The cytoskeletal organization of epithelial cells in culture

Cytoskeleton organization of cultured normal epithelial cells (epithelium of newborn mouse kidney, mouse and rat hepatocytes) was studied using electron microscopy of platinum replicas. These cells in culture were firmly connected with each other and formed multicellular islands. Pseudopodial activity was observed only at the free edges of marginal cells of the islands. Cytoskeleton in the vicinity of such active edges included several structurally different zones. The most peripheral zone contained dense actin meshwork. More inner "sparse" zone contained loose actin filament network. Next zone in the same direction was the lamella proper. It contained individual microfilaments and their bundles or meshwork patches. Microtubules and intermediate filaments were also present in the lamella proper. The characteristic feature of the central (endoplasmic) region of the marginal cells of the islands was the presence of the submembranous microfilament sheath. Microfilaments in the sheath were densely packed. Individual fibers were visible along a significant distance. The inner cells in the epithelial islands had no zonal organization of the cytoskeleton. The endoplasmic microfilament sheath occupied the whole dorsal cell surface in these cells. Different epithelia studied here had some variations in the relative width of cytoskeletal zones. The organization of cytoskeleton in the epithelial cells has many features in common with that in fibroblasts. Possible mechanisms of establishment of the zonal cytoskeletal organization in both the cell types are discussed.     

Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes

Summary In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20° C in 5% CO₂:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 μM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.     

Collagen synthesis and deposition during mammary epithelial cell spreading on collagen gels

Mouse mammary epithelial cultivated on collagen gels demonstrate active spreading as the cells form monolayers. In this novel system, initiation of cell spreading is preceded by de novo synthesis of type IV collagen. The newly synthesized collagen is partitioned such that after 48 hr, approximately 24% is found in the culture medium, 35% is intracellular, and 41% is deposited in the extracellular matrix of the developing epithelium. Cultures deprived of serum failed to spread and to synthesize collagen. Proline analogues were shown to inhibit cell spreading and to suppress collagen synthesis in a dose-dependent manner. Cytochalasin D inhibition of F-actin elongation was shown to prevent cell spreading but not to suppress total collagen synthesis. During cytochalasin D treatment, inhibition of cell spreading was shown to result from failure to deposit or to maintain deposited collagen in the epithelium extracellular matrix. The data indicate that synthesis and extracellular deposition of a major basal lamina component (viz. type IV collagen) must precede and then accompany epithelial cell spreading in collagen gel culture. It is suggested that the microfilament apparatus, through some hypothetical integral membrane protein, can anchor extracellular type IV collagen, which then provides a necessary condition for cell spreading.     

Demonstration of contractility of circumferential actin bundles and its morphogenetic significance in pigmented epithelium in vitro and in vivo

Each pigmented epithelial cell bears circumferential actin bundles at its apical level when the pigmented epithelium is established in eyes in situ or in culture in vitro. Well-differentiated pigmented epithelia in culture were treated with a 50% glycerol solution containing 0.1 M KCl, 5 mM EDTA, and 10 mM sodium phosphate buffer, pH 7.2, for 24 h or more at 4 degrees C. When the glycerinated epithelium was transferred to the ATP solution, each cell constituting the epithelium began to contract. The epithelium was cleaved into many cell groups as a result of contraction of each cell. The periphery of each cell group was lifted to form a cup or vesicle and eventually detached from the substratum. However, those cells that had not adhered tightly and not formed a monolayer epithelium with typical polygonal cellular pattern contracted independently as observed in the glycerinated fibroblasts. Contraction of the glycerinated pigmented epithelial cells was inhibited by N-ethylmaleimide but not by cytochalasin B. ITP and UTP also effected the contraction of the glycerinated cells, but GTP and ADP did not. Ca2+ was not required. This contractile model of pigmented epithelium provides a useful experimental system for analyzing the function of actin in cellular morphogenesis.     

大珠母贝外套膜表皮细胞的超微结构

利用透射电系统地观察大珠母贝的外套有皮细胞，结果表明，细胞可分为５种，即柱状表皮细胞、凸细胞、电子透明大粒细胞、电子稠密粒细胞和电子透明小粒细胞。它们在不同区域的分布、形态和数量变化与外套膜的功能分化密切相关，尤其是与贝壳组分的分泌有关。结缔组织中也分布着许多闰细胞和电子稠密粒细胞，它们可作变形运动进入表皮层。     

Scanning Electron Microscopy of Cell-Surface Changes in Methylnitrosurea (MNU)-Treated Rat Bladders in vivo and in vitro

Scanning electron microscopy has been used (1) to characterize epithelial cells of bladders from normal rats and from rats treated with a single initiating but non-carcinogenic dose of 2 mg methylnitrosurea (MNU), 24 h and 6 weeks after treatment; and (2) to compare morphological aspects of epithelial differentiation in organ culture of bladder explants taken from untreated and MNU-treated rats at these time intervals.
There are marked differences in vivo between the surface organization of normal urothelium and urothelium undergoing reversible hyperplasia following MNU treatment. Maturation of the normal rat bladder epithelium in vivo is shown to be related to a series of well-defined cell-surface changes readily identified by SEM. By contrast the maturation response is perturbed in the hyperplastic epithelium; the cells lose their ability to differentiate normally and form instead an excess of stubby globular microvilli which project from the cell surface.
In organ culture, maturation of normal bladder epithelium (both in re-epithelialized areas of the explant and in areas of epithelial outgrowth over cellulose acetate substrates) can be also related to a series of cell surface changes showing close similarities to those in vivo. However, epithelial maturation remains defective in organ cultures of bladders from MNU-treated animals. The closely parallel behaviour of the bladder epithelium in vivo and in vitro in both normal and treated tissues underlines the potential value of the bladder organ culture system for studying the comparative biology of hyperplastic development produced by a single initiating dose of MNU and suggests it will be useful with which to study carcinogenesis following multiple doses of MNU.     

Mathematical Modeling of the Impact of Actin and Keratin Filaments on?Keratinocyte Cell Spreading

Keratin intermediate filaments (IFs) form cross-linked arrays to fulfill their structural support function in epithelial cells and tissues subjected to external stress. How the cross-linking of keratin IFs impacts the morphology and differentiation of keratinocytes in the epidermis and related surface epithelia remains an open question. Experimental measurements have established that keratinocyte spreading area is inversely correlated to the extent of keratin IF bundling in two-dimensional culture. In an effort to quantitatively explain this relationship, we developed a mathematical model in which isotropic cell spreading is considered as a first approximation. Relevant physical properties such as actin protrusion, adhesion events, and the corresponding response of lamellum formation at the cell periphery are included in this model. Through optimization with experimental data that relate time-dependent changes in keratinocyte surface area during spreading, our simulation results confirm the notion that the organization and mechanical properties of cross-linked keratin filaments affect cell spreading; in addition, our results provide details of the kinetics of this effect. These in silico findings provide further support for the notion that differentiation-related changes in the density and intracellular organization of keratin IFs affect tissue architecture in epidermis and related stratified epithelia.     

Differentiation and growth potential of human ovarian surface epithelial cells expressing temperature-sensitive SV40 T antigen

The epithelial ovarian carcinomas arise in the ovarian surface epithelium (OSE) which is the mesothelial covering of the ovary. Studies of human USE have been hampered by the small amounts and limited lifespan of this epithelium in culture. OSE cells expressing SV40 large T antigen (Tag) or the HPV genes E6 and E7 have increased growth potentials but lack some of the normal characteristics of OSE. In this study, we used conditional SV40 Tag expression to produce OSE cells with increased proliferative potentials but relatively normal phenotypes. Primary OSE cultures from three women, one of whom had a BRCA1 mutation, were infected with a temperature-sensitive Tag construct (tsTag), and from these, 28 monoclonal and four polyclonal lines were isolated. The effects of temperature changes were examined in two monoclonal and two polyclonal lines. At the permissive temperature (34 degrees C), these cell lines underwent 52-71 population doublings (PD) compared to 15-20 PD for normal OSE. Nuclear SV40-Tag and p53 expression, demonstrated by immunofluorescence, showed that tsTag was uniformly present and biologically active in all lines. At 34 degrees C, culture morphologies ranged from epithelial to mesenchymal. The mean percentage of cells expressing the epithelial differentiation marker, keratin. varied between lines from 20 to 97%. Collagen type III, a mesenchymal marker expressed by OSE in response to explantation into culture, was present in 24-43% of cells. At 39 degrees C, tsTag was inactivated by 2 d while nuclear p53 staining diminished to control levels over 2 wk. Over 3 d. the cells assumed more epithelial morphologies, keratin expression reached 85-100% in all lines and collagen expression increased significantly in two lines. The cultures with the BRCA1 mutation expressed the most keratin and the least collage n III at both temperatures. As indicated by beta-galactosidase staining at pH 6.0, changes leading to senescence were initiated at 39 degrees C by 6 h and were present in all cells after 24 h. However, the cells underwent 1-3 population doublings over up to 1 wk before growth arrest and widespread cell death, thus providing an experimental system where large numbers of OSE cells with different genetic backgrounds and growth potentials can be studied without the concurrent influence of Tag.     

Regeneration of the Cornea in Adult Newts: Overall Process and Behavior of Epithelial Cells

Regeneration of the cornea in adult newts was studied by means of light- and electron-microscopic techniques. We focused our analysis particularly on the behavior of epithelial cells during the initial process of wound healing after we had excised a central disk about 0.5 mm in diameter through the entire thickness of the cornea. Fine fibrous material, assumed to be fibrin, appeared within 30 min to form an acellular layer of mucous consistency which sealed the wound opening completely. The cut edge of corneal epithelium moved centripetally on this layer by coordinate movement of individual epithelial cells. Almost all cells of the remained epithelium were completely rearranged within 5 h after excision. Some desmosomes among the epithelial cells persisted during the process of cellular rearrangement. Thus, the wound opening was covered completely within 24 h by the epithelium alone without cell proliferation. Cytochalasin B or D completely inhibited movement of the corneal epithelium on the stroma in conditions in vitro, suggesting active participation of intracellular contractile microfilaments in such movement of the epithelium. Active growth of cells in the epithelium started on day 3 and the epithelium recovered its normal thickness by day 10 after excision.
After the recovery of the epithelium, keratocytes moved out from the wounded edge of the remained corneal stroma. These keratocytes actively proliferated in the wound area under the newly formed epithelium and participated in the stromal reconstitution, which proceeded gradually for more than 5 weeks.     

Virus replication in infected epithelial cells is coupled to cell shape-responsive metabolic controls

The cell shape of monkey epithelial cells was varied from flat to spheroidal by gradually reducing the substrate adhesiveness with poly (HEMA) films of increasing thickness. The decrease in cell spreading is accompanied by a dramatic response in cellular macromolecular metabolism in the nucleus. Within 14 to 16 hr, DNA and RNA syntheses are inhibited by more than 95%, while the level of protein synthesis is reduced by only twofold after 24 hr in spheroidal-suspension culture. When epithelial cells, spread to various degrees, are infected with SV40 or herpes virus a parallel inhibition of virus replication and cellular macromolecular metabolism occurs. However, VSV can proliferate in the metabolically active cytoplasm of epithelial cells in which nuclear activity is inhibited owing to alterations in cell shape. The results suggest that the metabolic restrictions imposed on epithelial cells, owing to changes in cell spreading, are a dominant phenomenon that cannot be overcome by virus infection. Rather, virus replication, which is dependent on the cellular metabolic machinery, is inhibited in parallel with the inhibition of cellular macromolecular metabolism.     

Effects of corticosteroid-induced apoptosis on airway epithelial wound closure in vitro

Damage to the airway epithelium is common in asthma. Corticosteroids induce apoptosis in and suppress proliferation of airway epithelial cells in culture. Whether apoptosis contributes to impaired epithelial cell repair after injury is not known. We examined whether corticosteroids would impair epithelial cell migration in an in vitro model of wound closure. Wounds (approximately 0.5-1.3 mm2) were created in cultured 1HAEo- human airway epithelial cell monolayers, after which cells were treated with up to 10 microM dexamethasone or budesonide for 24 h. Cultured cells were pretreated for 24 or 48 h with dexamethasone to observe the effect of long-term exposure on wound closure. After 12 h, the remaining wound area in monolayers pretreated for 48 h with 10 microM dexamethasone was 43+/-18% vs. 10+/-8% for untreated control monolayers. The addition of either corticosteroid immediately after injury did not slow closure significantly. After 12 h the remaining wound area in monolayers treated with 10 microM budesonide was 39+/-4% vs. 43+/-3% for untreated control monolayers. The proportion of apoptotic epithelial cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end labeling both at and away from the wound edge was higher in monolayers treated with budesonide compared with controls. However, wound closure in the apoptosis-resistant 1HAEo-.Bcl-2+ cell line was not different after dexamethasone treatment. We demonstrate that corticosteroid treatment before mechanical wounding impairs airway epithelial cell migration. The addition of corticosteroids after injury does not slow migration, despite their ability to induce apoptosis in these cells.     

Aspects of cell proliferation kinetics of the inner dental epithelium during mouse molar and incisor morphogenesis: a reappraisal of the role of the enamel knot area.

First lower E-14 and E-16 mouse molars and E-13 lower incisors were cultured in vitro and either sequentially or continuously labelled with BrdU (5-bromo-2'-deoxyuridine). The behaviour of the non-cycling inner dental epithelial cells emerging from the enamel knot area of the molars was analysed by 3D (three dimensional) reconstructions of serial sections. These cells, as well as slow cycling cells underwent a coordinated temporo-spatial patterning leading to their patchy segregation at the tips of the forming cusps. In incisors (in vitro and in vivo), non-cycling cells were also present in the inner dental epithelium of the enamel knot area. However, these cells were not redistributed during incisor morphogenesis. These non-dividing inner dental epithelium cells of the enamel knot area which are either redistributed or not according to the tooth type specific morphogenesis might represent the organizers of morphogenetic units (OMU), the cusps.     

Expression of vimentin by rabbit corneal epithelial cells during wound repair

Summary Intermediate filaments of epithelial cells generally consist of specific combinations of keratins. However, cultured epithelial cells from certain tissues and some epithelial tumors have been shown also to express vimentin. In the present study, the expression of vimentin by epithelial cells in healing corneal wounds (partial thickness penetrating wounds) and in tissue culture was analyzed. Both immunohistochemical and immunotransblot analyses indicated that although vimentin was not detected in the normal rabbit corneal epithelium in vivo, cultured rabbit corneal epithelial cells co-express keratins and vimentin. At 1 day post-wounding, vimentin was not detectable in the epithelial cells that had covered the denuded stroma. However, at 2 days post-wounding, the epithelium at the base of the epithelial plug immunoreacted with both anti-vimentin and antikeratin monoclonal antibodies. Immunotransblot analyses of the extracts of the epithelial plugs confirmed the presence of vimentin (M_r=58k). The 58k band was not detected in the extract of normal rabbit corneal epithelium. At day/5, vimentin was no longer detectable in the epithelium. This study demonstrated that corneal epithelial cells transiently co-express vimentin and keratins in vivo during wound healing and in tissue culture. The time-course of the transient expression of vimentin suggests that the vimentin expression in the epithelial cells during healing is not linked to cell proliferation or to the centripetal migration of the epithelium during early stages (first 24 h) of healing, but may be linked to cell-matrix interactions or the migration of basal cells in the upward direction at the following stage of healing.     

Aspergillus fumigatus causes in vitro electrophysiological and morphological modifications in human nasal epithelial cells

The role of the airway epithelium in the development of invasive aspergillosis in immunocompromised hosts has rarely been studied although patients at risk for this infection frequently have epithelial damage. We developed an in vitro model of primary culture of human nasal epithelial cells (HNEC) in air-liquid interface, which allows epithelial cell differentiation and mimics in vivo airway epithelium. We subsequently tested 7-day and 24-hour Aspergillus fumigatus filtrates on the apical side of HNEC to know whether A. fumigatus, the main species responsible for invasive aspergillosis, produces specific damage to the epithelial cells. The results were compared with those obtained with non-pathogenic filamentous fungi. Seven-day culture filtrates of A. fumigatus and Penicillium chrysogenum induced electrophysiological modifications whatever the fungus tested. In contrast, only 24-hour A. fumigatus filtrates induced a specific decrease in transepithelial resistance, hyperpolarization of the epithelium, and cytoplasmic vacuolization of HNEC compared with both A. niger and Penicillium chrysogenum. The inhibition of the A. fumigatus effects with amiloride suggests that the 24-hour fungal filtrate acts through sodium channels of HNEC. These early modifications of the epithelial cells could facilitate colonization of the airways by A. fumigatus. To know whether the molecules involved are specific to A. fumigatus or simply produced more rapidly than by other filamentous fungi warrants further investigation. In this perspective, the primary culture of HNEC represents a suitable model to study the interactions between airway epithelial cells and A. fumigatus.     

Spreading of porcine kidney epithelial cells under normal condition and under the effect of inhibitors of energy metabolism. I. Dynamics of spreading

In the present work dynamics of cell spreading on a solid substratum has been investigated in normal conditions and under ATP-synthesis inhibitors. Sodium aside (20 mM), which blocks ATP-synthesis in mitochondria, and N,N-dicyclohexylcarbodiimide (DCCD) (100 mM), which blocks both ATP-synthesis and glycolysis, were ATP-synthesis inhibitors of choice. In the range from the moment of cell plating to 24 h three stages of cell spreading could be distinguished according to the dynamical change of the average projected cell area. At the first stage, within 1.5 h in the control culture the cell area increases rapidly. The slowing of spreading occurs at the second stage, within 1.5-4.0 h. The third stage is characterized by a slow but pronounced increase in cell spreading, which ceases in 24 h. Under inhibitory treatment, the pattern of cell spreading during the first 4 h is essentially the same as in control conditions. The subsequent DCCD action results in cell spreading inhibition; sodium aside, on the contrary, accelerates the spreading. The cell shape analysis has demonstrated that even as early as in 0.5 h the first small polarized cells appear simultaneously with the nonpolarized cells. In control cells, the share of polarized cells increases in almost 2 h, conversely, under drug actions the process of polarization begins earlier to be more pronounced in the presence of sodium aside. Thus, it has been shown that the spreading of PK cells does not require any additional ATP-synthesis. At early stages in normal conditions and under inhibitory treatment the picture of cell spreading is the same. A complete inhibition of ATP-synthesis slow down the process of cell spreading. However, an activation of these processes was observed in cells with low content of ATP, resulting from glycolysis retaining.     

Association of mesenchymal cells and immunoglobulins with differentiating epithelial cells

Background

Mesenchymal-epithelial interactions play an important role in the physiology and pathology of epithelial tissues. Mesenchymal cells either associate with epithelium basement membrane [pericytes and perivascular monocyte-derived cells (MDC)] or reside within epithelium (MDC and T cells). Although intraepithelial mesenchymal cells were suggested to contribute to the epithelium physiology, their association with particular steps in differentiation of epithelial cells, interactions among themselves, and their fate remain unclear. We studied epitopes of mesenchymal cells and their products (immunoglobulins) in stratified epithelium of uterine ectocervix, which is one of the prototypes of complete cellular differentiation from stem into the aged cells.

Results

Perivascular CD14 primitive MDC associated with basal (stem) epithelial cells. Thy-1 pericytes of microvasculature secreted intercellular vesicles, which associated with Ki67 postmitotic epithelial cells expressing MHC class I. Intraepithelial T cells showed an association with veiled type MDC [dendritic cell (DC) precursors] among parabasal cells, and exhibited fragmentation after entering intermediate (mature) epithelial layers. Mature DC secreted CD68 and exhibited fragmentation after reaching mid intermediate layers. Binding of IgM was detected at the top of each layer: in the upper parabasal, upper intermediate, and most surface epithelial cells. IgG was confined to the entire superficial layer.

Conclusions

These data suggest that the phylogenetically and ontogenetically developed hierarchy of mesenchymal cells (MDC, pericytes, T cells) and immunoglobulins (IgM, IgG) accompanies differentiation of epithelial cells from immature into the mature and aged phenotype. Further studies of an involvement of mesenchymal cells in the regulation of tissue homeostasis may bring novel approaches to the prevention and therapy of tissue dysfunctions characterized by permanent tissue immaturity (muscular dystrophy) or accelerated aging (degenerative diseases).     

Variations in shape-sensitive restriction points mirror differences in the regeneration capacities of avian and mammalian ears

When inner ear hair cells die, humans and other mammals experience permanent hearing and balance deficits, but non-mammalian vertebrates quickly recover these senses after epithelial supporting cells give rise to replacement hair cells. A postnatal decline in cellular plasticity appears to limit regeneration in mammalian balance organs, where declining proliferation responses are correlated with decreased spreading of supporting cells on artificial and native substrates. By culturing balance epithelia on substrates that differed in flexibility, we assessed spreading effects independent of age, showing a strong correlation between shape change and supporting cell proliferation. Then we made excision wounds in utricles cultured from young and old chickens and mice and compared quantified levels of spreading and proliferation. In utricles from young mice, and both young and old chickens, wounds re-epithelialized in <24 hours, while those in utricles from mature mice took three times longer. More cells changed shape in the fastest healing wounds, which accounted for some differences in the levels of proliferation, but inter-species and age-related differences in shape-sensitive restriction points, i.e., the cellular thresholds for shape changes that promote S-phase, were evident and may be particularly influential in the responses to hair cell losses in vivo.     

The fine structure of the planarianPolycelis tenuis ijima

Summary The fine structure of the pharynx is presented and demonstrates that the pharyngeal epithelial system is a continuous one. The epithelial lining of the pharyngeal cavity with its characteristic fibrous secretory bodies merges with the outer pharyngeal epithelium at the point of anchorage of the pharynx. A few of these cells are insunk, the nuclei occurring beneath the underlying muscular layers. The nature of the outer epithelium changes towards the free end of the pharynx; the cells become ciliated and in contents come to resemble the inner epithelium which it joins at the tip.The gut cells merge at a transitional zone with the inner pharyngeal epithelium and at this point both bear microvilli and contain rod-shaped apical bodies. Some of these cells are also insunk. Towards the mouth the epithelium shows a greater degree of insinking and exhibits microapocrine secretion. Both inner and outer epithelia bear sense receptors which are concentrated at the lip.At the point of pharyngeal insertion, the sub-epithelial tissue resembles planarian parenchyma, but is rich in gland cells. These glands open on to the outer epithelium especially towards the free end of the pharynx.This research was supported by the Scientific Research Council. Grant No. B/RG/086.     

The influence of extracellular matrix components on the proliferation and migration of inner cell mass-derived parietal endodermal cells.

Reichert's membrane is a basement membrane deposited on the inner surfaces of rat and mouse trophectodermal (TE) cells beginning at the blastocyst stage of embryonic development that may play a role in the migration of the parietal endodermal (PE) cells to form an inner lining to the TE. The abilities of various glycoproteins present in Reichert's membrane to support PE cell migration and replication in vitro were examined by isolating inner cell masses (ICMs) from Day 5 rat blastocysts (Day 1 = day of vaginal plug) and culturing them (24-72 h) either on surfaces that had been precoated with collagen IV, fibronectin, or laminin or on thin (1-2-mm) gels of Matrigel (a tumor cell-derived basement membrane preparation) or type I collagen. Time-dependent changes in the area occupied by each ICM on the culture surface and the number of migrating cells per ICM were quantified by morphometric analysis. Type IV collagen, the basement membrane-specific collagen, supported ICM attachment and the outward migration (overall increase of approx. 60-fold in mean ICM area occupied on the culture surface) and proliferation (cell doublings following every 24 h of culture) of laminin-containing PE-like cells. These effects were not altered by the inclusion of exogenous fibronectin or laminin in the culture medium. Collagen IV coating concentrations as low as 0.16 micrograms/ml supported PE cell attachment and migration, and maximal responses were seen with a coating concentration of 0.63 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and culture of bovine oviductal epithelial cells for use in the anatomy and physiology laboratory and undergraduate research

This article presents methods for the isolation and culture of epithelial cells from the bovine oviduct for use in both research and the teaching laboratory and provides examples of ways that an oviductal cell culture can be incorporated into an undergraduate research program. Cow reproductive tracts are readily available from area butchers, and the procedure for isolation of the epithelium is simple and inexpensive. The cells can be observed immediately after isolation or can be cultured for up to 72 h under simple conditions for observation over several days. For experimental use, epithelial cells are cultured in standard cell culture medium, where they continue to divide and actively secrete substances into the medium. The ease with which the tissue can be collected and cells isolated makes the oviductal epithelium ideal for use in both the teaching laboratory and research projects in which undergraduates serve as investigators.