Human mesothelial cells (HMC) cover a variety of serosal surfaces and have been shown to rest upon an underlying subcellular basement membrane in vivo. Bovine corneal endothelial cells produce an extracellular matrix (ECM) in vitro that mimics HMC subcellular basement membrane and was found to modulate HMC adhesion, morphology and proliferation in vitro. Our results indicated that within minutes after plating, a high percentage (>80%) of HMC firmly attached to ECM. Active cellular migration and subsequent proliferation were observed leading to the formation of a well-organized closely apposed cell monolayer. However, when cells were plated on plastic, the rate of cell attachment was much lower and the proliferative rate of HMC grown on plastic also was strikingly lower (exponential doubling time 4·3 days) than that of cells grown on ECM (exponential doubling time 2·4 days). Cells upon reaching confluency on plastic were markedly enlarged as compared to confluent cells grown on ECM. These observations corroborated differences in final cell density where it was noted that HMC cultured on ECM demonstrated a 10fold greater final cell density as compared to cells grown on plastic. Results from these studies illustrate the fact that phenotypic expression as well as proliferative responsiveness of HMC can be modulated by adhesive interactions with preformed ECM.

The serous membranes lining the major body cavities consist of a single layer of highly flattened mesothelial cells resting upon an underlying subcellular basement membrane. Mesothelial cells provide a non-adhesive surface that facilitates the movement of visceral organs (Andrews & Porter, 1973) and acts as a permeability barrier, which regulates fluid and solute transport between the systemic circulation and body cavities (Cunningham, 1926; Courtice & Simmonds, 1954). The basement membrane separates mesothelial cells from the underlying supportive tissue and functions as an extracellular scaffold that maintains orderly tissue structure (Vracko, 1974). The composition of the basement membrane present beneath mesothelial cells in vivo has not been characterized. However, on the basis of studies with basement membranes from a variety of cell types (Hay, 1981), it is most probably composed of collagens, glycosaminoglycans, glycoproteins and proteoglycans arranged in a biochemically complex and intricately arranged three-dimensional structure.

At present, few in vitro studies have been undertaken on isolated human mesothelial cells (HMC). The long-term maintenance of these cells is hampered by the inability to culture human mesothelial cells (HMC) easily on plastic on a routine basis (Castor & Naylor, 1969; Singh et al. 1978). Limited growth of these cells has been achieved by using specific growth factors (Connell & Rheinwald, 1983), conditioned medium and fibroblast feeder layers (Wu et al. 1982), as well as specific attachment factors (Lechner et al. 1985).

Cell-substratum interactions have been reported to be important for cell growth and differentiation of both normal and malignant epithelial cells (Hay, 1981; Vlodavsky et al. 1980). Such cells generally attach, grow and maintain their differentiated functions better when extracellular matrix (ECM) or isolated ECM components are used as the substratum rather than plastic (Wilde et al. 1984; Jozan et al. 1982). The relevance of cell—substratum interactions in HMC growth and morphology has not been fully ascertained. This paper demonstrates the effect of ECM-coated culture dishes, produced by bovine corneal endothelial cells (BCEC), on the cellular adhesion, morphology and growth of isolated HMC. It has been demonstrated that the ECM produced by BCEC is composed of interstitial collagen types I—III and basement membrane collagen types IV and V (Tseng et al. 1981, 1983). Also present are proteoglycans comprised primarily of heparan sulphate and dermatan sulphate (Robinson & Gospodarowicz, 1983; Nevoei al. 1984), as well as glycoproteins such as elastin, fibronectin and basement-membrane-specific laminin (Gospodarowicz et a/. 1981). It has also been shown by Gospodarowicz et a/. (1977) that the ECM produced by BCEC in vitro is analogous to the ECM associated with these cells in vivo.

HMC cultured on ECM may provide a relevant model that may help to elucidate further the mechanisms by which human tumour cells interact with the mesothelium to form metastatic implants (Niedbala et al. 1985). In addition, mesothelial cells cultured in this manner may aid in investigating the growth, repair and transformation of the mesothelium.

Isolation of human mesothelial cells

Human mesothelial cells were derived from peritoneal effusions of an ovarian cancer patient, since it is well known that ascites fluid is an excellent source of large quantities of viable HMC (Cunningham, 1926; Domagala & Koss, 1979). Ascitic fluid containing a high percentage of mesothelial cells, as determined by cytological analysis of cytospin preparations, was centrifuged (500g) for 10 min. The cell pellet was resuspended in Roswell Park Memorial Institute (RPMI) 1640 culture medium supplemented with 20% foetal calf serum (FCS), fungizone (2·5 µml-1), and gentamycin (50µml-1) (Gibco, Grand Island, NY). Cells were plated onto 10cm plastic culture dishes (Falcon, Becton, Dickinson and Co., Cockeyville, MD) for 24h at 37°C (95 % air/ 5 % CO2). During this time interval a high percentage of HMC adhered to the plastic, while the majority of tumour cells and other inflammatory cell types in general did not become adherent. Tumour cells and other cell types were easily removed from the adherent HMC by gentle washing with warm medium (37°C). HMC were subsequently dissociated from the plastic substrate by a brief exposure (2-3 min) to 0·5 % trypsin and 0’02% EDTA at 37°C. Cells were washed, pelleted by centrifugation, replated onto plastic culture dishes and incubated for 6h at 37°C. Adherent HMC were then maintained as described by Wu et al. (1982) with the following modifications: a 1:1 (v/v) mixture of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20 % FCS, hydrocortisone (0·4 μgml-1) and cell-free medium, that had been conditioned by 3T3 mouse fibroblasts in exponential growth for 48–72 h, was used. Under these selective culture conditions it was observed, by phase-contrast microscopy, that HMC grew progressively as primary cultures.

Preparation of ECM-coated culture dishes

Primary cultures of bovine corneal endothelial cells were established from calf eyes and were maintained in culture as described by Gospodarowicz et al. (1977). After 6–8 days, post-confluent BCEC monolayers were solubilized by washing cultures with sterile distilled water and briefly exposing cells to 0·02M-NH4OH. This resulted in the lysis of BCEC and exposure of the underlying, structurally intact ECM, which was firmly and uniformly attached to the culture dishes. ECM-coated culture dishes were carefully washed with PBS and used immediately, or stored in PBS containing antibiotics at 4°C for no longer than 2 weeks.

Adhesion assay

Serially passaged human mesothelial cells (P2) at a concentration of 2×106 to 5×106cellsml-1 were prelabelled with 0-·25 mCi of 51Cr-labelled sodium chromate as described previously (Crickard et al. 1983; Niedbala et al. 1985). Radiolabelled HMC were added to either 16mm ECM-coated or plastic culture dishes. At selected times non-adherent cells were removed by aspiration and the cultures were carefully washed three times with warm PBS (37°C). The remaining, firmly adhering cells were released by trypsinization and cell-associated radioactivity was measured using a gamma counter (Searle model 1185, TM Analytic Inc., Elk Grove Village, IL).

Karyotyping of cells

HMC grown on ECM-coated culture dishes were exposed to colcemid (10μgml-1) and incubated for 8h at 37 °C (95% air/5% CO2). Cells were dissociated from culture dishes by trypsinization, washed and pelleted. Cells were processed for karyotypic analysis, as described by Crickard et al. (1983).

Cytological analysis

Cytology slides were prepared using a Cytospin centrifuge (Shandon Southern Instruments, Inc., Sewichley, PA) and immediately fixed in 70% ethanol for staining with haematoxylin and eosin.

Cell proliferation

Exponentially grown HMC maintained under previously defined conditions, were harvested by trypsinization, washed and pelleted. Cells were plated at an initial density of 1×104 cells/35mm culture dishes with or without an ECM. Triplicate cultures were trypsinized and cell number was determined electronically with a Coulter Counter (Coulter Electronics Inc., Hialeah, FL) every other day. The morphological appearance of these cultures was observed by phase-contrast microscopy.

DNA-flow cytometry

Cell suspensions containing 2×106 cells ml-1 in RPMI 1640 were stained with a DNA fluorochrome, 4’-6-diamidino-2-phenylindole (1 μgml-1), in the presence of 0·2% Triton X-100 (Taylor, 1980). Human blood leucocytes were used as a diploid standard. The DNA ploidy index was calculated as the ratio of the G1 peak channel of cell suspension to that of the internal diploid cell standard. Flow cytometry was carried out using a ICP 22A flow cytometer (Ortho Instruments, Westwood, MA) as described by Frankfurt (1983).

Indirect immunofluorescence studies

Cells were cultured on glass coverslips in the presence and absence of ECM, fixed in absolute methanol at — 20°C for 5 min and then in acetone for 5 min. After fixation, the cells were stained for indirect immunofluorescence, as described by Asch et al. (1981). Experiments were undertaken with a mouse monoclonal human anti-keratin (AE-3), described by Woodcock-Mitchell et al. (1982), and goat antisera raised against mouse mammary fibroblast vimentin, recently shown to be cross-reactive with human fibroblast vimentin (Asch & Asch, 1986); fluorescein-conjugated sheep anti-mouse and rabbit anti-goat immunoglobulin (IgG) were used as secondary antibodies. Coverslips were mounted with phosphate-buffered glycerol (pH 8’6) containing p-phenylenedia-mine. Cells were examined for indirect immunofluorescence with a Zeiss epifluorescence photomicroscope II and a 40x plan Neofluor objective with oil immersion. Photomicrographs were made using Kodak Tri-X film exposed at ASA1200.

Characterization of mesothelial cell primary cultures

Under the selective culture conditions used in this study, a relatively homogeneous mesothelial cell population was isolated as characterized, according to the following criteria.

Cytological analysis

Cytospin preparations of dispersed mesothelial cells were stained with haematoxylin and eosin for cytological analysis. In all cases, typical cytological features of normal cells were noted and it was concluded from cell preparations that a single population of proliferating cells was present. Occasionally, multinucleated reactive mesothelial cells were observed but the percentage of these cells in primary cultures was limited to less than 5 %.

Flow cytometry and karyotypic analysis

A diploid population of cells constituted the majority of the proliferating cell population as determined by cytogenetic analysis (Fig. IB). A modal chromosome number of 46 with a modal range of 42·48, equivalent to 85 % of the total proliferating cell population, was observed. DNA flow cytometry indicated the presence of a single population of diploid cells using human leucocytes as an internal diploid standard (Fig. 1A). A total of 30000 to 50000 cells was analysed for each histogram and a ploidy index equivalent to unity was observed, corroborating results from cytogenetic studies.

Immunofluorescence microscopy

HMC, cultured to a semiconfluent state on either glass or ECM-coated glass coverslips, were found to contain large amounts of keratin organized in a dense, filamentous network, as defined by reaction with keratin antibodies. All of the cells reacted positively, regardless of the substrates used (Fig. 2). Keratin filaments were distributed throughout the cells despite changes in cell shape and size, which were dependent on the growth substrate. Cells showed bundles of keratin filaments, which were organized concentrically in perinuclear regions (Fig. 2C,D). The positive reactivity of these cells to a variety of keratinspecific antibodies demonstrates their epithelial nature. In contrast, human mesothelial cells when cultured on plastic or ECM-coated culture dishes expressed relatively low levels of immunoreactivity to goat antiserum directed against vimentin (Fig. 3A,B).

In addition to the studies carried out above we have used a monoclonal antibody (F36/22) that recognizes an antigen determinant expressed by ovarian carcinoma cells but not by mesothelial cells (Croghan et al. 1984). This monoclonal antibody was used to screen primary mesothelial cell cultures as well as subsequent cultures, periodically, for the presence of tumour cells. The results were compared with those from studies of the reactivity of cells to an irrelevant control monoclonal antibody of the same subclass using indirect immunoperoxidase staining. In all cases cultures the same subclass using indirect immunoperoxidase staining. In all cases cultures were consistently negative for the expression of F36/22 antigen determinant with staining levels similar to that of irrelevant controls. The sensitivity of this probe for ovarian carcinoma cells has been recently described (Niedbala et al. 1985) in the presence of mixed cultures of mesothelial cells grown on ECM following the addition of ovarian tumour cells. In these studies single ovarian tumour cells could be readily identified by the intense immunoreaction product associated with tumour cells. Finally, mesothelial cells when injected subcutaneously into nude mice were not observed to be tumorigenic with initial inocula up to 5×107 cells per animal, following 5 months of observation.

Rate of cell attachment

In order to quantify the attachment of HMC to ECM versus plastic, HMC were prelabelled with 51Cr and seeded onto these respective substrata (Fig. 4). Timedependent attachment to these substrata was determined by measuring the radioactivity associated with adherent HMC as a function of total cellular radioactivity (adherent and non-adherent fractions). At early times HMC adhered to ECM more rapidly and more firmly than to plastic. Greater than 80% of the seeded HMC attached to ECM after 15 min. In contrast, less than 25 % of HMC attached to plastic within 15 min. The amount of time required for the firm attachment of 50% of mesothelial cells was defined as the adhesion index for 50% of cells (AI50). For plastic an AI50 of 28 min was observed whereas for ECM it was markedly faster with an AI5Q of 9 min. At later times (>6 h) the percentage of cells that became firmly adherent to plastic approached that of cells exposed to ECM (Fig. 4).

Effect of substratum on cell morphology and culture organization

Marked differences in the morphological appearance of HMC grown on plastic- and ECM-coated culture dishes were observed using scanning electron microscopy (Fig. 5). HMC cultured on plastic became considerably enlarged and a high percentage (25%) was observed to be multinucleated within 72 h. In addition, HMC cultured on plastic showed a greater degree of cellular overlap in comparison to cells grown on ECM. In contrast, HMC plated on ECM adopted a highly flattened cellular morphology composed of tightly packed non-overlapping cells, which covered the entire surface of the culture dish. In some cases, complex intercellular junctional complexes mediated via interdigitating cell processes were observed when HMC were cultured on ECM. Cells could be maintained on either substratum for as long as several months following confluency with minimal alterations in their respective cell morphologies and viability, as determined by their subsequent proliferative capacity. Interestingly, upon dissociation of cells grown on plastic and reseeding on ECM, HMC immediately adopted the morphological characteristics described above for HMC maintained originally on ECM.

Cell proliferation studies

Comparative studies were undertaken to determine the growth rate of HMC plated at a low density on plastic versus that of ECM-coated culture dishes (Fig. 6). Cells maintained on ECM divided more rapidly with a mean exponential doubling time of 2·4 days. Cells grown on plastic did proliferate under the conditions used but at a significantly slower rate (mean doubling time 4·3 days). Differences in final cell density were also observed between the two substrata after 10 days of growth. HMC grown on ECM had a final cell density 10-fold higher than that of cells grown on plastic. These results were consistent with the morphological appearance of confluent cultures maintained on plastic versus ECM (Fig. 5). Cells grown on ECM were composed of tightly packed closely apposed cells, whereas those grown on plastic were strikingly larger and exhibited considerably more cell overlap.

The role of cell-substratum interactions in regulating cell proliferation and morphogenesis both in vitro and in vivo has been demonstrated in a number of laboratories (Hay, 1981 ; Kleinman et al. 1981 ; Vracko, 1974). Mesothelial cells have been shown to rest upon a basement membrane in vivo (Odor, 1954). Cellular contact with the basement membrane may directly modulate the morphogenesis and proliferation of the mesothelium. Likewise, in order to proliferate and express their normal phenotype, mesothelial cells maintained in tissue culture may require not only nutrients and growth factors, but also an appropriate physical substratum to which they can attach and then proliferate.

This study takes advantage of the fact that bovine corneal endothelial cells (BCEC) retain their ability to secrete basally an extensive ECM in vitro. This ECM, although bovine in origin, has been shown to conform to the chemical criteria (Kefalides, 1980) and biological criterion (Kleinman et al. 1981) of a basement membrane, in general, and serves as a substratum to which cells can attach, and on which they can migrate and divide both in vitro and in vivo. BCEC ECM has been found in this study to support HMC growth in vitro and to serve as a substitute for the basement membrane underlying HMC/w vivo. In addition, HMC may modulate the preformed BCEC ECM by producing their own collagens and mucopolysaccharides, as described for other diploid cell types (Madri et al. 1983; Kato & Gospodarowicz, 1985), and thus become capable of modulating their own growth and morphology by the production of a hybrid matrix. Preliminary studies indicate that the HMC used in this investigation are capable of depositing extracellular matrix components onto plastic. These studies were carried out using monospecific antibodies directed against basement-membrane-specific laminin and collagen type IV in conjunction with immunofluorescence (unpublished data). These observations corroborate the findings of Rennard et al. (1984), which demonstrated that rat pleural mesothelial cells can produce matrix components in vitro.

In this study it was observed that HMC attached rapidly to ECM as compared to plastic. Cellular attachment to ECM involves a complex series of interactions with a variety of adhesive and polyionic structural macromolecules present in the ECM (Yamada, 1983; Kefalides, 1980). Interestingly, the ability of HMC to attach to plastic approached that of ECM at later times, indicating that perhaps adherent HMC can condition the plastic culture surface, making it more favourable to supporting further attachment.

Close contact of HMC with ECM may increase HMC responsiveness to growth factors present in the culture medium, which are not observed when cells are maintained on plastic (Gospodarowicz et al. 1980). This could explain the observation that HMC plated at a low density on ECM proliferated more rapidly and attained a higher final cell density as compared to cells grown on plastic. The results described here also indicate that mesothelial cultures maintained on plastic and passaged at a low cell density rapidly lose their normal morphological phenotype (Baradi & Hope, 1964), while cultures maintained on ECM retain it. The ECM may therefore stabilize normal phenotypic expression of HMC, since a simple change of substrate from plastic to ECM may restore the sensitivity of cells to factors present in the culture medium, resulting in enhanced proliferation and a striking change in cellular morphology. Similar results have been observed for a number of other cell types (Gospodarowicz & Tauber, 1980; Bethea et al. 1982; Kato & Gospodarowicz, 1985).

The mechanism by which the ECM exerts its ‘permissive’ effect on HMC proliferation is still a subject for hypothesis. Gospodarowicz et al. (1978) have speculated that cell shape may be instrumental in making cells responsive to serum factors and mitogens to which they cannot respond unless they adopt an appropriate shape. Alternatively, the ECM may contain small amounts of growth factors and other mitogens, which may be sequestered within its macromolecular structure, thereby resulting in enhanced cellular proliferation as compared to other substrates. Recently, Gospodarowicz et al. (1983) attempted to address this question by exposing the ECM to a variety of conditions that are known to destroy the activity of a number of mitogens. In short, the growth-promoting effect of the treated ECM was equivalent to that of untreated ECM, suggesting that endogenous growth factors may not be responsible for its growth-promoting effect and that other mechanisms related to its structural composition may be involved.

Recent studies by Connell & Rheinwald (1983) have indicated that EGF when added to HMC in the presence of serum resulted in the induction of cellular proliferation and in the acquisition of a fibroblastoid morphology, which was reversible upon removal of EGF. Interestingly, HMC when cultured in the presence of EGF were observed to have increased amounts of vimentin and decreased absolute amounts of cytokeratins. In our studies HMC adopted an epithelial cellular morphology, with high levels of cytokeratins and low levels of vimentin being observed when ECM was used as a growth substrate despite an increase in cellular proliferative capacity. Our observations are in agreement with in situ observations in which HMC were shown to possess epithelial morphology, high keratin and low vimentin content (LaRocca & Rheinwald, 1984).

Preliminary studies on EGF treatment of HMC over a wide range of concentrations (0-5—50ngml-1) have failed to demonstrate considerable morphological and/or proliferative differences when HMC are maintained on plastic versus ECM. For this reason it is believed, on the basis of our results, that two independent mechanisms may account for differences in HMC proliferative rates, one being mediated by the growth substrate and another via soluble factors.

The results presented in this study demonstrate the importance of human mesothelial cell—extracellular matrix interactions in maintaining normal cell morphology, behaviour and proliferative responsiveness in vitro. In addition, we have been successful in isolating four other mesothelial cell lines from peritoneal effusions of ovarian cancer patients. In all cases the observations were similar to those described here. Thus, the culture system described in this paper may represent a model system in which to investigate the mechanisms by which human tumour cells interact with the mesothelium to form metastatic foci (Niedbala et al. 1985). It also may permit the study of growth, repair and transformation of the mesothelium in vitro.

The authors thank Dr E. Nava for her assistance with cytological analyses, Dr B. Asch for help with immunofluorescence studies, and Dr O. Frankfurt for performing flow cytometry. This work was supported in part by grants from the National Cancer Institute (USA), CA-13038, CA-42898, CA-24538 and CA-32767.

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