Cytotechnology 7: 137-149, 1991. 9 1991 KluwerAcademicPublishers. Printedin the Netherlands. Review Article

Explant organ culture: A review James H. Resau a,1, Kosaku Sakamoto 2, John R. Cottrella, Eric A. Hudson a and Stephen J. Meltzerb,c,2 Departments of Pathology a and Medicine (G.I. Division) b, School of Medicine, and Graduate Program in Molecular and Cell Biology c, University of Maryland at Baltimore, Baltimore, MD 21201, and Veterans Affairs Hospital, Baltimore, MD 21218, U.SA. Received 27 November 1991; acceptedin originalform 3 December 1991

Key words: Organ culture, human, epithelium, colon Abstract Organ explant culture models offer several significant advantages for studies of patho-physiologic mechanisms like cell injury, secretion, differentiation and structure development. Organs or small explants/slices can be removed in vivo and maintained in vitro for extended periods of time if careful attention is paid to the media composition, substrate selection, and atmosphere. In the case of human tissues obtained from autopsy or surgery, additional attention must be paid to the postmortem interval, temperature, hydration,and cause of death. Explant organ culture has been effectively utilized to establish outgrowth cell cultures and characterize the histiotypic relationships between the various cell types within an organ or tissue.

Introduction Tissue culture has often been used generically or figuratively to include all in vitro techniques, though the term "tissue culture" refers to the in vitro maintenance of a tissue such as aortic intima (Yu et al., 1990), primary carcinomas (Resau et al., Siegfried et al., 1991) or esophageal mucosa (Resau et al., 1990). Organ/explant culture can be defined as an in vitro technique that maintains whole or portions of organs in culture using specialized media, substrates, and atmospheres (Carrel and Lindberg, 1938; Defries and Franks, 1976; Jones et al., 1981; Freshney, 1987). Explants are oriented into a culture vessel and

bathed with media, often utilizing rocking chambers, platforms or roller bottle instrumentation to increase diffusion of nutrients into the tissue mass. In contrast to cell culture which implies the relatively homogeneous isolation and culture of specific types of cells in defined media and under controlled conditions, organ culture utilizes slices or small segments of organs or tissues which contain multiple cell types. One of the most common methods utilized to establish primary cell cultures is to put a small segment of an organ or tissue from which the cells originate into a culture vessel and facilitate the cellular migration of the cells of interest from the explant by selection of media and substrate.

1j. Resau is a visiting scientistat the NCI-LMO-DCEin Frederick,MD 21702, U.S.A. 2K. Sakamotois a visiting scientistfrom the Departmentof Surgery, GunmaUniversitySchool of Medicine,Maebashi,Japan

138 Examples of cultured cells established by this method include endothelial cells, pancreatic duct epithelium, bronchial cells and keratinocytes. Organ culture offers several significant advantages for studies of cellular biology. These include preservation of the histiotypic relationships among cells of an organ without any disturbance of the cellular or tissue architecture which is caused by enzymes, chemicals or mechanical separation. During the culture of organ/explants, this relationship remains intact and the effect of the adjacent basement membranes and cells on each other can be accurately evaluated in the explants. Examples of studies on the pattern and processes of growth and differentiation of such organs include mouse (Calvert and Micheletti, 1981), human (Menard and Arsenault, 1985), and rat intestine (Quaroni, 1985).

Prototypes of explant organ culture methodology Fetal exocrine pancreas evolving from a mass of pluripotent epithelial cells into the system of enzyme-secreting acinar cells and connecting ductal cells has been described by several investi-

gators (Parsa et al., 1969a,b; Spooner et al., 1977; Githens et al., 1980, 1989). Fertilized eggs have been allowed to develop in vitro into specialized stages (blastula, morula, etc) and mesenchymal, ectodermal, and endodermal tissues (Fujinaga et al., 1991; McKiernan and Bavister, 1990; Eyestone and First, 1989; Ellington e t al., 1990). Currently, considerable research is underway in the differentiation and development of embryos as part of scientific studies relating to in vitro fertilization, preservation of endangered species, and artificial insemination/implantation (Cuthbertson and Beck, 1990; Vigier et al., 1989; Mackay and Smith, 1989). A prototypic model that is particularly suitable for organ culture is the culture of epithelia containing basal or stem cells that migrate and differentiate in a vectored fashion. Examples of this type of culture model include skin keratinocyte maturation (Bell et al., 1981; Parenteau et al., 1991), gastrointestinal columnar cells (Moyer 1983; Moyer et al., 1985, 1990) and esophageal squamous epithelium (Resau et al., 1988, 1990) (Table 1). Interestingly, several biotechnology corporations are promoting models of skin epithelium (Clonetics; Marrow Tech, San Diego, CA) that recapitulate the epidermis/dermis relationship of human skin. The actual relation-

Table 1. Examples of successful maintenance of organ/explants in vitro and is by no means all inclusive.

Tissue

Method/Assay

Reference

1. Bronchus 2. Colon 3. Breast 4. Esophagus 5. Skin 6. Intestine 7. Uterus 8. Bladder 9. Kidney 10. Prostate 11. Thyroid 12. Pancreas 13. Liver 14. Ovary 15. Testes & Ovary 16. Aorta 17. Embryos

Morph/Biochem Morph/Biochem Morph/Kinetics Morph/Kinetic Morph Morph/Biochem/Kinetic Morph/Biochem Morph/Kinetic Morph/Kinetic Morph/Biochem/Kinetic Morph/Immunohistochem Morph/Kinetic

Barrett et al.; Schiff et aL; Stoner et al. Autrup et al.; Shamsuddin et aI.; Lipkin et al. Strum and Resau Resau et al.; Vocci et al.; Parenteau et al.; Bell et al., Chapman et al. Trier; Smoot et al.; Moyer Chang et al. Rahman et al.; Heatfield et al.; Reedy and Heatfield; Knowles et al. Briere et al.; Weller et al. Sanefuji et al. Hullett et al. Jones et al.; Parsa and Marsh; Githens et al. Barr et al.; Costantino et aI. Siemens and Auersperg; Vigier et al. Mackay and Smith Barrett et al.; Yu et al., Constantindes and Robinson EUington et al.; Cox et al.

Morph Morph Morph Morph

139 ships already exist in skin explants and models have been described for the long-term maintenance of this all important organ (Chap'man et al., 1989). Adult differentiated epithelia have been established and cultured for extended periods of time in vitro. Normally, mammalian tissues are removed from anesthetized animals, oriented onto the surface of an unmodified, scraped, or coated culture vessel with or without a specialized substrate and cultured in serum supplemented or defined media. Examples of this type of study include the pancreas (Resau et al., 1983); trachea (Barrett et al., 1976); gastrointestinal epithelium (Autrup et al., 1978a,b; Browning and Trier, 1969), bladder (Reedy and Heatfield, 1987; Heatfield et al., 1980; E1 Garzawi et al., 1982; Rahm a n et al., 1987; Knowles et al., 1983); liver (Ban" et al., 1991); and pancreas (Anderson et al., 1981; Hasty and Murrell, 1978; Jones et al., 1977) (Table 1).

Human tissue culture protocols One can ethically utilize human material that is available from diagnostic material obtained as a part of surgery, autopsy, or organ donation. These types of human tissues are ordinarily used in protocols similar to those pioneered by Harris and Trump (Harris et al., 1978; Trump and Harris, 1979). When utilizing human material one must be careful to consider several key factors that are especially important in improving the viability of these tissues. Factors that affect explant viability include the cause of death, original health status of the individual, post-mortem interval, temperature, and hydration of the material (Constantindes and Robinson, 1969; Biasco et al., 1990; Costantino et al., 1990; Eastwood and Trier, 1973; Fluge and Asknes, 1981a,b). Intestinal cells were originally thought to undergo rapid cell death and necrosis of the explants shortly after the initiation of culture (Levine et al., 1970). Browning and Trier (1969) demonstrated that one can maintain small intestinal epithelial explants for 24 hrs. They empirically noticed the importance of not

maintaining the explant tissues completely submerged in the culture media. Modifications of standard collection procedures can improve the viability of specific epithelium (Reiss and Williams, 1979, 1984; Reiss et al., 1983). Smith et al., (1991) have shown how the use of mucolytic agents and selection of viable areas will dramatically improve the viability of cultures. Wargovitch et al. (1983), Trump et al. (1983), and Newmark et al. (1984) have demonstrated the effect of various ion concentrations and calcium in particular on cells in culture. Selection of specific antibiotics for microbial organisms that are unique or typically associated with an organ or tissue will similarly improve the viability of cultures. Extensive washes and perfusions of cultures with physiologic solutions has similarly improved the viability of cultures (Smoot et al., 1990).

Applications of explant organ culture Since many pathologic lesions arise in organs and involve more than one tissue or cell type (e.g., cancer, diabetes, etc.) toxicologic and pathologic processes are especially well suited for study and analysis in organ culture models. Typically one places a well preserved and viable explant of the organ in culture and exposes the explant with an appropriate dosage of the toxicant or carcinogen and characterizes the morphologic response (Autrup et al., 1978), pattern and amount of cell injury/toxicity (Costantino et al., 1990), DNA binding (Kahng et al., 1979; Stoner et al., 1982; Schut et al., 1984), metabolism (Autrup et al., 1977, 1978) and cellular or molecular biologic changes. Since many human in vivo toxicologic studies are ethically impossible, many agents, toxic compounds and experimental pharmacologic chemicals (Costantino et al., 1990), carcinogens (E1-Gerzawi et al., 1982) and known or suspected human toxicants are especially suitable to organ culture. Organ culture protocols can be used in clinical or diagnostic procedures. The original methods for the preservation and transportation of organs and tissues owes a lot to the pioneering work of

140 Carrel and Linberg (1938) who originally described the methodology for physiologic media, perfusion of nutrients, and the effect of temperature and hydration. Interestingly they felt that the organs "inside" the body were not ordinarily exposed to light and therefore the culture work should be done in the dark and with "all black material." Clinical applications for the short term culture of organs and tissues include toxicologic assays for the effect of possible chemotoxic drugs in the treatment of cancer (Lipkin et al., 1989) and other chemical applications (Browning and Trier, 1969; Fluge and Aksnes, 1981a,b). Lipkin et al. (1989) utilized organ culture to assess the effects of calcium on the proliferation and differentiation of colonic mucosal cells in hopes of predicting the potential therapeutic applications or response of individuals with a familiar or genetic predisposition to the development of proliferative colon syndromes. This group's observations indicated that calcium may inhibit the proliferation of the colonic mucosal cells and may reduce one's potential risk of developing colonic carcinomas. Schiff and Moore (1985) similarly used tracheal explant organ cultures to define and measure the effects of retinoid compounds on the tracheo-bronchial epithelium.

Molecular genetic applications of organ culture There are numerous unique advantages from the molecular genetic standpoint of organ culture experiments using cultured material to compare with time zero frozen tissue or cell lines. Assays requiring uptake of radionuclides by living cells, such as 35S-methionine or 3H-thymidine, can be done in organ culture, and may be superior to cell lines when appropriate duplication of the cell's in vivo environment is desired. Molecular studies which can be performed on organ culture preparations include immunoprecipitation for the precise determination of quantitative protein expression levels, tritiated thymidine uptake to determine cell proliferation rate, in situ hybridization to detect specific R N A expression by individual cell

types (Chang et al., 1990; Sompayrac and Danna, 1990; Tecott et al., 1988), and in situ polymerase chain reactions (PCR), along with standard molecular assays such as Southern, Northern and Western blotting, DNA sequencing, DNA cloning, reverse transcriptase (RNA-based) PCR, and differential screening or subtraction cloning. Current techniques that have direct application in molecular and cell biologic assays of cellular pathophysiology also include DNA flow cytometry for ploidy values (Reedy et al., 1991).

Technical considerations Evaluation of explant culture viability Traditional assays of viability are often not directly applicable to organ culture models and require modification. The most common viability assays include trypan blue (TB) (Schrek, 1936), neutral red (NR) (Borenfreund and Puemer, 1986), and lactate dehydrogenase (LDH) (Danpure, 1984). The first two assays utilize attached ceils or single cells in suspension and determine viability by the incorporation of a dye through an endocytic process (NR), or determine the percentage of nonviable cells on the basis of the diffusion of the dye (TB) into cells with non-viable membrane pumps. LDH is a cytosolic enzyme that is released from the cytosol of injured or dead ceils and the media or serum values are used to predict injury. TB and NR viability measurements are dependent on physical factors such as dye concentration, incubation temperature, and media osmolality. Organ cultures are not single cell suspensions by definition and have not been evaluated with either N-R or TB alone. L D H is more applicable but one must consider as well the total pool of potential releasable LDH since absolute values are not necessarily predictive of injury. For example a rapidly growing culture will have a considerable number of cells, a certain percentage of which will terminally differentiate and die. Although the LDH value in the media from these cultures may be quite high, the relative value may be low. Incorporation of labelled precursors and markers

141 of cell division have been used effectively. Studies of cell injury and repair as described by Keenan et al. (1982) have been similarly observed in organ culture models (Albright et al., 1989; Vocci et al., 1984; A.rsenault and Menard, 1983; Biasco et al., 1990; Defries and Franks, 1977; Lipkin et al., 1989). Ordinarily, morphology is the assay or method of choice to evaluate viability of explant cultures (Barrett et al., 1976; Kedinger et al., 1974; Knowles et al., 1983; Cottrell and Resau, 1985; Resau et al., 1987). Unfortunately, once explants are prepared for histologic analysis, they are by definition fixed and no longer viable and available for subsequent analysis. Cytopathology utilizes exfoliated cells or scrapings to evaluate the tissue on the basis of individual cells. This clinical technique has direct application to organ culture and has been used to evaluate tissues in organ culture (Resau and Jones, 1984; Resau and Albright, 1986). Resau and Albright (1986) in hamster tracheal organ culture and Resau et al. (1991) with primary lung carcinomas have shown how one can make permanent cytopathology preparations by preparing imprint smears and evaluating them for the degree of differentiation and viability. Culture vessels

Plastic petri dishes (e.g. 60 mm) are the most commonly used labware for explant culture. Spe-

cial dishes with spacers for mesh supports and media are also available. Some investigators have scratched the petri dish so that explant tissues do not float during rocking (Shamsuddin, 1981), while others have placed explant fragments on stainless steel mesh or grids, or alternated supporting substrates (Browning and Trier, 1969, Kedinger et al., 1974, Eastwood and Trier, 1973; Defries and Franks, 1977, Reiss and Williams, 1979, Schiff, 1975) (Table 2). Each dish ordinarily contains small pieces of tissue placed on substrates. These substrates facilitate the attachment of the explants to the culture vessel as well as promoting the maintenance of the differentiated phenotype. The typical response of an explant to organ culture media and conditions is quite similar to the wound response observed in vivo (Keenan et al., 1982). Epithelial wound repair consists of a sequential pattern of injury, cell flattening, migration, proliferation, and finally differentiation and restitution of the tissue histology. Substrates like collagen (Leighton et al., 1968), fibronectin (Lechner and LaVeck, 1985), Gelfoam | sponge (Resau et al., 1983, 1984), and fibrin foam (Schiff, 1975) have positive effects on cell growth and differentiation. One can identify the anatomical orientation of the specimen and follow the outgrowth pattems of the cells within and on the surfaces of the substrates. Figure 1 demonstrates this feature of epithelial explants. The Gelfoam

Table 2. An aid in identifying typical substrates used in organ culture - by no means all encompassing.

Substrate

Reference

1. Gelfoam Sponge 2. Fibrin foam 3. Basement Membrane Proteins 4. Chick Embryo Extract Plasma Clot 5. Glass Cover Slips 6. Membrane Filters 7. Matrix 8. Feeder Layers 9. Special Media 10. Nylon Mesh 11. Wire Mesh 12. Hyperbaric Oxygen 13. Tissue Slices

Resau et al.; Leighton et al. Schiff Lechner and LaVeck Penso and Bolducci Penso and Bolducci Parsa et al. Githens et al.; Moyer et al.; Leighton Siegfried et al. Calvert and Micheletti Chapman et al. Chang et al. Hullet et al. Krumdieck et al.

142 sponge matrix "floats" in the media and assumes an appropriate level that is optimal for the growth of the epithelial cells at an atmosphere/explant/ media interface. The cells in the "viable zone" have well preserved organelles and are easily discriminated from the zone of non-viable cells which have morphologic features of necrosis. The viable outgrowth cells are readily identified in the matrix of the sponge and, with time, for many organs may assume a histiotypic pattern characteristic o f ducts (Resau et al., 1983) or intestinal lumens (Resau et al., 1988). Culture apparatus Explant containing dishes are routinely placed in a controlled atmosphere sealed chamber (Bellco Glass Co., Wineland, NJ), which is filled with an atmosphere consisting of 50% 02, 45% N2 and 5% CO2 gas mixture. The chamber is then placed onto a Rocker Platform (Bellco Blass Co.) and oscillated 5 to 10 times per minute. The whole apparatus is usually placed into an incubator or culture cabinet warm r o o m to keep the temperature inside the chamber at 37~ (Autrup et al., 1977; Barrett et al., 1976; Han et al., 1987). A complete description of the types of dishes and culture vessels can be found in the Freshney's reference (1987).

Colon explant organ culture methodology

Fig. 1. Mouse intestinal explant culture. Mouse small intestinal explants were maintained on Gelfoam sponge rafts. Explants were covered by a uniform layer of columnarepithelial cells by 14-30 days (a). Areas of the epithelium were more papillary than others (b). On the border of the explant outgrowth and the sponge raft epithelial cells were easily identifiedmigrating into the sponge (c). In certain of those areas the epithelial cells were

As a paradigm o f explant organ culture methodology, colonic explants are presented for the final segment of this review. The first long-term maintenance o f colon explant culture was reported by Autrup et al. (1978a,b) and Shamsuddin et al. (1978). Generally, colon explants lose their normal architecture during extended (4 to 9 weeks) periods o f in vitro culture. H u m a n explants retain their cryptic architecture for 3 weeks of culture (Sakamoto et al., 1991). B e y o n d that period, cryptic structure is gradually lost and the epitheobservedto pile up on the sponge and migrate along the surface of the raft (d).

143 lial cells on the surface of explant tissue are replaced by a monolayer of columnar or cuboidal cells mixed with mucus producing cells. The period of explant culture available so far does not appear to be long enough to observe morphological changes of carcinogenic transformation. An effort to maintain viable explants even longer than in vitro culture period has been achieved by xenotransplanting the short-term cultured explants into athymic nude mice, which produced 89 days survival of human colon xenoplants by Valerio et al. (1981), and 147 days by Sakamoto et al. (unpublished observation).

1977), chilled to 4~ and supplemented with antibiotics, is often used for tissue transport. The shorter the interval between tissue collection and initiation of explant culture, the more successful the culture. Since the influx of extracellular calcium [Ca2+]e plays an important role in developing irreversible cell injury (Trump et al., 1983), calcium- and magnesium-free Hanks' balanced salt solution (HBSS) has also been utilized for rinsing the tissue before culture initiation (Yu et al., 1990).

MeSa

The colon is opened along the Taenia libera and washed extensively with chilled HBSS. The mucosal layer is separated from the underlying submucosal and muscle layer and cut into small explants. The colon contents, if present, are gently squeezed out and removed and the clean explants are put into 4~ L15 medium supplemented with antibiotics. The explants are extensively washed and then cut into smaller fragments of about 3 to 5 mm 2 in size. The explant fragments are subsequently placed onto presoaked sponge substrates with their mucosal surface positioned upwards. Figure 2 shows an example of sequential change of LDH activity in the media in human colon explant culture and typical morphology of the explant after 21 days is illustrated in Fig. 3.

Colon organ culture medium (pH 7.2-7.4) (e.g., CMRL 1066; RPMI 1640, etc.) usually includes heat inactivated fetal bovine serum, insulin (100 gg/ml), hydrocortisone 21-sodium succinate (1.0 gg/ml), L-glutamine (2.0 mM), and antibiotics. During the initial period of explant culture, the medium contains additional antibiotics (e.g., gentamycin (50 gg/ml); fungizone (250 gg/ml)). Since differentiated colon epithelial cells consume methionin to produce mucin substances, Autrup (1980) fortified the L-methionin concentration in the media. Other investigators have shown evidence that high concentrations of calcium, e.g., 1 to 2 mM, may inhibit epithelial cell growth and induced a nonreversible terminal differentiation (Newmarket al., 1984; Wargovich et al., 1983a,b). Moyer's group (1983, 1985, 1990) empirically selected a combined formulation of L-15 and low calcium MEM. Other basic media used by various investigators for colon explant culture have included: Eagle's minimal essential medium (MEM) with Earle's or Hanks' salts (Deschner et al., 1963, 1966; Lieb and Lisco, 1966), Dulbecco's modified MEM (DMEM) with high glucose, Ham's F12, Morgan's 199 medium, Trowell's T8, Waymouth's MB 752/1, and Williams medium E. The primary purpose of the tissue transport-rinse medium is to prevent further cell damage, reverse certain effects of ischemia and control bacterial contamination during transport. Leibovitz's medium (L-15; Leibovitz,

Preparation of intestinal explants

Application of explant methodology to colonic physiology study Nygard and Berglindh (1989) cultured rabbit colonic biopsy specimens and investigated the time course profile of uptake of radiolabelled leucine, thymidine and arachidonic acid for at least 48 h. Incorporation of leucine into tissue protein was linear during 48 h culture. The explants were stimulated With Ca ++ ionophore A23187 and/or phorbol 12-myristate 13-acetate at various time points. They observed a different time-dependent prostaglandin E2 production profile between normal and inflamed mucosa.

144

LACTATE DEHYDROGENASE ACTIVITY IN CULTURE MEDIA

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Fig. 2. Lactate dehydrogenaseactivity in human colon explant culture media. The culture media were sampled separately at medium change. Liberated cells and cell debris were removed by centrifugation.Each bar represents mean + SD of pentaplicate values. Reproduced from Sakamoto et aL (1991) with permission from Karger AG, Basel, Switzerland. A p p l i c a t i o n to colon c a r c i n o g e n e s i s study

Colon explant culture carcinogenesis has been summarized by Reiss et al. (1983). Long-term explant culture o f human colon was originally developed to study the metabolism of suspected environmental carcinogens directly in the target organ. Cultured human colon can metabolize procarcinogens from various chemical carcinogens such as polynuclear aromatic hydrocarbons, Nnitrosamines, and dialkylhydrazine into functional carcinogens which bind to the human cells (Autrup et al., 1977; 1978a). Shamsuddin and Trump cultured colon explants from Fischer 344 rats that were pretreated in vivo with azoxymethane (Shamsuddin and Trump, 1981). After four weeks of culture, the explants were exposed to a single dose of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) dissolved in dimethyl sulfoxide (DMSO). The explants treated with M N N G demonstrated atypical morphological and cytochemical changes identical to neoplastic transformation. These changes were characterized by abnormal nuclei, increased

Fig. 3. An exampleof human colon explant. Typicalexample of human colon explant morphology at 21st day of culture is illustrated in contrast to 0-time control. Well-organized gland structure is very similar to that of 0-time control. The mucous substance is rich in goblet cells, x160. Scale bar, 100 pm. Reproducedfrom Sakamoto et al. (1991) with permission from Karger AG, Basel, Switzerland.

nuclear-cytoplasmic ratio, hypercellularity, and epithelial papillary projections into the lumen o f the crypts. Ultrastructural changes in the M N N G treated explants also showed features typical o f adenocarcinoma. These features were observed as early a s 1 week after the M N N G treatment and increase in degree and frequency as time progressed. After 9 weeks, mucus substances contained in the crypts were mostly sialomucin. The first report o f animal colon explant culture in conjunction with 1,2-dimethylhydrazine (DMH) pretreatment was described by Defries and Franks (1976). They observed an increased crypt mitotic index in the explants from D M H

145 pretreated mice. Recently, Sakamoto et al. ( 1991) reported a 3-step transformation model that could be applied to human colon carcinogenesis study. They described the long-term maintenance of human and rat colon mucosa and the morphological transformation of rat colon mucosa by using a method which combines organ culture, exposure to xenotropic murine sarcoma virus in vitro, and xenotransplantation into nude mice. In order to correlate this to the human colons that are presumed to be initiated because of repeated exposure to environmental carcinogens, they pretreated the experimental rats with low dose of D M H and used the colon before obvious morphological changes became evident. They demonstrated neoplastic transformation in the xenoplant tissue that was recovered from the nude mice, while normal appearing cryptic tissue was recovered from the control untreated xenoplants. Application to inflammatory bowel disease study Mahida et al. (1991) demonstrated that mononuclear cells isolated from mucosa with active inflammatory bowel disease produced increased amounts of interleukin 1 beta over controls (Mahida et al., 1989). Based on their work, they tried to elucidate the mechanism of inhibitory effect of 5-aminosalicylic acid on inflammatory bowel disease (IBD), and carried out explant organ culture using biopsy specimens from inflamed colonic mucosa of patients with IBD. Increased concentrations of interleukin 1 beta and thromboxane B2 are accepted to be parameters for the active state of IBD. These substances are suggested to be mediators of inflammation and tissue damage. The interleukin 1 beta value was significantly higher in the patients group than the normal control group not only in the mucosal biopsy tissues before culture but also in the explant tissues and media after 24 h culture. The value of thromboxane B2 correlated with interleuldn 1 beta. Addition of 5-aminosalicylic acid as well as dexamethasone in the culture media with therapeutic dose concentrations inhibited production of interleukin 1 beta.

Summary Explant organ culture is an appropriate and wellestablished model for the study of mechanisms of carcinogenesis, secretion, proliferation, and differentiation. There are specialized media, substrates, and vessels available for use and considerable published references document the applications, capabilities, and usefulness of the techniques.

Acknowledgements The authors wish to thank the Department of Pathology's Publications staff at the University of Maryland for the preparation of this manuscript. The authors also wish to thank the Pathologists and Surgeons at University of Maryland Medical Center; Veterans Administration Medical Center, Baltimore; Harbor Hospital Center; Baltimore County General Hospital; St. Agnes Hospital, and Union Memorial Hospital for their support. The Human Tissue Resources is supported in part by a grant from the NDRI for a Remote Site Facility. Portions of this work were supported in part by Grant #PDT-419 from the American Cancer Society, the Crohn's and Glitis Foundation of America, the Franck C. Bressler Research Fund, and a Graduate Research Assistantship]Special Research Initiative Support Grant from the School of Medicine Designated Research Initiative Fund. This is contribution No. 3151 from the Pathobiology Laboratory of the University of Maryland.

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