Mucin synthesis and secretion by cultured tracheal cells: effects of collagen gel substratum thickness

Summary We previously demonstrated that human tracheobronchial epithelial (TBE) cells synthesize mucin and form mucous granules in culture when they are maintained on a collagen gel (CG) substratum, but not on a plastic tissue culture surface or a thin collagen-coated surface (Wu et al., Am. J. Respir. Cell Mol. Biol., 3:467–478; 1990). This observation led us to examine the effects of CG thickness on cell growth and differentiation in primary human/monkey TBE cell cultures. Using the same CG preparation, culture dishes with different thicknesses of CG substratum were prepared. In general, equivalent degrees of cell attachment and proliferation were observed in all cultures maintained on a collagen gel, independent of the thicknesses of CG substratum. However, a greater degree of mucin synthesis and secretion by the cells was observed as the thickness of the CG substratum was increased. Cultures maintained on a thick collagen gel (1 mm) exhibited greater apical membrane complexity, more pseudostratification, and more mucous granules than did cultures maintained on a thin CG substratum. The optimal culture surface for airway mucous cell differentiation contains more than 1-mm thickness of collagen gel substratum.     

Temporal and spatial distribution of type XII collagen in high cell density culture of periosteal-derived cells.

Periosteal-derived cells of young chicks have been reported to possess the potential to undergo terminal differentiation into osteogenic or chondrogenic phenotypes under high cell density culture conditions. In this culture, the temporal and spatial distribution of type XII collagen was immunocytochemically assessed using a monoclonal antibody. These high-density plated cells first formed a multilayer of fibroblast-like cells, in which type I and XII collagen were evenly distributed throughout the full thickness of the culture. With time, the top portion of the culture differentiated into bone tissue, while cells below this top layer differentiated into hypertrophic chondrocytes. In this transition, type XII collagen was temporally and spatially colocalized primarily with type I collagen: the top portion of bone layer was positive for both type I and XII collagens, whereas their staining intensity in the bottom portion decreased with time in culture. Using this antibody, type XII collagen was also found in developing embryonic chick tibiotarsus. These observations, taken together, suggest that type XII collagen production is a characteristic property of bone-forming cells.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and alpha 2 beta 1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize alpha 2 beta 1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Cell lines with developmental potential restricted to mesodermal lineages isolated from differentiating cultures of pluripotential P19 embryonal carcinoma cells

Embryonal carcinoma (EC) cells are developmentally pluripotential cells which can be induced to differentiate in cell culture to form a wide variety of cell types. To investigate the lineage relationships between cells of different types, we set out to isolate cell lines with multiple but restricted developmental potentials from differentiating cultures of P19 cells, a line of EC. By selecting for differentiated cells capable of anchorage-independent growth, we isolated cell lines which differentiated in high density cultures to form at least two cell types; myocytes that resembled fetal skeletal muscle cells and loose connective tissue cells that secreted large amounts of type I collagen. These results suggest that skeletal myocytes and connective tissue share a common precursor and that stem cells with limited but multiple developmental potentials can be isolated from differentiating cultures of P19 cells.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize α2β1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

FINE STRUCTURE OF DIFFERENTIATING MOUSE PANCREATIC EXOCRINE CELLS IN TRANSFILTER CULTURE

Fine structural observations have been made in the 11-day embryonic mouse of exocrine cells in pancreatic epithelium developing in tissue culture transfilter from salivary gland mesenchyme of the 13-day embryonic mouse. After 2 days in culture, the exocrine cells show increased cytoplasmic density, abundant ribosomes in aggregate or "rosette" form, and expanded profiles of rough-surfaced endoplasmic reticulum. After 3 and 4 days in culture, the cells exhibit continued expansion of the profiles of endoplasmic reticulum, increased amounts of Golgi membranes, and large areas of light density (prozymogen granules). After 5 days in culture, dense zymogen granules are present in the most highly differentiated cells. In addition, at the filter-epithelial surface, at 2 days, small fibers can be discerned which, after 4 days in culture, show obvious periodicity and are thought to be collagen. The significance of these changes, in relation to the mesenchymal effect, to the onset of specific synthesis and to the stabilization of differentiation is discussed.     

Culture of large vessel endothelial cells on floating collagen gels promotes a phenotype characteristic of endothelium in vivo

The vascular endothelium in vivo is a remarkably quiescent cell layer that displays a highly differentiated and tissue-specific phenotype. Once established in culture, endothelial cells (EC) are phenotypically different from their in situ counterparts, displaying altered gene expression, increased mitotic index, and decreased cell density. To determine whether manipulating the microenvironment of cells in vitro would lead to a more differentiated phenotype, we cultured bovine aortic EC on floating collagen gels. EC cultured to confluence on floating gels for 24 or 48 hr display mitotic indices nearly identical to those of quiescent endothelium in vivo, nearly two log orders lower than that of EC cultured to confluence on plastic, and cell density on floating gels also resembles that observed for endothelium in vivo. Culture of EC on floating gels leads to decreased expression of platelet-derived growth factor-B, fibronectin, and fibronectin isoform ED-B, and increased levels of connexin40, relative to cells cultured on plastic. We conclude that culture of bovine aortic EC under standard culture conditions results in a phenotype reminiscent of development and/or wound healing, and that culturing them on a floating collagen gel leads to a more differentiated phenotype, reminiscent of that observed for large vessel EC in vivo.     

Culture of endodermal stem/progenitor cells of the mouse tongue

The tongue represents a very accessible source of tissue-specific epithelial stem cells of endodermal origin. However, little is known about the properties of these cells and the mechanisms regulating their proliferation and differentiation. Foxa2, an endodermal marker, is expressed throughout the tongue epithelium during embryonic development but becomes confined to a minority of basal cells and some taste bud sensory cells in the adult tongue. Using a previously described line of transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of a human keratin 5 promoter region (Krt5-eGFP), we have isolated a subpopulation of cells in the basal epithelial layer of the mouse tongue with a high efficiency of generating holoclones of undifferentiated cells in culture with a feeder layer. Krt5-GFP(hi) cells can both self renew and give rise to differentiated stratified keratinized epithelial cells when cultured on an air-liquid interface.     

Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha_s1-casein, lactoferrin (Lf), and alpha-lactalhumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf scrum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 μg/ml medium, which was 2–3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for eryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin. and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.     

Cytodifferentiation in the accessory glands of Tenebrio molitor. XI. Transitional cell types during establishment of pattern

The bean-shaped accessory glands of male Tenebrio consist of a single-layered epithelium which is surrounded by a muscular coat. The epithelial layer, which produces precursors of the wall of the spermatophore, contains eight secretory cell types. Each secretory cell type is in one or more homogenous patches, and discharges granules which form one layer of the eight-layered secretory plug. Maturation begins in cell types 4, 7, and 6 on the last pupal day. A newly identified cell (type 8) in the posterolateral epithelium matures last. Cells of individual types mature in synchrony, and their secretory granules “ripen” in a sequence that is characteristic for each type. As the secretory cells of each patch mature, unusual short-lived cells appear at interfaces between patches. In some cases the secretory granules in these boundary cells have ultrastructural features which are mixtures of the definitive characteristics of granules in adjacent cell types. The transitional cell types disappear at 3–4 days after eclosion. Intermediate cell types are absent in the mature gland and boundaries between the patches are distinct. The transitional cells may form granules of intermediate structural characteristics as a dual response to cellular interaction with adjacent and previously differentiated secretory cells.     

The cell and the organism: the role of subdivisional cell replication in the development and maintenance of the multicellular organism

Evidence of new cell formation from nucleoli (subdivisional cell replication) was observed in vitro and in vivo. Mouse melanoma cells, human fibroblasts, and rat mast cells were observed in tissue culture with phase contrast time-lapse cinematography. Evidence of subdivisional cell replication seen in tissue culture was supported by observations of mast cells, cervical epithelial cells, melanoma cells, keratinocytes, and fungal spores in vivo. Indirect evidence for subdivisional cell replication was the presence of differentiated form and function in nuclei and nucleoli. Synergism between subdivisional replication and mitotic replication (subdivisional expansion) is believed to be a key to morphogenesis, whereby cellular and subdivisional zones act as biologic "molds". It is believed that subdivisional cell replication has a key role in maintenance of differentiated form in multicellular organisms, as well as in morphogenesis.     

Development of chicken epidermis cultured with embryo extract

The epidermis from 11-day-old chick embryo shank skin was cultured with 11-day-old chick embryo extract. The growth and the differentiation of the epidermis in culture were studied histologically, electron microscopically and with polyacrylamide gel electrophoresis of keratin proteins. The epidermis cultured with the chick embryo extract proliferated and stratum structures developed simultaneously with the increase in epidermal cell layers. Finally, a keratinized layer was observed after 10 days in culture. Electron microscopic observations revealed that tonofilaments were produced after 2 days in culture and increased thereafter with culture time, becoming condensed with desmosomes. Keratohyaline granules were observed in 7-day cultures. These keratinization characteristics occurring during culture showed the general characteristics of the alpha stratum observed in the skin of in ovo embryos during the early stages of development. However, the development of peridermal and subperidermal granules was poor and the stratum granulosum, which develops at the late stages between the stratum intermedium and the stratum corneum, was not observed. Polyacrylamide gel electrophoresis of S-carboxymethylated keratin proteins showed that the keratin protein band patterns of the culture differed from those of in ovo skin epidermis.     

Synthesis of extracellular matrix glycoproteins by a differentiated thyroid epithelial cell line

We examined the synthesis of extracellular matrix macromolecules by the differentiated rat thyroid epithelial cell line FRTL-5. As shown by electron microscopy, the extracellular material produced by these cells is deposited at the basolateral surface and focally organized in the form of a basement membrane. Biochemical and biosynthetic studies demonstrated that laminin, type IV collagen, and fibronectin are synthesized and deposited in the culture monolayer. Secretion of fibronectin into the culture medium also occurred. By immunofluorescence we observed some peculiarities in the distribution patterns of the basement membrane glycoproteins; while fibronectin and laminin had an almost superimposable distribution, type IV collagen displayed a rather different pattern. Type IV collagen and laminin localization at sites where extracellular material was detected was confirmed by immuno electronmicroscopy using the protein A-colloidal gold technique. The results indicate that under appropriate culture conditions the differentiated thyroid epithelial cell line FRTL-5 synthesizes, secretes and organizes an extracellular matrix where some basement membrane glycoproteins are present.     

Embryo development of Corticium candelabrum (Demospongiae: Homosclerophorida)

Abstract. Corticium candelabrum is a homosclerophorid sponge widespread along the rocky Mediterranean sublittoral. Scanning and transmission electron microscopy were used to describe the gametes and larval development. The species is hermaphroditic. Oocytes and spermatocytes are clearly differentiated in April. Embryos develop from June to July when the larvae are released spontaneously. Spermatic cysts originate from choanocyte chambers and spermatogonia from choanocytes by choanocyte mitosis. Oocytes have a nucleolate nucleus and a cytoplasm filled with yolk granules and some lipids. Embryos are surrounded by firmly interlaced follicular cells from the parental tissue. A thin collagen layer lies below the follicular cells. The blastocoel is formed by migration of blastomeres to the morula periphery. Collagen is spread through the whole blastocoel in the embryo, but is organized in a dense layer (basal lamina) separating cells from the blastocoel in the larva. The larva is a typical cinctoblastula. The pseudostratified larval epithelium is formed by ciliated cells. The basal zone of the ciliated cells contains lipid inclusions and some yolk granules; the intermediate zone is occupied by the nucleus; and the apical zone contains abundant electron-lucent vesicles and gives rise to cilia with a single cross-striated rootlet. Numerous paracrystalline structures are contained in vacuoles within both apical and basal zones of the ciliated cells. Several slightly differentiated cell types are present in different parts of the larva. Most cells are ciliated, and show ultrastructural particularities depending on their location in the larvae (antero-lateral, intermediate, and posterior regions). A few smaller cells are non-ciliated. Several features of the C. candelabrum larva seem to support the previously proposed paraphyletic position of homoscleromorphs with respect to the other demosponges.     

Culture of differentiated and undifferentiated type II cells from fetal rat lung

We have developed a relatively simple and reproducible method for the isolation and culture of both differentiated and undifferentiated type II cells from fetal rat lung. The technique involves an initial period of explant culture in serum and hormone free medium, followed by enzymatic dissociation of the explants, differential adhesion to remove fibroblasts, incubation of the cell pellet to promote aggregation of the type II cells and monolayer culture of the type II cells. The type II cells form clusters which are surrounded by scattered fibroblasts. When the technique was performed with three differential adhesion steps, cultures contained 86.0 +/- 1.4% type II cells. To obtain a higher degree of purity and greater yield, two differential adhesions followed by gentle trypsinization of the cultures which selectively removes the isolated fibroblasts was performed. This resulted in cultures with 89.4 +/- 1.7% type II cells. The differentiated fetal type II cell cultures were prepared from 19-day fetal rat lungs which were initially maintained in explant culture for 48 h. These differentiated cells demonstrated the characteristic morphologic features of type II cells including lamellar bodies and microvilli. Undifferentiated fetal cells were prepared in a similar manner from 18-day fetal rat lung maintained in explant culture for 24 h. These cells did not contain intracellular osmiophilic granules; the appearance of these granules could, however, be induced by hormones. For this reason they are considered to be pre-type II cells. The viability of the cultured cells was 97%. Both the differentiated and undifferentiated fetal type II cells specifically bound the Maclura pomifera lectin, a type II cell surface marker. The phospholipid profile of the fetal cells was similar to that of adult rat type II cells; the differentiated fetal cells, however, synthesized less phosphatidylcholine than the adult cells did, but more than the undifferentiated fetal cells. The differentiated fetal cells secreted phosphatidylcholine at a basal rate of 0.6% +/- 0.1% during a 90-min incubation. There was dose-dependent stimulation of phosphatidylcholine secretion after exposure to terbutaline. Maximum stimulation (76%) was observed at a concentration of 10 microM. This culture system provides a valuable model for studies of the maturation of the undifferentiated fetal type II cell and surfactant metabolism and secretion in the differentiated fetal type II cell.     

Changes in cells's secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus; type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear "lucent" after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded "lucent" fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the "lucency" of the fibrils indicated that they were composed of collagen I, whereas the "lucency" of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.     

Ultrastructure of the nonhuman primate vaginal mucosa: epithelial changes during the menstrual cycle and pregnancy

Ultrastructural changes in the vaginal epithelium of the rhesus monkey during the menstrual cycle and pregnancy were studied by scanning and transmission electron microscopy. During the menstrual cycle, the epithelium was keratinized but varied in thickness. Cells of the basal and parabasal layers were polyhedral in shape but as they differentiated they accumulated glycogen and filaments. Cells in the intermediate layers had keratohyaline and membrane-coating granules. Cells in the superficial layers had a thickened cell envelope, abundant keratin filaments, electron-dense intercellular material, and focal tight junctions. The epithelial surface had numerous microridges and numerous adherent bacteria; bacteria were rare on desquamating cells. The epithelium remained keratinized for about the first month of gestation, then underwent "mucification." The cells contained abundant granules and Golgi apparatus. Concomitant with this transformation, bacteria were no longer adherent to the epithelial surface and the surface cells had microvilli instead of microridges. The epithelial changes during pregnancy were roughly associated with the changing pattern of steroid hormone secretion during gestation.     

Ultrastructure of the epidermis of the lizard (Lacerta vivipara) at the resting stage of the sloughing cycle

The ultrastructure of the epidermis of the lizard ( Lacerta vivipara ) one day after sloughing is described. The non-keratinized layers of the epidermis are essentially similar in structure to those of amphibians and mammals. The cells of the basal layer are not however separated from each other by the large spaces described in the amphibian (Farquhar & Palade, 1965). The middle layers of the epidermis at this stage of the sloughing cycle produce neither the characteristic mucous granules found in amphibians nor the keratohyalin granules of mammals. A small number of granules corresponding in size and location to the "Odland bodies" of both mammalian and amphibian epidermis are, however, present. The intermediate layer cells also contain a number of bodies similar in appearance to those described by Farquhar & Palade as lysosomes in amphibian skin. These structures are both osmium iodide and acid phosphatase positive. Unlike the condition in amphibians and mammals, the cytoplasm of cells in the layer immediately beneath the keratinized strata is honeycombed with small vesicles, and contains large irregular vacuoles of uncertain content. Certain nonkeratinizing elements within the epidermis are tentatively interpreted as nerve terminations. Two morphologically distinct keratinized strata can be distinguished, the inner stratum consisting of flattened cells similar to those of the stratum corneum of mammalian epidermis; individual cell outlines cannot be distinguished in the outer stratum, which has a structure similar to that of avian feather keratin. A shallow surface zone of the outer keratinized stratum has been identified as the Oberhautchen. This consists of longitudinally disposed leaflets or laminae which are responsible for the sculptured pattern of the epidermal surface. The observations reported here provide a basis for analysis of changes occurring at other stages of the sloughing cycle.     

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins

Summary A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture Unlike what is commonly observed on glass or plastic where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state. Supported by grants from the Italian Assciation for Cancer Research (AIRC).     

An air-liquid interface promotes the differentiation of gastric surface mucous cells (GSM06) in culture

The gastric surface epithelium is situated at an air-liquid interface because the luminal surface of the alimentary tract is in continuity with the air phase. However, the effects of this microenvironment on the gastric epithelium remain unclear. The aim of this study was to clarify the effects of an air-liquid interface on gastric epithelial cell biology. Gastric surface mucous cells (GSM06) were cultured at an air-liquid interface. Cultured cells were examined by histology, histochemistry, and transmission electron microscopy. When the cells were cultured at an air-liquid interface, the surface cells on the collagen gel became tall columnar and secreted periodic acid-Shiff-positive substances at the apical surface. These cells indicated many mucous granules in the apical cytoplasm and organized the basal lamina at the contact side with the gel. In contrast, under immersed condition, the surface cells showed immature features. This is the first report of an air-liquid interface promoting the differentiation of gastric surface mucous cells in a reconstruction culture of the gastric surface epithelial layer, suggesting that an air-liquid interface may function as a crucial luminal factor to maintain the homeostasis of gastric mucosa.