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The Prostate 32:43–48 (1997)
Rat Prostate Explants in Serum-Free Organ
Culture: A Comparison of Two Media and
Gas Mixtures
Xuan Khai Nguyen-Le,1 Jacques Corcos,2* and Normand Brière1
1
Département d’Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de
Sherbrooke, Sherbrooke, Québec, Canada
2
Department of Urology, McGill University, Montreal, Québec, Canada
BACKGROUND. Urologists are looking for a way to easily discriminate between aggressive
and very slow-growing prostate tumors. A sound way to appreciate such developing activities would be to identify an appropriate cell marker in prostate explants maintained in a
defined culture system.
METHODS. Different biological parameters were compared in rat prostate explants cultured
for 5 days in rich CMRL or basic Leibovitz’s L-15 medium, unsupplemented with serum,
under a mixture of either 95% air/5% CO2 or 50% N2/45% O2/5% CO2.
RESULTS. DNA synthesis was somewhat similar with the two-gas combination, but was
higher in explants maintained in L-15 medium than in CMRL. Hence, L-15 medium and the
95% air/5% CO2 mixture were selected. Under these defined conditions for 5 days, cells were
still able to synthesize DNA and proteins while preserving their morphological integrity and
maintaining alkaline and acid phosphatase activities.
CONCLUSIONS. Since the present culture system works well in a controlled environment
and under such minimal conditions, it appears to be a reliable and promising model that will
provide basic data and allow the study of hormones and growth factors involved in prostatic
tissue growth. It might eventually permit the identification of a cell marker. Prostate 32:43–48,
1997. © 1997 Wiley-Liss, Inc.
KEY WORDS:
prostate; culture conditions; enzyme activities; DNA synthesis; protein
synthesis
INTRODUCTION
The recently introduced ‘‘watchful waiting’’ concept has further increased the controversy surrounding prostate cancer management. This ‘‘nontouch’’ approach is supported by existing biological variations
in the progression of prostate neoplasms. Some of
these tumors are aggressive and require correspondingly drastic treatment, but most grow very slowly,
raising doubts about the need for treatment.
Urologists are looking for a way to easily discriminate between these two classes of tumors characterized by different prognoses. A sound way to appreciate such developing activity would be to find an appropriate cell marker. To identify such a marker,
maintenance of live prostatic explants outside their
© 1997 Wiley-Liss, Inc.
normal in vivo environment appears to be the first
logical step. Moreover, the culture medium should
ideally contain a minimum of components and be devoid of serum, hormones or growth factors, etc.
The aim of the present work was to compare basic
data on rat prostates in a culture medium (CMRL)
previously used by other investigators for prostate
culture to those obtained with a minimal medium
(Leibovitz’s L-15) currently employed in our labora-
*Correspondence to: Dr. Jacques Corcos, now at the Department of
Urology, Jewish General Hospital, 3755, Chemin de la Côte Ste.Catherine, Montreal, Québec H3T 1E2, Canada.
Received 8 May 1996; Accepted 21 June 1996
44
Nguyen-Le et al.
tory for culturing the kidney [1] and intestine [2]. To
the best of our knowledge, the latter medium has
never been used for culturing prostatic tissue.
man scintillation system (Beckman Instruments, Fullerton, CA). All results were expressed as disintegrations per min (dpm) per mg of DNA.
MATERIALS AND METHODS
Enzyme Activities
Culture
At each culture time interval (0 and 5 days), approximately 25 explants from the same organ were
pooled and homogenized in ice-cold redistilled water
in a Polytron Omnimixer (Brinkman Instruments,
Rexdale, Ontario, Canada) for 1 min at half-maximal
speed. Alkaline phosphatase and acid phosphatase activities were measured according to the techniques of
Eichholz [7] and Seitz and Aumüller [8], respectively.
The activity of each enzyme was expressed as mmoles
of substrate hydrolyzed per min (international units,
IU) per g of protein.
The prostate was removed from anesthetized adult
male Wistar rats weighing about 200–250 g. It was cut
into explants (2 × 2 mm) and deposited on lens paper
(Canlab Supplies, Ltd., Montreal, Quebec, Canada)
covering a stainless steel grid overlying the central
well of an organ culture dish (Falcon Plastics, Los Angeles, CA). The explants (6–9/dish) were maintained
in culture medium devoid of serum for 5 days at 37°C
in a controlled humidified atmosphere. To prevent
contamination, garamycin (40 mg/ml) and mycostatin
(40 mg/ml) were added to the medium. The culture
media were renewed every 2 days.
Culture Conditions
Two culture media of known composition (GIBCO,
Burlington, Ontario, Canada) were tested. Leibovitz’s
L-15 medium contains minimal nutrients and is devoid of serum and glucose, but is enriched with Dgalactose (900 mg/l). CMRL-1066 medium (Connaught Medical Research Laboratories, Willowdale,
Ontario, Canada), which is much more complete, was
previously used for explant culture of rat prostates. A
humidified mixture of 95% air/5% CO2 [1] was compared to 50% N2/45% O2/5% CO2 as employed by
Heatfield et al. [3] for long-term explant culture of
normal human prostates in CMRL medium.
DNA and Protein Synthesis
To assess cell proliferation, DNA synthesis was
evaluated by culturing explants in the presence of 3Hthymidine added to the culture medium (5 mCi/ml;
77.8 Ci/mmol; New England Nuclear Corporation,
Boston, MA). Protein synthesis was determined following incubation in the presence of 3H-leucine (8
mCi/ml; 60 Ci/mmol). Both radioactive precursors
were added during the last 4 hr of culture. After incubation, DNA and proteins were extracted as described by Malo et al. [4]. DNA content (in mg DNA/
mg tissue) was evaluated by the procedure of Giles
and Myers [5] using calf thymus as standard. Protein
content (mg protein/g tissue) was estimated according to the method of Lowry et al. [6], with bovine
serum albumin (BSA) as standard. Incorporation of
radioactive precursor into DNA and proteins was
quantified by counting 0.5 ml of the filtrate in a Beck-
Statistical Analysis
The results were expressed as mean ± SEM, and
statistical significance of the difference between means
was determined by Student’s t-test. The level of significance was fixed at P < 0.05.
Morphology
At days 0 and 5 of culture, some explants were
fixed overnight in ice-cold 2.8% glutaraldehyde and
washed in cacodylate buffer (0.1 M)-sucrose 7.5%, pH
7.4, postfixed in 1% OsO4 buffered with 0.1 M cacodylate, dehydrated in ethanol, and embedded in Epon
812. For light microscopy, semithin sections (1 mm)
were prepared and stained with toluidine blue. For
electron microscopy, thin sections were stained with
uranyl acetate and examined in a Philips 300 electron
microscope. For scanning electron microscopy, some
explants, following fixation in glutaraldehyde, were
cryofractured at mid-dehydration. The ethanolinfiltrated tissues were critical point-dried, using liquid CO2 without prior treatment with amyl acetate.
Afterwards, they were gold-coated and examined in a
Stereoscan 120 scanning electron microscope at 15–
20 kV.
RESULTS
Culture Conditions
To determine the best conditions for the maintenance of rat prostate explants in culture, two culture
media and two gas mixtures were tested.
In a humidified mixture of 95% air/5% CO2 (Fig. 1),
uncultured explants (day 0) displayed higher DNA
synthesis following 4 hr of incubation in the presence
of 3H-thymidine added to Leibovitz’s L-15 than when
Rat Prostate Explants in Organ Culture
Fig. 1. In a humidified mixture of 95% air/5% CO2, the level of
DNA synthesis is higher in prostate explants cultured in L-15 than
in CMRL at the beginning of culture (day 0 + 4 hr of 3H-thymidine
incorporation) and after 5 days. Symbols indicate a significant difference. Level of significance was fixed at P < 0.05.
the precursor was added to CMRL (1,333 ± 13 vs. 459
± 109 dpm/mg DNA; P < 0.0005). After 5 days of culture, DNA synthesis was significantly decreased, an
observation reported previously in other culture systems. The reduction was rather similar in L-15 (64%)
and in CMRL (67%). However, DNA synthesis remained significantly higher in L-15 than in CMRL (481
± 65 vs. 153 ± 44 dpm/mg DNA; P < 0.005).
In a mixture of 50% N2/45% O2/5% CO2 (Fig. 2),
3
H-thymidine incorporation into DNA was also significantly higher in explants maintained in L-15 than
in CMRL on day 0 (741 ± 162 vs. 318 ± 35 dpm/mg
DNA; P < 0.025) as well as on day 5 of culture (424 ±
71 vs. 169 ± 42 dpm/mg DNA; P < 0.01). In explants
maintained in L-15 for 5 days, DNA synthesis was not
significantly different with both gas mixtures (Fig. 1,
481 ± 65 vs. Fig. 2, 424 ± 71 dpm/mg DNA). When the
tissues were cultured in CMRL, the level of 3Hthymidine incorporation into DNA after 5 days was
also quite similar with the two-gas mixtures (Fig. 1,
153 ± 44 vs. Fig. 2, 169 ± 42 dpm/mg DNA).
In summary, no significant difference in DNA synthesis was observed with the two-gas combinations.
However, the level of 3H-thymidine incorporation
was higher in explants maintained in L-15 than in
CMRL. Hence, L-15 and 95% air/5% CO2 were selected for continuation of the study.
Morphology
Prostate explants kept in L-15 presented some visible and progressive changes during culture; they
were softer, flattened, and shrunken, and they appeared smaller. These alterations were more apparent
45
Fig. 2. In the gas combination of 50% N2/45% O2/5% CO2,
incorporation of 3H-thymidine into DNA was significantly superior
in explants maintained in L-15 than in CMRL, at the beginning of
the culture and after 5 days.
after 5 days of culture. Within 2 weeks, necrosis was
observed in the explants which now appeared very
pale.
At the light microscopic level, in uncultured explants, the gland acini were composed of a large lumen lined with a simple, high columnar epithelium
(Fig. 3). After 5 days of culture, the glandular epithelium changed from a columnar to a cuboidal shape,
resulting in apparent enlargement of the lumen (Fig.
4), which often contained cell remains dispersed in
secretory substances.
At the electron microscopic level, the structural features of cytoplasmic organelles such as nuclei, mitochondria, Golgi apparatus, and rough-surfaced endoplasmic reticular elements looked normal at the
beginning of culture plus 4 hr of incubation in L-15
supplemented with 3H-thymidine. The apical cytoplasm was filled with dense secretory vesicles, and the
plasma membrane formed numerous microvilli (Fig.
5). The number and various lengths of these membrane specializations were better appreciated by scanning electron microscopy. After 5 days in serum-free
L-15, the microvilli were preserved, but the number of
secretory vesicles had decreased, while free ribosomes
were more abundant (Fig. 6).
In summary, rat prostate explants can be maintained for at least 5 days in L-15, a minimal culture
medium devoid of serum or hormones. In fact, these
explants, kept under minimal and poor nutrient conditions, were still able to incorporate 3H-leucine and
synthesize proteins, although at a limited rate, after 5
days of culture (Fig. 7), suggesting functional endoplasmic reticulum machinery. Also, the activities of
alkaline phosphatase, a marker of microvillus differentiation in epithelial cells, and acid phosphatase, a
prostate lysosomal marker, were shown to be main-
46
Nguyen-Le et al.
Fig. 3. Light micrograph of rat prostate section from an uncultured explant. Each gland acinus comprises a large lumen (L) lined
by a simple high columnar epithelium. Secretory units are surrounded by connective tissue elements. ×225.
Fig. 4. Prostate explant cultured for 5 days in L-15, a basic
serum-free medium. The epithelium appears rather cuboidal (arrow). Small dark cells and debris are observed around some acini
and in the lumen. ×225.
tained (at approximately 100 and 75 IU/g protein, respectively), indicating the preservation of certain functional enzymes during the culture period. Considered
together, these results demonstrate that the above culture model enables the maintenance of normal rat explants for periods long enough to study the influence
of growth factors on the proliferation and differentiation of prostate tissues. One of these regulators might
eventually be used as a marker of progression in prostatic carcinogenesis.
probably resulting from inappropriate culture conditions [19].
It is noteworthy that Bologna et al. [20] succeeded
with short-term cultures of human prostatic carcinoma in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum, hydrocortisone, and insulin. Another group of investigators [3] maintained
normal human prostatic explants for 24 weeks in
CMRL medium in a mixture of 50% N2/45% O2/
5% CO2.
A common denominator with most of these models
is the use of rich media supplemented with fetal serum. Because of its unknown composition, serum is
considered undesirable in investigations where the respective influence of possible growth factors has to be
determined. For these reasons, various combinations
of chemicals and factors have been devised to replace
serum in culture systems [21].
To study the influence of individual regulators on
nephrogenesis, a model has been developed in our
laboratory enabling the maintenance of human fetal
kidney in culture [1]. The novelty of this system resides in the known composition of basic Leibovitz’s
DISCUSSION
Over the years, different models have been tried to
keep prostate explants in various culture media, such
as Parker-199 [9], Waymouth-199 [10], Trowell T-8
[11], PFMR-4 [12], and PFMR-4A [13], either supplemented with serum or serum-free. Although interesting studies have been performed with animal [14,15]
and human [16–18] prostatic cells or tissues, the prostate has been very difficult to culture for long timeperiods. The problems most frequently observed were
abnormal fibroblast proliferation and tissue necrosis,
Rat Prostate Explants in Organ Culture
Fig. 5. In uncultured explants, the cell apical plasma membrane
is characterized by numerous microvilli (M). Dark secretory granules (arrows) are present in the apical cytoplasm. ×1,500.
Fig. 6. In explants cultured for 5 days in L-15, the morphological
integrity of cells was apparently well-preserved. This electron micrograph shows microvilli, granular endoplasmic reticulum elements, free ribosomes, secretory granules, and a junctional complex (arrow). ×15,000.
L-15 medium, which is devoid of glucose, serum, or
hormones. Thus, all conditions are defined and kept at
a minimum for growth and differentiation. This system has been useful in assessing the direct effects of
many growth factors such as epidermal growth factor
[22] as well as hydrocortisone [23], insulin, and transferrin [24].
The main objective of the current study was to
47
Fig. 7. Cells in prostatic explants are still able to incorporate
3
H-leucine after 5 days in L-15, although at a reduced level. Asterisk indicates significant difference (P < 0.01) when compared to
day 0 explants.
verify whether our culture system, used for kidney
explants, with its numerous advantages, could be applied to prostatic tissue. L-15 medium proved to be
superior to CMRL for DNA synthesis, as determined
by 3H-thymidine incorporation, but no significant difference was observed between the two-gas combinations. Thus, a mixture of 95% air/5% CO2 was selected
for practical reasons, while L-15 was retained for its
numerous advantages discussed above.
The use of L-15 without the addition of serum or
hormones permitted us to maintain rat prostatic explants in culture for at least 5 days without significant
changes in overall architecture and ultrastructural features. The cells incorporated 3H-leucine, synthesized
proteins, and maintained their acid phosphatase activity level during the culture period. Although 3Hthymidine incorporation decreased between days 0–5
because culture conditions were kept at a minimum
for growth and differentiation, as discussed above and
elsewhere [25], the viability of the explants was manifested by their capacity to still synthesize DNA. The
observation that the activity of alkaline phosphatase, a
differentiation membrane marker of epithelial cells,
was not significantly modified, suggests the maintenance of a functional epithelium.
CONCLUSIONS
The proposed organ culture system permits the
preservation of morphological and functional features
48
Nguyen-Le et al.
of rat prostate explants in a serum-free medium for up
to 5 days. Since it isolates tissues from multiinteractions of the organism and since it is achieved in a
controlled environment under minimal conditions for
differentiation without hormonal supplements, ours
appears to be a reliable and promising model that will
provide basic data and allow the study of growth factor and hormone effects on growth and functional activities of the prostate.
REFERENCES
1. Brière N: Human foetal kidney explants in serum-free organ
culture. Anat Embryol (Berl) 176:105–114, 1987.
2. Ménard D, Arsenault P: Explant culture of human fetal small
intestine. Gastroenterology 88:691–700, 1985.
3. Heatfield BM, Sanefuji H, Trump BF: Long-term explants culture of normal human prostate. In Harris CC, Trump BF, Stoner
GD (eds): ‘‘Methods in Cell Biology,’’ Baltimore: Academic
Press, 1980:121–194.
4. Malo C, Arsenault P, Ménard D: Organ culture of the small
intestine of the suckling mouse in a serum-free medium. Cell
Tissue Res 228:75–84, 1983.
5. Giles KW, Myers A: An improved diphenylamine method for
the estimation of deoxyribonucleic acid. Nature 206:93, 1965.
6. Lowry OH, Rosebrough NF, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193:265–
275, 1951.
7. Eichholz A: Structural and functional organization of the brush
border of intestinal epithelial cells. III. Enzymatic activities and
chemical composition of various fractions of tris-disrupted
brush borders. Biochem Biophys Acta 135:475–482, 1967.
8. Seitz J, Aumüller G: Cytochemistry and biochemistry of acid
phosphatases: I. Cytochemistry and isoelectric focussing of acid
phosphatases of the rat ventral prostate. Histochemistry 67:99–
111, 1980.
9. Johansson R: RNA, protein and DNA synthesis stimulated by
testosterone, insulin and prolactin in the rat ventral prostate
cultured in chemically defined medium. Acta Endocrinol
(Copenh) 80:761–764, 1975.
10. Waymouth C: Normal and benign human prostatic epithelium
in culture. JNCI 22:1003–1009, 1959.
11. Simmett JD, Morley AR: Factor controlling growth of prostatic
epithelium. A comparison of mitotic activity in mice of different
ages in vivo and in organ culture. Exp Cell Res 46:29–36, 1967.
12. Lechner JF, Babcock MS, Marnell M, Narayan KS, Kaighn ME:
Normal human prostate epithelial cell cultures. In Harris CC,
Trump BF, Stoner GD (eds): ‘‘Methods in Cell Biology,’’ Baltimore: Academic Press, 1980:195–225.
13. Peehl DM: Serial culture of adult human prostatic epithelial
cells. J Tissue Cult Methods 9:53–60, 1985.
14. Lasnitzki I, Mizuno T: Role of the mesenchyme in the induction
of the rat prostate gland by androgens in organ culture. J Endocrinol 82:171–178, 1979.
15. Martikainen P: Maintenance of adult rat ventral prostate in organ culture. Anat Rec 218:166–194, 1987.
16. Webber MM: Growth and maintenance of normal prostatic epithelium in vitro. A human cell model. Prog Clin Biol Res 37:
181–216, 1980.
17. Malinin TI: Establishment of primary cell cultures from normal
and neoplastic human prostate gland tissue. Prog Clin Biol Res
37:161–180, 1983.
18. Merchant DJ, Clarke SM, Ives K, Harris S: Primary explant culture: An in vitro model of the human prostate. Prostate 4:523–
542, 1983.
19. Peehl D: Culture of human prostatic epithelial cells. In: ‘‘Culture
of Human Prostatic Epithelial Cells,’’ Wiley-Liss, Inc., 1992:159–
180.
20. Bologna M, Vicentini C, Festuccia C, Muzi P, Napolitano T,
Biordi L: Short-term tissue culture of prostatic carcinoma
samples provides useful biological parameters related to patient
prognosis. Eur Urol 15:243–247, 1988.
21. Barnes D, Sato G: Serum-free culture: A unifying approach. Cell
22:649–655, 1980.
22. Bertrand L, Brière N, Ferrari J: Epidermal growth factor (EGF)
influences DNA synthesis in human fetal kidneys maturing in
serum-free organ culture. Biofactors 1:313–317, 1988.
23. Bertrand L, Brière N: Effect of hydrocortisone on the maturation
of human foetal kidney explants in serum-free organ culture.
Biochem Cell Biol 67:121–127, 1989.
24. Brière N, Ferrari J, Chailler P: Insulin and transferrin restore
important cellular functions of human fetal kidney in serumfree organ culture. Biochem Cell Biol 69:256–262, 1991.
25. Brière N, Chailler P: Minimal growth factor requirements of
human fetal kidney in serum- and glucose-free culture. Biofactors 4:55–61, 1992.
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