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  and intestine . 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  and Seitz and Aumüller , 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  was compared to 50% N2/45% O2/5% CO2 as employed by Heatfield et al.  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. . DNA content (in mg DNA/ mg tissue) was evaluated by the procedure of Giles and Myers  using calf thymus as standard. Protein content (mg protein/g tissue) was estimated according to the method of Lowry et al. , 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 . It is noteworthy that Bologna et al.  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  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 . 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 . 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 , Waymouth-199 , Trowell T-8 , PFMR-4 , and PFMR-4A , 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  as well as hydrocortisone , insulin, and transferrin . 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 , the viability of the explants was manifested by their capacity to still synthesize DNA. 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