The Use of Tissue Slices for Pharmacotoxicological Studies

The Report and Recommendations of ECVAM Workshop 20^1,2

Reprinted with minor amendments from ATLA 24, 893-923

Peter H. Bach,³ Alison E.M. Vickers⁴, Robyn Fisher⁵, Andreas Baumann⁶, Eva Brittebo⁷, David J. Carlile⁸, Henk J. Koster⁹, Brian G. Lake¹⁰, Florence Salmon¹¹, Thomas W. Sawyer¹², and Greg Skibinski¹³
³Interdisciplinary Research Centre for Cell Modulation Studies, Faculty of Science and Health, University of East London, Romford Road, London E15 4LZ, UK; ⁴Sandoz Pharma Ltd, 4002 Basel, Switzerland; ⁵Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ 85724, USA; ⁶Institut für Pharmakokinetik, Schering Aktiengesellschaft, 13342 Berlin, Germany; ⁷Department of Pharmacology and Toxicology, SLU Biomedical Centre, 751 23 Uppsala, Sweden; ⁸Department of Pharmacy, University of Manchester, Oxford Road, Manchester M13 9PL, UK; ⁹Solvay Duphar, C.J. van Houlenlaan 36, 1380 DA Weesp, The Netherlands; ¹⁰BIBRA International, Woodmansterne Road, Carshalton, Surrey SM5 4DS, UK; ¹¹Crop Protection Animal Metabolism and Residue Chemistry, BASF Aktiengesellschaft, 67114 Limburgerhof, Germany; ¹²Medical Countermeasures Section, Defence Research Establishment Suffield, Medicine Hat, Alberta T1A 8K6, Canada; ¹³Department of Surgery, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK

¹ECVAM - The European Centre for the Validation of Alternative Methods. ²This document represents the agreed report of the participants as individual scientists.

Address for correspondence: Prof. P.H. Bach, IDRCCMS, Faculty of Science and Health, University of East London, Romford Road, London E15 4LZ, UK

Address for reprints: ECVAM, TP 580, JRC Environment Institute, 21020 Ispra (VA), Italy

Preface

This is the report of the twentieth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1).

The workshop on The Use of Tissue Slices for Pharmacotoxicology Studies was held in Angera, Italy, on 27-31 May 1996, under the co-chairmanship of Peter Bach (University of East London, UK) and Alison Vickers (Sandoz Pharma, Switzerland). The objective of the workshop was to assess our current state of knowledge regarding the uses of tissue slices, particularly precision-cut slices.

Tissue slices complement other in vitro techniques, and are intermediate between the widely used cell culture methods and the much more complex perfused organ systems. The use of tissue slices can provide valuable pharmacotoxicological data. Following a decade of revived interest in the application of tissue slices, the aim of the workshop was to undertake a critical assessment of what had been achieved to date, and to identify ways in which the full potential of tissue slices could be realised during the next decade.

Introduction

The use of tissue slices in in vitro investigations was initially reported in the 1920s (2). Since then, slices have been prepared from various organs, most often by using "free-hand" methods or, more recently, using simple "slicers", such as the Stadie-Riggs microtome. Even with these aids, it is not easy to obtain highly reproducible data, especially for the novice. In addition, the quality of the slices produced generally restricts investigations to relatively short-term experiments, lasting for a few hours with liver, kidney, heart, etc., although slices obtained from the brain and lung are viable for longer periods.

Despite these limitations, tissue slices have been widely used, and viable material from many different organs (brain, kidney, liver, lung, cardiac and smooth muscle, lymphoid tissue, placenta, mammary gland, adrenal, thyroid, and gastrointestinal tract) and species (man, non-human primates, all laboratory and domestic animals, and some exotic species) have provided valuable biochemical, physiological, pharmacological, and toxicological data.

There are several important factors which make the use of tissue slices attractive (Table I). The major advantage is that slices contain all of the cell types present in the organ in vivo, in their normal spatial relationships, and with the potential for normal intercellular communication and cellular interactions. The presence of different cell types in slices enables both regional and cell-specific effects to be investigated in vitro.

Table I: Major Advantages of Using Tissue Slices

Represents complexity of the intact organ
Methodology allows an easier cross-species comparison
Potential to use many organs from the same donor
Facilitates histological evaluation as an alternative to, or as a complement to, biochemical tests
Able to co-culture slices derived from different organs
Possible to investigate regional toxicity (that is, proximal v distal; periportal v pericentral) and metabolism
Possible to use human tissue efficiently

The methodology for preparing slices is similar, regardless of the species or organ from which they are derived. This facilitates comparisons between different species; it is straightforward to prepare slices for multi-organ comparisons in laboratory animals, larger species, and man. Furthermore, the methodology maximises the use of the available tissue, which is essential for domestic animals, primates and man, where only a limited amount of material is typically available. The "economy" of slice preparation is illustrated by the potential to obtain about 75 slices from one adult rat liver, and 10-20,000 slices from an adult human liver (if several laboratories were involved in the preparation). Each slice (8-10mm in diameter) represents sufficient tissue to study metabolism, cell biology, and toxicology. Finally, tissue slices can be prepared from treated animals, and from animal and human organs with pathological lesions.

Regulatory guidelines are now beginning to encourage the use of in vitro data. Thus, industry is making greater use of in vitro methods, and is sponsoring their development and evaluation.

The "Renaissance" in Tissue Slicing

The use of tissue slices has undergone a major revival in the last ten years due to the introduction of instrumentation which makes it possible to cut very thin tissue slices reproducibly, and to incubate these slices using surface culture techniques (3-16). Slices produced by using these new instruments are less damaged than those prepared with "free-hand" methods, and the use of appropriate media and novel incubation systems has enabled the prolonged maintenance of viable tissue slices from a wide variety of organs (for example, liver slices have been maintained for up to five days; 13, 14). It is important to ensure that tissues incubated for extended periods are carefully evaluated by as many sensitive parameters as possible, to ensure that tissue viability is adequately defined.

Devices for Slice Preparation

Slices have been produced by using a number of methods, ranging from "free-hand" procedures to fully automated slicing machines. Both brain and lung slices which are viable for long periods can be prepared by using simple methods (for example, free-hand). The strengths and weaknesses of the most widely used methods are outlined in Table II.

Table II: Advantages and Disadvantages of the Different Systems Available for Producing Tissue Slices

Slicing system	Disadvantages	Advantages
Free-hand slicers	Poorly reproducible slice thickness Depends on expertise	Inexpensive Accessible Successful in the hands of experienced researchers
Stadie-Riggs	Poor reproducibility Compresses tissue	Inexpensive Accessible Successful in the hands of experienced researchers
Mcllwain chopper	Compresses tissue Thick slices Slice size generally not consistent unless tissue core is used	Relatively inexpensive Quick and reproducible (with care) between experiments Works well for brain and lung tissue
Precision-cut slicers	More expensive More labour intensive More maintenance Requires training	Reproducible thin slices in large numbers Tissue is cut under physiological, oxygenated, buffer Less tissue compression Slice production parameters easily controlled More amenable to longer term culture

Free-hand Slices

Much of the early work on tissue slices was conducted with slices which had been prepared by using a hand-held razor blade or scalpel (Appendix 1).

Guided Slices

The uniformity of slice thickness can be improved by using various mechanical aids when preparing the slices, as detailed below.

Space-separated Razor Blades

Several devices have been described which employ simple spacers between razor blades as the basis for selecting slice thickness. These may have two or more blades, and may be of a simple or complex design.

Dermatome

The dermatome consists of a simple guide adjacent to a scalpel or razor blade which helps to maintain the cutting edge at a fixed level from the surface. It has been widely used for producing split skin preparations for plastic surgery.

Stadie-Riggs Microtome

The Stadie-Riggs microtome (17) is the simplest device for trying to ensure reproducible slice thickness. It consists of a microtome in a brass base (which can be used as a cooled heat sink to reduce the tissue temperature) which lifts the tissue above a flat cutting surface of the block.

Mechanical Slicers

Mechanical slicers have become increasingly popular, since they enable better slices to be produced, more easily and rapidly. The major features of the two newest precision-cutting tissue slicers (the Krumdieck® and the Brendel-Vitron® slicers) are listed in Table III.

Table III: Comparison of Krumdieck and Brendel-Vitron Tissue Slicers

	Krumdieck Slicer	Brendel-Vitron Slicer
Cost	> $10,000	< $5,000
Cutting blade	Oscillating	Rotating
Mechanics	Complex	Simple
Maintenance	Complex	Simple
Level of automation	Fully automated	Semi-automated
Sterilization	Autoclave and cold	Cold only

McIlwain Tissue Chopper

The McIlwain tissue chopper® (18) was the first mechanical device that enabled the thickness of tissue slices to be pre-set (from 100-1000µm), and which produced tissue slices which were adequately reproducible between experiments. The slicer consists of a mechanically operated chopping device which is brought down on a moving platform that holds the tissue. It has been used most extensively for the preparation of brain and lung slices. Many of the references cited in Appendix 1 relate to the use of the McIlwain tissue chopper.

Precision-cut Tissue Slicers

The application of precision-cut tissue slices for pharmacology and toxicology studies has opened up many new possibilities for using tissues such as liver, kidney, lung and heart (3-16). These slicers are capable of producing relatively thin tissue slices of consistent thickness from a range of organs and from any species.

(a) Krumdieck tissue slicer. This was the first precision-cut tissue slicer to be described, in 1983 (3); it is marketed as a fully automated mechanical slicer, which uses an oscillating blade and circulating buffer flow which carries the slices away from the cutting region. Oxygenation and cooling accessories are available.

(b) Brendel-Vitron tissue slicer. This instrument encompasses many of the same concepts as those of the Krumdieck slicer, including cutting slices in a circulating buffer, which can be cooled. The Brendel-Vitron slicer incorporates a rotating blade, where the whole cutting surface is used, and also limits the mechanical parts to those of simplest function. Oxygenation and cooling of the circulating buffer is a standard part of the basic unit.

There are also a number of mechanical dermatomes available.

Precision-cut Tissue Slices

Although the workshop concentrated mainly on applications of precision-cut tissue slices, it is recognised that conventional tissue slices are still widely used, and that their use provides valuable data and has contributed considerably to our knowledge of cell biology. There is little doubt that these conventional techniques will continue to be widely used (Appendix 1).

Practical Considerations When Using Precision-cut Tissue Slices

The use of precision-cut tissue slices has been reviewed on several occasions (4-14). Precision-cut tissue slices represent a complementary technique to monolayer cell cultures; both of these methods have inherent strengths and weaknesses (Table IV).

Table IV: Comparison of the Strengths and Weaknesses of Tissue Slices and Monolayer Cultures

Strengths	Weaknesses
Tissue Slices
Easier comparison between species and different organs All cell types are relevant to the in vivo situation Cells in their normal architectural relationships Intercellular communication Cellular interactions Morphological studies possible	New technology still being developed, including the optimisation of conditions for phenotypic expression of each cell type Viable cell enrichment not possible Lack of specific markers for measuring target-selective cell toxicity
Primary Cells
Well-established, well-characterised, widely used Viability for up to 4 weeks with special techniques (for example, collagen sandwiches) Defined cell type interaction studies possible Co-culture studies possible (for example, effects of mediators) Viable cell enrichment possible	Only preselected cells can be studied Isolation of cells can be technically challenging and time-consuming Cell damage during isolation (use of proteolytic enzymes) Cross-species comparisons difficult Most studies on solid supports Cellular interactions more difficult to study

Much of the current experience with precision-cut tissue slices is with liver slices. This serves as a basis for developing procedures for using slices from other tissues, including kidney, lung, intestine, spleen, and heart. Specific applications for each of these are considered below.

Availability and Storage of Tissue Prior to Slicing

Fresh tissue (for example, from experimental animals) should be used immediately after necropsy. If it is necessary to transport organs prior to slicing, they should, if possible, be perfused with University of Wisconsin, V-7 or Eurocollins solutions, or an appropriate balanced salt solution supplemented with mannitol (for example, Sack's buffer) and glucose, at 4°C. If the slices are to be incubated for long periods (>8 h), antibacterial (for example, gentamicin, streptomycin) and antifungal (for example, fungizone) agents should be added. Appreciable changes in osmolality between transport, slicing, and incubating should be avoided.

Preparing Tissue Cores

Tissue cores (normally 8 or 10 mm in diameter) can be prepared by using a motor-driven coring tool (for example, a variable speed motor) or by hand. It is preferable to use a motor-driven tool. It is possible to make cores by hand from small laboratory animal (rat and mouse) tissues, and some researchers also use this approach with tissues taken from larger species (dog, pig, human). In the latter case, care should be taken to select appropriate anatomical regions (for example, the inner or outer cortex of the kidney), to avoid any major blood vessels, and not to cut cores perpendicular to their longitudinal axis. The tissue to be cored should be placed on a suitable support (for example, on wax or filter paper) to avoid damaging the coring tool, which needs to be sharpened regularly.

Preparing Tissue Slices

Tissue slicing is normally performed in cold medium (temperatures of 4-12°C have been used successfully), although some laboratories have reported good results with media kept at room temperature. The slicing buffer is normally gassed with carbogen (95% O2/5% CO2) and may consist of a balanced salt solution (for example, Krebs-Henseleit buffer) with added glucose or another energy source, tissue culture medium or cold preservation medium.

The cutting speed (the rate at which the tissue core moves across the blade) is critical, and differs for each organ and species. For less compressible tissues (for example, human liver), up to 200 slices may be prepared in 30 minutes. However, with softer tissues (for example, rat liver) a slower slicing rate should be employed, to avoid tissue compression. Finally, care should be taken to ensure that the tissue slicer blade is sharp, and is changed as necessary.

Slice Thickness

Slices should be cut at a thickness which facilitates rapid nutrient and waste exchange; the choice of thickness is influenced primarily by the purpose for which the slices will be used (for example, slightly thicker slices are used for cryopreservation). Slice thicknesses appropriate for using different tissues for metabolic studies are given in Table V.

Table V: Optimal Thickness of Precision-cut Slices from Various Tissues for Metabolism Studies

Organ	Optimal thickness (µm)^a
Liver^{b, c}	200-250
Kidney^b	200-250
Heart^b	250-350
Lung^b	500-700
Spleen^d	200-350

^aOptimal slice thickness may vary according to the incubation method used.
^bData from Parrish et al (12).
^cWith liver slices, the limits of usable slice thickness are 150/180-300µm.
^dData from Hoffmann et al (95) and Skibinski et al (submitted for publication).

Measuring thickness is an essential aspect of slice use, and can be undertaken directly on the Krumdieck slicer, or microscopically. An automatic gauge is available for the Krumdieck slicer, which allows slice thickness to be measured immediately after the slice has been cut. Thus, cutting parameters can be optimised within minutes without needing to handle the slices. The use of indirect measures of slice thickness (for example, protein determination, slice wet weight) can be used for routine monitoring of the slicing procedure, providing that calibration curves have been generated for the specific conditions employed. Slice weight may be particularly useful as the most rapid means of assessing slice thickness (R. Fisher, unpublished information).

Handling Tissue Slices

Slices have to be handled with care, during both preparation and incubation, since they are sensitive to compression and mechanical damage. Tissue slices should be removed carefully from the slicer, and then either floated, or gently transferred with an appropriate tool (for example, a paintbrush or a spatula), onto the incubation mesh or into the incubation device.

Incubation of Liver Slices

The successful incubation of liver slices requires the selection of an appropriate incubation system, incubation medium, and gas phase. Different applications may require variations in the experimental conditions in order to achieve optimal results. Studies using liver slices can be broadly divided into three categories:

Short-term - slices may be maintained for 8 h or less in a simple balanced physiological salt solution supplemented with a suitable energy source (for example, glucose, fructose) or tissue culture medium. These conditions are often suitable for metabolic studies (4-16).
Intermediate-term - slices incubated for longer periods require a nutrient-rich tissue culture medium designed to optimise their viability. Slices can thus be maintained for up to 24 h for investigating the toxicity and metabolism of compounds. The slices can also be exposed to chemicals for a short time and then maintained for up to 24 h to assess the effects of acute exposure (8-14).
Long-term - slices maintained for 24 h or more in tissue culture medium enable the effects of longer exposures to chemicals to be investigated, and enzyme induction studies to be conducted (11-14).

Several incubation systems are available for the maintenance of tissue slices (Table VI); the choice of the most appropriate system depends upon the incubation time and medium, the specific application, and the tissue type and species (12-16). In experimental protocols requiring short-term (up to 12 h) incubations, the slices can be maintained in oscillating flasks or submerged in oscillating or rotating multi-well plates. There is evidence that the use of wide-diameter wells (12-well plates) without a mesh can enable slices to be maintained for 72 h (C. Ruegg, unpublished information). For applications where longer term incubations are desirable, the slices can be suspended at the air-liquid interface on solid supports, or rotated through medium into a gas phase on a rotating platform.

Table VI: Characterisation of Incubation Systems Currently Used for Precision-cut Liver Slices^a

Culture	Incubation Systems	Incubation Time	Application Type
Surface	Dynamic organ culture	up to 5 days	Short- and long-term studies, including metabolism and kinetics, xenobiotic-induced toxicity, enzyme induction
	Rocker platform (Netwell inserts)	up to 24 h	Short- and long-term studies, including metabolism and toxicity
Submersion	Orbital shaker (no mesh; 24 well plates)	12-24h	Metabolism, kinetic, and toxicity studies
	Orbital shaker (no mesh; 12 well plates)	12-72 h	Metabolism, kinetic, and toxicity studies
	Stirred wells (with mesh, 6 or 12well plates)	12-24	Metabolism, kinetic, and toxicity studies
	Shaking flask	up to 12 h	Short-term metabolism and toxicity studies

^aBased on data from references 4, 7, 8, and 12-15.

Consideration should be given to the slice-to-medium ratio. For many systems this is about one slice/ml medium; this has been used for long-term incubations. Many different incubation media and additions have been described in the literature, consisting either of balanced salt solutions or conventional tissue culture media (for example, those routinely used for the culture of primary hepatocytes or other cells). Examples of buffer systems and tissue culture media which have been shown to be suitable for the long-term incubation of liver slices are given in Table VII. The use of poorly nutritive buffers may be a critical factor limiting long-term viability; the use of richer media should be recommended as a standard approach for improving the systems. In general, fetal calf serum, insulin, and a glucocorticosteroid are added to the media, as well as antimicrobial agents to help prevent contamination.

Table VII: Suitable Culture Media for Liver Slices

Medium	Additions^a	Applications^b
Balanced salt solution (e.g. Krebs-Henseleit buffer)	Glucose, fructos	Short-term culture (e.g. metabolism studies)
Tissue culture medium (e.g. RPMI 1640, Waymouth's, William's medium E)	Fetal calf serum (5-10%), hormones (insulin, hydrocortisone), antibacterial gentamicin, streptomycin, penicillin), antifungal (fungizone)	Short- and long-term culture (e.g. metabolism, toxicity, enzyme induction studies)

^aFor short-term incubations with tissue culture medium, not all of the additions recommended for long-term incubations may be necessary.

^bDepending upon the application, the concentrations of the added constituents may need to be varied (for example, for steroid metabolism studies, the concentrations of hormone supplements may need to be reduced).

With care, it is possible to prepare slices from animal tissues under sterile conditions. While the use of low concentrations of antibiotics is usually appropriate, antifungal agents such as fungizone affect cell membranes even at fairly low concentrations. It is also possible that any added antimicrobial compounds could interfere with the test chemicals.

Liver slices have also been incubated for extended periods in an atmosphere of 95% air/5% CO₂. The data available suggest that, for liver slices, a high oxygen concentration (95% O₂/5% CO₂), such as that achieved with several commercially available incubators (for example, the Vitron Dynamic Organ Incubator® [13, 14]), gives higher and longer term viability.

Liver slices are normally incubated in control medium for periods of up to 2 h prior to exposing them to test compounds. The culture medium can then be replaced, to remove any surface debris from the slices and any enzymes (for example, lactate dehydrogenase [LDH], alanine aminotransferase [ALT]) which have been released from damaged cells. This is especially important if free enzymatic activities are to be used as a marker for cell injury. If this is not the case, unnecessary changing of the medium can have detrimental effects on slice viability. Some researchers change the medium every 24 h to avoid pH differences. For metabolism studies, a short preincubation period (for example, 10-30 minutes) with a change of medium is recommended whereas, for toxicity studies, a longer preincubation period (for example, 1-2 h) will allow the liver slice K⁺ levels to return to normal.

A period of preincubation with the final buffer system to be used is necessary because cold preservation media have different ionic balances to most of the incubation media used. In addition, the antioxidant molecules present in preservation solutions may need to be eliminated before exposure of the slices to test chemicals, due to the possibility of interactions/interference.

The lung warrants special comment. The air-liquid biphasic system used for inhalation toxicology studies enables the testing of gaseous pollutants and compounds dissolved in the aqueous phase, and exposure to gaseous substances which contain particulate material is possible. Using appropriately modified dynamic flow rotating chamber incubation systems, there is free access to the gaseous phase and the size of the particulate fractions can be controlled (J.P. Morin, unpublished information).

For optimal viability, lung slices are best cultured in serum-free medium. Biochemical and functional characteristics can be maintained for at least 48h in dynamic, biphasic air-medium incubation systems with a high oxygen concentration (> 20%, although very high oxygen concentrations cause loss of viability from 24h onwards). Lung viability parameters include those described for other tissues, and cell-type specific measures, such as polyamine transport, surfactant synthesis and secretion, and detoxification pathways (J.P. Morin, unpublished information).

Endpoints

It is important to ascertain the viability of the tissue throughout the generation, maintenance, and use of slices. This is particularly critical when developing or standardising procedures, or with long-term slice use (13, 14). Among other things, assessing viability enables the investigator to: (a) assess the status of the tissues received (especially important with human organs); (b) judge the technical quality of the procedure being used; and (c) assess the maintenance of tissue integrity throughout the incubation.

The criteria used for assessing viability are diverse, and include markers for cell viability and organ-specific functionality tests (Table VIII). Selection of the most appropriate tests depends upon the proposed use of the slices, and on the degree of confidence in the results which is required. In general, the greater the number of complementary endpoints of viability/functionality measured, the greater the confidence in the data generated. The use of positive and negative controls is essential, as is a knowledge of the strengths and weaknesses of monitoring biophysiological indicators of cellular function.

Table VIII: Tests for General Cell Viability and Cell Function

Cell Viability

ATP content
Protein synthesis
K⁺ retention
Enzyme leakage (LDH, ALT, AST)
MTT, neutral red

Organ-specific Functionality Tests

Liver:	Gluconeogenesis Urea synthesis Regiospecific enzymes Testosterone hydroxylation Glutathione concentration Heat shock proteins
Kidney:	Organic ion transport Glutathione concentration Regiospecific enzymes
Spleen:	Immunoglobulin synthesis Cytokine production
Lung:	Surfactant production
General:	Histological assessment of target organs mRNAs to organ-specific proteins Antibodies to organ-specific proteins Cell-specific markers

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase.

For metabolic profiling of substrates of unknown metabolic fate, it is necessary to determine metabolic competence by undertaking parallel studies with established marker substrates (for example, 7-ethoxycoumarin, which undergoes both Phase I and II metabolism). For kinetic studies, the maintenance of linear rates of metabolite production with time is an indication of metabolic stability.

Use of Precision-cut Tissue Slices to Study Xenobiotic Metabolism and Toxicity

Investigation of the complete metabolic profile of a xenobiotic and any associated metabolism-mediated toxicity requires a system which reflects the in vivo situation. Precision-cut liver slices provide such an in vitro model, and have been used to study a wide range of xenobiotics, xenobiotic-induced toxicity and the effects of xenobiotics on liver enzymes (Table IX). In common with hepatocyte cultures, the levels of total cytochrome P450 and associated enzyme activities in liver slices decline with time (19, 20).

Table IX: Examples of Pathways of Xenobiotic Metabolism in Precision-cut Liver Slices

Xenobiotic	Phase I pathways	Phase II pathways	References
4-Aminobenzoic acid	-	N-Acetylation	21
Coumarin	Aromatic and lactone ring hydroxylation, ring opening	Glucuronidation, sulphation	22
Cyclosporin A	Aliphatic hydroxylation and demethylation	-	23, 24
Diazepam	Aromatic and alicyclic hydroxylation	-	25
7-Ethoxycoumarin	O-Deethylation	Glucuronidation, sulphation	9, 22
Ondansetron	N-Demethylation, aromatic hydroxylation	Glucuronidation, sulphation,	26
Paracetamol	-	Glucuronidation, sulphation, GSH conjugation	27
Somatostatin analogue	-	Aglycone formation	28
Tolbutamide	Benzylic hydroxylation	-	29

Use for Studying the Metabolic Fates of Xenobiotics in Various Species

A wide range of xenobiotics are metabolised in liver slices derived from experimental animals and humans. The data available on liver, kidney, and lung suggest that all known pathways of Phase I and Phase II xenobiotic metabolism are active in liver slices (Table IX; 19-78).

In general, good in vitro/in vivo correlations have been observed for the qualitative and semi-quantitantive metabolism of xenobiotics in liver slices obtained from the different species used during drug development (22-24, 28, 30, 32). Some xenobiotics have been shown to be metabolised to products which bind covalently to intracellular macromolecules (for example, protein, DNA).

Use for Deriving Data on Xenobiotic Metabolism Kinetics

Liver slices can be used to determine the intrinsic clearance (V_max/K_m) of a compound, which in turn can be used to predict in vivo behaviour. Rates of metabolism can be determined under conditions of linearity for time and enzyme content (slice thickness and/or number). Enzyme kinetic models used in other in vitro systems are applicable to tissue slice experiments (25, 29, 32).

In general, there are good correlations between the rank orders of intrinsic clearance values obtained by using liver slices and those determined in other in vitro systems and in vivo. Comparison of the available rate data for slices, expressed in terms of hepatocyte number (accounting for the presence of non-parenchymal cells) does, however, indicate that metabolic rates in liver slices are consistently lower than in isolated hepatocytes (25, 26, 33, 34). This discrepancy appears to be due to the delayed uptake of the substrate into all of the cells of the liver slice, the fact that some of these cells will not be metabolically active, and the lack of penetration, especially if the centre of the slice is necrose.

Use for Studying Xenobiotic-induced Toxicity

Precision-cut tissue slices have been employed for studying xenobiotic-induced cytotoxicity and genotoxicity. Alterations in cellular functions may prove to be useful surrogate markers for monitoring the development of compound-induced adverse effects (11). Cytotoxicity may be assessed by using histological and biochemical endpoints. For example, histologically, in keeping with in vivo findings, bromobenzene produced centrilobular necrosis in liver slices from phenobarbitone-treated rats (35).

Biochemical markers include: (a) leakage of proteins and enzymes; (b) intracellular ATP and K⁺ contents; and (c) inhibition of protein synthesis. Using such markers, the toxicities of a wide range of xenobiotics, including halogenated volatile anaesthetics (36-46), chloroform (47, 48), allyl alcohol (35), bromobenzene derivatives (35, 49, 50), carbon tetrachloride (51-53), cocaine (54, 55), cresol isomers (56), diquat (57), ethanol (58), dichlorobenzenes (59-61), S-(1,2-dichloro-vinyl)-L-cysteine (52-66), valproic acid (67, 68), allylamine (69), doxorubicin (69), and paracetamol (acetaminophen; 27), have been investigated in rodent or pig tissue slices. Xenobiotic-induced genotoxicity has been demonstrated by using the unscheduled DNA synthesis technique (70, 71), and by measuring the formation of DNA adducts by ³²P-postlabelling (72, 73).

The toxicities of metabolically activated compounds, such as 2-acetylaminofluorene (70), 2-aminofluorene (72, 73), nitrofurantoin (74), acrolein (74), dichlorobenzenes (59-61, 75), and valproic acid (68, 76), have been assessed in human tissue slices. The toxicities of heavy metals, such as cisplatin (77, 78), mercuric chloride (79-81), arsenic (80), and chromium (81), have been assessed in rodent tissues. There are interactions between the uptake of arsenic, cadmium, chromium, and mercuric salts in rabbit renal tissue (82). There are also data which enable cross-species comparisons between rodents and human tissues to be assessed for cisplatin (78) and mercuric chloride (79).

Liver slices have been used to demonstrate interactions between volatile anaesthetics and albumin; these studies indicate that the slices are highly representative of the perfused organ and have comparable metabolic rates to those found in vivo (44, 45). Precision-cut tissue slices have been prepared from rat neonatal livers, and the metabolism of volatile anaesthetics has been compared to that in slices obtained from adult animals (46).

Use for Studying the Modulation of Liver Enzyme Activities

The ability to culture liver slices for periods of 3-5 days (13, 14, 19, 20, 83) has enabled studies on the induction of liver enzymes to be undertaken. Measurements of mRNA levels and marker enzyme activities, and Western blotting, have demonstrated that CYP 1A, 2B, 3A, and 4A sub-families are inducible in liver slices (19, 20, 84). In addition, known rodent peroxisome proliferators have been shown to increase the numbers of peroxisomes, and to induce both peroxisomal and microsomal fatty acid oxidising enzyme activities, in liver slices (20, 83), in common with results obtained in vivo and in studies with primary hepatocyte cultures.

Preparation and Incubation of Precision-cut Tissue Slices from Extrahepatic Tissues

Metabolism and toxicity studies have also been conducted with precision-cut kidney, lung, heart, spleen, adrenal, and intestinal slices (punched out from pieces of tissue). For example, intestinal slices have been used for studying xenobiotic metabolism (23, 24); heart (12, 69), kidney (62-65, 77-82, 85-90), and lung (74, 91-93) slices have been employed for studying xenobiotic-induced toxicity. Compared with liver and kidney slices, less effort has been made to optimise the experimental procedures for the preparation and use of such slices. The preparation of tissue slices from non-hepatic tissues may require changes in the methods used with liver slices, such as embedding in agarose, different optimal slice thicknesses, different incubation systems, and media, or different gas phases. When using these slices, markers of organ-specific functions may be helpful for establishing optimal culture conditions.

Lung

An agarose-instilling technique has been developed for the preparation of precision-cut slices from rodent and human lung (74, 91-93). Infusion of the lungs with an agarose preparation (of low gelling point) in tissue culture medium, followed by embedding in additional agarose, has met with some success. Once the agarose has solidified, the slices are cut and the infused agarose provides a solid support, effectively preventing the collapse of the alveolar architecture and subsequent necrosis throughout the lifetime of the slice culture (74, 92, 93). Standardisation of the results relative to slice weight are inappropriate where irregular tissues have been embedded in agarose. The use of the McIlwain tissue chopper for preparing lung slices is referred to in Appendix 1.

Intestine

For optimum viability of intestinal slices, warm ischaemia time should be minimised. It is possible to leave the muscle attached to the mucosal layer, and use the slice mucosal side down on roller culture inserts, or to strip away the muscle before coring the tissue. It is important to wash intestinal slices 6-8 times to help prevent contamination of the medium, which otherwise can occur within 12-24 h. Intestinal slices have primarily been used for biotransformation studies (23, 24).

Kidney

The tissue source plays an important role in dictating the type of slices which can be prepared. The size of the kidneys in larger species and in humans facilitates the preparation of pure outer or inner cortical tissue or medulla whereas, with rats and mice, the whole cortex and outer medulla are included. The organs are decapsulated, and kidney slices are prepared and used as described previously for liver slices. It is preferable to culture kidney slices in a highly oxygenated atmosphere.

Precision-cut renal slices enable the individual different anatomical regions of kidneys from larger animals to be studied (for example, inner and outer cortex), by using cores of tissue cut along the papillary-cortical axis. Slices from different species have been used to investigate the metabolism of therapeutic agents and the mechanisms of nephrotoxicity of some chemicals. The uses of precision-cut renal tissue slices have been reviewed (12, 15, 85, 87).

Heavy Metals

Renal slices from the rabbit have been used to show the sensitivity of the proximal tubule to mercuric chloride (86, 87), potassium dichromate, and hypoxic conditions (87). Tissue slices lend themselves to analysing multiple exposures to metals, by using proton-induced X-ray emission; interactions between different metals have been demonstrated with this technique (94). Heavy metals such as mercury, cadmium, chromium, and arsenic are rapidly taken up (maximally between 3-6 h) into rabbit renal cortical slices (82, 94), where the presence of one metal substantially alters the uptake of another metal. For example, HgCl₂ was shown to hinder the uptake of K₂Cr₂O₇, NaAsO₂, and CdCl₂ (in that order), while these metal salts each facilitated the uptake of HgCl₂. In addition, incubation with GSH reduced the toxicity of HgCl₂ and NaAsO₂ (80). The acute effects of mercuric chloride (10-100 µM) have also been assessed in human kidney cortical slices (79), which are of similar sensitivity to those from the rat.

Therapeutic Agents

Only limited nephrotoxicity of cephaloridine and gentamycin has been observed in rabbit renal slices (88), which may be due to the unique nature of the in vitro exposure. The differential toxicities of a series of platinum analogues was shown to correlate well with their clinical toxicities (78), and with cellular platinum levels (79). The acute effects of cisplatin (0.25-1.0 mM) have been studied in human kidney cortical slices (79). Cisplatin was more nephrotoxic in human slices than had previously been found in rabbit slices, although both species showed similar sensitivities to mercuric chloride. Celiptium (another anti-cancer drug) toxicity is related to its uptake by the organic cation transport system (89). Data are also available on the renal metabolism of cyclosporin A and its derivative SDZ IMM 125 (23, 24), somatostatin analogue (28), and tropisetron (an anti-emetic 5-HT3 antagonist; 30) in rat, dog, and human kidney slices.

Organic Chemicals

The target selectivity, biotransformation, and mechanism of toxicity of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) have been studied (63-66); the morphological changes parallel those reported in vivo. More recently, it has been shown that DCVC-induced lipid peroxidation can be inhibited by a variety of antioxidants, but that this has a minimal effect on its cytotoxicity, suggesting that lipid peroxidation occurs as a consequence of the primary insult (62). Studies with 1-benzylquinolinium have emphasised the importance of the cation transport system for quaternary ammonium compounds (90).

Lymphoid Tissue

Slices of spleen and other lymphoid tissues (lymph nodes, Peyer's patches, tonsils) of both mouse and human origin have been used for immunobiology studies. Incubation is preferably carried out in Netwells, on a rocking platform, although incubation on solid supports, and in microperfusion chambers, has also been described (95).

Unstimulated human spleen slices spontaneously synthesise and secrete higher levels of immunoglobulin than do splenocyte suspension cultures run in parallel (95). There are also marked differences in the cytokine secretion profile between explants and suspensions (G. Skibinski, P. Hoffmann, A. Radbruch & K. James, submitted for publication). These differences are attributable to maintenance of the intact architecture in tissue slices compared with loss of the stromal elements during preparation of the suspension cultures. The activities of the lymphoid slices can be manipulated by various exogenous stimuli, which offers the possibility of using the system for a wide range of immunological, including immunotoxicological, studies.

Heart

To prepare cardiac tissue slices, the heart is first perfused with calcium-free Krebs-Henseleit buffer, and the ventricles are then separated from the atria at the atrioventricular junction. The tissue of interest should be laid flat and cored, and the slices prepared immediately in Sack's cold preservation solution. Cores should then be produced sequentially and sliced, to limit the spontaneous cardiac contractions which make slicing more difficult (12, 69, 96, 97). The co-culture of rat liver and myocardial slices has been used to demonstrate the metabolite-mediated cardiotoxicity of allyl alcohol (97).

Morphology

The maintenance of morphological integrity is one of the most important indicators of the viability of tissue slices, and is an important way of detecting target cell injury. Morphological assessment of tissue slices offers a suitable link to in vivo studies. The use of conventional light and electron microscopy is, however, time-consuming, and therefore its application to tissue slices has been limited. Structural integrity has been shown to be well-maintained in renal (63-66, 81, 85, 86), cardiac (93, 96), and liver slices (27, 70). Morphology has also formed the basis for assessing unscheduled DNA synthesis (70).

One of the great advantages of precision-cut tissue slices is that the biochemical and molecular alterations can be correlated with pathological events. Slices have been used to demonstrate the regio-selective centrilobular injury caused by paracetamol (acetaminophen) in the hamster, but not in the rat (27), the same species sensitivity as is observed in vivo. In addition, heavy metals, such as mercury and dichromate (81, 85, 86), and DCVC, cause selective proximal tubule necrosis (63-66). Electron microscopy can be used to show how one cell is affected whereas an adjacent, morphologically different, cell is not (63). Light microscopic autoradiography with ³⁵S-DCVC demonstrated its selective localisation in the injured cells (63). These pathomorphological features are indistinguishable from the lesions caused by the same chemicals in vivo.

Laser scanning confocal microscopy (98), digital imaging, and the use of fluorescent probes offer the potential to determine biophysiological changes in different cellular compartments of living cells as they are actually occurring.

Tissue slices offer the possibility of identifying pathophysiological changes in the organ and species of choice in vitro. This is a unique feature, since it is not possible to demonstrate site-specific lesions by using cell culture methods, and it is also difficult to identify these in perfused organs. The relative scarcity of reports on such studies does, however, demonstrate that pathomorphology is technically more difficult and resource-intensive than the assessment of biochemical changes.

Application for Studying Environmental Agents and Agrochemicals

Interactions between the uptake of mercury, cadmium, chromium, and arsenic (82), and the toxic effects of mercury (78-80), arsenic, (80) and chromium (81) individually, have been assessed by using tissue slices. The metabolism and toxicity of a diverse range of organic molecules of environmental relevance have also been studied (35, 47-53, 56, 57, 59-65, 70-75, 83, 92).

Trout liver slices have been used for investigating the metabolism of ethoxycoumarin, biphenyl, and 1-naphthol (99), and for studying the toxicities of allyl formate and allyl alcohol (99), and of cadmium sulphate and lead acetate (100). In future, tissue slices from relevant species could be used for assessing the metabolism and potential toxicities of pesticides and other environmental contaminants, in a similar manner to their current successful application for investigating specific effects of therapeutic agents.

The Use of Human (and Other Rare) Tissues

The use of human tissues in vitro represents a good way to bridge the gap when trying to extrapolate from results obtained in vitro and in animal studies in vivo to the human situation. The comparison of metabolism and target cell toxicity in tissue slices derived from humans and animals provides a basis for more-focused cross-species investigations, and aids the design of better, more predictive, in vivo studies. The advantages of this approach appear to have been recognised by the regulatory authorities in the USA, who now accept submissions containing in vitro as well as in vivo data. At this time, the situation in terms of the acceptance of such data by European regulatory agencies is not clear. The US regulatory authorities are strongly encouraging the use of in vitro test systems based upon human tissues (current FDA Draft Guidance for In Vitro Drug Metabolism Studies).

Availability of Human Tissues

Human tissues can be obtained from organ donors, as biopsy material from surgical procedures, and as cadaver material. Experience in the USA shows that it is possible to procure and distribute tissue (and tissue slices) for biomedical research purposes (101). There are distinct advantages to using non-transplantable material (Table X), since the use of cadaver material is limited by the significant loss of drug metabolising enzymes which typically occurs due to the protracted periods which normally elapse between death and the use of the tissue (102-105). Human cells from most organs have been cultured (105).

Table X: Advantages and Disadvantages of Human Tissue Obtained from Different Sources

Type of Tissue	Advantages	Disadvantages
Non-transplantable tissue	Large samples available Multi-organ tissue may be available Warm ischaemic time < 5 min (but could be as long as 1 h depending on the surgical technique) Tissue procured as if it were going to be transplanted Serological tests are required Tissue functions well-preserved Clinical history and relevant lifestyle factors may be well-documented	Cold ischaemic time varies from 1-72 h. Conflict of interest with transplant programmes Legal and ethical considerations need to be harmonised at a European level
Cadaver tissue	Tissues may be easier to procure	Warm ischaemic time > 30 min, which is detrimental to liver, kidney, heart, intestine, and spleen cells Serological test results not normally available
Surgical "waste", tumour resected, or biopsied tissue	>Warm ischaemic time < 5 min Can be rapidly processed after collection Clinical history and relevant lifestyle factors may be well-documented Donor can give fully informed consent	Serological test results not normally available Only small amounts of tissue available Ethical requirements may be more difficult to meet May be difficult to differentiate between diseased and normal tissue
Diseased tissue	Essential to better understand disease processes Donors may participate in bequesting of tissue	Identification of appropriate in vitro conditions to keep tissue representative of diseased organ may be difficult May be difficult to prepare slices

There are different laws within Europe regarding the acquisition of human tissues for transplantation and biomedical research (103, 104). Ethical considerations/requirements for the procurement of tissues also differ, but informed consent is a normal prerequisite when viable tissue is to be used for biomedical research. Some of the legal, ethical and sociological implications of using human tissues have been considered (103, 104). Sources of human tissues from non-profit-making organisations which undertake a cost-recovery commercial operation and provide material for bona fide researchers are listed in Table XI.

Table XI: Organisations Providing Fresh and Frozen Human Tissues and Tissue Slices

Organisation	Address	Telephone/Fax Numbers
Anatomical Gift Foundation	P.O. Box 879, Woodbine, GA 31569-0879, USA	Tel: 001 912 576 5889 Fax: 001 912 576 3727
Anatomical Gift Foundation of Arizona	1313 N. 2nd Street, Suite #2, Phoenix, AZ 85004-1776, USA	Tel: 001 602 528 3715 Fax: 001 602 528 3717
Association of Human Tissue Users (AHTU)	8320 S. Wentworth Road, Tucson, AZ 85747, USA	Tel: 001 520 647 7412 Fax: 001 520 647 7416
Human Cell Culture Centre (HCCC)	P.O. Box 879, Woodbine, GA 31569, USA	Tel: 001 301 572 4605 Fax: 001 301 572 9160
International Institute for the Advancement of Medicine (IIAM)	15 E. Uwchlan Avenue, Suite 406, Exton, PA 19341, USA	Tel: 001 610 363 3600 Fax: 001 610 363 3605
International Institute for the Advancement of Medicine (IIAM)	Maurice Shock Medical Sciences Building, P.O. Box 138, University of Leicester, Leicester LE1 9HN, UK	Tel: 44 (0)116 2523047 Fax: 44 (0)116 2525680
National Disease Research Interchange (NDRI)	1880 JFK Boulevard, 6th Floor, Philadelphia, PA 19103, USA	Tel: 001 215 557 7361 Fax: 001 215 557 7154

Human tissues should, if possible, be perfused with, and subsequently placed in, an appropriate cold preservation (Viaspan) solution. Non-transplantable human tissues should be procured as if they were going to be used for transplantation, to ensure maximum cell viability. It is important that all human organs are carefully assessed for viability prior to use.

While it is mandatory to have appropriate serological information on the risk of contagious diseases (human immunodeficiency virus [HIV], hepatitis A, B, and C, syphilis, etc.) from non-transplantable tissues, this is less often the case for surgically procured material, and it is very unlikely that the information will be available when using tissues taken from cadavers.

It is important to remember that human tissues represent a biohazard, and that appropriate safety precautions should always be followed when handling them.

Scarce Tissues

Tissue slices are the most efficient and economic way to obtain viable cells for in vitro investigations, while at the same time providing mixed cell types representative of the intact organ. Similarly, tissues from non-human primates, domestic and farm animals, carcinogen-exposed animals, and exotic species are scarce, and cost and other implications indicate that the use of tissue slices should be the preferred option for in vitro investigations. Multiple organs can be obtained from a single animal, and tissue slices could be prepared and then cryopreserved from each organ.

The potential to undertake many investigations with tissue slices procured from a single organ, or from multiple organs taken from a single animal, is important. It enables many new chemical entities to be screened in vitro by using tissues obtained from relatively few animals. In addition, the number of compounds which subsequently undergo testing in animals in vivo is considerably reduced.

Organisations such as the International Institute for the Advancement of Medicine (IIAM) and the Human Cell Culture Centre (HCCC) procure non-transplantable human tissues, and also process these into slices for distribution to a number of researchers throughout the world. This can help to maximise the distribution of slices from fresh tissues, but there is still a need to establish banks of well-characterised and cryopreserved tissue slices, so that limited human material can be utilised to the greatest extent possible.

Tissue slices derived from large organs have the potential to provide experimental systems which represent specific regions (for example, the renal cortex or the medulla). A single human liver is an immense resource in terms of the many thousands of slices which can be produced. For this reason, it is of great importance that appropriate cryopreservation methods are developed and used.

Cryopreservation

There is a need to perfect the cryopreservation of tissue slices. The long-term stability of frozen slices, together with the potential to distribute material to all parts of the world, means that rare resources such as slices derived from humans, non-human primates, domestic animals and, for example, laboratory animals treated with carcinogens, can be used by many researchers over a number of years. With the increase in organ transplantation activities and in the demand for human tissues for research, there are likely to be relatively few human organs available for research purposes in the future. The successful cryopreservation of human tissues, in the form of slices, would enable the more efficient utilisation of non-transplantable human tissues. In addition, the need to reduce animal use wherever possible, and the requirement to use alternative methods, means that there is a need for slices derived from animal tissues to be cryopreserved successfully and then stored for future use, so that the use of all available animal tissues can be maximised.

Various cryopreservation methods have been described, which use slow or fast freezing, as well as vitrification (freezing without the formation of ice crystals); the cryopreservation process has been controlled with or without the aid of computer devices. Each approach has advantages and limitations. The use of relatively low and non-toxic concentrations of cryoprotectants for slow rate freezing requires sophisticated, computer-controlled, equipment, whereas the simpler method of flash freezing for vitrification has disadvantages which include the high, and potentially toxic, concentrations of cryoprotectants which are used.

Human, monkey, dog and pig liver slices, and human and dog kidney slices, have been cryopreserved by using various conditions (106-109). Cellular viability has been assessed using K⁺ retention and protein synthesis, in addition to organ-specific endpoints, such as 7-ethoxycoumarin metabolism, gluconeogenesis and urea synthesis for liver, and organic ion transport for kidney. Cryopreserved liver and kidney slices retain between 60-90% of their pre-frozen slice viability, depending upon which particular indicator is used.

Recently, lower cryoprotectant concentrations have been used successfully for the cryopreservation of rat and monkey liver slices (109). This approach has also been successful with human liver slices (de Kanter, submitted for publication), and with slices from a range of other species (hamster, dog, guinea pig, and rabbit; de Kanter & Koster, unpublished). Cell viability in these studies was assessed by measuring urea synthesis, alanine aminotransferase leakage, and cytochrome P450-dependent testosterone hydroxylations; slice functionality was always at least 70% of the values measured in freshly prepared slices. These results still need to be reproduced in different laboratories, to ensure that protocols for the cryopreservation of slices are standardised and sufficiently robust to be readily transferable between laboratories.

While cryopreservation is essential for maximising the use of tissue slices, the relevance of data derived from biological material which exhibits <100% viability needs to be carefully assessed. It seems likely that not all cell types in heterogeneous organs (such as the kidney) will freeze in exactly the same way during cryopreservation. This needs to considered and checked carefully. For long-term studies, the most sensitive viability parameters need to be defined.

It is already possible to establish tissue banks, containing characterised slices from various species, where 200-300 slices have been cryopreserved at a time, stored in liquid nitrogen for up to one year, and then thawed rapidly and incubated in tissue culture medium for up to 24 h. However, there is a need to automate the cryopreservation methods to encompass freezing batches of a thousand or more slices (to maximise the use of tissues from larger animals, including human liver tissue). There is also a need to optimise the freezing and thawing conditions, to allow slices to be incubated for more than 24 h.

It is important to ensure that the precious and bequested resource of human tissues, and tissues from other larger animals, is available to all researchers who have bona fide reasons to use it as an alternative to sacrificing more animals. This material needs to be well-characterised (by using standard, validated assays) and, ideally, to be cryopreserved for banking purposes.

Validation

The reliability and relevance of tissue slice technology for specific purposes or endpoints needs to be fully documented, if this approach is to be established as a useful tool to reduce, refine, and replace animal studies and complement or replace other in vitro systems (110). Liver slices are the best studied and characterised. To date, limited, informal, interlaboratory studies have been undertaken (30-32), to help standardise and optimise procedures for assessing the transferability of the technique and the further optimisation of conditions. The results appear to be reproducible between laboratories (30-32), but there has been no concerted effort to undertake the formal assessment of a defined set of chemicals, in a blind trial, as a route towards regulatory acceptance. It is clear that the validation of tissue slice technology is at least partially dependent upon the willingness of laboratories to share their existing expertise, adopt common conditions and procedures to make tissue slice data comparable (Table XII), and actively participate in standardised, multi-laboratory exercises, such as those used to validate other in vitro methods.

Table XII: Standardisation Criteria

Parameter	Criteria to be Standardised
Oxygen tension	Use of high oxygen concentration or air; type of support
Energy status	Substrates used for energy production
Medium selection	Type of medium and additions
Incubation	Submerged versus surface culture; type of support
Endpoint selection	Biochemical (cell selective or non-cell selective) or morphological
Species/strain	Sensitive v resistant, human tissue v animal tissue
Test chemicals	Solvent used to solubilise chemicals

Conclusions and Future Possibilities

Tissue slices have been used successfully for over 75 years to define fundamental processes in biochemistry, physiology, pharmacology, and toxicology, and there has been a recent resurgence in their use. This has occurred because of the availability of improved methodologies for preparing uniform, precision-cut, slices, and the development of optimised media and incubation methods, which maintain viable slices for extended periods in the presence of nutrient or gas exchange. These slices are composed of all cell types, as they occur in vivo, and they can be used for investigating species differences in biological processes and biotransformation, and regio- and cell-specific target organ damage. Much of the current use of slices is focused on liver tissue, but other tissues are being used increasingly. The preparation of tissue slices has facilitated and maximised the use of rare tissues, such as those from humans and non-human primates.

It is not possible to predict how the availability of human tissue will develop in the future. The immense value of tissue slices in reducing and refining the use of animals is strongly dependent upon the regular availability of "high quality" (healthy; low ischaemic time) human tissue. The growing trend for multi-organ human donors means that there could continue to be an adequate supply of tissue for the preparation of tissue slices, provided that there is significant public awareness that the donation of tissue can benefit both transplantation and medical research.

The scientific advantages of using precision-cut tissue slices are already considerable, and will increase during the next five years, including:

Better markers of individual cell-type integrity. In order to improve the phenotypic expression of cells, maximise the duration of viability, and identify target cell toxicity, better markers of cellular integrity (which are amenable to mass analysis) are required, which will allow screening procedures to be automated.
Long-term viability. Conditions will be developed for each of the tissues of interest, which enable the phenotypic expression of more of the cell types present to be maintained for longer (beyond five days). In the case of liver, kidney, etc., the slices will express activities and functions comparable to the in vivo situation. The parameters to be controlled, in particular, will be the incubation system, the choice of medium, oxygenation, and the addition or pulsing of hormones, nutrients and other factors to help ensure extended cellular viability.
Additional tissues. In addition to the liver, kidney, lung, intestine, heart, and lymphoid tissue, the other major organs (such as endocrine organs, stomach, muscle, and skin) and tumours will be studied by using slices, and systems will be optimised for relevant cell expression and prolonged viability for each tissue type.
Other species. The numbers of species that have been investigated will be extended beyond conventional laboratory and domestic animals and humans. This will facilitate a broader application of slices, to assess the likely impacts of environmental chemicals on natural fauna.
More applications. Most of the applications have focused on biotransformation, with some toxicological studies also being undertaken. In the future, precision-cut slices will be used for a broad range of cellular and molecular biology studies, which are likely to include investigations of biophysiological and cell signalling processes which depend upon intact cell-cell interactions, immune-mediated effects, and interactions between cells and chemicals. Tissue slices also make it possible to investigate mechanisms of xenobiotic-induced alterations in cell function (including carcinogenic events), and to undertake cross-species comparisons of mechanisms, to establish the potential human risk. When human tissues can be used, better animal/human correlations can be established.
Long-term cryopreservation. It is expected that cryopreserved tissue slices from large organs which provide abundant material for immediate use will be able to be stored for long periods (more than 2-3 years), to yield slices which, when thawed, retain all, or the vast majority, of their biological characteristics (>90%), or which have been optimised to enable specific applications to be undertaken.
Better understanding of slice biokinetics. Observed differences between intrinsic clearances in isolated hepatocytes and slices suggest that additional studies are required to understand the relationship between the rate and extent of uptake, physicochemical properties, and the metabolic rate of compounds. This is fundamental for the validation of all investigations using slices. It may also enable the overall prediction of in vivo clearance from liver slice data.
Multi-organ interaction. The co-incubation of, or transfer of medium between, slices from different organs will enable investigation of those toxic effects which arise as a result of interactions between different organs.
The study of diseased human and animal tissue. As knowledge about the biology of tissue slices increases, and culture times are prolonged, so other biomedical disciplines will make use of slice technology, to help discover how new drugs affect diseased organs, and how diseased tissues interact with therapeutic agents and environmental toxins.
Metabolic interactions and toxicity. Precision-cut liver slices have the potential to be used for studying interactions between xenobiotics. They could be used to obtain kinetic data on the inhibition or induction of Phase I or II xenobiotic metabolism, and to assess mechanisms of multiple exposure toxicities. In addition, it should be possible to treat animals, and then expose the tissue slices obtained to different chemicals to study possible interactions.
Receptor and cell signalling. Conventional tissue slices have been widely used for understanding receptor responses. Precision-cut tissue slices can also be used to study ligand-receptor interactions, receptor regulation, cell signalling, and intercellular communication in healthy and diseased tissues. Slices can also be used to study the key processes that are involved in cell-cell interactions within the intact architecture of the tissue; this may provide an insight into the possible long-term consequences of acute injury.

Recommendations

In order to realise the full potential of tissue slice technology, there is a need for a multi-disciplinary approach to addressing the following issues.

Wider Applicability of Slice Technology

It is apparent from the published data, that precision-cut slices offer considerable advantages which complement those of other in vitro methods. This technology is being used by industry. Nevertheless, it is recognised that some researchers have invested time and resources in slice technology, but have not been able to reproduce the published data, especially with respect to prolonged slice viability (beyond 72h). There are aspects of the methods used which, even though they may have been adequately described, have subsequently been modified or have not been completely followed. Care should be taken to use methods which have been optimised with respect to slice viability.

ECVAM should encourage the establishment of a European tissue slice users group or network which enables rapid transfer of the appropriate technology.
ECVAM should ensure a suitable level of training within Europe, to facilitate rapid adoption of the most up-to-date techniques and procedures.
There is a need to develop standard operating procedures (SOPs) which fully describe all of the aspects of the preparation of cores and slices, and their use and cryopreservation, and to make this information widely available. The FRAME/ERGATT INVITTOX databank, which has now been transferred to ECVAM, could be one way to achieve this.
There is a need to identify and use the most sensitive cell viability parameters which are deemed appropriate for characterising optimum conditions and for ensuring that cell functionality is well-described for both short- and long-term incubation periods.
Assessment of slice morphology and histochemistry is important with respect to slice viability; these techniques offer the potential to define cell-selective damage. For assessing cell injury, there is a need to combine morphological and histochemical assessments with the use of biochemical markers.

Optimising the Methodology

More systematic investigations need to be undertaken to ensure the maintenance of slice architecture and cell-specific functions during long-term incubations (> 5 days).

There is a need to develop culture media which specifically maintain the phenotypic expression of different cell types.
There is a need to extend the period for which tissue slices are viable during organ culture (> 5 days), and to adapt tissue slice technology so that sterile conditions can be easily maintained.

Cell-/Organ-specific Markers

Whereas non-specific markers of cellular injury (lactate dehydrogenase and K⁺ leakage, MTT reduction, etc.) are widely used for monocultures, they could provide misleading data with systems in which there is cellular heterogeneity.

More specific biochemical markers of cell viability need to be developed, covering all aspects of slice preparation, and for each species and tissue combination, to enable standardisation.
A panel of cell-specific markers are needed for assessing the integrities of individual cell types in terms of histological and biochemical changes. These should be species-independent where possible, to allow for cross-species comparisons. Where they are species-dependent (for example, monoclonal antibodies), the markers should be developed for all of the major species of interest. Such markers should also be applied in in vivo studies, for comparative purposes.
There is an important need to identify those markers which are amenable to automation, for mass screening purposes, without compromising the scientific quality of the data.

Slices as an In Vitro Tool for Biotransformation Studies

The ease of liver slice preparation increases the speed with which cross-species comparisons can be undertaken for both biotransformation and hepatotoxicity. The use of human tissues enables the focus to be on biotransformation pathways relevant to humans. Important aspects are: identifying and characterising the major metabolites; checking for metabolite activities (pharmacological and toxicological); and selecting species for investigational studies or toxicological registration studies. In addition to the use of slices for metabolic profiling, kinetic determinations of intrinsic clearance need to be understood and defined.

Drug uptake by liver slices and slices from other tissues should be fully characterised.
Biokinetic modelling should incorporate data on tissue slice distribution and metabolism.
Relationships between the physicochemical properties of compounds and their biokinetic behaviour in slices should be investigated.
Establishing the key factors which determine the pathways of xenobiotic metabolism in tissue slices will ensure that the full relevance of the data obtained is understood. This will be facilitated by: (a) providing full details of the techniques and procedures which have been used, thereby increasing the likelihood of acceptance of the methods by industry and regulatory agencies; (b) encouraging the publication of data on in vitro/in vivo comparisons of metabolic and toxicity profiles for different species; and (c) establishing a database incorporating in vitro data obtained with slices, to compare with in vivo metabolic profiles of compounds for which the therapeutic and toxicological effects are well established.
Strategies must be developed to increase the use of human tissue slices, and the use of tissue slices from other important species, such as dogs and non-human primates.

Application to Agrochemicals and Environmental Agents

Investigations with tissue slices are able to improve our understanding of environmental processes, by enabling studies to be undertaken with tissues from rare species, including wild animals. The acceptance of tissue slice data for regulatory purposes (pesticides) depends upon the validation of the methodology and the quality of the data submitted.

Laboratories working on agrochemical problems and environmental contaminants should consider the use of tissue slices as a possible alternative (or a complementary tool) to difficult in vivo studies (especially for interspecies comparisons); they are encouraged to publish their data.
The quality criteria and validation steps necessary for subsequent acceptance of tissue slice data for the registration of agrochemicals must be the same as for pharmaceuticals. In this respect, ECVAM could support the development of a document detailing the minimum criteria which must be met.
In the field of inhalation toxicology, effort should be made to develop methods which are suitable for testing environmental gaseous contaminants, such as automobile exhaust fumes, industrial smokes, and aerosols.

Better Use of Rare/Scarce Tissues

The widest possible availability of tissues should be achieved, to avoid a monopoly and their commercial exploitation. The availability of human tissues for research and testing is not harmonised across Europe, where the legal and ethical constraints differ between countries, as does the infrastructure for their procurement and use. The availability of cryopreserved slices from human organs would provide a means to optimally use this scarce and valuable tissue. The availability of other scarce tissues, such as those from non-human primates, carcinogen- or other toxin-treated animals, genetically manipulated laboratory animals, normal domestic and wild animals, and those exposed to environmental pollutants and toxins, would help to refine investigations in biomedical, veterinary, wild-life conservation, and environmental research.

The legal implications of, and ethical attitudes to, using non-transplantable, surgical, and autopsy human tissues throughout Europe should be accurately documented.
Appropriate, harmonised, legislation within the European Union should be developed, to expedite the uniform availability of human tissues, as a way of ensuring their more widespread use (111).
The establishment of a European Tissue Repository would help to ensure that fully characterised material was available from a single source to all bona fide researchers.

Cryopreservation

The unpredictable availability of non-transplantable and surgically resected human tissues means that there is an urgent need for methods for cryopreserving this material for banking and distribution once it has been fully characterised.

ECVAM is encouraged to provide a central source of information which will help to optimise the cryopreservation of human tissue slices (from hepatic and extrahepatic tissues), thereby providing material which is viable after thawing, and which remains viable for a reasonable incubation period. There is a need for standardised tests for demonstrating tissue viability.
Optimised methods for the cryopreservation of slices obtained from all organs and species need to be developed.
Methods for the high throughput cryopreservation of tissue slices should be developed, to ensure that maximum use is made of donated and non-transplantable human tissues, tissues from other large animals, and tissues from rare or chemically exposed species.

Publications and Databases

The literature on tissue slices is diffuse; thus, the advances which have already been made and could be adapted to precision-cut tissue slices are not always easily identifiable. Similarly, a great deal of very valuable data is held within pharmaceutical companies, and is therefore not readily accessible. The lack of availability of such information is likely to hinder the further use of tissue slices and validation of the methodology.

An electronically accessible database which documents all aspects of the use of tissue slices should be established. This would aid the validation process.
Companies using tissue slices should be encouraged to contribute relevant, unpublished information to this database.

Validation and Regulatory Acceptance of Tissue Slices

The use of tissue slices is being encouraged by regulatory authorities in the USA; data generated by using tissue slices are acceptable in regulatory submissions to the US Food & Drug Administration, and tissue slices are being used in studies being conducted under the auspices of the US National Toxicology Programme. The situation in Europe is less clear, and can only be harmonised when academics, industrial scientists, and regulatory authorities exchange and discuss information relating to the use of tissue slices.

ECVAM is encouraged to establish a forum in which such an exchange can take place, to ensure that the European requirements for new drug and agrochemical registrations are reconciled with the need to refine and reduce the numbers of animals used.
Publication of in vitro/in vivo comparisons, so that the predictivity of the data obtained by using tissue slices can be evaluated, is strongly encouraged.

Use of Diseased Tissues

Most of the research published to date on tissue slices has involved the use of human tissues which are considered to be almost "normal". It is widely appreciated that it is difficult to isolate hepatocytes from cirrhotic, fibrotic or fatty livers. Thus, very little is known about the changes in intracellular processes which occur in humans as various diseases develop.

The potential uses of slices derived from diseased tissues should be identified, and methods should be developed which will enable determination of the aberrant cellular processes occurring in these tissues; the appropriate conditions under which the tissues should be kept to maintain the "disease status" need to be identified. These diseased tissues should be used to gain a better understanding of changes in receptors and cell signalling processes which underlie disease processes, and how these could possibly be altered to slow down, or prevent, degenerative processes.

Acknowledgements

We are indebted to the following colleagues for access to unpublished data and for their critical input into this report: K. Akiyama, B.A. Bardsley, K. Brendel, R.D. Combes, D.A. Early, A.J. Gandolfi, E. George, Z. Gregus, J. Groten, J.P. Guichard, M. Hall, J.B. Houston, P. Howroyd, K. James, C.L. Krumdieck, G. Langley, L.H. Lash, N. Marchant, E.J. Massaro, J.P. Morin, P. Mutch, D.K Obatomi, P. Olinga, R. Owen, G.M. Pacifici, and C.E. Ruegg.

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The Use of Tissue Slices for Pharmacotoxicological Studies

The Report and Recommendations of ECVAM Workshop 201,2

Appendix 1

Appendix 2

The Report and Recommendations of ECVAM Workshop 20^1,2