Reconstitution of the malignant phenotype of genitourinary cancer in gradient culture

Summary The diagnosis of cancer is made on histologic examination by recognizing the characteristics of the malignant phenotype, i.e. abnormal cells in abnormal groupings, often in abnormal locations. Histophysiologic gradient culture reconstitutes conditions that meet the spatial imperatives of tissues in nature. A variety of carcinomas arise in the genitourinary system involving both glandular and stratified epithelium. To be considered here are the contrasting polarizations of proliferation of normal and neoplastic rat urothelium, the continuity of sheets of epithelium in nature, the poorly understood stable and unstable interepithelial boundaries, and the formation of organoid endocrinelike tissue in histophysiologic gradient culture of normal human amnion epithelium. This work was supported by research grant CA-14137 from the National Cancer Institute, Bethesda, MD, grant 1793 from the Council for Tobacco Research, and grants from the American Fund for Alternatives to Animal Research.     

Structural biology of epithelial tissue in histophysiologic gradient culture

Summary Epithelial cells proliferate, forming organized tissues, when positioned in the lumen of a thin-walled, transparent, elongated cylindrical, cystlike culture chamber. The closed chamber, 2.5 mm in diameter and 25 mm long, bathed in medium, incubated with continuous gentle agitation, enables the inoculum to exchange metabolites including oxygen by diffusion across the thin, nylon filament-reinforced collagen membrane wall of the chamber. After periods of culture of a week or more, using inocula derived from urothelium, the inner surface of the cystic chamber is lined by a stratified epithelium. Proliferation of cells is seen in the basal zone, which is attached to the collagen substrate. The development of the model is briefly described. Some of the applications of the procedure are illustrated using cell lines, chick embryo tissues, and clinical tissues. Implications of the procedure are considered for studying categories of tissue biology, e.g., problems of aging, neoplasia, and toxicology.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Summary Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continuous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing or luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasinal myoepithelial cells, characterized by myofilaments and plasmalemmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over T₀ values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes. The work was supported by USPHS Grants CA-05388 and CA-05045 from the National Cancer Institute, DHEW.     

Exploring processes of organization of normal and neoplastic epithelial tissues in gradient culture

The biology of animal cells is often studied in individual cells or in sheets of cells. The relevance of such studies to the intact animal is unclear, since the spatial conditions encountered by cells in animals is one of dense three-dimensional masses of cells, with limits to migration, and with gradients both of diffusion of metabolites and morphologic maturation. These spatial requisites have gradually been met in culture. A brief account describes sponge matrix culture for three-dimensional growth and unilaminar, bilaminar, and radial histophysiologic gradient cultures. Some of the common neoplastic abnormalities of surface epithelial issues are considered. Proposals for investigating the histokinetic mechanisms regulating some epithelial tissue processes are suggested. In the most recent development of gradient culture methods, a thin transparent collagen membrane is intrinsically strengthened by producing a waffle membrane pattern for histophysiologic gradient culture.     

Factors affecting the differentiation of epithelial transport and responsiveness to hormones

Under standard culture conditions, epithelial cells grow with their basal surface attached to the culture dish and their apical surface facing the medium. Morphological and functional markers are located in the appropriate plasma membrane, and transepithelial transport occurs in a variety of cultured epithelia. As a result of the polarity of the cells and the presence of tight junctions between cells, on standard tissue culture dishes there is restricted access of growth medium to the basolateral surface of the epithelium, which is the surface at which nutrient exchange normally occurs. Greater differentiation of epithelial cultures can be achieved by growing primary cultures or continuous cell lines on permeable surfaces such as porous bottom cultures dishes in which the porous bottom is formed by a filter or membrane of collagen, or on floating collagen gels. In many cultures, differentiation varies with the time after the culture was seeded. Certain chemicals that accelerate differentiation in nonepithelial cells also accelerate the differentiation of epithelial cultures. Ultimately, defined media and specific substrates for cell attachment should lead to further differentiation of epithelia in culture.     

Effects of retinoic acid on the growth and morphology of hamster tracheal epithelial cells in primary culture

Hamster tracheal epithelial cells were grown in primary culture on a collagen gel substrate in hormone-supplemented serum-free Ham's F12 medium with 10(-8) M retinoic acid (RA+), or without retinoic acid (RA-). On days 1 and 2, the colonies were composed of large (secretory) cells and lesser numbers of small (basal) cells; ciliated cells were rare. At these times, cell number, thymidine incorporation, and total labelling indices (small and large cells, combined) were similar in RA+ and RA- cultures, but the large cells became flat in RA- medium on day 2. On days 3-5, thymidine incorporation and total labelling indices were less in RA- than RA+ cultures, and on days 4-6, cell numbers were decreased in RA- cultures. On day 3, the large cells of the RA- colonies had flattened further and clusters of small basal cells had formed. On day 4, the RA+ colonies were composed of densely-packed cuboidal secretory cells, small basal cells were inconspicuous; the total labelling index was about 27%. The RA- colonies were composed of large flat secretory cells and numerous small basal cells which were clustered in groups; the total labelling index was about 7%. Since large and small cells could be discriminated by size in RA- colonies, a labelling index was generated based on cell size. On days 2, 3 and 4, the labelling index of the small basal cells in the RA- colonies was 44%, 43% and 24% respectively, whereas the labelling index of the large secretory cells fell rapidly over the same period (56%, 14% and 2%). On days 5 and 6, the cuboidal secretory cells in the RA+ cultures had differentiated further and the cells were stratified focally. Some new ciliated cells had formed on day 6. In RA- cultures, mucous granules were not observed in the large flat cells and ciliated cells were not seen. The total labelling indices were 11% and 0.35% in RA+ cultures, and 0.5% and 0.25% in RA- cultures on days 5 and 6, respectively. The study shows that the target cell for vitamin A in the hamster tracheal epithelium is the secretory (mucous) cell. When retinoic acid was deficient, the secretory cells flattened and their capacity to divide was greatly diminished. Since the basal cells continued to replicate when the secretory cells did not, the population density of the basal cells increased disproportionally, which could be interpreted erroneously as a "basal cell hyperplasia".(ABSTRACT TRUNCATED AT 400 WORDS)     

Radial histophysiologic gradient culture chamber: Rationale and preparation

Summary Histophysiologic gradient culture methods reconstitute important spatial relationships that occur in nature between a parenchyma and its supporting stroma. At the epithelial-stromal interface, epithelia are firmly attached to the stromal substrate, initiation of renewal takes place, and metabolites are exchanged by a process of diffusion between epithelium and substrate. Other spatial imperatives characteristic of stratified epithelium are high density of cells, gradients of maturation, and continuity of epithelia along the entire course of the stromal-parenchymal interface. In radial gradient culture these relationships of epithelial cells, and supporting substrates are reconstituted. The culture chamber consists of a thin-walled cylinder, 2 to 3 mm in diameter and 3 cm long. The wall is a transparent collagen membrane in whose substance is embedded a reinforcing nylon mesh. To prepare a culture, one end of the cylinder is ligated, 1 or 2 particulate inocula are inserted in the open end of the cylinder, guided toward the ligature, and the open end is ligated. Subsequently, during incubation in a container with medium, the explants attach and proliferate. Proliferation and migration result in the cylinder being completely lined by a complex organoid tissue with structural characteristics of the original tissue. The tissue patterns in radial gradient culture of two human cell lines, RT-4, a bladder cancer, and 87×50, and ovarian cancer, are illustrated.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing of luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasional myoepithelial cells, characterized by myofilaments and plasmalemmmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over TO values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes.     

Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences

Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an understanding of the possible roles of cell-extracellular matrix interactions and the requirements for meeting polarity needs of the epithelium. When cells are cultured on floating collagen gels they assemble a basal lamina-like structure composed of laminin, collagen (IV), and heparan sulfate proteoglycan at the interface of the cells with the stromal collagen. To assess the role of these components, an exogenous basement membrane containing these molecules was generated using the mouse endodermal cell line, PFHR-9. This matrix was isolated as a thin sheet attached to the culture dish, and mammary cells were then plated onto it. It was found that cultures on attached PFHR-9 matrices expressed slightly higher levels of beta-casein than did cells on plastic tissue culture dishes, and also accumulated a large number of fat droplets. However, the level of beta-casein was approximately fourfold lower than that in cultures on floating collagen gels. Moreover, the beta-casein made in cells on attached matrices was not secreted but was instead rapidly degraded intracellularly. If, however, the PFHR-9 matrices with attached cells were floated in the culture medium, beta-casein expression became equivalent to that in cells cultured on floating stromal collagen gels, and the casein was also secreted into the medium. The possibility that floatation of the cultures was necessary to allow access to the basolateral surface of cells was tested by culturing cells on nitrocellulose filters in Millicell (Millipore Corp., Bedford, MA) chambers. These chambers permit the monolayers to interact with the medium and its complement of hormones and growth factors through the basal cell surface. Significantly, under these conditions alpha 1-, alpha 2-, and beta-casein synthesis was equivalent to that in cells on floating gels and matrices, and, additionally, the caseins were actively secreted. Similar results were obtained independently of whether or not the filters were coated with matrices.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pre-natal innervation of the human female genital tract

In the human fetus of 14 weeks, ganglia on either sides of the Müllerian uterovaginal canal contained two types of cells. In the 16th week, axons invaded the basal zone of the stratified squamous epithelium at the sides of the upper vagina. In the 20th week, vesicular nuclei typified the large neurons in the midportion of the cervico-vaginal ganglion. During the 22nd week, capsulated ganglia invaded the wall of the upper vagina forming three concentrically disposed strata. Non-capsulated clusters invaded its lamina propria. At the 24th week, axons were shaded after reaching the superficial zone of the stratified vaginal epithelium. In the 28th week, satellites surrounded the mature neurons and sheath cells enveloped the axons. Ganglia invaded the splitted muscle layer of the upper vagina at 30 weeks. Intraepithelial fibres invaded the whole thickness of the endometrium, the columnar epithelium of the cervix and uterine tube at 40 weeks. Nerve cells were detected among the basal epithelial cells of the lower vagina and its subepithelial plexus.     

Retinoids alter the direction of differentiation in primary cultures of cutaneous keratinocytes

The effects of vitamin A on the morphological expression of differentiation were studied in cell cultures of cutaneous keratinocytes from the newborn rat. The cells were first cultivated in a medium containing 0.11 mM calcium until a confluent monolayer had been formed. Stratification and terminal differentiation were then triggered by raising the calcium concentration of the medium to 1.96 mM ('normal' culture). The rise in the concentration of calcium was coupled with the addition of retinol (RL) of retinoic acid (RAC) to the medium to produce an excess of vitamin A (high-retinoid culture). Delipidized serum was used to produce a deficiency of vitamin A (low-retinoid culture). The tissue organization and the ultrastructure of the keratinocytes in the stratified culture were the same as those seen in conventional cultures and skin explants. These stratified cultures expressed the morphological features of the epidermis of intact skin. The addition of RL or RAC to the medium enhanced features characteristic of the secretory epithelium, such as the formation of an extensive endoplasmic reticulum, an enlargement of the Golgi zone, and an increase in the number of vacuoles. At the same time, the addition of retinoids diminished features characteristic of the terminal differentiation of the stratified squamous epithelium, such as stratification and keratinization. Deficiency of vitamin A in the medium resulted in a culture with many differentiated layers. The differentiated cells of the low-retinoid cultures contained densely packed tonofilaments and synthesized products that reacted with the monoclonal antibody AE2 that is specific for keratin peptides which are markers of epidermal differentiation. In the cell culture system that is presented here, an excess of retinoids redirected epithelial differentiation from a stratifying and keratinizing epithelium towards a secretory epithelium. This system is a useful tool for elucidating the mechanisms responsible for the effect of vitamin A on the differentiation of epithelial cells.     

The presence of G1 and G2 populations in normal epithelium of rat urinary bladder

The cell population kinetics of transitional epithelium of the rat urinary bladder was analysed by (3H) thymidine autoradiography and pararosanilin Feulgen DNA cytofluorometry. By flash and 72 h continuous DNA labelling, the generative cells of the transitional epithelium were found to be well localized in the basal layer, and it was postulated that che cells produced by cell proliferation in the basal layer would migrate towards the surface, maintaining direct attachment to the basement membrane by anchorage of a cellular process. Analyses of normal and wounded transitional epithelium revealed that 58.8% of all basal cells are G0 cells in G1 phase (G1-population), and 59.0% of the remaining basal cells reside in prolonged (75.1-108.0 h) G2 phase, preserving the ability to divide (G2-population). The cell cycle time of the generative basal cells including the long G2 phase was calculated as 129.1-162 h. All the cells existing in upper layers were found to be also G0 cells in G1 phase, with the DNA amounts of 2C class. No polyploid cells could be detected except for 2C-2C binucleated cells in the superficial layer. The existence of a G2-population may serve for the urgent need of cell incrementation to repair cell loss as the cells in G2 phase can divide without the time-delay needed for DNA synthesis. The rat transitional epithelium, which is composed exclusively of proliferating and potentially proliferative cells, will have much greater capability to repair damage than stratified squamous epithelia.     

A cell line, ROSE 199, derived from normal rat ovarian surface epithelium

A cell line, ROSE 199, derived from rat ovarian surface epithelium (ROSE) formed papillary structures which resembled, histologically, serous papillary cystadenomas of borderline malignancy seen in the human ovary. Crowded cultures produced two layers of cells separated by a thick layer of collagen fibers. Such cultures shed viable cells into the growth medium, while no cells were shed by short-term ROSE cultures. The resemblance to ovarian tumors exhibited by ROSE 199 cells in culture, reinforces the hypothesis that the common epithelial tumors of the ovary are derived from the ovarian surface epithelium. ROSE 199 cells, while retaining their epithelial morphology and ultrastructural characteristics, express stromal activity such as abundant collagen production. Perhaps this ability to express epithelial and stromal behavior is a contributing factor to the ready neoplastic transformation of the ovarian surface epithelium.     

Expansion and long-term culture of differentiated normal rat urothelial cells in vitro

The objective of this study is to establish a reliable cell culture system for the long-term culture of rat urothelial cells (RUC), in which the cells multiply in vitro and form stratified polarized urothelium. Urothelial cells were harvested by the enzymatic digestion of the urothelium exposed by the eversion of resected rat bladders. Primary cultures were initiated in keratinocyte serum-free medium (KSFM) for selective proliferation of urothelial cells. Subsequently, the cells were propagated in a mixture of conditioned medium (CM) derived from Swiss 3T3 cell culture supernatant and KSFM (CM-KSFM). Mean population doubling time was 13.8 +/- 0.9 h. RUC were successfully maintained for 18 passages over a period of 4-5 mo. Detailed investigations of culture conditions showed that CM-KSFM yielded a differentiated multilayer structure. The stratified urothelial sheets measuring 4 x 6 cm2 could be formed and then detached using dispase. Cytokeratin pattern in both the cultured urothelial monolayer and engineered stratified layers was similar to those seen in vivo, as assessed with monoclonal antibody against cytokeratin 17. Ultrastructural morphology showed microvilli, basal cell layer, and desmosomes between adjacent cells in the stratified urothelium.     

Autoradiographic analysis of hormone-independent development of the mouse vaginal epithelium in organ culture

Summary Epithelial development was studied in organ cultures of vaginas from ovariectomized mice. In defined medium without estrogen, basal cell proliferation and formation of a stratified squamous epithelium occurred spontaneously in a high percentage of cases. During this “spontaneous” development, the length of the cell cycle and duration of DNA synthesis were the same as those occurring in vivo in response to estrogen. RNA synthesis was also stimulated. Thus, spontaneous development in vitro appears to resemble estrogen-induced development in vivo both qualitatively and quantitatively. In vitro, estrogen was found to have no detectable effect on the atrophic epithelium. In addition, estrogen was unable to halt degeneration of the fully developed epithelium on explants that had been estrogen-primed in vivo. The results suggest that both hormone-dependence and ability of hormones to induce a response are dependent on cell environmental factors. Supported by Grant 1 PO 1 CA 11536 from the National Cancer Institute, and Grant 1 PO AM 15515 from the National Institute of Arthritis and Metabolic Diseases.     

Basal lamina glycoproteins laminin and type IV collagen are assembled into a fine-fibered matrix in cultures of a teratocarcinoma-derived endodermal cell line

Extracellular matrix glycoproteins synthesized and deposited by a mouse teratocarcinoma-derived endodermal cell line (PYS-2) in culture were analysed by metabolic labelling and immunochemical methods, and the matrix structure was studied by immunofluorescence and electron microscopy. PYS-2 cells secreted two major high-molecular weight glycoproteins, laminin and type IV collagen, which were deposited in apparently unprocessed form under the cells into a lamellar matrix composed of a loose network of fine fibrils and attached dense grains. The cells did not synthesize detectable amounts of fibronectin, but the matrix was found to bind fibronectin from the culture medium. The matrix structure was sensitive to bacterial collagenase indicating a role for type IV collagen in matrix integrity. The PYS-2 matrix which contains defined basal lamina glycoproteins provides possibilities for in vitro studies on the organization of deposited basal lamina components.     

Differentiation and maturation of cultured fetal rat jejunum.

To determine whether differentiation and maturation of mammalian intestinal mucosa require influences available only in vivo or whether they can occur in vitro, fetal rat jejunum was cultured in chemically defined medium using organ culture methodology. Segments of jejunum from 18-day fetal rats were cultured in modified Liebowitz L-15 medium in room air at 37°C. Segments harvested after 24, 48, and 72 hr of culture were examined by light and electron microscopy. Uncultured jejunum from 18-day fetuses had either no or very few rudimentary villi and was lined largely by undifferentiated stratified epithelium. Goblet cells were not seen. In contrast, villi were present in the majority of 24-hr cultures, and simple columnar rather than stratified epithelium predominated. After 48 and 72 hr, villi were present in over 90% of cultured jejunal segments and stratified epithelium had disappeared. Goblet cells were seen in jejunal segments cultured 48 hr or longer in 47 of 74 fetuses. Electron microscopy further documented progressive differentiation of the epithelium during culture. Microvilli increased in number and height, a terminal web developed in the apical cytoplasm and the number of apical vesicles, mitochondria and formed elements of endoplasmic reticulum increased in absorptive cells. Jejunal lactase and alkaline phosphatase activity increased nine- and sevenfold, respectively, during 72 hr of culture, while the activity of the mitochondrial enzyme, ornithine carbamoyl transferase, increased fourfold. These observations indicate that jejunum from 18-day fetal rats can be cultured in vitro for at least 72 hr in chemically defined medium and that, during culture, maturation of the jejunal mucosa takes place with the appearance of villi, conversion of stratified to columnar epithelium and differentiation of individual epithelial cells.     

The isolation and characterization in vitro of normal epithelial cells,endothelial cells and fibroblasts from rat urinary bladder

Epithelial cells, microvascular endothelial cells, and fibroblasts have been isolated in culture from normal urinary bladders of Fischer rats. Normal epithelial cells were cultured most efficiently when transitional epithelial sheets were plated on to collagen-coated roller flasks. The epithelial sheets were obtained by two micro-dissection techniques. In the first method, the epithelium was peeled as a large coherent sheet from the submucosal connective tissue following subepithelial injection of a collagenase solution, and after incubation of the bladders in the same enzyme solution. Epithelial sheets with intact basal cell layers were essential for culture success. On collagenous matrices, epithelial differentiation was similar to that in vivo. The in vitro transitional epithelium was composed of three cell layers, namely superficial, intermediate, and basal cells. Basal cells were attached to newly synthesized basal lamina by means of hemidesmosomes. Superficial cells were sealed at their apical lateral membranes by a junctional complex, i.e. a terminal bar. Asymmetric luminal membrane plaques were not apparent. In the second method, the epithelium was separated from the underlying connective tissue after collagenase-trypsin digestion of everted urinary bladders. Although the digest consisted mainly of epithelial cells, these rarely survived the first passage when plated on conventional plastic growth surfaces. After the third culture week, epithelial cells usually died and slowly growing colonies of fibroblasts or large flattened epitheloid cells became apparent. Epitheloid cells were identified by their typical ultrastructure as endothelial cells, showing Weibel-Palade bodies and pinocytotic caveolae. These cells were reactive with antiserum against factor VIII. The free surface of monolayer cultures was non-thrombogenic when incubated in the presence of platelets. Fibroblasts were isolated from heavily contaminated epithelial cell cultures after differential trypsinization. These three cell types represent the normal control cells of an in vitro tumor model for the study of invasiveness. All three cell types are involved in the formation and functional maintenance of the epithelial-stromal junction. The study of cell-cell and cell-matrix interactions may provide important clues for the understanding of tumor invasiveness, a process that starts at the epithelial-stromal junction and proceeds with its destruction.     

Outgrowth in vitro of cervical epithelium separated from the uterovaginal anlage of newborn mice

Summary After gentle trypsinization, the pseudostratified columnar Müllerian epithelium that lines the uterine cervix of newborn mice could be separated from the enclosing stromal tissue. Pure epithelial tubes explanted in vitro and were allowed to grow in a standard medium for 3–4 days forming a confluent colony of rather closely-fitting cells. The cell sheet was studied by a preparatory technique that allows examination of a large number of cells with preserved intercellular spatial orientation. Attempts were made to identify cultured cells according to the morphology of cell types in the cervicovaginal epithelium in vivo.Electron micrographs revealed that, close to the explant, the cultured cell sheet exhibited several features similar to the Müllerian epithelium in vivo. Outside these central areas of the colony was a broad transitional zone consisting of thin platelike cells distinguished by an abundance of microfilaments. At the periphery of the colonies, bulky cells possessing microvilli and a vacuolated cytoplasm tended to overlap adjoining platelike cells. These bulky cells had a morphology resembling that of the superficial cells seen in the upper vagina and common cervical canal of immature and diestrous animals. The epithelial development in the cultures apparently simulated the transformation in vivo from a pseudostratified Müllerian epithelium in the newborn to a stratified epithelium resembling that of the uppermost vagina and common cervical canal of immature animals. Judged by morphological and cytochemical criteria, the Müllerian cells in the outgrowth obviously had many changed features. It thus seems questionable whether the cells grown in vitro are comparable with the corresponding cells in vivo when used for experiments requiring the controlled conditions of the culture environment.Supported by grants from the Norwegian Research Council for Science and the Humanities and from the Norwegian Cancer Society