long-term organ culture of human uterine endocervix1...prolapsus uteri. '' lost by...

[CANCER RESEARCH 38. 3723-3733, November 1978]0008-5472/78/0038-OOOOS02.00

Long-Term Organ Culture of Human Uterine Endocervix1

Walter Schurch, ' Elizabeth M. McDowell, and Benjamin F. Trump2

Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland 21201

ABSTRACT

Expiants of human endocervix have been maintained inorgan culture for 24 weeks. The epithelium was viable forthe entire duration of culture, as demonstrated by ultra-structural morphology and mitotic activity. The epitheliumof the expiants retained a near normal morphology for 2to 4 weeks. The only changes observed were decreasedmucus secretion in columnar cells, focal epidermoidmetaplasia, and an increase in autophagic vacuoles.Subsequently, a slowly progressive transformation of thecolumnar epithelium into a metaplastic epithelium wasobserved, with loss of mucus-secreting columnar cellsand ciliated cells and the appearance of cuboidal andflattened epidermoid cells, forming often two to threelayers. Metaplasia began at the top of the papillae andridges and extended progressively downwards into theclefts. Nevertheless, in a few areas deep in the clefts,columnar cells retained evidence of mucus secretionduring the entire duration of culture.

Epidermoid metaplasia appeared to develop in the ex-plants by transformation of columnar mucous cells intocuboidal and flattened cells with epidermoid characteristics. This hypothesis is supported by ultrastructural observations that showed mucus secretion and early keratini-zation in the metaplastic cells. Mitotic figures were observed with transmission electron microscopy for up to 24weeks, and all dividing cells contained mucous granules.

INTRODUCTION

Epidermoid and columnar epithelia of the human uterinecervix have been little studied in vitro, in spite of their readyaccessibility for sampling and the frequent occurrence ofpremalignant and malignant lesions arising within them.Results of in vitro studies of human cervical epithelia,benign and neoplastic, have recently been reviewed indetail (27, 28). Previously, normal cervical epithelia havenot been maintained in organ culture for more than 3 weeks(2, 5, 8, 9). This apparently limited life span restricted theexperimental potential of the organ culture model of thehuman cervix.

The present study represents one aspect of our programof investigations of human carcinogenesis in which weobtain tissues either at surgery or "immediate autopsy" (25,

26) and culture them in vitro. Methods have already beendescribed for long-term organ culture of the human bron-

1This work was supported by the Department of Pathology. University ofMaryland School of Medicine. This is Contribution 552 from the CellularPathobiology Laboratory.

2 To whom requests for reprints should be addressed.3 Present address: Department of Pathology, HÃ´telDieu Hospital, Mon

treal. Quebec, Canada.Received May 3, 1978; accepted July 28, 1978.

chial epithelium (3) and the pancreatic ductal epithelium(14). The cultured expiants have been used to study metabolic responses to carcinogens (11-13). Organ culture hasthe advantage over cell culture in that stromal and epithelialarchitecture and the normal cellular relationships remainpreserved. In this paper we present results of long-termorgan culture of the human uterine endocervix. The endo-cervical epithelium was maintained in culture for 5.5months, and individual cells were shown to be viable forthe entire duration of culture, as demonstrated by ultra-structural morphology and mitotic activity. Expiants retained a near normal morphology for 2 to 4 weeks ofculture. Subsequently, columnar cells became progressively transformed to a cuboidal or flattened configuration,forming occasionally a multilayered epidermoid epithelium,although a few columnar cells were observed in some areasup to 24 weeks of culture.

MATERIALS AND METHODS

Specimens. Expiants from the human endocervix wereobtained from 18 patients who underwent hysterectomiesfor various uterine diseases and from 2 immediate autopsies(Table 1). Immediately after removal the uterus was dissected under aseptic conditions. The endocervical expiantswere obtained by opening the whole uterus from the external cervical os up to the fundus. The mucosal surface of theendocervix was cleaned with a jet-wash of cold L-15 (17)(Grand Island Biological Co., Grand Island, N. Y.) from asyringe. The expiants were removed; each consisted ofepithelium with 2 to 3 mm of the underlaying stroma. In thisway 6 to 9 expiants of 0.5 to 1 sq cm each were obtainedfrom each endocervix. Particular attention was paid toobtaining expiants high in the endocervical canal, awayfrom the squamocolumnar junction or the transformationzone, in an attempt to select areas free of marked metaplastic changes.

Culture. Expiants were placed in cold L-15 for transportation to the culture laboratory and were then culturedaccording to the technique used in our laboratory for long-term organ culture (3, 14). Expiants with the epitheliumfacing the gas-medium interphase were placed into 60-mmplastic Petri dishes (Falcon Plastics, Oxnard, Calif.) with 3to 5 ml of culture medium composed of CMRL-1066 (GrandIsland Biological Co.) containing hydrocortisone hemisuc-cinate (0.1 /xg/ml; Schwartz/Mann, Orangeberg, N. Y.),recrystallized bovine insulin (1 ng/m\; Schwartz/Mann),penicillin G 100 units/ml), streptomycin (100 /Â¿g/ml),am-photericin B (1 /Ltg/ml) (Grand Island Biological Co.), 2 rriML-glutamine (Grand Island Biological Co.), and 5% heat-inactivated fetal calf serum (Grand Island Biological Co.).Culture dishes were placed in a controlled atmospherechamber and gassed with 45% 0,, 50% N,, and 5% CO,. Thechamber was then placed on a rocker platform at 5 to 10

NOVEMBER 1978 3723

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W. SchÃ¼rchet al.

Table 1Age, diagnosis, source, and duration of culture of endocervical

expiants from patients used in this study

Case1234567891011121314151617181920Age(yr)3940491845374845346119544030374442324934Immedi-Diagno- Hysterec- ate au-

sis tomytopsyMU"+MU+MU+HI+MU+MU+MU+MU+MU+AE+HI+AHE+MU+MU+MU+MU+MU+P+MU+MU

+Wk

cultured43266"4Â»4Â»12121010164Â»20242020202024

" MU, myoma uteri; HI, head injury; AE, adenocarcinoma ofendometrium; AHE. adenomatous hyperplasia of endometrium; P,prolapsus uteri.

'' Lost by contamination.

cycles/min, which caused the medium to flow intermittentlyover the epithelial surface. Expiants were incubated at36.5Â°,and the medium was replaced 3 times a week.

Morphology. Morphological studies were made on eachcase at zero time, i.e., before culture, and from the culturedpieces at regular intervals. In the early experiments culturedexpiants were sampled daily. As experience was gainedwith this system, samples were taken twice per week, thenweekly, and finally every month. Zero-time samples andcultured expiants were fixed in 4F-1G4 (18) and were proc

essed for LM, TEM, and SEM. All 3 techniques wereperformed on the same specimen in every instance.

LM. At zero time a sample was taken from each case atthe squamocolumnar junction and/or the transformationzone of the anterior and posterior lip of the cervix as well asfrom the lower one-third of the endocervix. This was done

in an attempt to assure that expiants for culture were takenfrom areas free of marked metaplastic changes. Sectionsfor LM were embedded in paraffin, cut at 3 to 5 Â¿Â¿m,andstained with hematoxylin and eosin and AB-PAS with andwithout diastase digestion (19). Prior to AB-PAS staining,paraffin sections fixed in 4F-1G were hydrated and soaked

overnight in a saturated solution of dimedone in order toblock free aldehyde groups introduced by glutaraldehydefixation. After removal of the specimens for LM and theexpiants for culture, the rest of the cervix and the uterinebody were routinely processed for diagnostic purposes. No

4The abbreviations used are: 4F-1G, mixture of 4% formaldehyde and 1%glutaraldehyde prepared with 200 mOsmol phosphate buffer; LM. lightmicroscopy; TEM. transmission electron microscopy; SEM, scanning electron microscopy; AB-PAS. Alcian blue (pH 2.5)-periodic acid-Schiff; DM.dissecting microscope; RER. rough-surfaced endoplasmic reticulum.

cervical pathology other than chronic cervicitis was foundin any of the material used in this study.

TEM. Samples taken at zero time and after differentintervals of culture were cut into relatively large pieces,measuring approximately 2x2x1 mm each, with theepithelium on the lateral side, and were fixed by immersionin 4F-1G for 24 hr and washed in 0.1 M sodium cacodylate

buffer containing 7% sucrose prior to postfixation in 1 or2% OsO4. The tissue pieces were dehydrated through aseries of alcohols and propylene oxide and were embeddedin Epon 812. Pieces were embedded with particular attention to their orientation in order to obtain sections of theepithelium perpendicular to the basal lamina. Large semi-

thin sections, 0.5 to 1.0 /urn, were stained with toluidineblue for selection of suitable areas for thin sectioning. Thinsections were double stained on copper grids with uranylacetate and lead citrate and examined in a JEOL 100 Belectron microscope.

SEM. Study of the mucosal surface of the cervix is oftenhampered by the presence of a thick layer of mucus thatprevents examination of the underlying epithelium. Inorder to overcome this problem, we used a relatively time-

consuming cleaning procedure that removed the mucuslayer with little or no damage to the epithelium. Afterfixation for 0.5 hr in 4F-1G, samples from the endocervix at

zero time and after culture were fixed with dissectingneedles on a wax plate and treated as follows. First, part ofthe mucus layer was removed under the DM by the use offine forceps. The pieces were then subjected to severalbursts of sucrose-cacodylate buffer from a syringe in order

to remove the residual mucus layer. The surface was examined by DM and, if necessary, the entire procedure wasrepeated. The tissue pieces were transferred to 4F-1G for

24 to 72 hr, washed in 0.1 M sodium cacodylate buffercontaining 7% sucrose, postfixed in 1% Os04 in 0.1 M s-

collidine buffer, and dehydrated in graded series of ethanol.When the dehydration was completed and the specimenwas in 100% ethanol, it was again examined by DM, andsmall residual fragments of mucus were removed withforceps. After completion of the critical point drying procedure with CO-2,the specimen was mounted on an aluminumstub with silver conducting paint. We carried out the finalstage of mucus removal by holding the stub and usingcompressed air, allowing very small mucus flakes, nowfriable, to be blown away. Larger mucus fragments werelifted off, without touching the epithelium, with a dissectingneedle while being viewed by DM. Finally, the specimenswere coated with gold by means of a Hummer sputteringdevice and examined in an AMR 1000 scanning electronmicroscope.

RESULTS

Morphology of the Endocervix at Zero Time. The normalmorphology will be presented only briefly, since it has beendescribed in detail by others (10, 21-23, 29).

As observed with LM, the epithelium of the endocervixconsists of a single layer of uniform mucus-secreting columnar cells (Fig. 2) and scattered ciliated cells. AB-PAS-

stained sections show variable amounts of neutral and acid

3724 CANCER RESEARCH VOL. 38



Organ Culture of Human Endocervix

mucosubstances in the supranuclear portion of individualcolumnar cells (Fig. 3). The nuclei are characteristicallybasally located and oval. Zero-time samples from the lowerthird of the endocervical canal show small areas of earlystages of focal epidermoid metaplasia (10), usually locatedat the tips of papillae, and sometimes also so-called "reserve cell" hyperplasia. The expiants used for culture were

therefore free of marked metaplastic changes, since theywere removed higher in the endocervical canal.

As observed with TEM, the typical mucus-secreting columnar cells have numerous tightly packed supranuclearmucous granules, fibrillar bodies, and a well-developedRER (Fig. 4). The close association of fibrillar bodies andmucous granules suggests that the former represent apeculiar filamentous form of storage of mucosubstances(22). The Golgi complex is prominent, in close juxtapositionto formed or forming mucous granules. The supranuclearcytoplasm is filled with the membrane-bound mucous granules, which causes an apical bulging at the luminal border.The luminal plasma membrane forms well-developed longmicrovilli which occasionally are branching. The nuclei areirregular in shape and frequently show indentations. Thebasal part of the columnar cells is attached by hemidesmo-somes to the basal lamina. The lateral surfaces of adjacentcells are connected by desmosomes and are mostlystraight, but complex interdigitations are also observed.Tight junctions are present near the luminal border of thecolumnar cells. In a few mucus-producing columnar cells,bundles of tonofilaments are observed. The ciliated endocervical cells contain well-developed cilia, composed ofciliary shafts, basal bodies, and cross-striated rootlets, aswell as long surface microvilli (Fig. 4). A few mucus-secreting columnar cells are also ciliated. Mitochondria of columnar mucous and ciliated cells show a slight swelling,presumably due to the anoxic period between ligation ofthe vessels and fixation.

SEM demonstrates the 3-dimensional architecture of theendocervical mucosa, which can best be seen at low magnification. The most striking features of the mucosa arelong ridges and finger-like papillae covered by columnarcells. Between the ridges deep clefts are observed, runningin oblique, transverse, and longitudinal directions. Athigher magnification, individual columnar and ciliated cellsare seen, covering the entire surfaces of the papillae,ridges, and all the intervening areas (Fig. 1). Columnar cellsare uniform in size, round or polygonal, presenting abulging surface covered by microvilli of variable length. Nointercellular junctions are observed between individual columnar cells. The number of ciliated cells varies greatly fromone area to another. Close to the squamocolumnar junction, ciliated cells seem to be fewer in number than thosehigher in the endocervical canal. In most of the zero-timesamples cut near the squamocolumnar junction, small focalareas of epidermoid metaplasia are seen. The SEM aspectof different stages of epidermoid metaplasia has beendescribed in detail (15). Most frequently, early stages ofmetaplasia are present, and these are usually located at thetips of papillae, although rarely at the surface of ridges aswell.

Organ Culture. Expiants from 20 caseswere cultured for2 to 24 weeks (Table 1). The epithelium was viable by

ultrastructural criteria for the entire duration of the culturein each case. In none of the cases was the culture interrupted because of nonviability of the epithelium.

Two- to 4-Week Culture. Whole expiants show a nearnormal morphology for 2 to 4 weeks, except at the edges,where the columnar cells become flattened and appear togrow around the expiant (Fig. 8).

As observed with LM, most of the papillae and ridges andall the intervening areas are covered by tall columnar cellswith basally located nuclei. In hematoxylin- and eosin-stained sections, the columnar cells show a slight eosino-philia of the cytoplasm (Fig. 6). The AB-PAS stain reveals aslight decrease in the amount of mucus in the supranuclearportion. At the tops of a few papillae and ridges, andinfrequently also in the clefts, the epithelial cells tend to becuboidal and sometimes also flattened. Evidence for mucussecretion is still present in these cells. Occasionally, smallareas of focal epidermoid metaplasia, composed of a well-organized multilayered epithelium, are found at the tips ofpapillae, most probably representing a prÃ©existentmetaplastic process.

TEM demonstrates only minor changes in columnar cellsat 4 weeks of culture. Mucous granules are smaller than atzero time, but fibrillar bodies are still present. There is anincrease in the number and size of autophagic vacuoles insome columnar cells. The initial mitochondrial swellingobserved at zero time is reversed, and mitochondria returnto normal morphology after 1 to 3 days in culture. Theintercellular space at the base of some columnar cellsappears to be widened. The basal lamina is continuous.Sections from early metaplastic areas are composed ofcuboidal mucus-secreting cells interspersed by flattenedcells. The latter also contain mucous granules near the cellsurface, and they have a well-developed RER and a prominent Golgi complex. Occasionally, columnar, cuboidal, andflattened cells contain bundles of tonofilaments within thecytoplasm.

SEM demonstrates that there is an excellent preservationof normal epithelium over almost the whole expiant up to 4weeks. Individual cells show a bulging surface with longmicrovilli (Fig. 5), which sometimes appear to be fused.Numerous well-formed ciliated cells are present (Fig. 5).Again, small areas of early epidermoid metaplasia are foundat the tops of some papillae and ridges. These areas arecomposed of cells with a slightly bulging surface coveredby microvilli of variable length, interrupted by the presenceof cells with a flat or slightly raised surface that is polygonaland covered by short microvilli (Fig. 7). No well-formedmicroridges are seen at this time of culture, but occasionally terminal bars are observed between individual metaplastic cells. At the lateral sides of papillae and ridges epidermoid metaplasia is infrequent. The cut edges of theexpiants are covered by flat elongated cells which show asurface ultrastructure similar to that found in early metaplastic cells.

Four- to 24-Week Culture. The changes observed duringthe first 4 weeks of culture are found to be slowly progressive up to 24 weeks.

As observed with LM, the metaplastic process extendsprogressively from the tops of papillae and ridges to theirlateral sides. Deep in the clefts, and less frequently also in

NOVEMBER 1978 3725



W. SchÃ¼rchet al.

the adjoining lateral sides of ridges, there is still a singlelayer of tall mucus-producing columnar cells (Figs. 10 and11). However, on the exposed surfaces of papillae andridges, cuboidal and flattened cells show an eosinophiliccytoplasm but are still covered by a thick layer of Alcianblue-positive mucus. After 8 weeks in culture the epitheliumon the surface of the large ridges is often composed of 2 to3 layers of cuboidal or flattened cells (Fig. 12). At this timethe stroma underlying the epithelium shows only a fewscattered spindle cells, which occasionally form activeproliferating foci near the epithelium.

As observed with TEM, the flat epidermoid cells on theexposed surface frequently contain bundles of tonofila-ments (Fig. 13), but they still show a well-developed PER, aprominent Golgi complex, and a few mucous granules nearthe cell surface (Fig. 17). The surface microvilli are usuallyshort. Columnar cells from areas deep in the clefts show anexcellent preservation up to 24 weeks (Fig. 18). Some ofthem contain a large number of autophagic vacuoles (Fig.18), whereas others retain a near normal morphology (Figs.9 and 14). An interesting feature is the progressive appearance of tonofilaments and sometimes also of well-formedtonofilament-desmosome complexes (Fig. 15) in most ofthe columnar cells maintained in culture from 12 to 24weeks (Fig. 18). Ultrastructural evidence of mucus secretionis visible, as shown by the presence of small mucousgranules located in the apical portion near the cell surfacein most of the columnar cells during the entire duration ofthe culture. The mucous granules contain a finely granularelectron-dense material (Figs. 16 and 18). As observed atzero time, a few columnar cells with apical mucous granules are found to be ciliated. In some sections the basallamina is incomplete and often has a fragmented or floccu-lent appearance (Figs. 9 and 13), whereas in other sectionsthe lamina is very thin but still continuous (Figs. 14 and 18).Another feature observed in long-term cultured expiants isthe presence of a relatively large number of mitochondriain both epidermoid and columnar cells (compare Fig. 4 withFig. 14). A total of 5 mitotic figures (Fig. 16) were observedin cells by TEM (4, 12, 18, 20, and 24 weeks), and in everycase apical mucous granules were present. In some cells indivision, short bundles of microfilaments were observednear the cell membrane. Cell division was not seen in anyother cell type during the entire study.

SEM reveals that the tops of the papillae and ridgesbecome progressively covered by cells that show a flatpolygonal surface with densely packed short microvilli witha tendency to form microridges. Terminal bars are nowmore apparent than they were when observed earlier. After12 weeks in culture almost all exposed surfaces, papillae,and ridges, including their lateral sides, are covered bycells with a flattened surface covered by short microvilli,some showing a smooth surface with only a few shortmicrovilli (Fig. 21). Between 12 to 24 weeks in culture, thesurface ultrastructure of flattened cells shows no furthersignificant changes. Well-formed densely packed micro-ridges, seen at zero time in the native epidermoid epithelium of the exocervix, are not observed. Focal areas of cellswith a bulging surface and long surface microvilli becomeprogressively smaller but are still found on the lateral sidesof ridges and deep in the clefts up to 20 weeks (Figs. 19 and

20). The surface contours of these cells, however, areslightly changed. They are often less uniform than whenthey were observed earlier in culture, and the surfacemicrovilli are usually of variable length. The number ofciliated cells decreases progressively, but they are observedin small numbers up to 20 weeks. The surface of ciliatedcells is slightly raised, with shorter microvilli, and thenumber of cilia seems to be decreased (Fig. 19). Some ofthe ciliated cells display a longitudinal surface shape.

DISCUSSION

The present study convincingly shows that the humanendocervical epithelium can be maintained in organ culturefor several months with preservation of viability as demonstrated by ultrastructural criteria and the presence of mitotic figures at regular intervals during the entire durationof culture. The increase in number and size of autophagicvacuoles seen in the epithelial cells of cultured expiants isconsidered to result from a sublethal injury (1). The columnar epithelium of whole expiants preserved a near normalmorphology for 2 to 4 weeks of culture. Afterwards, a slowlyprogressive transformation of the columnar epithelium occurred, with progressive loss of mucus-secreting columnarcells and ciliated cells, and appearance of cuboidal cellsand noncornifying epidermoid cells forming a multilayeredmetaplastic epithelium. In a few areas, however, columnarcells with ultrastructural evidence of mucus secretion wereobserved during the entire duration of the culture. Themetaplastic changes seen in cultured expiants followed asimilar pattern to that seen with the SEM (28) and bycolposcopy in vivo (6, 7). Similar observations were madein short-term organ culture of the human endocervix (9).The tops of papillae and ridges, where the metaplasticprocess started, are more exposed to the gas-mediuminterphase and to the intermittent flow of the medium overthe surface of the expiants, whereas the epithelium in theclefts remains protected for a longer time by the secretedmucus. The intermittent flow of the medium over the surface of the epithelium probably removes the mucus andexposes the epithelium to the gas-medium interphase,initiating the metaplastic process.

An important factor in the interpretation of the changesoccurring in cultured expiants is the type of the epitheliumat the start of culture. It has been demonstrated (10) thatvarying degrees of epidermoid metaplasia occur in almostall cervices. The area most often involved is the transformation zone of the squamocolumnar area. Epidermoidmetaplasia can also be observed on tops of papillae andridges as well as in clefts, particularly in the lower third ofthe endocervical canal (10). Areas of epidermoid metaplasiaat the tops of some papillae and ridges found at zero timebecame, after 4 weeks of culture, more and more frequent,and they covered almost all exposed surfaces after 12weeks. It is very unlikely that the progressively appearingmultifocal epidermoid metaplasia on exposed surfaces represents an extension of prÃ©existentmetaplastic foci. Wecan thus assume that the developing and progressively extending epidermoid metaplasia is culture-induced ratherthan an extension of an originally prÃ©existentprocess.

Concerning the origin of the metaplastic cells, 3 major





hypotheses are being considered, all of which were recentlyreviewed (16). Two of these theories insist that the newcells formed arise from cells already present in the epithelium, while the third places the origin of progenitor cells inthe subepithelial stroma. As we will now discuss, epider-moid metaplasia in the organ culture model seems todevelop by direct transformation, most probably involvingcell division of columnar mucus-producing cells, to cuboi-dal and flattened epidermoid cells with formation of anoncornifying epithelium, showing epidermoid differentiation. This hypothesis is supported by ultrastructural differentiation of mucus-producing columnar cells and the observation of the 5 cells in division which showed JEMevidence of mucus production. No undifferentiated epithelial cells were observed at any time during the study, whichcould account for the replacement of columnar cells by anew cell population. The direct transformation of columnarmucus-producing cells to flat epidermoid-like cells hasbeen considered impossible (20).

During culture we observed a gradual transformation inthe shape and also in the cytoplasmic differentiation ofmucus-secreting columnar cells. While there was a progressive decrease in the number and size of apical mucousgranules, columnar cells became cuboidal and then flat. Inaddition, the mucus-producing cells of all shapes frequentlycontained bundles of tonofilaments as well as a prominentGolgi complex and a well-developed RER. This means thatthe mucus-producing cells of various shapes all showultrastructural features of mucus secretion and epidermoiddifferentiation. This might result from a modulation in thestructure and function of mucous cells. It would be premature to conclude that the histogenesis of epidermoid metaplasia in the human cervix in vivo follows the same sequences as proposed for the in vitro system. However,direct conversion of columnar mucus-secreting cells intocuboidal and flattened epidermoid cells was recently proposed as the mechanism for the histogenesis of epidermoidmetaplasia of the human and hamster tracheobronchialepithelium (4, 24).

We believe that the long-term organ culture model of thehuman endocervix represents a useful in vitro system fortesting metabolic and morphological responses to potentialchemical and biological carcinogens.

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25. Trump, B. F., Valigorsky, J. M.. Dees, J. H., Mergner, W. J., Kim. K. M.,Jones. R. T.. Pendergrass, R. E., Garbus. J.. and Cowley, R. A. CellularChanges in Human Disease. Human Pathol..4 (Part 1): 89-109.1973.

26. Trump, B. F., Valigorsky, J. M.. Jones, R. T., Mergner, W. J., Garcia, J.H., and Cowley, R. A. The Application of Electron Microscopy andCellular Biochemistry to the Autopsy: Observation on Cellular Changesin Human Shock. Human Pathol., 6: 499-516, 1975.

27. Wilbanks, G. D. In Vitro Studies on Human Cervical Epithelium, Benignand Malignant. Am. J. Obstet. Gynecol., Â»2?:771-778. 1975.

28. Wilbanks. G. D., and Fink. C. G. Tissue and Organ Culture of CervicalEpithelium â€”¿�Physiologicaland Preinvasive. In: J. A. Jordan and A.Singer (eds.), The Cervix, pp. 429-441. Philadelphia: W. B. Saunders,Co., Ltd., 1976.

29. Williams, A. E., Jordan, J. A., Allen, J. M.. and Murphy, J. F.The SurfaceUltrastructure of Normal and Metaplastic Cervical Epithelia and Carcinoma in situ. Cancer Res.. 33: 504-513, 1973.

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W. SchÃ¼rchet al.

Figs. 1 to 4. Endocervical mucosa at zero time.Fig. 1. Scanning electron micrograph showing the surface of columnar cells, which is bulging and shows long surface microvilli. Several scattered

ciliated cells are present. â€¢¿�4,000.

Fig. 2. Light micrograph from the surface of an endocervical ridge. The epithelium is composed of a single layer of tall columnar cells with basally locatednuclei. H & E, x 400.

Fig. 3. Light micrograph from asimilar area as shown in Fig. 2. The supranuclear portion of the columnar cells is filled with mucus. Nuclei are not stained.AB-PAS, x 400.

Fig. 4. Transmission electron micrograph showing several mucus-producing columnar cells and a single ciliated cell. Mucous cells have basally locatedindented nuclei the supranuclear portion of which is filled with numerous mucous granules. Two fibrillar bodies are visible in a mucus-producing columnar

cell (FB). The epithelial cells are separated from the underlying stroma by a continuous basal lamina (BL). Uranyl acetate and lead citrate, x 7,500.

Fig. 5. Four-week culture. Scanning electron micrograph from the surface of a ridge showing well-preserved epithelial cells with a bulging surface andlong surface microvilli. Numerous ciliated cells are present. A single cell with a flattened polygonal smooth surface is visible (arrow), x 3,000.

Fig. 6. A 4.5-week culture. Light micrograph from a villus showing well-preserved columnar cells forming a single layer. There is increased eosinophiliaof

the supranuclear cytoplasm of individual columnar cells. H & E, x 400.Fig. 7. A 4-week culture. Scanning electron micrograph from the top of a villus with early epidermoid metaplasia. Most of the cells show a polygonal

flattened or slightly raised surface with microvilli of variable length. No distinct terminal bars are visible between individual cells. A single cell with a bulgingsurface and long microvilli (single arrow) and a flattened ciliated cell (double arrow) are present, x 3,000.

Fig. 6. A 3-week culture. Light micrograph from the edge of an expiant. The cut undersurface of the expiant is covered by a cellular outgrowth of flat epidermoid cells (single arrow). In the underlying cleft the epithelium is tall columnar (double arrow) or low columnar (arrow head). H & E, x 540.

Fig. 9. A 12-week culture. Transmission electron micrograph from a cleft showing tall columnar cells with long surface microvilli and mucous granules(MG) near the cell surface. Within the cytoplasm there are numerous mitochondria, a prominent Golgi complex (G) in close juxtaposition to forming mucousgranules, and a well-developed RER. The basal lamina (BL) is discontinuous. Uranyl acetate-lead citrate, x 6,000.

Fig. 10. A 12-week culture. Light micrograph from a cleft. The epithelium is composed of tall columnar cells and cuboidal cells. H & E, x 400.

Fig. 11. A 12-week culture. Light micrograph from the same area as shown in Fig. 10. Tall columnar cells contain AB-PAS-positive mucous granules. AB-PAS, x 640.

Fig. 12. A 12.5-week culture. Light micrograph from the top of a ridge showing multilayered epidermoid metaplasia. Epon semithin section stained withtoluidine blue, x 640.

Fig. 13. A 12.5-week culture. Transmission electron micrograph from the same area as shown in Fig. 12. Flat epidermoid cells show short surfacemicrovilli and contain within the cytoplasm bundles of tonofilaments (arrows). The basal lamina (BL) is fragmented. Uranyl acetate and lead citrate, x 8,000.

Fig. 14. A 20-week culture. Transmission electron micrograph from a cleft. The tall columnar cell contains apical mucous granules (MG), numerousmitochondria, and a few myelin figures. The intercellular space at the basal part is slightly widened and the basal lamina (BL) is continuous. Uranyl acetate-lead citrate, x 6,000.

Fig. 15. An 18-week culture. Transmission electron micrograph showing part of a columnar cell with mucous granules (MG ) and tonofilaments convergingto adesmosome (arrow). Uranyl acetate-lead citrate, x 14,000.

Fig. 16. A 20-week culture. Transmission electron micrograph from a columnar cell in division with apical mucous granules that contain a finely granularelectron-dense material (MG). Uranyl acetate and lead citrate, x 14,000.

Fig. 17. An 18-week culture. Transmission electron micrograph of a flat epidermoid cell from the top of a ridge. Mucous granules (MG) and bundles oftonofilaments (arrows) are visible within the cytoplasm. Uranyl acetate-lead citrate, x 16,000.

Fig. 18. A 24-week culture. Transmission electron micrograph from a cleft showing tall columnar cells with apical mucous granules (MG) and numerousbundles of tonofilaments (arrow) within the cytoplasm. Autophagic vacuoles (AV) are present in the basal part of the columnar cell. The basal lamina (BL)is continuous. Uranyl acetate-lead citrate, x 8,000.

Fig. 19. A 20-week culture. Scanning electron micrograph from a cleft. A single ciliated cell, interspersed among epithelial cells with a bulging surfaceand long microvilli, is visible, x 3,000.

Fig. 20. A 20-week culture. Scanning electron micrograph from a cleft showing several cells with a bulging surface and microvilli of variable length. Nointercellular junctions are visible, x 7,500.

Fig. 21. A 20-week culture. Scanning electron micrograph from the top of a ridge. The cells show a polygonal flat surface with short microvilli (arrow) witha tendency to form microridges (double arrow). Terminal bars are observed between individual cells, x 3,000.





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1978;38:3723-3733. Cancer Res Walter Schürch, Elizabeth M. McDowell and Benjamin F. Trump Long-Term Organ Culture of Human Uterine Endocervix

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