Chapter 2 Tissue Culture of the Human Uterine Cervix

METHODS IN CELL BIOLOGY, VOLUME 21B

Chapter 2 Tissue Culture of the H u m a n Uterine Cervix GEORGE D. WILBANKS, EDMUND LEIPUS, AND DAVID TSURUMOTO Department of Obstetrics and Gynecology, Rurb Medical College, Rnsb Presbyterian St. Luker Medical Center, Cbicago, Illinois

I. Introduction . . . . . . . . . . . . . . . . . . . . . A. S c o p e . . . . . . . . . . . . . . . . . . . . . . B. Review of Methodology and Results . . . . . . . . II. Materials and Methods . . . . . . . . . . . . . . . . . A. Materials for Tissue Culture . . . . . . . . . . . . . B. Methods for Tissue Culture . . . . . . . . . . . . . III. Results and Discussion . . . . . . . . . . . . . . . . IV. Perspectives . . . . . . . . . . . . . . . . . . . . . A. Current Applications and Findings . . . . . . . . . B. Futurestudies . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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29 29 30 31 31 38 40 47 47 49 50

I. Introduction

A. Scope Most reports in the literature on the behavior of human uterine cervical tissues in vitro have focused mainly on abnormal material. For a review of cervical neoplasia in vitro, see Wilbanks and Fink (1976). These reports have generally lacked practical information on the tissue culture of normal cervical cells and have usually mentioned them briefly as controls for comparison with neoplastic cells. In addition, information on only a relatively small number of normal biopsies has been reported. This chapter will review the literature stressing normal human cervical culture and describe briefly the experience of this labora29 Copyrighr @ 1980 by Academic Press. h. AU rights of nprodwtim in any form ~ a c r v c d .

ISBN 0-12-5641400

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GEORGE D. WLBANKS et

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tory with normal human cervical cells in tissue culture in an attempt to fill in this apparent void. Results of routine cell culture, preliminary results on the effects of hormone addition, and preliminary results of organ cultures using porcine skin substrates will be presented.

B. Review of Methodology and Results A summary of the literature on the tissue culture of normal human uterine cervix is presented in Table I. A variety of techniques have been used to initiate and maintain cultures from cervical biopsies of adult patients. Early cultural techniques (Gey et al., 1952; Southam and Goettler, 1953; Grand and Ayre, 1954; Moore, 1955, 1956; Bromelow and Schaberg, 1957; Mellgren et al., 1962; Mulligan, 1962; Simeckova et al., 1962), which met with some success, utilized plasma clots and hanging drops on glass surfaces in several types of biological fluids. As they became available, chemically defined media with serum supplements (Mellgren et al., 1962; Simeckova et al., 1962; Cummins and Ross, 1963; Richart, 1964a,b; Nordbye, 1969; Wilbanks and Richart, 1966; Wilbanks, 1969; Wilbanks and Shingleton, 1970; Balduzzi et al., 1972; Fink et al., 1973; Schurch et al., 1978) and various biologic substrates (Mellgren et al., 1962; Auersperg and Worth, 1966; Balduzzi et al., 1972; Fink ef al., 1973) were later used with resulting increased cell proliferation and enhanced maintenance of organ tissue architecture. Accurate assessment or comparison of data from these studies is difficult because the number of possible experimental variables to explain divergent results from apparently similar studies is too great. The problems associated with the use of serum-supplemented media (Milo et al., 1976; Keay, 1978), and the formation of toxic photoproducts from the breakdown of riboflavin and tryptophan in tissue culture media by ordinary fluorescent room light (Wang, 1976), further complicate interpretation. For example, some studies reported the growth of epithelial cells from normal areas of abnormal cervices having known neoplastic lesions, while others reported results with normal cervices from patients who were pregnant or who had other benign lesions of the uterus. In addition, some investigators separated the epithelial layer from the underlying stroma in an attempt to grow only epithelial cells and reported that cultures of pure epithelium or fibroblasts were obtained based on the standard histopathology or electron microscopy of only a small number of samples. Some investigators have subjectively graded colonial growth as poor, good, or excellent while others have measured the area of outgrowth around primary explants-even though in two areas of the same size the number of individual cells in one could be 3 to 10 times that in the other. Quantitation of the number of hormone-induced mitotic figures within an area of outgrowth has also been reported, though the stimulatory effect of background levels of hormones present originally in the serum supplement

2.

TISSUE CULTURE OF THE HUMAN UTERINE CERVIX

31

was not known and could not be compared between different studies. And finally, some studies have reported cell counts on primary cultures even though not all of the cells were of cervical origin (or perhaps not even of human origin) and were probably leukocytes and cells comprising blood vessels. There has been little quantitative information on the routine cell culture of normal cervical tissue, the expected results, and the length of culture viability. One reason might be that no one has succeeded in obtaining a continuous line of either epithelial or fibroblast cells from the cervix. In most instances cultures were maintained for a few passages, and then the cells died. Thus long-term serial subculture has not been reported and practical methodology is lacking in this area. Organ culture has recently been used to study endo- and ectocervical cells in normal tissues (Balduzzi el al., 1972; Fink et al., 1973; Schurch et al., 1978). In these studies whole sections of tissue have been explanted with the stromal and epithelial layers intact. The tissue was placed either on a plastic or glass surface with the addition of growth medium, with or without mechanical rocking (Schurch et al., 1978), or on a wire grid in specially designed organ culture dishes filled with growth media. In some studies a biological substrate, such as pigskin, collagen gel, or agar, was placed in between the tissue and the grid in an attempt to bring nutrients and moisture to, and remove wastes from, the tissues. These methods have met with limited success and relatively short-term studies (up to 6 months) have been performed with them to date (Schurch et al., 1978).

II. Materials and Methods A.

Materials for Tissue Culture

Growth media used in this report consisted of the following: Eagle’s basal medium (BME), RPMI- 1640, or BioLab’s modified Dulbecco’s medium (BioLabs, Inc.) plus 15 to 20% fetal bovine serum (FBS) (BioLabs, Inc.), penicillin and streptomycin (P and S) [Grand Island Biological Co. (GIBCO)], each at 100 units and 100 pg/ml, respectively, and fungizone (Squibb) at 5.0 pglml. Balanced salt solution (BSS) was composed of the following: Hank’s balanced salt solution base, P and S or gentamicin (Schering) or kanamycin (Bristol) each at 100 units or 100 pg/ml, 10 p,g/ml fungizone, glutamine at 0.292 mg/ml, and sodium bicarbonate to bring the pH to 7.2. The trypsin-EDTA solution was composed of the following: 0.25% trypsin [ 1:250 stabilized for tissue culture (GIBCO)] and 0.1% ethylenediaminetetraacetic acid [EDTA (Sigma)] in Hank’s balanced salt solution. The pH was adjusted to approximately 7.5 with sodium bicarbonate. Hormones included insulin (Sigma 15500), j3-estradiol (General Biochemicals

TABLE I TISSUECULTURE OF NORMALHUMAN UTERINECERVIX' Number of normal cervical biopsies

Number of cultures

Gey et ol. (1952)

No data given

No data given

Moore (1952)

No data given

11

Southam and Goettler (1953)

No data given

3

No data given

No data given

Investigator

seeded

t4 W

Culture methods or techniques employed Explants grown in chick plasma clot, bovine embryo extract, and human placental cord serum Chick embryo plasma clot; hanging drop; under perforated cellophane; embryo extract, placental cord WNm, and balanced salt solution Plasma clot; 50% human ascitic fluid, 45%BSS,and5%chick e m b o extract Plasma clot; equal parts of 50% solution chick embryo extract, fowl plasma, and human placental sem

Growth

characteristics

Additional comments

Difficulty in initiating growth. Rapid differentiation

No quantitative data

Limited growth in five cultures in 4-5 days; one could be subcultured; all died out at 40-45 days

Age range of ptients was 23-42 years; two were pregnant

1: epithelial, poor outgrowth; 1: fibroblastic; 1: no growth

10% of cultures grew

out in 10 days

Moore (1955)

16

16

Chick embryo plasma clot

Four cultures showed small amount of epithelial cell growth; one culture had moderate epithelial growth

Moore (1956)

51

51

Same as Moore (1955)

Bromelow and Schaberg (1957)

No data given

I

Mellgren et al. (1962)

No data given

No data given

Mulligan (1962)

8

192 explants

Organ culture; 25% human plasma, 16% horse serum, 12% human fetal brain press juice, in Hank’s BSS On rat collagen; and in chick plasma clot; culture medhm was Eagle’s supplemented with human serum, adult or cord (26%) Chicken plasma clot

w W

Eight biopsies from pregnant patients; eight from nonpregnant. Normal areas of Ca patients grew better than those from normal patients

10 of 23 cultures from

normal and pregnant patients grew; 23 of 28 normal arras from Ca or C I S patients grew well to excellent Poor growth or maintenance

Obtained cells after 10-20 days

Cultured normal areas of cervices with neoplasms; dissected epithelium from stroma

Growth obtained in 68 explants for up to 15-29 days

Biopsies from parous women

~

(continued)

TABLE I (continued)

Investigator Sirneckova et al. (1%2)

Number of normal cervical biopsies

Number of cultures seeded

40

No data given

w

Cummins and Ross (1963)

At least 2

No data given

Richart (1964a,b)

4

No data given

Culture methods or techniques employed Monolayer cultures in roller tubes or in plasma clot; medium 199 and 10% bovine serum In m e x glass petri dishes; medium consisted of phosphitebuffered base containing fructose, glucose, Eagle vitamins, lactalbumin hydrolysate, or Medium 199, and 10% adult human serum and 10% fetal bovine serum Dissection of epithelial layer from stroma; explantation under perforated cellophane in Eagle’s MEM and 15% fetal bovine serum

Growth characteristics

Additional comments

Poor to fair growth in all biopsies

Maintained colonial growth for up to nine subcultures; fibroblasts mainly resulted

Monolayer cultures of pure epithelial cells which could be subcultured one or more times with a trypsin versene solution

Unpredictable success rate

W

Auersperg and Worth (1966)

8

No data given

Nordbye (1969)

11

No data given

Wilbanks and Shingleton (1970); Shingleton and WilbanJts (1974)

8

40

Same as Richatt ( 1964a)

Balduzzi et al. (1972)

7

7

Organ culture of endoand ectocervix with underlying stroma; on 8% Noble agar containing Ham’sF-12basalmedium and 10% newborn bovine serum

Vesterinen er al. (1980)*

Explantation to glass, cellulose sponge, and collagen gel; culture medium was Waymouth’s and 10% fetal bovine serum Biopsies were minced and trypsinized to yield single-cell suspensions; seeded in Puck’s Medium E2a

Cell outgrowth began 4 to 14 days; ceased 9 to 35 days, with differentiation

“Secondary” epithelioid cells at 14 to 56 days with dying off at 21 to 100 days

After 3 weeks, epithelial cells formed pavementlike sheet; fibroblasts were in lamelfated pattern and swirls; at 5 weeks it was difficult to distinguish fibroblasts from epithelia Monolayers of mostly squamous epithelia; limited culture life; could not be subcultured

Number of subcultures were not reported

Showed that in vitro cells compared favorably with in vivo tissues by morphologic criteria for up to 21 days

Five cultureshad good morphology for up to 23 days; stromal degeneration at 11 to 12 days

(continued)

TABLE I (conn’nued)

Investigator

Fink et al. (1973)

Number of normal cervical biopsies

Number of cultures seeded

No data given

No data given

W

m

Chaudhuri et al. (1974)

Not known; mixed data on normal ’ with abnormal lesions; 25 abnormal patients

92

Culture methods or techniques employed Organ culture of endocervix; epithelium and underlying stroma placed on thin slab of agar-gelled medium (1.4% agar in Eagle’s BME, no serum supplement) supported by a wire grid Epithelium was separated from underlying stmma in 28 cultures; in 64 cultures, they were left intact; grown in MEM or McCoy’s 5A plus 20% fetal bovine serum and 2 mM glutamine; subcultured using trypin

Growth

characteristics

Additional comments

Held up well for 8 days. Columnar cell pyknosis at 10 days; observed progression of changes of squamous metaplasia seen in vivo

In 26/28 cultures of epithelium alone, epithelial cells grew but with fibroblast contaminationat 5-7 weeks; with intact biopsies, 33/64 had epithelial growth but were overgrown with fibroblasts after 5 weeks. Epithelial cells survived6-16 weeks and could not be subcultured whereas fibroblasts could be subcultured several times

Optimum pH for growth of epithelial cells was 7.2 and that for fibroblasts 7.6

Wilbanks (1975)

166

No data given

Method of Richart ( 1964a)

Good growth obtained in 140/166 biopsies.

Three spontaneous cell Brdar et al. (1977)

No data

No data

given

given

Ishiwata et al. (1978)

15

484

Schurch et al. (1978)

20

No data given

Growth media consisted of Dulbecco modified Eagle’s medium supplemented with 10% fetal bovine serum Epithelial layer planted in culture; Ham’s F-12, 10% fetal bovine serum, with and without 17P-estradiol

4 w

Organ culture of upper one-third of endocervix along with 2-3 mm underlying stroma; culture medium: CMRL1066, hydrocortisone hemisuccinate, insulin, glutamine, and 5% heat-inactivated fetal bovine serum, on rocker platfom

“Ca, carcinoma; MEM, minimal essential medium; CIS, carcinoma in situ.

* Reference published after submission of manuscript.

transformations Occurred Monolayers; could be subcultured at least five times

69% or 333/484 explants produced squamous cells; 21/484: mixture of squamous cells contaminated with fibroblasts; 141484 were pure fibroblasts; 97 did not grow out (20%) Cultures held up well for 24 weeks, by morphologic criteria

Measured colonial outgrowth and mitotic index. No cell line established

Metaplastic epithelia covered all exposed surfaces within 12 weeks

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GEORGE D. WILBANKS

et al.

802OO), progesterone (Schering), testosterone (Matheson, Coleman & Bell TX 95), hydrocortisone (Sigma H-4001),and cortisone (Sigma C-2755). Porcine skin substrates were tissue culture quality porcine skin (Bum Treatment Skin Bank of Phoenix, Arizona). Activated charcoal powder was “Norit A” (Matheson, Coleman, & Bell). Instruments and equipment included # 11 surgical knife blade with holder, plastic petri dishes, 100 x 15 mm and 60 X 15 mm (Lux), plastic screw cap tubes, 16 x 125 mm (Falcon), culture flask, 25 cm2 (Falcon), organ tissue culture dishes, 60 x 15 mm (Falcon), organ culture grids (Falcon), dissecting microscope (American Optical, Model #56M-1), dissecting forceps (Matheson), eye scissors (E. Weck & Co.), B-D Cornwall Pipettor (Becton, Dickinson, and Co.), and a water-jacketed C 0 2 incubator (National Appliance Co., model # 3331).

B. Methods for Tissue Culture 1. HUMANUTERINE CERVICAL TISSUE All human cervical tissues used in the authors’ study were derived from patients who had undergone hysterectomies for various benign conditions not related to cervical neoplasia, usually symptomatic pelvic relaxation, uterine fibroids, etc. Papanicolaou smears were negative. After a portion of the tissue was removed for histologic examination, adjacent areas of the cervix were selected for culture, and the epithelial layer as well as the underlying stroma were cut out with sharp surgical scissors. The pieces, approximately 0.5 X 0.5 X 0.3 cm (thickness), were placed in a plastic screw cap tube and then washed seven times with BSS. After the last wash, the media was removed and the pieces placed in a 100 X 15-mm plastic petri dish. While viewing each piece of tissue through a dissecting microscope (magnificationat 70 X ) and holding the specimen in place with dissecting forceps, the epithelial layer, a light yellowish, cheese-like material, was gently separated from the stroma, using light scraping movements, with a surgical knife. Soaking the tissue in media containing at least 10% FBS for several hours or overnight in the refrigerator sometimes aided in separating the epithelial layer, but often reduced its growth potential. After dissection, the pieces of epithelium were transferred to a 60 X 15-mm plastic petri dish where they were minced with sharp eye scissors, in 1 .O ml of growth medium. When the pieces were 1 mm or less in size, the suspension was further diluted with growth media and 1.0 ml was transferred to each 25-cm2 culture flask. The optimal number of explants per flask ranged from 80 to 100 and up to six flasks were seeded from a single cervix. The cultures were incubated in a humidified atmosphere of 5 % c02-95% air at 37°C.

2.

TISSUE CULTURE OF THE HUMAN UTERINE CERVIX

39

The use of small volumes of media when culturing primary cervical tissues was very critical. The explants were not submerged in medium, but bathed on all sides by it, otherwise attachment and outgrowth did not occur. After attachment and commencement of growth, the cultures were fed two times per week, with increasing amounts of growth media added as the cells filled the flasks. Feeding of large numbers of cultures was accomplished using a Cornwall pipettor instead of time-consuming hand pipetting. Subculturing of cells was accomplished by removing the growth media, adding 2.0 ml of trypsin-EDTA solution per 25-cm2 flask for 30 seconds, and then incubating the flask at 37°C in a 5% C02-95% air, humidified atmosphere until the cell sheet flowed from the flask. The cells were then resuspended in growth medium, diluted 1:2, and planted in fresh flasks.

2. ORGANCULTURE Organ cultures of human cervical epithelium on pigskin substrates were maintained using RPMI-1640 and 15% fetal bovine serum as growth medium. The method used was that reported by Freeman et al. (1976). Briefly, pigskin, dermis side up, was placed on a wire grid in an organ culture dish (Falcon). Pieces of stripped epithelium with a thin underlying stromal layer were usually oriented stromaside down on the pigskin. Medium was added until the level touched the underside of the pigskin and was changed twice weekly. Incubation was in a humidified atmosphere of 5 % C02-95% air at 37°C. 3.

MARMOSET

UTERINE CERVICAL TISSUES

Cervical tissue from the uterus of a marmoset monkey was explanted into tissue culture and subcultured using basically the same method as for the human tissue except that, because of its small size (1 mm), no stripping off of the epithelial layer was done and the entire cervix was cultured. Because this type of tissue was heavily contaminated at the outset, the whole cervix was soaked in growth medium containing two to three times the regular amount of antibiotics for several hours at room temperature. Extra washings before mincing were also effective in controlling microbial outgrowth. 4.

PREPARATION OF

HORMONE STOCKSOLUTIONS

Stock hormone solutions were made by dissolving the hormone in either absolute alcohol or acetone depending upon hormone solubility. Aliquots of the stock solutions were added to growth media to yield the working concentrations, over and above that already present in the FBS, listed in Table 111. These

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GEORGE D. WILBANKS

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concentrations also depended upon the solubility of dissolved hormone in the aqueous growth media.

5. CHARCOAL TREATMENT OF FETALBOVINE SERUM Fetal bovine serum, 500 ml, control No. R 60804 was absorbed for 16 hours at room temperature with powdered charcoal (0.25% w/v). After removal of charcoal the serum was filter sterilized. The volume after this treatment was 400 to 450 ml and the pH was 7.03. 6.

RADIOIMMUNOASSAY OF STEROID HORMONES

The amount of steroid hormones in FBS before and after charcoal treatment was determined by radioimmunoassay (Abraham, 1974).

III. Results and Discussion In a review of the literature on the tissue culture of normal cervical tissues, the problem of what constitutes growth in the cultures becomes readily apparent. Terms such as “poor,” “fair,” “good,” and “excellent,” and later, “confluent monolayer,” have been used to describe the way the tissues grow in vitro (Wilbanks, 1975). In this report, growth refers to both small and large patches of cells, with or without explants, or colonial growth, to formation of confluent monolayers, all detected by visual scanning of culture vessels with low power, light microscopy and medium power, phase microscopy. From Table II,slightly less than 50% of attempted cultures showed growth. Of these (282 cultures), 85% grew or could be maintained for up to 1 year, while 15% could be grown for greater than 1 year. In contrast, Ishiwata et al. (1978) obtained growth in 80% of 484 individual explants. This apparently higher success rate could be accounted for by chance alone since their sampling was considerably smaller than ours. The results in the present report were obtained from approximately 48,000 to 60,000 individual explants. On the other hand, their cultural conditions may have been more suitable, though such detailed information was not presented in their report. Extended viability in culture could not be correlated with a particular age group since almost identical age groups were represented in each culture interval. The age range was 23 to 71 years. Likewise, once overtly toxic lots of fetal bovine serum were eliminated there was no,difference in growth-promoting ability of various lots of FBS. For instance, those lots of FBS that were used in cultures that grew for greater than 1

2.

41

TISSUE CULTURE OF THE HUMAN UTERINE CERVIX

TABLE II OVERALL GROWTHPERFORMANCE OF NORMAL, HUMANCERVICAL CELLSIN TISSUECULTURE Culture interval

Number of patient biopsies

< I Month 1-6 Months 6- 12 Months > 12 Months No growth

2 47 25 23 63

4 (1.0%) 167 (28.0%) 69 (12.0%) 42 (7.0%) 315 (52.0%)

100

597 (100%)

Total

Total number of cultures (% of total)

Number of subcultivations 1

1-10

1-8 1-19 -

Age range of patients biopsied, in years 27 -42 23-57 26-7 1 25-71 25-71

year were also used in cultures with no growth at all. Thus the growth potential of human cervical cells in vitro may vary greatly from cervix to cervix and from explant to explant from a single cervix. Primary explants usually began growing out at 1-3 days to 1 month (Figs. 1 and 2). The reader is referred to Wilbanks (1975) for a detailed account of the fine structure of normal cervical cells in culture. Cultures of pure epithelium were rare and most were contaminated with fibroblasts from the stroma, the degree depending on the dissection technique. In most cultures these contaminants would eventually overgrow the epithelium. Epithelial cells had a tightly packed cobblestone appearance, while fibroblasts grew in parallel arrays and whorls. In most cultures that grew from 6 months to greater than 12 months, the fibroblasts would die out after three or four passages and a new type of large, bipolar cell would emerge. In very dense, confluent cultures, these cells would become epithelioid, while they would appear as “fat” fibroblasts in newly planted cultures (Fig. 3). Total viable counts performed on these cells ranged from lo4 to lo6 per confluent 25-cm2 flask. The longest interval a culture survived was for 3.5 years and 19 subcultivations. The effects of insulin and steroid hormones were tested in a small number of cultures selected at random. The preliminary results are presented in Table III. The treated cultures grew or were maintained in culture from less than 1 month to 12 months. Since no treated cultures grew for more than a year, this group of cultures did less well than untreated controls which grew for more than 1 year. There were no obvious toxic effects of the hormones. There were eight cultures in which there was no outgrowth. The small number of cultures tested have not allowed us to arrive at any firm conclusions about the effects of hormones on cervical cells at this time. Background levels of steroid hormones in one lot of fetal bovine serum were determined by standard radioimmunoassay (Abraham, 1974). Insulin determina-

42

GEORGE D. WILBANKS

et al.

FIG. 1. Phase contrast photomicrographs of primary normal human cervical epithelium in culture for 5 days. Bar represents 40 pm.

tions were not performed. Progesterone, hydroxyprogesterone, estradiol-l7& androstenedione, dehydrotestosterone, testosterone, hydroxycortisone, and dehydroepiandrostane sulfate were assayed and their concentrations in FE3S are listed in Table IV. Attempts were made to remove these steroid hormones from FBS with char-

2.

TISSUE CULTURE OF THE HUMAN UTERINE CERVIX

43

FIG.2. Primary normal human cervical epithelium in culture for 7 days. Bar represents 80 pm.

44

GEORGE D. WILBANKS

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FIG.3. Norman human cervical cells two weeks after second subcultivation. Note possible epithelioid replacement cells (see text). Bar represents 80 pm.

coal treatment (Heyns et al., 1967). The results of these attempts are presented in Table IV. The charcoal was effective in removing most of the progesterone, all of the dehydrotestosterone, all of the testosterone, and a considerable amount of the hydroxyprogesterone. Heat inactivation of FBS caused all of the dehydrotestosterone to be converted to testosterone. The effects of this “stripped” serum, with or without hormones added back at known concentrations, on cervical cell cultures are now being investigated. It was not known whether the background amounts of hormones in our serum were responsible for the effects of the added hormones. Milo et al. (1976) found inhibitory levels of progesterone in untreated FBS and these levels could explain the generally short longevity of an entire series of cultures from three patients biopsied. The lack of a striking difference between hormone-treated and untreated cultures suggests that inhibitory levels were already present in the FBS and that the effect could not be further increased because of already saturated hormone receptor sites. On the other hand, the one untreated culture that survived for greater than 12 months (Table 111) and three subcultivations may be significantly different, suggesting that the levels of added hormones were detrimental. Nevertheless, the small number of biopsies and cell cultures tested does not allow any definitive conclusions. Unlike human cells, the marmoset cells formed an apparently continuous line which is presently at passage 50 (Figs. 4 and 5). After a 1:2 dilution at the time of

2.

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TISSUE CULTURE OF THE HUMAN UTERINE CERVIX

TABLE IU OVERALL EFFECTOF HORMONE TREATMENT ON THE GROWTHPERFORMANCE OF NORMAL, HUMAN CERVICAL CELLSIN TISSUECULTURE" Number of cultures treated with hormone (number of serial subcultivations after hormone treatment) Culture interval Hormone and concentration Insulin 5 &ml 5 mg/ml P-Estradiot 1-2 pg/ml Progesterone 1-2 pg/ml Testosterone 1-2 pg/ml Hydrocortisone I x 10-4 M Cortisone 1 x 10-6M No Hormone Biopsy 113 Biopsy 129 Biopsy 274

<1 Month

>I2 Months

No growth

1-6 Months

6-12 Months

0 2 (2P)

3 (2P) 3 (2-3~)

0 0

0

1 (2P)

0

2 (IP)

2 (1P)

0

4

0

6 (IP)

5 (1-2P)

0

1

4 (1P)

2 (1P)

0

0

2

0

3 (IP)

0

0

0

0

3 (IP)

0

0

0

0 1 (IP)

0 0 5 (IP)

2 (2P) 0

1 (3P)

0 0 0

0

0

1 (7P)

0 0

1

"Cells were at subculture one or two at the beginning of treatment. Hormones were added at the time of explantation or subculture or up to 10 days later. Those receiving hydrocortisone or cortisone were switched to growth medium without added hormone after 2 months. Untreated control cultures were derived from the same patient biopsies as those cultures receiving added hormone.

subcultivation, confluent monolayers were formed within 3 to 5 days. These monolayers could be maintained for up to 1 month, if refed twice weekly with fresh growth media containing 10% FBS. This cell line appears to be purely epithelial without any contaminating fibroblasts. Unlike the human cells, it can be frozen down in liquid N2using 10%glycerol or dimethyl sulfoxide (DMSO). However, neither the human nor the marmoset cells could be cloned in soft agar. In organ cultures of separated cervical epithelium, the epithelial cells migrated over, and often digested, the porcine skin, leaving circular cleared, translucent areas in their wake. These cleared areas served to monitor cell outgrowth. heliminary histologic observations revealed that the layered growth of squamous cells was similar to that found on H and E sections of normal tissues in vivo. Ectocervical epithelium was maintained from 5 days to 1 month before degeneration and sloughing of the tissue layers were observed (see Figs. 6, 7, and 8).

46

et al.

GEORGE D. WILBANKS

TABLE IV STEROID HORMONE CONTENT OF FETALBOVINE SERUM USED IN OF CERVICAL CELLS

THE

TISSUECULTURE

Hormone concentrations (nglml) in fetal bovine serum

Hormone

Untreated

P Progesterone 17P: Hydmxyprogesterone E2: Estradiol-17p A: Androstenedione DHT: Dehydrotestosterone T Testosterone CpF: Cortisol D-S: DHEA sulfate"

0.148 0.104 0.145 0.069 0.049 0.096 0 0

Charcoal treated

Untreated; heat-inactivated at 56°C for 1 hour

0.06-0.088 0.080 0 0.063 0 0

0.202 0.110 0.166 0.088 0 0.169

0

0

0

0

Dehydroepiandrosteronesulfate.

FIG. 4.

Seven-day-old primary marmoset cervical cell culture. Bar represents 80 pm.

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TISSUE CULTURE OF THE HUMAN UTERINE CERVIX

47

FIG. 5 . Five-day-old culture of marmoset cervical cells at fortieth subculture. Bar represents 80 P-n.

Certain lots of porcine skin did not support outgrowth and the resulting clearing effect in the substrate; these were actually toxic and rapid degeneration of the cervical tissues occurred. It is suggested that lots be pretested before large-scale experimentation is launched using porcine substrates.

IV.

Perspectives

A . Current Applications and Findings Human uterine cervical tissues in cell or organ culture have been used to study a diverse group of biologic problems. Many of these studies center around carcinogenesis in this organ, as well as its normal physiology. Balduzzi et al. (1972) and Birch er al. (1976) used organ cultures of normal endocervix and ectocervix with and without the underlying stroma to show that herpes simplex virus type 2 (HSV-2) could infect and replicate in these tissues. Fink et al. (1975) used organ culture to study celIuIar events of metaplasia in the uterine cervix. Wilbanks and Fink ( 1976) and Marczynska et al. (1980) reported unsuccessful attempts to transform normal adult cervical epithelium of both human and marmoset origin in cell culture using UV-irradiated or unirradiated HSV-2, and/or incubation at 42"C, although primary hamster embryo and rat embryo cell

48

GEORGE D. WILBANKS et al.

FIG.6. Hematoxylin and emin-stained section of normal human cervical epithelium and stroma (HCx) on porcine skin (PS) maintained in organ culture for 2 weeks. Bar represents 40 pm.

FIG. 7. Hematoxylin and eosin-stained section of normal human cervical epithelium and stroma (same biopsy as in Fig. 6) on organ culture grids for 2 weeks. Note lack of cohesiveness of epithelial cells. Bar represents 40 pm.

2.

TISSUE CULTURE OF THE HUMAN UTERINE CERVIX

49

FIG.8. Hematoxylin and eosin-stainedhistological section of a biopsy of normal human cervical tissue (same biopsy as in Figs. 6 and 7). Bar represents 40 pm.

cultures became transformed under these conditions. Vesterinen et al. (1977)induced a persistent latent herpesvirus infection in human ecto- and endocervical epithelial cells using adenine arabinoside (ara-A). The inhibiting effects of human leukocyte interferon on normal cervical cell cultures have recently been reported by Brdar et al. (1977). Ishiwata et al. (1978) have shown the stimulatory effect of low concentrations of estradiol-17p on growth and differentiation of normal cervical squamous cells in culture. Rowinski et al. (1978) cultured normal fibroblasts of the human uterine cervix with various lesions and examined their nuclear features using image analysis. They found a wide variation (as much as 10-fold) in the size of the nucleus which could not be correlated with the type of lesion involved.

B . Future Studies Studies involving long-term effects on cervical tissues in vitro, e.g., carcinogen studies, should be performed in cell culture since only by this method can cells be maintained long enough for possible effects to be seen. Even then, a large number of biopsies must be sampled in order to obtain cultures that can be grown for a year or more. Organ culture should be used for relatively short-term studies. Short-term effects of drugs, physical, or biological agents, etc. seem to be suitable topics for study in organ cultures.

50

GEORGE D. WILBANKS

et al.

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