Perfusion hepatocyte isolation

The best procedure for hepatocyte isolation is the 2-step collagenase perfusion technique. Freshly isolated human hepatocytes are now also available from commercial vendors. However, the quality of the liver may be affected by the transportation period. One needs to ensure that the hepatocytes are near confluent and that no significant attachment occurs during the culturing period. 

Numerous in vitro and/or in situ models have been developed to investigate drug metabolism. In order of decreasing complexity, they are the isolated perfused liver, isolated liver slices, hepatocytes in coculture with epithelial cells or bacteria, hepa-tocytes in suspension and in primary culture, subcellular hepatic microsomal S9 fractions and high-speed pellet microsomes. The field of metabolism is immense and will not be covered in depth here. However, one should have a basic understanding of the benefits and drawbacks of each one of these methods. ... 

Treatment of rat hepatocytes isolated by in vitro coUagenase perfusion with doxorubicin increased catalase but decreased Mn superoxide dismutase mRNA expression (Rohrdanz et al. 2000). Doxorubicin almost completely inhibited RNA synthesis and induced hpid peroxidation as measured by the accumulation of malondialdehyde in the medium. 

The question arose next was which organ or tissue is responsible for potassium liberation into the blood in intoxicated animals. To answer the question, we perfused the enterotoxin into isolated organs or tissue. As shown in Fig. 2, concentration of potassium in perfusates from isolated lungs and lower extremities showed no obvious change after the perfusion of the enterotoxin. However, the concentration of potassium in perfusate from isolated liver showed a remarkable increase after the perfusion of the enterotoxin. The leakage of potassium from the liver occurred within a minute and finished 5 min after the perfusion of the toxin (Fig. 3). The amount of the potassium liberated from the liver was estimated to be about 133 pmol, which is enough to raise plasma level of potassium to more than lethal level. In addition, activities of transaminases in the perfusate from the enterotoxin-treated liver increased following to the elevation of potassium concentration (Fig. 3). The results indicated that potassium is liberated from hepatocytes, and that the enterotoxin makes a small pore ... 

Of particular interest in brevetoxin research are the diagnosis of intoxication and identification of brevetoxins and their metabolites in biological fluids. We are investigating the distribution and fate of radiolabeled PbTx-3 in rats. Three model systems were used to study the toxicokinetics and metabolism of PbTx-3 1) rats injected intravenously with a bolus dose of toxin, 2) isolated rat livers perfused with toxin, and 3) isolated rat hepatocytes exposed to the toxin in vitro. 

These studies represent the first report of the metabolism of brevetoxins by mammalian systems. PbTx-3 was rapidly cleared from the bloodstream and distributed to the liver, muscle, and gastrointestinal tract. Studies with isolated perfused livers and isolated hepatocytes conflrmed the liver as a site of metabolism and biliary excretion as an important route of toxin elimination. [ H]PbTx-3 was metabolized to several compounds exhibiting increased polarity, one of which appeared to be an epoxide derivative. Whether this compound corresponds to PbTx-6 (the 27,28 epoxide of PbTx-2), to the corresponding epoxide of PbTx-3, or to another structure is unknown. The structures of these metabolites are currently under investigation. 

Data from both in vivo and in vitro systems showed PbTx-3 to have an intermediate extraction ratio, indicating in vivo clearance of PbTx-3 was equally dependent upon liver blood flow and the activity of toxin-metabolizing enzymes. Studies on the effects of varying flow rates and metabolism on the total body clearance of PbTx-3 are planned. Finally, comparison of in vivo metabolism data to those derived from in vitro metabolism in isolated perfused livers and isolated hepatocytes suggested that in vitro systems accurately reflect in vivo metabolic processes and can be used to predict the toxicokinetic parameters of PbTx-3. 

Miyauchi, S., Sawada, Y., Iga, T., Hanano, M., Sugiyama, Y., Comparison of the hepatic uptake clearances of fifteen drugs with a wide range of membrane permeabilities in isolated rat hepatocytes and perfused rat livers, Pharm. Res. 1993, 10, 434-440. 

Since a high yield isolation procedure of rat hepatocytes was described in 1969 [6], hepatocytes have become the model of choice for drug transport studies in the liver in vitro [7]. With this procedure, isolated hepatocytes from many species have been prepared, including hepatocytes from rat, mouse, chicken, dog, fish, hamster, pig, cow, sheep and monkey liver (for an extensive review see reference [8]). Before 1976, only relatively small numbers of human hepatocytes could be isolated, due to the use of non-perfusion techniques [9]. Bojar et al. [10] were the first to use a perfusion technique on human livers which greatly enhanced the yield of hepatocytes. In principle the procedure that is now commonly used, is based on the one described by Seglen [6] for rat hepatocytes. Either a biopsy wedge with intact capsula on... 

Digoxin uptake into rat hver shces showed a temperature-dependent component, compatible with the involvement of carrier-mediated uptake mechanisms. Quinine markedly inhibited the uptake of digoxin, in contrast to its diastereomer quinidine, which only slightly inhibited the digoxin uptake in rat liver slices. This stereoselective inhibition is in line with results obtained in isolated rat hepatocytes and isolated perfused rat hvers [90,91]. These results were also found after cryopreservation of the slices, indicating that carrier-specific phenomena can be studied after cryopreservation [92]. 

Sugiyama and Kato [63] described a model for the receptor-mediated endocytosis of the polypeptide hormones epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in isolated perfused rat liver and in isolated rat hepatocytes, to estimate the efficiency of dmg targeting using these polypeptide hormones as potential drng carriers. 

LIVER Use of isolated perfused liver in studies of biological transport processes, 192, 485 measurement of unidirectional calcium ion fluxes in liver, 192, 495 preparation and specific applications of isolated hepatocyte couplets, 192, 501 characterizing mechanisms of hepatic bile acid transport utilizing isolated membrane vesicles, 192, 517 preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes, 192, 534. 

Because of the need for isolation techniques applicable to other species, Hoogenboom et al [18] investigated the isolation of hepatocytes from porcine livers and developed an enzymatic method with closed-loop and open-loop perfusion stages for the perfusion of liver lobes. We investigated a method to perfuse the whole organ to receive a great amount of hver cells (up to 25-10 cells per pig liver) and a viability higher than 90%. 

The so-called UCLA bioartificial liver involves the direct hemoperfusion of microencapsulated porcine hepatocytes in an extracorporeal chamber (Eigure 7.3). Since it permits perfusion with whole blood, it has an advantage over the hollow fiber technique that has to be perfused with plasma. The hepatocytes are isolated from pig livers and microencapsulated in an alginate-polylysine membrane. Microencapsulated hepatocytes are approximately 300 to 700 pm in diameter. 

Isolate hepatocytes by collagenase perfusion of liver biopsy... 

T.C. Orton, A.E. Sorman, D.N. Crisp, A.P. Sturdie, Dynamics of xenobiotic metabolism by isolated rat hepatocytes using a multichannel perfusion system. Xenobiotica 13 743, 1983. 

Using an erythrocytes-containing medium for perfusion one has to take into account the putative involvement of the erythrocytes themselves with respect to uptake of the candidate compound. Therefore, not only the erythrocyte-free perfusate but also the erythrocytes fraction should be included in the analysis of the candidate compound, separately. In our hands the model of isolated perfused liver is metabolically active for up to 3 hours and during this time no decline in hepatic metabolic activity becomes obvious. However, bile flow declined during the perfusion experiments. Therefore, our total perfusion time of isolated livers in our standard experimental setup is limited to 2 hours. The tissue level of the candidate compound analyzed after 2 hours in the liver is a measure for the total amount of compound in the whole organ. This does not necessarily mean the presence of the candidate compound in hepatocytes but additionally in the capillary and biliary space of the liver. 

The slice technique was already introduced by Otto Wartburg in 1923 and commonly used in in vitro liver research until isolated hepatocytes and isolated perfused liver preparation were introduced and optimized (Olinga 1998). Previous methods of slicing using the Stadie Riggs (Stadie 1944), Vibratomc (Smith 1985), or hand made slice technique (Forster 1948) suffered on a rapid preparation of thin tissue slices of uniform thickness and dimension under conditions that minimize trauma to the live tissue (Krumdieck 1980). 

Hepatocytes - both freshly prepared or cryopreserved - are commercially available e.g. Gen test, IVT, Xenotech or prepared in-house. Isolation of the animal hepatocytes follows after a two step collagenase perfusion of the liver via the vena portae in situ (Seglen 1976) or via several blunt-end cannulae inserted into vessels available on the cut surface of pieces of human liver obtained from resection. Liver cells are gently scraped out into suspension buffer and washed twice to three times by centrifugation to remove cell fragments and non-vital cells. Hepatocytes are used immediately or cryopreserved for further use. 

CRITICAL ASSESSMENT OF THE METHOD Human hepatocytes (fresh or cryopreserved) are now commercially available e.g. from BD/Gentest, In Vitro Technology or Xenotech. However, the quality, stability and availability of the commercial preparation remain questionable (Mandan 2002). Isolation (and cultivation) of hepatocytes is still time- and labintensive and needs to be optimized for livers of every different animal species (De Graaf 2002). Metabolism studies in hepatocytes might be a good compromise between perfused livers and subcellular fractions such as microsomes, since the complete cellular machinery is available. Nevertheless, some pitfalls have to be taken in account ... 

---