Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel

In Vitro Cell. Dev. Biol. 26:722-730, July 1990 �9 1990 Tissue Culture Association 0883-8364/90 $01.50+0.00

SERUM-FREE CULTURE OF ENRICHED MOUSE ANTERIOR AND VENTRAL PROSTATIC EPITHELIAL CELLS IN COLLAGEN GEL

TIMOTHY TURNER, HOWARD A. BERN', PETER YOUNG ANDGERALD R. CUNHA,

Cancer Research Laboratory and Department of Integrative Biology, University of California, Berkeley, California 94720 (H. A. B.) and Department of Anatomy. University of California,

San Francisco, California 94143 (T. T., P, Y., G. R. C.)

(Received 5 March 1990; accepted 23 March 1990)

SUMMARY

Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1:1 (vol/vol), supplemented with bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5a-dihydrotestosterone, cortisol, putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture. Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics. Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male mice. These tissue recombinants showed distinctive prostatic histologic characteristics (alvcoli and ducts lined with cuboidal or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258, epithelial cells of mouse origin were distinguishable from stromal cells of rat origin.

Key words: prostate; mouse; epithelial cells; primary culture; serum-free media; collagen gel.

INTRODUCTION

Various in vitro techniques have been employed to analyze the events occurring in vivo. Cell culture has proven to be a powerful method for studying and evaluating the many aspects of a particular cellular element under defined conditions. Specifically, in vitro cell culture is contributing to a better understanding of the factors involved in the growth and differentiation of the prostate gland.

Primary cultures of prostatic epithelial cells have been hampered by two fundamental problems: (a) the degeneration of epithelial cells with time in culture and (b) the concurrent outgrowth and domination of the cultures by fibroblasts (3,35,45). Difficulties such as these in the study of mammary epithelial growth were overcome by the development of a collagen gel culture system by Yang et al. (55). The separation of epithelial cells from stromal cells was carried out using various enzymatic combinations. Epithelial cell isolation was further enhanced in the mammary gland (56), as well as in the prostate {43), by the addition of a density gradient

' To whom correspondence should be addressed.

722

centrifugation step. This approach has allowed mammary epithelial growth in primary culture in serum-containing (49,56) and serum-free culture media (27). It has also been successfully implemented in the culture of mouse vaginal and endometrial epithelia by Iguchi et al. {24,25) and Uchima et al. (50) and rat ventral prostatic epithelium by O'Conner and Sinha (42) and Kawamura and Ichihara 130).

Prostatic development in vivo has been shown to depend on epithelial-mesenchymal interaction {13-15). The urogenital sinus mesenchyme (UGM) plays an important role in the induction of prostatic growth and development. Its significance in this process is illustrated by the following observations: UGM induces cell growth when recombined with adult prostatic ducts (41) and induces prostatic development when recombined with epithelium of the urogenital sinus, neonatal vagina, and embryonic and adult urinary bladder (11,12,15,16). Recently, the epithelial-mesenchymal recombination technique has been integrated with the collagen gel cell culture system to examine the loss or re-expression of hormonal responsiveness and normal morphology by vaginal and uterine epithelial cells previously cultured (8).

In the present study, the collagen gel culture system and tissue recombination techniques were adopted to establish enriched mouse prostatic epithelial cell cultures,

MOUSE PROSTATIC EPITHELIAL CELLS 723

to establish a serum-free environment capable of sustaining multifold growth, and to identify the cultured cells as mouse prostatic epithelial cells.

MATERIALS AND METHODS

Cell dissociation. Norma' ~nterior (AP) and ventral (VP) prostates were dissected from 2- to 3-mo-old intact male BALB/cCRGL mice, pooled separately, and kept in Medium 199 (GIBCO, Grand Island, NY) on ice. The glands were finely minced, resuspended in Medium 199, and centrifuged at 800 rpm for 5 min. The supernatants were discarded and the pellets resuspended in 50 ml Medium 199A (M199A) [Medium 199 supplemented with bovine serum albumin fraction V (BSA) (Sigma Chemi- cal, St. Louis, MO), 1 mg/ml; vitamin E (Sigma), 1 ~g/ml; catalase (Sigma), 100 U/ml; and superoxide dismutase (DDI Parmaceutical, Mountain View, CA), 10 ~g/ml] with 0.04% DNase (Sigma), collagenase [CLS I I I , 400 or 300 U/ml for the AP or VP, respectively (Worthington, Freehold, NJ)], and 0.1% hyaluronidase (Sigma). These solutions were swirled on a gyratory water bath shaker at 150 to 175 rpm at 37 ~ C for approximately 3 to 4 h or until observations under an inverted phase contrast microscope revealed good epithelial-stromal separation. The cells were collected by centrifugation at 800 rpm for 5 min and washed once with Medium 199. To aid in further dissociation of stroma from epithelium, the pellets were resuspended in 50 ml Medium 199A with pronase (250 U/ml, Sigma) and gently swirled on a gyratory water bath shaker for 15 to 30 rain. The cells were collected by centrifugation, washed in Medium 199, and enriched for prostatic epithelial cells by adding the cells to a preformed Percoll (Pharmacia, Piscataway, NJ) density gradient. This solution was centrifuged at 3000 rpm for 20 min; the bottom fraction of the gradient, consistently found to contain predominantly epithelial elements, was collected and washed in Medium 199. The washed pellet was resuspended in 12 ml Dulbecco's modified Eagle medium:Ham's F12 medium (D:H, both from GIBCO), 1:1 (vol/vol) with 0.04% DNase and vortexed for 3 to 5 s. The cells were allowed to settle, and the supernatant was removed in an attempt to eliminate any stromal elements released during vortexing. The pellet was suspended again in 12 ml 0.04% DNase and the larger cell clumps (probably containing stromal contaminants) were allowed to settle. The supernatant was retained and the larger cell clumps were discarded. The cells were collected by centrifugation, the pellet was resuspended in 1 ml 0.04% DNase in D:H, and the mixture was placed on ice until plated in collagen gels. Cell number was estimated by mixing 1 vol cell suspension with 9 vol 0.02% crystal violet in 0.1 M citric acid and counting the stained nuclei in a hemacytometer.

Culture procedure. Collagen solution and gels were prepared as originally described by Michalopoulos and Pitot (37). Briefly, 1 g rat tail collagen fibers was sterilized in ethanol overnight and dissolved in 300 ml acetic acid in sterile distilled water, 1:1000 (vol/vol); the supernatant after centrifugation at 10 000 Xg for 30 min provided the stock collagen solution. Eight volumes stock solution were

mixed with 2 vol 10X Waymouth medium (GIBCO):0.34 M NaOH (2:1; vol/vol), keeping pH and osmolarity of the final mixture within the physiologic range. This mixture was kept on ice to prevent immediate gelation. Cells were added to the cold gelatin mixture; 0.5 ml, containing 0.5 to 2.0 X 105 cells, was placed on 0.3 ml gelled collagen in each well of Falcon 24-multiwell plates (Falcon, Oxnard, CA) and allowed to gel at room temperature. After gelation, the collagen was overlain with culture medium and the cells were cultured for 10 or 14 d. Cultures were analyzed on Days 0, 2, 4, 6, 8, 10, 12, and 14. DNA was assayed by transferring the collagen gels (containing cell colonies) to 10 X 75-mm glass tubes (Fisher Scientific Co., Pittsburg, PA); the cells were either recovered from the gels at this point or the gels were immediately frozen and cells recovered from the gels at a later time. Cells were recovered from the collagen gels by dissolving the gels by the addition of 50 /A 25% glacial acetic acid (52). The cells were then pelleted, and the pellet was extracted with 70% ethanol, dried, and stored until used for DNA assay.

The cells were cultured in a medium consisting of HEPES-buffered (20 raM) D:H medium, containing NaHCO3 (1.2 g/liter), penicillin (50 U/ml, Sigma) and streptomycin (37 U/ml, Sigma). This medium was supplemented with BSA (10 mg/ml), insulin (In; 10 /~g/ml, Sigma), epidermal growth factor (EGF; 10 ng/ml, Collaborative Research, Waltham, MA), transferrin (Tf; 10 /~g/ml, Sigma), cholera toxin (CT; 10 ng/ml, Sigma), 5a-dihydrotestosterone (DHT; 1 ~g/ml, Sigma), ovine prolactin (PRL; 5 /~g/ml, NIH-P-S.11), trace element cocktail [TE: 1 gl/ml, consisting of 1 mM each of MnCI~ '4H20 , NazSiO3"9H~O, (NH4)6MoTOz4"4H20, NH4VO3, NiCI2"6H~O,SnCI2"2H20, and 2 mM of H2SeO~, gift from S. Nandi], cortisol (F; 5/~g/ml, Sigma), putrescine (10/~g/ml, Sigma), and ovine pituitary basic fibroblast growth

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x

d z 2 �84

O

T ODH /

TO D2 D4- D6 D8 DIO

FIG. 1. Growth of ventral prostatic epithelial cells cultured in collagen gel matrices and nourished with a defined medium consisting of the D:H medium supplemented with In, EGF, BSA, Tf, DHT, PRL, CT, and TE (referred to as SF medium). TO represents the initial plating density; DH refers to the unsupplemented medium after 10 d in culture. Time points were taken on Days D2, D4, D6. D8, and D10. Estimates of cell number were derived from an average of three experiments, three wells per experiment. Mean of the triplicate determinations for each point in an experiment was considered as a single determination. Vertical lines indicate SEM.

724 TURNER ET AL.

factor (FGF; 200 ~g/ml, gift from D. Gospodarowicz}. All cultures were incubated at 37 ~ C in 95% air:5% CO2. The serum-free medium was changed every 2 d. The DNA content was determined in triplicate wells for each point by a fluorometric assay (23) utilizing BALB/cfC3H mammary tumor epithelial cells counted in a hemacytometer as a standard. The mean of the triplicate determinations for each point in an experiment was calculated and considered as a single determination. Each experiment was repeated 3 times (unless otherwise stated); the mean and standard error of the mean (SEM) were calculated from these values.

Cultures for light microscopy were fixed in Bouin's fluid, dehydrated in ethanols, and embedded in paraffin. These embedded samples were sectioned at 7/~m and stained with hematoxylin and cosin. For transmission electron microscopy, cultures were fixed at room temperature for 2 h in 1% formaldehyde:3% glutaraldehyde:0.1 M sodium eacodylate, pestfixed for 1 h in 1% OsO4/0.1 M sodium eacodylate, stained ] to 2 h e n bloc with saturated uranyl acetate dehydrated in ethanol, and embedded in Spurr's resin. Thin sections stained with uranyl acetate and lead citrate were examined in a Siemens Elmiskop 103 electron microscope.

Tissue preparation for recombination and transplantation. Embryonic urogenital sinuses were taken from 17- to 18-d Sprague-Dawley rat fetuses and separated into urogenital sinus mesenchyme (rUGM) and epithelium by incubation in 1% trypsin (1:250, Difco Laboratories, Detroit, MI) in calcium- and magnesium-free Hanks' balanced salt solution (University of California, San Francisco Cel[ Culture Facility) for 2 h at 4 ~ C (16). The rUGM obtained was used for recombination with cultured prostatic epithelium.

Prostatic epithelium was separated and cultured for 10 d in collagen gels as previously described. Epithelium for recombination was obtained by incubating the epithelium-containing collagen gels with 1% collagenase for 30 to 45 rain at 37 ~ C. Isolated epithelial colonies were removed and washed in calcium- and magnesium-free Hanks' balanced salt solution.

Recombination of rUGM and mouse prostatic epithelium followed the procedure of Cooke et al. (8). Briefly, rUGM and cultured prostatic epithelium were recombined by placing pieces of rUGM on a solidified agar medium and then transferring isolated epithelium with a drawn-out Pasteur pipette onto the rUGM (11). The recombinants were allowed to adhere overnight in a humidified incubator and then grafted under the renal capsule of an intact male BALB/c athymic mouse. After 4 wk of growth in the host, the transplants were prepared for histologic examination.

The heterospecific recombinant samples were fixed in buffered formalin, embedded in paraffin, and sectioned at 6 /~m. To distinguish mouse epithelium from rat mesenehyme, Hoechst dye 33258 was used as described by Cunha and Vanderslice (17). Briefly, formalin-fixed paraffin sections were stained with 4 pg/ml Hoechst 33258 in Hanks' balanced salt solution without phenol red, rinsed in running water, mounted in McIlvaine's buffer at pH 5.5, and sealed with clear lacquer. Slides were examined by fluorescence microscopy to distinguish rat

and mouse cells by their unique patterns of nuclear fluorescence: mouse cell nuclei contain several small discrete fluorescent bodies, which are absent from rat cell nuclei.

RESULTS

Development of serum-free culture conditions, Initial- ly, normal, freshly dissociated ventral prostatic epithelial cells were cultured in collagen gel matrices and nourished with a defined medium consisting of the D:H medium supplemented with In, BSA, EGF, CT, Tf, DHT, PRL, and TE. This medium will be referred to as sermn-free (SF). The growth patterns of ventral prostatic epithelial cells were monitored by viewing the cultures in an inverted phase contrast microscope, and growth was quantified later by measuring the total DNA content per well as a function of time. The SF medium was only capable of maintaining a slight increase above the initial seeding density during a 10-d culture period (Fig. 1). Around Days 6 to 8 the cultures began to show considerable cell death and colony degeneration, as observed under the phase microscope.

Cortisol, putrescine, and FGF were added to the SF medium in an attempt to increase cell number and cell viabil- ity over the culture period. This combination of components will be referred to as the SF plus (SFq-) medium. Growth studies of mouse ventral prostatic epithelial cells nourished with different combinat ions of media (SF-F; SFq-minus putrescine and FGF; SF4- minus FGF) were examined over 14 d in culture (Fig. 2). In all three combinations there was a maintenance of the initial seeding density for the first 4 d. The cell number

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SF + F + Putrescine

2- ~2~ SF + F + Putrescine + FGF

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FIG. 2. Growth of ventral prostatic epithelial cells cultured in collagen gel matrices and nourished with a defined medium consisting of the D:H medium supplemented with SF components plus F; SF plus F and putrescine; SF plus F, putrescine, and FGF. TO represents the initial plating density; DH refers to the unsupplemented medium after 14 d in culture. Time points were taken on Days D2, D4, D6, D8, D10, DI2, and D14. Estimates of cell number were derived from an average of three experiments, three wells per experiment. Mean of the triplicate determinations for each point in an experiment was considered as a single determination. Vertical lines indicate SEM.


increased in each medium tested on Days 6 through 14. Cells cultured in S F + displayed the most growth over the culture period: an eighffold increase in cell growth over 14 d in culture. The small clumps of cells embedded in the collagen gel matrix produced three-dimensional outgrowths which extended throughout the gel during the culture period (Fig. 3 a,b). Histology of these out-

r--q SF + F F-721 SF + F + Putrescine

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FI6. 6. Growth of anterior prostatic epithelial cells cultured in collagen gel matrices and nourished with a defined medium consisting of the D:H medium supplemented with SF components plus F; SF plus F and putrescine; SF plus F, putrescine, and FGF. TO represents the initial plating density; DH refers to the unsupplemented medium after 14 d in culture. Time points were taken on Days D2, D4, D6, D8, D10, D12, and D14. Estimates of cell number were derived from the mean of triplicate wells for each point. Vertical lines indicate SEM.

FIo. 3. a, Appearance of a ventral prostatic epithelial cell colony embedded in the collagen gel matrix and nourished with SF+ medium after 2 d in culture�9 b, Three-dimensional outgrowth of ventral prostatic epithelial cell colony embedded in the collagen gel matrix and nourished with SF+ medium after 8 d in culture. )<50.

FIG. 4. Histology of ventral prostatic epithelial cell colony grown in collagen gel matrix for 10 d and nourished with SF+ medium. H&E. )< 130.

FIO. 5. Electron microscope observation of ventral prostatic epithelial cells cultured in collagen gel matrix for 10 d and nourished with SF+ medium. Note the appearance of a lumen ~Lt, secretory vesicles iS), tight junctions ~TI, and desmosomes tD), characteristic features of epithelial cells. IElectron microscopy by Susan Hamamotot X16 400.

growths showed the formation of acinuslike structures (Fig. 4). Electron microscope observations of these cultures revealed secretory vesicles, desmosomes, and fight junctions, characteristic features of epithelial ceils (Fig. 5j. Signs of cell death and colony degeneration were observed in cells cultured with S F + from Days 8 to 14.

The growth potential of the SF medium and the three different medium combinations mentioned above was tested on mouse anterior prostatic epithelial cells in culture ~Fig. 6). Cells cultured in the four different media displayed a maintenance of the initial seeding density for the first 4 d of culture. All cultures continued to increase in cell number from Days 6 through 10. Cells cultured in all media except SF were able to maintain their maximum growth for the duration of the culture; cells cultured in unsupplemented SF decreased after Day 10 until cultures were terminated. Cells grown in all media increased at least eighffold in cell number through the culture period; S F + medium supported the most growth during culture. Anterior prostatic epithelial cells grown in the collagen gel matrix produced three-dimensional outgrowths similar to outgrowths observed in epithelial cells from the ventral prostate (Fig. 7D. Histologic sections of outgrowths of anterior prostatic epithelial cells showed the formation of lobular structures (Fig. 8j. Colony degeneration generally began after Day 8 in culture.

Recombination experiments. After 10 d of culture, collagen gels containing numerous anterior and ventral prostatic epithelial colonies were incubated in collagenase. The isolated prostatic epithelial cells were recombined with rUGM and grown for 4 wk under the renal capsule of an intact male nude mouse. Both types of recombinants grew vigorously during the 4-wk incubation period as was evident by the increase in size of the transplants at the time of their removal for histology.

726 TURNER ET AL.

Histologic observation of the recombinants revealed the normal, distinctive prostatic characteristics of each respective gland. Recombinants consisting of cultured anterior or ventral prostatic epithelial cells + rUGM showed alveoli and duets lined with cuboidal or columnar epithelium and surrounded by a thin fibromuscular

stroma IFigs. 9 and 10). The lumina frequently contained eosinophilic material, histologic evidence o| secretory activity (Figs. 9 and 10).

When histologic sections of recombinants were stained with Hoechst 33258, cells of mouse origin were distinguishable from rat cells (17). Both recombinants


displayed no stromal contamination in the cultured epithelium as all epithelial components were of mouse origin and all stromal elements were of rat origin (Figs. 11 and 12).

D ISCUSSION

The collagen gel culture system has permitted a variety of cell types used in primary culture to remain viable and demonstrate growth over many days in culture (see Introduction). The collagen matrix enables the particular cell type to be embedded in an environment that mimics, to some extent, its natural surroundings, thus allowing the cells to grow in a three-dimensional fashion, often similar to that found in vivo. This study utilized the collagen gel culture system to grow mouse prostatic epithelial cells in a sertun-free environment capable of sustaining growth.

When cultured prostatic epithelial cells were recombined with rUGM and transplanted under the renal capsule of an intact male nude mouse for a period of time, the recombinants displayed histologic characteristics typical of AP or VP from intact male mice. Morphologi- cally, this was apparent by the occurrence of numerous alveoli and ducts lined with cuboidal or columnar epithelium or both and surrounded by a connective tissue stroma, with secretion evident in the htmen {Figs. 9 and 10). The connective tissue cells surrounded each duct in close apposition to the epithelium. The Hoechst staining method of Cunha and Vanderslice (17), which distin- guishes tissues of mouse and rat origins by differences in their nuclear fluorescent staining patterns, was employed to prove that the epithelium in the transplants originated from the mouse prostatic cultures. The nuclei of the mesenchymal cells stained homogeneously, the pattern characteristic of rat tissue, whereas the punctate pattern d nuclear fluorescence displayed by epithelial cells was characteristic of mouse tissue ~17) (Figs. 11 and 12).

Both anterior and ventral prostatic epithelial cells embedded in col lagen gels grew well in the established S F + medium. Within 10 d of culture, the cell number increased sevenfold over the original seeding density. In this medium, the cultured prostatic epithelial cells seemed healthier than cells cultured in other media. Histologically, both prostatic epithelial cells cultured in collagen gels showed the lobulelike arrangement (Figs. 4 and 8) seen i n similarly cultured rat ventral prostatic epithelial cells by O'Conner and Sinha (42). Ultrastructur- ally, ventral prostatic epithelial cells possessed secretory

vesicles, tight junctions and desmosomes, characteristics associated with epithelial cells (Fig. 5).

The successful growth of prostatic epithelial cells derives not only from the use of the S F + medium, but

FIG. 11. Fluorescence microscope observation of a section of cultured mouse anterior prostatic epithelial cells + rUGM stained with Hoechst 33258. Mouse cells show a punctate pattern of nuclear fluorescence {arrows), whereas nuclei of rat stromal ceils show homogeneous fluorescence. X350.

FIG. 12. Fluorescence microscope observation of a section of cultured mouse ventral prostatic epithelial cells -4- rUGM stained with Hoechst 33258. Mouse cells show a punctate pattern of nuclear fluorescence ~arrows), whereas nuclei of rat stromal cells show homogeneous fluorescence. X350.

FIG. 7. Three-dimensional outgrowth of anterior prostatic epithelial cell colony embedded in the collagen gel matrix and nourished with SF+ medium after 8 d in culture. X85.

FIG. 8. Histology of anterior prostatic epithelial cell colony grown in collagen gel matrix for 10 d and nourished with SF+ medium. H&E. X130.

FiG. 9. Histology of transplanted recombinant consisting of cultured mouse anterior prostatic epithelial cells + rat urogenital sinus mesenchyme (rUGML Note the prostatic characteristics: alveoli and duets lined with cuboidal or columnar epithelium and surrounded by fibromuseular stroma. Also note that some lumens contain seeretorylike material. H&E. X245.

FIG. 10. Histology of transplanted recombinant consisting of cultured mouse ventral prostatic epithelial cells + rUGM. Note the prostatic characteristics: alveoli and ducts lined with cuboidal or cohunnar epithelium and surrounded by fibromuseular stroma. Also note that some lumens contain secretorylike material. H&E. X245.

728 TURNER ET AL.

also from the utilization of collagen gel as a three- dimensional matrix and the inclusion of M199A in the cell dissociation process. The significance of collagen gel matrices for epithelial cell culture has been reviewed by Yang and Nandi (54). M199A was used during the enzymatic dissociation process owing to its successful enhancement of epithelial cell growth in mouse and rat mammary cultures (D. Wallace, R. Guzman, and J. Richards, personal communication). It is believed that the antioxidants in M199A (vitamin E, catalase, and superoxide dismutase) play a principal role in decreasing the formation of harmful free radicals during the enzymatic dissociation process (J. Richards, personal communication).

Several investigators have reported successful growth of normal epithelial cells in serum-free media in primary cultures in collagen gel ( see Introduction). The factors chosen to constitute the present serum-free medium are a combination of hormones and growth factors which have been shown to be essential in the maintenance or growth or both of prostatic epithelial cells or other epithelial cell types in serum-free or serum-containing cultures. In several studies, In and EGF (24,27,53) or In, EGF, and glucocorticoid (2) are common constituents of the media. These three factors were found to he essential for rat prostatic epithelial cell proliferation (36). McKeehan et al. (36) have reported conditions that support the rapid and sustained proliferation of isolated normal rat prostatic epithelial cells in the absence of serum. Their results revealed direct mitogenic effects of In, EGF, glucocorticoid, CT, PRL, and an unknown pituitary-derived factor, later defined as prostatropin (9). The proliferation of adult human prostatic epithelial cells under serum-free conditions has been shown to be dependent on the addition of CT, EGF, a pituitary extract and F to a basal medium (44). Recently, Nishi et al. (40) have demonstrated the growth of rat dorsolateral prostatic epithelial cells in a serum-free nutrient medium supplemented with In, CT, EGF, dexamethasone, and FGF. Each of these factors or related ones has been in- cluded in the S F ~ medium herein.

Insulin is necessary in most epithelial cell culture systems. It, and other members of the insulin family of peptides, are known to have potent growth-promoting effects (48). In mammary epithelial cultures, evidence has suggested that In may mediate its growth-promoting effects through interaction with insulinlike growth factor receptors (26). Specific receptors for In have also been shown to be present on the rat prostatic epithelial cells (4). Due to the recent demonstration of insulinlike immunoreactivity in the epithelium of the rat prostate gland, it has been suggested that there may be some local In or insulinlike protein synthesis in this organ (47).

Epidermal growth factor has been shown to be a potent mitogen for the basal cell layer of various epithelia of ectodermal origin as well as a delayer of senescence for a variety of cultured cells of ectodermal and mesodermal origin (for review, s e e 21). Glucocorticoid regulates EGF receptor concentration; it also promotes the release of autocrine growth factors into the culture medium by fibroblasts (for review, s e e 10). CT exerts its effects in

most cells by increasing the accumulation of intracelluiar cyclic AMP (for review, s e e 38).

Considerable evidence suggests that PRL is an active participant in the regulation of prostatic growth and function. It acts synergistically with androgen in the support and stimulation of prostatic growth and metabolism. In hypophysectomized rats, exogenous androgens were incapable of restoring full prostatic growth unless accompanied by exogenous PRL (6) which may help androgens cross the plasma membrane (20L

The pituitary extract that McKeehan et al. t36) found to be essential for prostatic epithelial cell growth in culture was later characterized as a beparin-binding growth factor referred to as prostatropin (9). Prostatropin is also identical to acidic FGF. Basic FGF, which is also synonymous with a prostatic growth factor (22), was chosen for the SF~ medium as the result of experiments indicating that acidic FGF and basic FGF have equal mitogenic activity for prostatic epithelial cells and compete for the same 130 kDa cell surface binding site (34).

The other components of the SF+ medium are also known to be important in primary culture. BSA, a commonly used component in serum-free media, has been shown to stimulate growth of the mammary epithelial cells (27). The serum-protein transferrin has been reported to be essential for serum-free culture of metanephric mesenchyme (18) and is a co-mitogen for ovarian cells (28). This protein may provide iron for the cell (29,31).

Trace elements were added to prostatic epithelial cell cultures to supplement other trace elements present in the medium (51). Elevated cellular polyamine levels have been found to be associated with growth processes (1,7). The diamine putrescine, used in the S F ~ medium, gives rise to the polyamine spermine. Spermine has been shown to enhance the survival of mouse prostatic epithelial cells in culture (51).

Although androgens are an essential factor in the growth and maintenance of the prostate in vivo (6; for review s e e ]4), the majority of the reports in the literature fail to show any positive growth-promoting effects on prostatic epithelial cells in vitro (5,32,33,36,40). It is believed that one potential indirect effect of steroid hormones could be the up-regulation of receptors for growth factors of cells in culture. The number of EGF receptors in T47D, MCF7 (19) and HeLa $3 (39) cells is increased after hormonal treatment of these cells with glucocorticoids and progestins, respectively. Recently, Schuurmans et al. (46) have suggested that androgen- responsive growth of LNCaP cells, derived from a human prostate tumor, was in part caused by an increase of EGF-receptor expression directly related to androgen exposure.

Although growth of rat prostatic epithelial cells has been reported using the collagen gel matrix in serum- supplemented medium (30,42), the present study successfully used this matrix to grow mouse prostatic epithelial cells in a chemically defined medium. The defined medium meets the requirements for prostatic epithelial


cells in vitro. It has been able to sustain growth of mouse anterior and ventral prostatic epithelial growth over 10 d in collagen gel culture. These culture conditions have allowed the multifold proliferation of cells, which after transplantation retained the characteristics of prostatic epithelial cells.

Although prostatic epithelial cell growth was evident for the length of the culture period, cell degeneration became apparent around Day 8 and continued until the cultures were terminated. The several differentiated functions expressed by some epithelial cells in culture lead one to suspect that different cell types within a heterogeneous populat ion might require a special combination of factors for growth, and a somewhat different set of factors for differentiation. If this is the case, then the cell degeneration observed may result from the lack of some factor{s) in the culture medium, needed to meet the possible changing requirements of the cells over time in culture. Because in this study the cell number never decreased to initial levels, the combination of factors comprising the S F + medium supports the growth and maintenance of at least some colonies; the absence of the correct combination of factors could result in the degeneration observed in other colonies.

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The authors thank Drs. R. Guzman and J. Richards and Ms. D. Wallace for their critical input in this study. We would also like to thank K. T. Mills, K. Takemura, E. Fisher, and T. Herndon for their capable technical assistance; S. Hamamoto and N. Lidicker for histology; and J. Underhill for photography. Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship GM08730 to T. T.

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Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel