• Molecules and Cells
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• 제42권1호
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• pp.45-55
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• 2019

The liver is involved in a wide range of activities in vertebrates and some other animals, including metabolism, protein synthesis, detoxification, and the immune system. Until now, various methods have been devised to study liver diseases; however, each method has its own limitations. In situ liver perfusion machinery, originally developed in rats, has been successfully adapted to mice, enabling the study of liver diseases. Here we describe the protocol, which is a simple but widely applicable method for investigating the liver diseases. The liver is perfused in situ by cannulation of the portal vein and suprahepatic inferior vena cava (IVC), with antegrade closed circuit circulation completed by clamping the infrahepatic IVC. In situ liver perfusion can be utilized to evaluate immune cell migration and function, hemodynamics and related cellular reactions in each type of hepatic cells, and the metabolism of toxic or other compounds by changing the composition of the circulating media. In situ liver perfusion method maintains liver function and cell viability for up to 2 h. This study also describes an optional protocol using density-gradient centrifugation for the separation of different types of hepatic cells, allowing the determination of changes in each cell type. In summary, this method of in situ liver perfusion will be useful for studying liver diseases as a complement to other established methods.

• Journal of Pharmaceutical Investigation
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• 제28권1호
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• pp.25-33
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• 1998

Intestinal absorption of ${\beta}-lactam$ antibiotics and angiotensin converting enzyme(ACE) inhibitors has been shown to use the carrier-mediated transport system. In vitro experiments have established that the efficacy of uptake by enterocytes depends on an inwardly directed proton gradient. It was suggested that benazepril was mediated by tripeptide transport system and that amoxicillin was transported by dipeptide transport carrier. The aim of this study is to assess the influence of amoxicillin on the intestinal absorption of benazepril using in vitro diffusion chamber and in situ single pass perfusion technique in the rat in order to elucidate whether the above transport systems are competitive or not. We obtained the gastrointestinal pemeability coefficient of amoxicillin, benazepril and both of them using in vitro diffusion chamber. And also the gastrointestinal absorption clearance of amoxicillin, benazepril and both of them using in situ single-pass perfusion method at steady state were calculated. Amoxicillin and benazepril were analyzed by HPLC. The results by the use of diffusion chamber in vitro indicated that the apparent intestinal permeability coefficient of benazepril was significantly(p<0.01) decreased by amoxicillin(45.2%) and vice versa significantly(p<0.01) decreased(89.1%). The results by the in situ gastrointestinal single-pass perfusion method indicated that the intestinal absorption clearance of benazepril was significantly(p<0.05) decreased by amoxicillin (40.2%) and vice versa significantly(p<0.05) decreased(54.8%). These results might suggest that they share the same peptide carrier pathway for oral absorption.

• KSBB Journal
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• 제13권4호
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• pp.411-417
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• 1998

The possibility of application of membrane type immobilized adsorbent to the fed-batch or perfusion culture system with anchorage-independent cells as well as batch system was investigated. The improvement in cell density and cell viability due to the combination of immobilized adsorbent with each culture system was evaluated for the investigation, and the optimum culture system employing immobilized adsorbent system was suggested based on the results. It was observed that the system with immobilized adsorbent showed better cell growth and cell viability than that without immobilized adsorbent in every operation system of batch, fed-batch, and perfusion. In case of batch system, 200% improvement of maximum cell density was observed in the system where ammonium chloride was added on purpose. And 50% improvement of maximum cell density was observed in the fed-batch system where ammonium ion accumulates significantly, while small increase in maximum cell density was observed in the perfusion system where dilution of waste byproducts exists. Especially, the fed-batch system showed the most significant improvement on cell growth because both compensation of nutrient and removal of ammonium ion occurred simultaneously in the system. Therefore a combined system of immobilized adsorbent and fed-batch operation could be suggested as an optimum system with in situ removal of ammonium ion.

• 대한의생명과학회지
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• 제6권4호
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• pp.229-235
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• 2000

췌장에서 분비하는 인슐린과 글루카곤의 자극-분비 coupling 과정은 주로 혈당 농도와 중추신경계에 의하여 조절되어진다. 본 연구는 두부에 포도당이 결핍되었을 때에 중추신경계가 췌장에서 인슐린과 글루카곤이 분비되는 패턴을 Sprague-Dawley 흰쥐를 대상으로 하여 살펴보았으며, 실험 방법은 in situ 뇌-췌장 관류법을 이용하였다. 관류액은 100 mg/dL glucose와 20 mM arginine를 포함한 Krebs-Ringer 완충액 (pH 7.4)으로 하였으며, 95% $O_2$-5% $CO_2$ 가스를 계속적으로 주입시키면서 5 ml/min의 속도로 30분간 정주하였다. 대조군은 cephalic glucopenia가 일어나지 않는 군으로 하였고, 실험군은 두 군으로 나누어서 GLP1군은 cephalic glucopenia가 0분에 일어나도록 하였고, CLP2군은 16분에 일어나도록 하였다. 문맥으로 유출되는 췌장의 effluent액에서 인슐린과 글루카곤 농도를 RIA법으로 측정하였고 호르몬의 분비 속도를 산출하여 분비동태 양상을 분석하였다. 결과에서 인슐린 분비량은 GLP1군에서 가장 낮아서 cephalic glucopenia에 의하여 다소 감소하는 경향이었으나, 세 군간에 통계적으로 유의적인 차이는 없었다. 인슐린의 분비동태 양상을 살펴보면 이봉성의 정규 양상을 보였으나, GLP1군에서 첫번째 peak (4 min)가 다소 둔화되는 현상을 보였다. 글루카곤의 분비동태 양상도 이봉성의 정규 양상을 보였으며, 특히 GLP1군에 있어서 0~15분간의 글루카곤 분비량은 cephalic glucopenia에 의하여 유의성 있게 (p<0.05) 증가하였다. GLP2군에 있어서 글루카곤 분비량은 관류 후 15~30분 사이에 중가하는 경향을 볼 수 있었으나 통계적인 유의성은 없었다. 따라서 頭部의 포도당 결핍은 글루카곤의 분비를 증가시키는 것으로 나타났고, 이러한 현상은 특히 관류의 early period에서 현저하였다.

• Journal of Chest Surgery
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• 제45권4호
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• pp.251-253
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• 2012

We report a redo coronary artery bypass grafting (CABG) in a 55-year-old man. Angina recurred 7 years after the initial surgery. Coronary angiography showed all patent grafts except a faint visualization of the in situ right gastroepiploic artery (RGEA) graft, which was anastomosed to the posterior descending coronary artery, associated with celiac axis stenosis. Redo-CABG was performed at postoperative 10 years because of aggravated angina and decreased perfusion of the inferior wall in the myocardial single photon emission computed tomography. The saphenous vein graft was interposed between the 2 in situ grafts used previously; the right internal thoracic artery and RGEA grafts. Angina was relieved and myocardial perfusion was improved.

• KSBB Journal
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• 제30권3호
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• pp.103-108
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• 2015

Production of foreign proteins by transgenic plant cell cultures has several advantages such as post-translational modification, low risk of product contamination and low-cost production and purification. However, target proteins are degraded by extracellular proteases existing in the media. A solution to this problem is the use of perfusion culture and ion exchange chromatography for the application of integrated bioprocess using in situ recovery. With this method, production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in this study. First, optimization of cell concentration during the induction phase for the production of hGM-CSF was examined. As cell concentration increased, the level of hGM-CSF was decreased due to the presence of extracellular proteases. Induction using sugarfree media produced 33% more hGM-CSF. The effects of pH on the binding of hGM-CSF to cationic and anionic exchange resins were also investigated. In terms of stability, optimal pH was found to be 5~7. In the case of using buffer exchange when CM-Sepharose was used as a cationic exchange resin, optimal pH for binding was 4.8 and adsorption yield was 77%. When DEAE-Sepharose was used as an anionic exchange resin, it was 5.5 (74%). Without buffer exchange, optimal pH was 4.6 (84%). From these results, an integrated bioprocess using in situ recovery with simultaneous production and separation of foreign protein in transgenic plant cell suspension cultures was found to be feasible.

• The Korean Journal of Physiology and Pharmacology
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• 제17권1호
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• pp.9-13
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• 2013

The aim of the present study was to examine the effect of micellar systems on the absorption of beta-lapachone (b-lap) through different intestinal segments using a single-pass rat intestinal perfusion technique. B-lap was solubilized in mixed micelles composed of phosphatidylcholine and sodium deoxycholate, and in sodium lauryl sulfate (SLS)-based conventional micelles. Both mixed micelles and SLS micelles improved the in situ permeability of b-lap in all intestinal segments tested although the mixed micellar formulation was more effective in increasing the intestinal absorption of b-lap. The permeability of b-lap was greatest in the large intestinal segments. Compared with SLS micelles, the effective permeability coefficient values measured with mixed micelles were 5- to 23-fold higher depending on the intestinal segment. Our data suggest that b-lap should be delivered to the large intestine using a mixed micellar system for improved absorption.

• Journal of Pharmaceutical Investigation
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• 제37권2호
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• pp.73-78
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• 2007

The aim of this study was to investigate the absorption properties of ketoprofen. The in-situ perfusion model has advantages over in vitro models as it provides intact lymphatic and blood flow circulation. The absorption properties of six different concentrations of ketoprofen have been studied in single pass in-situ rat intestine model. $^{14}C-PEG$ 4000 was used as a permeability marker and the possibility of an energy dependent contribution to ketoprofen absorption was also Investigated using the metabolic inhibitor sodium azide. Three different concentrations of sodium azide were studied to examine its effect on absorption of ketoprofen from the rat intestine. The findings of this study suggest that mono-carboxylic type drugs like ketoprofen cause permeability changes in the intestine. This is shown by the increase in absorption of $^{14}C-PEG$ 4000 as the concentration of ketoprofen is increased. However, the trend for ketoprofen permeability is to decrease over the concentration ranges. It was observed that the Papp values for ketoprofen with sodium azide shows a trend towards reduction in the amount of ketoprofen absorbed from the rat intestine which was significantly different (p<0.05) from that of ketoprofen with sodium azide 3.0mM. This indicates that sodium azide has an affect on the absorption of ketoprofen. The pH of all the perfusion solutions was altered to ${\sim}pH\;6.7$ by the buffering capacity of the small intestine secretions. The results suggest that mechanisms other than passive diffusion may be involved in ketoprofen absorption. This would be consistent with the involvement of active transport or saturatable processes in the absorption of drugs containing monocarboxylic acid group, as has been previously suggested from in vitro data.

• Macromolecular Research
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• 제9권4호
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• pp.197-205
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• 2001

Platelet adhesion onto elastic polymeric biomaterials was tested in vitro by perfusing human whole blood at a shear rate of 100 sec$\^$-1/ for possible verification of mechanisms of initial platelet adhesion perfusion of blood on the polymeric substrates was performed after treatments either with or without pre-adsorption of 1% blood plasma, and either with or without residence of the protein-preadsorbed substrate in phosphate buffered solution. The surfaces employed were elastic polymers such as poly(ether urethane urea), poly(ether urethane), silicone urethane copolymer, silicone rubber and poly(ether urethane) with the anti-calcifying agent hydroxyethane bisphosphate. Each polymer surface treated was exposed in vitro to the dynamic, heparinized whole blood perfused for upto 6 min and the surface area of platelets initially adhered was measured by employing in situ epifluorescence video microscopy. The blood perfusion was performed on the surfaces treated at the following three different conditions: directly on the bare surfaces, after protein pre-adsorption and after residence in buffer for 3 days of the surfaces protein pre-adsorbed for 2 h. The effects of blood plasma pre-adsorption on the initial platelet adhesion was surface-dependent. The amount of the adsorbed fibrinogen and the surface coverage area of the adhered platelets were dependent on the surface conditions whether substrates were bare surfaces or protein pre-adsorbed ones. To test an effect of possible morphological (re)orientations of the adsorbed proteins on the initial platelet adhesion, the polymeric substrate pre-adsorbed with 1% blood plasma was immersed in phosphate buffered solution for 3 days and then exposed to physiological blood perfusion. The surface area of the platelets adhered on these surfaces was significantly different from that of the surfaces treated with protein pre-adsorption only. These results indicated that platelet adhesion was dependent on the surface property itself and pre-treatment conditions such as blood perfusion without any pre-adsorption of proteins, and blood perfusion either after protein pre-adsorption or after subsequent substrate residence in buffer of the substrate pre-adsorbed with proteins. Understanding of these results may guide for better designs of blood-contacting materials based on protein behaviors.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.272-272
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• 1996

While ranitidine is well known to be absorbed rapidly, the underlying cause of variable bioavailability in intra- and inter-subjects has not been clarified yet. Intestinal permeability is a key controlling factor for oral absorption of highly soluble drugs, In the present study, intestinal ferfusions have been conducted to determine the intestinal permeabilities(Peffs) of ranitidine in the rats, dogs and humans and compared to the estimated fractions of dose absorbed (FAs) in humans. A new in vivo methodology, using a regional segmental perfusion technique, has been used in the dogs and humans. In situ single-pass perfusion experiments have been performed in the rats. In the dog and human studies, perfusion experiments have been conducted on two periods to determine the intrasubject variability, There was low significant intrasubject variation as compared to intersubject variation. The Peffs of ranitidine were 33%, 51%, and 45% inthe rats, dogs and humans, respectively. The FAs were approximately the same for all three species models, suggesting rats and dogs are good animal models for estimating the oral absorption of ranitidine in humans. In addition, the estimated extent of absorption of this drug is consistent with the average bioavailability, indicating that ranitidine has permeability-limited absorption characteristics. Supported by FDA Grant FD01462.