regulates phosphorylation and dephosphorylation through mediation of protein kinase A. These results suggest that VTG production is highly dependent on cAMP state in hepatocytes because of its highly phosphorylated nature.

• 약학회지
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• 제34권5호
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• pp.341-347
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• 1990

The anti-hepatotoxic activity of Panax ginseng was studied using galactosamine (GalN)-induced cytotoxicity in primary cultured rat hepatocytes. Panax ginseng was fractionated into dammarane glycosides and protein fractions. The dammarane glycosides was further fractionated into panaxadiol and panaxatriol glycosides fractions. The protein fraction was further fractionated into four groups according to the molecular weight; larger than 10,000 dalton, between 5,000 and 10,000 dalton, between 1,000 and 5,000 dalton and between 500 and 1,000 dalton. A significant lowering action on the elevated glutamicpyruvic transaminase (GPT) activity in the culture medium of hepatocytes treated with 1.5 mM GalN was noticed with all four protein fractions studied at the concentration of both $50\;{\mu}g/ml$ and $100\;{\mu}g/ml$. However, the effect of dammarane glycosides fractions was not significant. It was noted that the addition of $100\;{\mu}g/ml$ of protein fractions smaller than 5,000 dalton significantly enhanced the syntheses of protein and RNA in the damaged hepatocytes induced by the treatment of 1.5 mM GalN. Dammarane glycosides fractions significantly enhanced protein synthesis at the concentration of $100\;{\mu}g/ml$ in the damaged hepatocytes by treatment of 1.5 mM GalN.

• Toxicological Research
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• 제3권1호
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• pp.1-8
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• 1987

In vitro induction of cytochrome 450 associated monooxygenase activities by phenobarbital (PB) and 3-methylcholanthrene (MC) was investigated in primary cultures of adult rat hepatocytes. PB and MC were added to the culture 24 hr after the initial plating of hepatocytes. A signiftcant increase of the activities of 7-ethoxycoumarin 0-deethylase and aryl hydrocarbon hydroxylase were observed in MC and PB treated culture. MC caused about 500% induction of the initial oxidation rates of both enzymes in 48 hr. However the PB maintained both enzyme activities close to the level of freshly isolated hepatocytes. Biphenyl 4-hydroxylase and aminopyrine N-demethylase activities were also induced by MC and PB. But the level of induction was less than that occuring with 7-ethoxycoumarin 0-deethylase and aryl hydrocarbon hydroxylase. When aflatoxin $B_1$ was added to the hepatocyte cultures which have been treated with MC or PB, it caused a significant increase of the unscheduled DNA synthesis at higher dose of aflatoxin $B_1$ as compared to those of untreated control hepatocyte cultures. The results suggest that microsomal enzyme activities can be selectively controlled preferably in hepatocyte cultures by the in vitro induction method. This principle may be useful for studying the metabolism and other toxicological studies.

• Archives of Pharmacal Research
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• 제26권1호
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• pp.64-69
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• 2003

To analyze vasopressin-induced $Ca^{2+}$ increase in liver cells, rat hepatocytes were isolated and attached to collagen-coated cover slips. Using fura-2, a $Ca^{2+}$-sensing dye, changes in intracellular $Ca^{2+}$ concentration by vasopressin were monitored. Results in this communication suggested that vasopressin-induced $Ca^{2+}$ increase were composed of both $Ca^{2+}$ release from internal $Ca^{2+}$ stores and influx from the plasma membrane. The $Ca^{2+}$ influx consisted of two distinguishable components. One was dependent on the presence of vasopressin and the other was not. SK＆F96365 blocked vasopressin-induced $Ca^{2+}$ influx in a dose-dependent manner. Vasopressin-induced $Ca^{2+}$ release from internal stores diminished in a primary culture of hepatocytes according to the culture time. However, changes in vasopressin-induced $Ca^{2+}$ influx across the plasma membrane differed from changes in the $Ca^{2+}$ release from internal stores, suggesting two separate signalings from receptor activation to internal stores and to the plasma membrane.

• 대한약리학회지
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• 제32권3호
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• pp.399-406
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• 1996

Thioacetamide는 non-genotoxic 발암제로서 세포단백의 변형을 초래하는 것으로 알려져 있으며 이것을 단기간 처리하면 핵소체의 비대를 초래하게 된다. 본 연구에서는 thioacetamide를 처리한 간세포를 in vivo와 in vitro 상태에서 관찰하였다. In vivo상태로서 thioacetamide를 쥐의 복강에 7일간 주사하면 (50mg/kg), 핵소체 비대와 B23 및 MAP kinase와 같은 신호전달분자들이 증가하는 것을 관찰할 수 있었다. In vitro 상태로서 쥐의 간장을 collagenase로 분리하여 유전자 치료에 사용될 수 있는 배양조건으로 간세포를 배양하여 핵소체를 관찰한 결과 핵소체 비대가 현저하였으며, B23의 양도 증가하였다. 본 실험의 결과로 미루어 볼 때, 간세포는 핵소체 비대 수용능력이 약 100배 이상이라고 할 수 있으며, thioacetamide 처리에 의하여 라이보좀 생성과 핵소체 증가 능력이 증폭되어 나타나는 것으로 사료된다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.305-306
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• 2003

To treat fulminant hepatic failure (FHF) patients, various extracorporeal bioartificial liver (BAL) systems have been developed. Several requirements should be met for the development of BAL systems: hepatocytes should be cultured in a sufficiently high density; their metabolic functions should be of a sufficiently high level and duration; and the BAL systems module should permit scaling-up and aseptic handling. Several investigators have found that freshly isolated primary hepatocytes can be cultured into three dimensional, tightly packed, freely suspended, multicellular aggregates, or spheroids. These specialized cell structures exhibited enhanced liver specific functions and a prolonged differentiated state compared to cells maintained in a monolayer culture. Cells in spheroids appear to mimic the morphology and ultrastructure of the in vivo liver lobule. The ability of hepatocytes to organize into three-dimensional structures was hypothesized to contribute to their enhanced liver-specific activities. In this study, the ammonia removal rate and urea secretion rate of pig hepatocytes spheroids encapsulated in Ca-alginate bead were determined. A packed-bed bioreactor with encapsulated pig hepatocytes was devised as BAL support system. The efficacy of the system was evaluated in vitro.

• Journal of Ginseng Research
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• 제19권2호
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• pp.108-113
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• 1995

Effect of ginseng polysaccharide fraction was examined for $CCl_4$-induced hepatotoxicity in vitro and in vivo. In $CCl_4$-injured primary cultured rat hepatocytes, treatment of the polysaccharide fraction (0.1, 0.3, 1.0 mg/ml) significantly Inhibited the release of LDH and GOT into the culture medium in a dose-dependent manner. Oral administration of the polysaccharide fraction (100, 200 mg/kg) inhibited the decrease of body weight and the increase of the ratio of liver to body weight in $CCl_4$-intoxicated rats. Elevation of GOT, GPT and ALP activity in the serum by $CCl_4$-induced hepatotoxicity was suppressed by administration of ginseng polysaccharide fraction. MDA levels increased in the serum as well as in the liver tissue by treatment with $CCl_4$ showed a tendency to be 연w in the rats given to the polysaccharide fraction. These results suggest that the polysaccharide fraction may be active substance responsible for antihepatotoxic effect of Panax ginseng.