Download - Optimal oxygen tension conditions for viability and functioning of precision-cut liver slices
Exp Toxic Pathol 2000; 52: 335-338URBAN & FISCHERhup://www.urbanfischer.de/journals/exptoxpath
Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, Germany
Optimal oxygen tension conditions for viability and functioning ofprecision-cut liver slices
C. DROBNER, R. GLOCKNER, and D. MULLER
With 3 figures
Received: June 2, 1999; Accepted: June 28, 1999
Address for correspondence: Prof. Dr. D. MULLER, Klinikum der FSU Jena, Institut fUr Pharmakologie und Toxikologie,D-07740 Jena, Germany; Fax: ++49(0)3641 938702, e-mail: [email protected]
Key words: Liver slices precision-cut; Cytochrome P450; Oxygen tension; 7-Ethoxycoumarin O-deethylation; Culturemedia.
Summary
Optimal oxygenation of culture media is important forthe successful use of liver slices as an in vitro tool forstudying liver function. For this reason the influence of 20,40, 70 and 95% O2 concentration on the viability andmetabolism of liver slices was investigated. The sliceswere incubated in the roller system at 37°C under continuous gassing for 2,24 and 48 hrs. Protein, DNA and potassium contents were maintained or even increased over timewithout influence by O2 concentrations. The albumin secretion of slices incubated at 40-95% O2 did not differ, butwas much lower at 20% 02. A slight non-significant decrease in albumin secretion after 24 hrs of cultivationcould be observed, whereas a much steeper decline wasfound in all groups after 48 hrs. Cytochrome P450 (CYP)dependent 7-ethoxycoumarin O-deethylation (ECOD) didnot differ between the various O2 concentrations, but declined from 2 to 48 hrs of incubation.
It can be concluded that O2 concentration of 20% is notsufficient to maintain all cell functions of incubated ratliver slices, wheras 40, 70 and 95% are useful O2 concentrations to retain all parameters investigated.
Introduction
Under basal metabolic conditions the liver consumes20-33 % of total O2 utilized by the organism (CATAPANOet al. 1996). A poor O2 supply has negative effects on theenergy metabolism of liver cells and may compromisetheir viability (CATAPANO et al. 1996). On the other hand,too high O2 concentrations may be toxic to the tissue,because the formation of reactive oxygen species leadsto cell injury (SANZ et al. 1996).
When in vitro models such as the isolated perfusedliver, precision-cut liver slices and isolated hepatocytesare used, oxygen tension must be carefully considered.NISHIKAWA et al. (1996) found that hepatocytes culturedat 10-50% O2 maintain their function very well, whereasat 5% and 90% O2 viability decreased. On the contrary,MIYAZAKI et ai. (199 I) found that even normoxic culturecondition is hyperoxic for hepatocytes, whereas a reduction of O2 tension (10%) in the primary culture increasedthe survival rate of hepatocytes. With liver slices the situation is more complicated because of an oxygen gradient between slice surface and inner cell layers, whichcan lead to limited O2 concentration and inhibitedmetabolic function in inner cells (LEEMAN et al. 1995,WORBOYS et al. 1997).
In contrast to isolated hepatocytes, tissue slices conserve the tissue architecture such that all cell types andcell to cell communications are present. Therefore theslice model has become a more and more used in vitrotool during the last years, e.g. for studies on drugmetabolism and its induction even in slices of humanorigin (e.g. DRAHUSHUK et al. 1998; LAKE et al. 1996;GLOCKNER et ai. 1999). A lot of studies exist regardingoptimal culture media and incubation systems (for review see OLINGA et al. 1997), whereas information aboutthe proper oxygenation of tissue slices has been rareuntil now. TOUTAIN et al. (1998) reported on a superiority of rat liver slice cultivation at 70% O2 over 20% concerning histology, whereas some biochemical parameters were similar at either oxygen concentration. PRICEet al. (1998) found only minor differences of some functions between culturing at 95 and 20% 02. Since toohigh and also too low oxygen tensions can be toxic for
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Results
Fig. 1. Protein, potassium and DNA content of rat liverslices after 2, 24 and 48 hrs incubation at O2 concentrationsof 20, 40, 70 or 95%. Arithmetic means and SEM areshown, n =6 for the groups 20% and 40%, n =9 for thegroup 70% and n = 3 for the group 95%.
Potassium concentration of rat liver slices incubatedin rollers for 2, 24 or 48 hrs was not influenced by theoxygen concentration and was stable until the 48th hour(fig. I). Also, DNA and total protein contents were neither influenced by incubation time nor by O2 concentration (fig. 1).
Liver slices are able to produce and secrete considerable amounts of albumin. Albumin secretion was equalat O2 concentrations of 40, 70 and 95%, but much lowerat 20% O2 (fig. 2). Independent of oxygen concentration,albumin secretion was relatively stable from the 2nd tothe 24th hr of incubation, whereas a marked declinecould be observed from the 24th to the 48th hr.
CYP-dependent ECOD in intact slices was not modified by O2 concentration (fig. 3). Considering the influence of incubation time, in the first series of experimentsECOD decreased only slightly (not significantly) fromthe 2nd to the 24th hr of incubation, but thereafter it de-
Material and Methods
hepatocytes, we determined the influence of a number ofO2 concentrations (20, 40, 70 and 95%) on the viabilityand metabolic function of rat liver slices. The sliceswere incubated for 2, 24 and 48 hrs using the dynamicorgan culture (SMITH et al. 1986). As a test for slice viability potassium retention was used, a parameter formembrane integrity, cellular energy and intactness of theNa+/K+-ATPase (FISHER et al. 1995 a). The differentiatedfunction of hepatocytes was established by measuringalbumin secretion and cytochrome P450 (CYP)-dependent biotransformation activity.
Chemicals: Krebs-Henseleit buffer (pH = 7.4) wassupplemented with HEPES (12.6 mM) and gentamicin(50 mg/ml) and gassed with a mixture of 20-95% 0/75-0% Ni5% CO2, Williams' Medium E (WME, Biochrom KG) was supplemented with I-glutamine (292 mgll),insulin (0.1 11M), gentamicin (50 mgll) and tylosin (100mg/I) and equilibrated with the respective gas mixture.
Preparation of slices: Liver slices were obtained frommale Han:WIST rats (33-40 days old), which were housedunder standardized conditions as described previously(MULLER et al. 1998). After the rats had been killed by decapitation in ether anaesthesia, livers were immediatelyperfused with cold Krebs-Henseleit buffer. Slices (thickness about 250 11m) were prepared with a Krumdieck tissue slicer from cylindrical tissue cores (diameter 8 mm).
Incubation of slices: For incubation in the rolIer system(Vitron Inc., Tucson, AZ) two slices were put on a stainless-steel insert, placed in an incubation vial containing1.8 ml of WME, continuously gassed with the above-mentioned gas mixtures at 37°C for 2-48 hrs. After 2 and24 hrs the medium was replaced by fresh WME. Theexperiments with O2 concentrations of 20, 40 and 70%(n =6) were performed at the same period of time, but itwas only possible to compare two gas contents in parallel(liver slices from the same animal). The investigation of95% O2 delivery had been carried out in many experimentsbefore, but for direct comparison an additional series wasperformed using 70 and 95% O2 in paral1el (n = 3).
Viability and metabolic function tests: Al1 parameterswere measured in duplicate slice samples. Protein, DNAand potassium were determined in slice homogenate. Forestimation of ECOD rate or albumin secretion two sliceseach were transfered from the rol1ers into 4 ml WME in 25ml-Erlenmeyer flasks. ECOD reaction was started by addition of substrate and stopped after 10 min, and the metabolite was measured fluorimetrically in the medium. Albuminconcentration was determined in the medium by ELISAafter 3 hrs additional incubation in flasks under gassingwith the appropriate gas mixture. Al1 methods have been recently described in detail (MULLER et al. 1998).
Statistics: Arithmetic means ± S.E.M. are given. Forstatistical evaluation the t-test for independent samples(slices from different livers) or paired samples (slices fromthe same liver) was used.
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For incubation of liver slices commonly 95% 0/5%CO2 or 95% air/5% CO2 are used (cf. OLINGA et al.1997). In two recent papers 95 vs. 20% and 70 vs. 20%02' respectively, were compared concerning viabilityand function of rat liver slices with differing conclusionsin dependence on the investigated parameters (PRICE etal. 1998; TOUTAIN et aL 1998, resp.). In our experimentswe tested a series of O2 concentrations between 20 and95%, to find out optimal incubation conditions for ourparameters in the roller incubator.
Slice viability over the incubation time of 48 hrs,proved by potassium retention, DNA and protein contents, was not influenced by the investigated gas mixtures containing 20, 40, 70 or 95% 02' The parameterswere in the range of those reported by MULLER et aL(1998), who also incubated rat liver slices in the rollersystem for 48 hrs gassed with 95% 02' In the shakenflask at 95% O2 similar results were obtained (unpublished). Our potassium concentrations correspond wellwith those of FiSHER et aL (1995b) at 95% 02' but incontrast to Fisher et aI., they were not decreased at 20%02' WRIGHT and PAINE (1992) cultivated liver slices at95% air/5% CO2 and found potassium contents comparable to ours, which remained stable over 24 hrs. In contrast to dynamic organ culture, incubation in shakenflasks at 20% O2 caused decreased potassium retentionto about 70% of initial values after 24 hrs and 60% after48 hrs (own unpublished results), which could be explained by worse diffusion than in the roller. The latter isalso true for incubation in well plates (FISHER at aL1995b).
PRICE et aL (1998) reported on 02-independence (95vs. 20%) of CYP-dependent ECOD in rat liver slices.We also found that ECOD was not influenced by thechosen oxygen conditions, but declined with incubationtime up to 48 hrs at all gas concentrations. In contrast toother parameters including other biotransformation reactions, however, absolute ECOD rates and their stability over longer incubation times vary among different series of experiments, e. g. in previous investigations nosignificant changes of ECOD activity between the 2ndand 48th hrs of incubation at 95% O2 could be detected(MULLER et al. 1998). The reason for the variability ofthis reaction is not clear.
Albumin is the major plasma protein, accounting for11-13% of the total protein, synthesized by the liver(IWAI et aL 1996). Therefore the amount of albumin secreted into the culture medium is a good indicator ofmetabolic activity and secretory capacity of the hepatocytes (SAAD et al. 1994). In our experiments the loweredalbumin secretion of slices incubated at 20% O2 is possibly due to hypoxic conditions within the tissue. It isknown that protein synthesis and especially that of albumin is sensitive to ischemic conditions (e.g. LINDELL etaL 1994). Decreased albumin synthesis is a feature ofliver response to injury and may reflect hepatocyte modulation by Kupffer-cell derived acute phase cytokines(KOWALSKI-SAUNDERS et aL 1992). BALLMER et aL
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Fig. 3. ECOD activity of intact rat liver slices after 2, 24and 48 hrs incubation at O2 concentrations of 20, 40, 70 or95 %, determined in two independent series. Arithmeticmeans and SEM are shown. * =significant difference tothe initial value of the same group (paired t-test, p ::; 0.05),x = significant differences to the next higher O2 content(independent samples t-test, p ::; 0.05).
clined more remarkably. In the second series ECOD ratewas very high after 2 hrs, but after 24 hrs it dropped tovalues comparable to the first series of experiments.
Discussion
Since too low and also too high O2 concentrationsmay cause liver cell damage (SANZ et aI. 1996), theproper oxygenation of cultured liver slices is of importance to provide enough O2 for hepatocytes in the centreof the slice and to avoid toxic concentrations in the outerlayers of the slice.
Fig. 2. Albumin secretion by rat liver slices after 2,24 and48 hrs incubation at O2 concentrations of 20, 40, 70 or95%. Arithmetic means and SEM are shown, n = 6 for thegroups 20% and 40%, n = 9 for the group 70% and n = 3for the group 95%. * =significant difference to the initialvalue of the same group (paired t-test, p ::; 0.05), x =significant differences to the next higher O2 content (independent samples t-test, p::; 0.05).
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(1992) observed that acute inflammatory diseases arecharacterized by a depression of serum albumin whileproduction of acute-phase proteins increases. The question remains, whether the assumed hypoxic conditionsin our experiments at 20% O2 are due to cultivation inthe roller system for 2, 24 and 48 hrs or due to the subsequent incubation in the shaken flask for 3 additional hrs.In the shaken flask the gas exchange is not so optimal asin the roller incubator, but albumin secretion cannot bedetermined reproducibly in rollers because of varyingcontact between tissue and medium (unpublished result). Therefore hypoxic conditions might be due to the3-hrs-incubation in Erlenmeyer flasks.
Alltogether, the results indicate that O2 concentrationof 20% is not sufficient to maintain all cell functions ofrat liver slices incubated in the roller system. 40, 70 and95% are useful O2 concentrations to retain all parameters to the same extent. Even at optimal O2 concentrations some functions decrease after 48 hrs incubation ofthe slices. But the results demonstrate that under appropriate conditions liver slices can be used for 24 hrs, atime which is sufficient for a lot of pharmacological andtoxicological investigations.
Acknowledgement: This work was supported by EUBiotec Programme, PL 962145. The technical assistanceof Mrs. H. GUDER and Mrs. H. MEUCHE is greatly appreciated.
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