Drug Targeting Organ-Specific Strategies

Drug Targeting Organ-Specific Strategies. Edited by G. Molema, D. K. F. Meijer Copyright © 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29989-0 (Hardcover); 3-527-60006-X (Electronic)

12Use of Human Tissue Slices in Drug Targeting Research

Peter Olinga, Geny M. M. Groothuis

12.1 Introduction

It has long been recognized that in vitro research can provide valuable information on basic mechanisms with respect to kinetics and efficacy of drug targeting concepts. Such in vitro research includes the use of isolated cells, cell lines and perfused organs. In this chapter the introduction of tissue slices into drug targeting research will be discussed.A brief history of the slice technique will first be given. Until now most research on the slice technique has been focused on the metabolism and transport of drugs and this topic will therefore be summarized before embarking on a discussion of the contribution of the tissue slice technique to the area of drug targeting research.

In vitro research began with organ culture of embryonic organ rudiments [1]. The slice technique, using slices of tumour and liver tissue, was performed as early as 1923 by Otto Warburg [2] and in the following years by H. A. Krebs [3], who investigated the metabolism of amino acids in liver slices of cats, dogs and rats. These liver slices were prepared manually with limited reproducibility and viability [4]. After a decline in the application of slices in liver research in favour of the use of isolated hepatocytes as well as isolated perfused liver preparation, the development of the Krumdieck slicer in the 1980s led to a ‘comeback’ of the technique enabling the production of reproducible and viable liver slices [5]. This technology induced a renaissance of the slice technology. The development of these in vitro preparations has been of paramount importance for research on human liver function. As most of the research with tissue slices concerned the liver, this chapter will focus on the use of liver as the target tissue and only briefly mention the use of slices from other tissues in the concluding section of the chapter.

The most abundant cell type in the liver is the hepatocyte, other cells in the liver are the non-parenchymal cells: Kupffer cells, the resident macrophages of the liver, endothelial cells and stellate cells. These cells have been discussed in more detail in Chapter 4.

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

are also extensively used in a variety of other research fields including pharmacology and toxicology.

Isolation from human liver of other cell types, such as Kupffer, endothelial and stellate cells has also been developed and extensively reviewed [29–31].

Although the isolated (human) liver cells have been shown to be valuable in the study of mechanisms of drug transport in drug targeting research, the isolation procedures involves digestion to disrupt the cell-to-cell contacts. Clearly, enzymatic digestion may also damage plasma membranes and transport systems therein. In addition, for hepatocytes the normal polarity is lost after isolation. For instance it has been reported that by using collagenase digestion to isolate hepatocytes, the amount of asialoglycoprotein receptor present on the membrane of the hepatocyte is reduced [32]. Recently, Ikejima et al. [33] showed that Kupffer cells isolated by the standard isolation procedure with collagenase and pronase had lost their CD14 receptor, presumably an important receptor in the uptake of lipopolysaccharide.

In contrast to isolated liver cells, no enzymatic digestion is necessary for the isolated perfused liver preparation. The isolated perfused liver preparation, extensively used to study rat liver functions [34], has also been employed with human liver tissue, but the application is limited by the fact that only pieces of tissue that are encapsulated with liver capsula can be used. The use of the isolated perfused human liver preparation has been discussed further in Chapter 4. Another in vitro liver preparation that can be used without the need for enzymatic digestion is the liver slice (Figure 12.1). One of the main features of slices is that the original architecture of the organ is retained in the slice.Therefore, in liver slices, the different cell types of the (human) liver; i.e. hepatocytes, Kupffer-, endothelial and stellate cells are still present in contact with their original matrix environment, which enables the function of all cell types present and their normal intercellular communication to be studied. In addition, studies on cell selective distribution of carriers and drug–carrier conjugates can be performed in liver slices, which is of major importance in drug targeting research. The slice technique itself can also be used on other solid organs such as kidney, lung, intestine and even brain; the use of slices from these organs will be described in the concluding section of this chapter, whilst the greater part of the chapter will describe the use of liver slices in drug targeting research.

12.2 Preparation of Liver Slices

Liver slices were initially prepared manually using razor blades or mechanical instruments such as the Stadie Riggs tissue slicer [4]. The reproducibility of the thickness of the slices at that time was largely dependent on the skills of the operator. Of note is the fact that the minimal slice thickness that could be produced was about 0.5 mm. This dimension appeared to limit the penetration of nutrients and oxygen into the inner cell layers: central necrosis in the slice occurred during incubation [35].

The introduction of the Krumdieck slicer enabled a more optimal and reproducible preparation of liver slices (Figure 12.1). With this technique the thickness of the slices is adjustable to a value as low as 100 µm.The slicing procedure itself is performed in a buffer assuring minimal trauma of the tissue. In addition, the Krumdieck slicer provides a rapid and automated

312 12 Use of Human Tissue Slices in Drug Targeting Research

production of slices with reproducible thickness. Recently, the so-called ‘Brendel slicer’ was introduced, which has largely the same characteristics as the Krumdieck slicer, but offers the advantage of more constant oxygenation. However, with this technique the slices have to be prepared manually [36]. Liver slices from both tissue slicers have been evaluated. No significant differences were observed in levels of protein, potassium, total glutathione (i.e. GSH and GSSG), reduced glutathione (GSH) and cytochrome P450 and activities of 7-ethoxyre- sorufin O-deethylase and 7-benzoxyresorufin O-debenzylase in freshly cut rat liver slices produced by either of the two tissue slicers [37].

To prepare liver slices with the Krumdieck slicer, cylindrical cores of tissue are first isolated from the liver specimens (Figure 12.1). These tissue cores are prepared preferably by advancing a sharp rotating metal tube into the liver tissue using a drilling press, thus assuring the preparation of accurately cylindrical cores. If a biopsy punch is used to prepare the cores, it is difficult to obtain a uniform cylindrical shape.

The cores are subsequently placed in the slicer, and the slicing procedure is performed by advancing the core over an oscillating knife in a controlled environment (Figure 12.1). Cold (4°C) Krebs–Henseleit buffer (pH = 7.4, saturated with 95% O2 and 5% CO2) supplemented with 25 mM glucose is commonly used in preparing the slices [35,38–40], but Williams’ medium E [41], Earle’s balanced salt solutions [37], Sacks preservation medium [42] and V-7 preservation buffer [43,44] are also used.

All these buffers have a glucose concentration of 25 mM, which seems to be essential for the viability of the slices.

The optimal thickness for liver slices, in order to retain their viability during culture, is approximately 175–250 m. Price et al. [45] reported that the optimal thickness of liver slices for drug metabolism studies should be 175 m. In slices thicker than 250 m the inner cell layers suffer from a lack of oxygen and substrates, and in slices thinner than 175 m the ratio of damaged cells in the outer cell layers to the living cell mass becomes unfavourable [36,43,44,46–48]. For cryopreservation slightly thicker slices were reported to give better results [40], although recent developments show that slices of approximately 200–250 m can also be successfully used for cryopreservation (unpublished observation).

12.3 Incubation and Culture of Liver Slices

12.3.1 Incubation Systems

Previously, liver slices were incubated in static organ cultures [1]. Hart et al. [49] cultured rat liver slices for 24 h spread out on wet filter paper, floating on top of the incubation medium. Several slice-containing vessels were placed in a box with saturated 95% O2 and 5% CO2 at 37°C. However, the slices employed were rather thick (approximately 0.3 mm) and only the upper cell layers (0.2 mm) in the slice contained viable cells. Together with the introduction of the Krumdieck slicer [5,46], a new incubation technique for slices, the dynamic organ culture system (DOC), was introduced [35]. The main characteristic of this system is the intermittent exposure of the slice to incubation medium and the gas phase. The DOC is in fact a modified version of the Trowell incubation system [1].

Meanwhile many incubation systems have been developed, mostly based on either DOC or culturing the slices in multi-well incubation systems [38,40–43,50–52] and all have been used in pharmacological and toxicological research [36]. The most remarkable phenomenon emerging from these studies is the observation that the liver slices can be cultured for up to 72 h with the maintenance of their biotransformation activities [43]. In contrast, the use of primary suspensions of hepatocytes for metabolic and transport studies is restricted to a few hours [25]. Culturing of the hepatocytes allows experiments to last for a longer period of time (up to approximately 5–7 days). The hepatocytes form monolayers and develop bile canalic- ular-like spaces in between the cells [20]. However, specific liver functions such as albumin secretion, transport activity and cytochrome P450 activity decrease considerably during incubation [53,54]. After 24 h of culturing, drug metabolism activity will already have decreased by about 50%. This is very likely due to de-differentiation on the level of gene transcription [55]. In recent years much effort has been put into the improvement of the culture conditions of hepatocytes by adding extracellular matrix components or by co-culturing with other cell types in order to maintain their differentiation status [54,56–63]. Although survival and functioning of these cells has been greatly improved, complete maintenance of differentiated isoenzymes patterns has not been achieved yet. In fact, the liver slices can be seen as the most natural co-culture system within the original matrix.

12.3.2 Evaluation of Incubation Systems

There are only a few studies published in which the various incubation systems for liver slices were evaluated. Smith et al. [35] showed that slices in dynamic organ culture maintain their viability, as measured by ATP and potassium concentration, up to 20 h. Connors et al. [64] used a 24-well incubation system, in which the medium was stirred with a magnetic stirrer. In this incubation system rat liver slices were cultured for 8 h and human liver slices for 9 h during which time a high potassium concentration was maintained in the slices. Connors et al. [65] reported that the 24-well incubation system and the dynamic organ culture gave similar metabolite patterns after 24 h of incubation with a somatostatin analogue. Vickers et al. [66] also used the 24-well incubation system for 24 h, but no viability parameters were described. Dogterom et al. [51] showed that in a 12-well culture plate, which is put on a gyratory shaker, rat liver slices maintain their viability up to 11 h as determined by potassium concentration and ATP content. However, an impairment of the rat liver slices in a 24-well incubation system on the gyratory shaker was seen after 11 h. This was explained by the insufficient agitation of the medium in the 24-well incubation system. Leeman et al. [41] described a modification of the dynamic organ culture: a netwell insert (200-m polyester mesh carrier) placed in the wells of a six-well culture plate on a rocker platform. In this system, as with the DOC, the slices are intermittently exposed to the gas phase, which in this system is 40% O2/5% CO2/55% N2 and to the medium. Using this incubation system, the 3[4,5-dimethyl-thiazole- 2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction, a test for the cellular reduction capacity both in mitochondria and extramitochondrially involving NADH and NADPH [67], was maintained in the slices for up to 72 h [41]. Simple incubation of slices in a 25-ml Erlenmeyer flask in a shaking water bath was reported by de Kanter et al. to be successful over a 24-h period [40].

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Brendel’s group compared two incubation systems, the roller system (dynamic organ culture (Figure 12.2)) and the 12-well plate culture (the plates were put on a gyratory shaker), with respect to their ability to maintain the functionality of rat liver slices over 72 h of culturing. The slices were evaluated with respect to ATP concentration, potassium retention, MTT reduction and protein synthesis, in addition to alanine transaminase (ALT) and LDH leakage. Metabolic function was investigated by oxidative O-deethylation of 7-ethoxycoumarin (7-EC) [43]. It was concluded that dynamic organ culture was superior to multi-well plate culture [43]. Recently, another comparison has been made between the DOC-system and 12well system, showing that the 12-well system was superior to the DOC-system with regard to the metabolism of xenobiotics following long-term incubations (> 24 h) [68]. However, this study was performed in 95% air and 5% CO2, which may have influenced the results obtained. A high oxygen percentage of at least 40% is essential for optimal incubation of liver slices, as will be described in more detail below. In addition, both sets of experiments were carried out using different incubation media, which also may have influenced the results obtained.

12.3.3 Incubation Systems for Human Liver Slices

Various incubation systems have also been tested using human liver slices, these include the 24-well plates with magnetic stirrers [38,66,69], the six-well plates in a shaker [52] and the DOC roller system [70,71], but no direct comparison has been made as yet. Human liver slices can be cultured for 72 h in DOC and maintain their ability to respond to specific inducers of cytochrome P450 such asmethylclofenapate and Aroclor 1254 [71].

12.3.4 Oxygenation and Culture Media for Liver Slice Incubation

In addition to the incubation system itself, the oxygen and nutrient concentration of the medium are also important for the viability of liver slices [46,47]. It appeared that a nutrientenriched medium containing bicarbonate maintained the slice viability better than a simpler medium such as Krebs–HEPES buffer [44]. It was shown that K+-retention, protein synthesis and LDH leakage was maintained in rat liver slices for 5 days in a Waymouth’s/bicarbonate medium in a dynamic organ culture system [44]. Oxygenation of the hepatocytes, especially those in the centre of the slice, has been a major concern. In this respect it is important to note that oxygen at too high a concentration may be toxic, due to tissue damage by oxygen radicals, whereas excessively low levels of oxygen may result in ischaemia. Both 95% air/5% CO2 and 95% O2/5% CO2 are commonly used. In long-term culture up to 5 days [43] the DOC system or DOC-related incubation systems are recommended, because of the intermittent exposure of the slice to culture medium and to the gas phase. This feature was claimed to be important for optimal gas exchange. In our experiments however, slices that were continuously submerged in medium performed equally well or even better than those in the DOC system or DOC-related system (six-well culture plate on the rocker platform [72]). It can be calculated that liver slices consume only 0.3–1% of the dissolved O2 per minute. This implies that, provided that the medium is continuously oxygenated, the availability of O2 is unlikely to be a limiting factor. In our laboratory we have carried out a study