Complete Serum-free Medium Research Articles

#2716 Selenoprotein-P1 (SEPP-1) in human proximal tubule cells after ischemic-reperfusion injury: an in-vitro model

Abstract Background and Aims Novel biomarkers, anticipating serum creatinine rise, are eagerly needed to have a speedy diagnosis of acute kidney injury (AKI) and thus to mitigate and to preserve kidney function with well-timed therapeutic plans. Recently, in a pilot study on patients undergoing elective major cardiac surgery with cardiopulmonary bypass (CPB), selenoprotein-p1 (SEPP-1) has emerged as a promising candidate plasmatic biomarker for an early AKI risk stratification. SEPP-1 increased particularly between 4th to 8th hour after CPB and with a strict relation with ischemia duration (Bolignano et Al. Rev. Cardiovasc. Med. 2022). SEPP1 is primarily secreted from liver and acts as a selenium transporter supplying tissues and organs with this trace mineral which elicits the activity of specific glutathione peroxidase selenoenzymes. Additionally, renal tubular cells are able to synthetize this protein but remain unknown if, during AKI, SEPP1 increases as defeat answer to renal cells damage or if it's hepatic SEPP1 that accumulates for a reduced renal glomerular filtration rate. The primary aim of this study was to investigate the release of SEPP-1 by renal tubular cells in an experimental model of AKI induced by ischemia-reperfusion and secondarily evaluate the effect of selenium supplying on renal cells injury. Method Human proximal tubular cells (PTC), HK2 cells and human embryonic kidney 293 cells (HEK293) were cultured in Keratinocyte-SFM a complete serum-free medium supplemented with human recombinant Epidermal Growth Factor (rEGF) and Bovine Pituitary Extract (BPE) (LGC Standards s.r.l., Milan, Italy) and in Eagle's Minimum Essential Medium (E-MEM) (LGC Standards s.r.l., Milan, Italy) containing 10% fetal bovine serum, respectively. The cells were pre-incubated with medium containing Sodium Selenite 100 nM for 24 h and then were treated for 24 hours with CoCl2 (Sigma Aldrich S.r.l., Milan, Italy) a chemical hypoxia inducer (500 µM for HK-2 and 250 µM for HEK-293). After that cell viability was evaluated by MTT and trypan bleu dye exclusion assay, while, protein expression of HIF (hypoxia marker) and SEPP1 was analyzed by western blot analysis. Results Ischemia-reperfusion was simulated by the exposure of HK-2 and HEK-293 to CoCl2 and confirmed by HIF expression. The expression of SEPP-1 increased significantly in HK-2 and HEK-293 after hypoxia stress (Fig. 1). Furthermore, the treatment with CoCl2 determined a decrease of cell viability (about 50% less) and Sodium selenite alone increased cell proliferation (about 15% more) in both cell lines. Interestingly cell growth after hypoxia augmented significantly if cell lines were pre-treated with sodium-selenite, highly in HK-2 cells (p < 0.001). Conclusion These preliminary data evidenced, in vitro, that AKI is able to induce renal tubular expression of SEPP-1 and probably SEPP-1 release in human after renal injury is not only a result of a reduced filtration of the hepatic released protein. Furthermore, SEPP-1 showed a possible protective role in AKI. In fact, AKI induced by ischemia-reperfusion, in our in vitro cellular model, was able to induce an increased expression of SEPP-1 in renal tubular cells suggesting a shielding mechanism against injury related to selenium transport. This hypothesis was supported by an exalted SEPP-1 expression after Sodium-Selenite adding to cells’ culture. Supplementary experiments, including functional molecular assays in vitro and in vivo studies will allow to better characterize the role of SEPP-1 in AKI.

Leishmania Parasite: the Impact of New Serum-Free Medium as an Alternative for Fetal Bovine Serum

Background:Flagellated protozoan of the genus Leishmania is the causative agent of vector-borne parasitic diseases of leishmaniasis. Since the production of recombinant pharmaceutical proteins requires the cultivation of host cells in a serum-free medium, the elimination of FBS can improve the possibility of large-scale culture of Leishmania parasite. In the current study, we aimed at evaluating a new serum-free medium in Leishmania parasite culture for future live Leishmania vaccine purposes. Methods:Recombinant L. tarentolae secreting PpSP15-EGFP and wild type L. major were cultured in serum-free (CSFM) and serum-supplemented medium. The growth rate, protein expression, and infectivity of cultured parasites in both conditions was then evaluated and compared. Results:Diff-Quick staining and epi-fluorescence microscopy examination displayed the typical morphology of L. major and L. tarentolae-PpSP15-EGFP promastigote grown in CSFM medium. The amount of EGFP expression was similar in CSMF medium compared to M199 supplemented with 5% FBS in flow cytometry analysis of L. tarentolae-PpSP15-EGFP parasite. Also, a similar profile of PpSP15-EGFP proteins was recognized in Western blot analysis of L. tarentolae-PpSP15-EGFP cultured in CSMF and the serum-supplemented medium. Footpad swelling and parasite load measurements showed the ability of CSFM medium to support the L. major infectivity in BALB/C mice. Conclusion:This study demonstrated that CSFM can be a promising substitute for FBS supplemented medium in parasite culture for live vaccination purposes.

Adaptation of the HEp-2 cell line to totally animal-free culture systems and real-time analysis of cell growth.

Routine cell culture demands the use of animal-derived products, mainly fetal bovine serumand swineor bovine trypsin. According to the 3Rs principle and to theEuropean Centre for the Validation of Alternative Methods, animal-free substitutes are strongly recommended for in vitro methods. In this study, theHEp-2 cell line was adapted to different totally animal-free culture systems, such as a serum-free complete medium (VP-SFM), human platelet lysate and a synthetic trypsin (TrypLE™ Express); afterward, cell growth was assessed with the xCELLigence instrument. Animal-free products provided promising results, with performances similar or preferable to the common reagents; thereforetheir use could be encouraged for bothethical and technical advantages.

Serum-free complete medium, an alternative medium to mimic androgen deprivation in human prostate cancer cell line models.

Almost all men who present with advanced prostate cancer (CaP) and some men who fail therapy for clinically localized CaP are treated with androgen deprivation therapy (ADT). CaP cell lines are used to identify and characterize new agents for ADT or investigate mechanisms of ADT resistance. CaP cell lines are maintained in culture medium that contains fetal bovine serum, which contains testosterone (T). Androgen deprivation experiments are performed using media supplemented with androgen-free serum, such as charcoal stripped fetal bovine serum (CS-FBS). However, CS-FBS composition varies from batch-to-batch and variations may impact experimental reproducibility. Serum free media (SFM) may provide a better defined alternative to media supplemented with CS-FBS (CSM). Cell growth of six human CaP cell lines was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Androgen levels were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). MTT assays showed 5 of 6 CaP cell lines grew after 6 days of culture in androgen- deprived SFM or CSM. LNCaP and VCaP growth was stimulated when cells were cultured in SFM or CSM supplemented with T. LNCaP, C4-2, LAPC-4, and VCaP cell growth was inhibited when cultured in SFM or CSM with T and bicalutamide. LC-MS/MS data showed LAPC-4 cells produced similar DHT levels when cultured in T-supplemented SFM or CSM. Dutasteride impaired T to DHT metabolism in LAPC-4. Media composition contributed to growth differences observed between CaP cells cultured in SFM or CSM. However, the differences in media composition did not impair CaP cell response to T-stimulated growth, bicalutamide growth inhibition, metabolism of T, or dutasteride efficiency. SFM can be used as a better defined alternative to CSM for androgen deprivation experiments.

Hyaluronan Constitutively Regulates Activation of COX-2-mediated Cell Survival Activity in Intestinal Epithelial and Colon Carcinoma Cells

Hyaluronan is a major component of the pericellular matrix surrounding tumor cells, including colon carcinomas. Elevated cycooxygenase-2 levels have been implicated in several malignant properties of colon cancer. We now show for the first time a strong link between hyaluronan-CD44 interaction and cyclooxygenase-2 in colon cancer cells. First, we have shown that increased expression of hyaluronan synthase-2 induces malignant cell properties, including increased proliferation, anchorage-independent growth, and epithelial-mesenchymal transition in HIEC6 cells. Second, constitutive hyaluronan-CD44 interaction stimulates a signaling pathway involving ErbB2, phosphoinositide 3-kinase/AKT, beta-catenin, and cyclooxygenase-2/prostaglandin E(2) in HCA7 colon carcinoma cells. Third, the HA/CD44-activated ErbB2 --> phosphoinositide 3-kinase/AKT --> beta-catenin pathway stimulates cell survival/cell proliferation through COX-2 induction in hyaluronan-overexpressing HIEC6 cells and in HCA7 cells. Fourth, perturbation of hyaluronan-CD44 interaction by hyaluronan oligomers or CD44-silencing RNA decreases cyclooxygenase-2 expression and enzyme activity, and inhibition of cyclooxygenase-2 decreases hyaluronan production suggesting the possibility of an amplifying positive feedback loop between hyaluronan and cyclooxygenase-2. We conclude that hyaluronan is an important endogenous regulator of colon cancer cell survival properties and that cyclooxygenase-2 is a major mediator of these hyaluronan-induced effects. Defining hyaluronan-dependent cyclooxygenase-2/prostaglandin E(2)-associated signaling pathways will provide a platform for developing novel therapeutic approaches for colon cancer.

Trafficking of a Secretory Granule Membrane Protein Is Sensitive to Copper

We explored the effect of copper availability on the synthesis and trafficking of peptidylglycine alpha-amidating monooxygenase (PAM), an essential cuproenzyme whose catalytic domains function in the lumen of peptide-containing secretory granules. Corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM were depleted of copper using bathocuproinedisulfonic acid or loaded with copper by incubation with CuCl(2). Depleting cellular copper stimulates basal secretion of soluble enzyme produced by endoproteolytic cleavage of PAM in secretory granules and transit of membrane PAM though the endocytic pathway and back into secretory granules. Unlike many cuproenzymes, lack of copper does not lead to instability of PAM. Copper loading decreases cleavage of PAM in secretory granules, secretion of soluble enzyme, and the return of internalized PAM to secretory granules. The trafficking and stability of the soluble, luminal domain of PAM and truncated membrane PAM lacking a cytosolic domain are not affected by copper availability. Taken together, our data demonstrate a role for copper-sensitive cytosolic machinery in directing endocytosed membrane PAM back to secretory granules or to a degradative pathway. The response of PAM to lack of copper suggests that it facilitates copper homeostasis.

The p14 Fusion-associated Small Transmembrane (FAST) Protein Effects Membrane Fusion from a Subset of Membrane Microdomains

The p14 Fusion-associated Small Transmembrane (FAST) Protein Effects Membrane Fusion from a Subset of Membrane Microdomains

Scavenger Receptor Class B Type I (SR-BI) Is Involved in Vitamin E Transport across the Enterocyte

Although cellular uptake of vitamin E was initially described as a passive process, recent studies in the liver and brain have shown that SR-BI (scavenger receptor class B type I) is involved in this phenomenon. As SR-BI is expressed at high levels in the intestine, the present study addressed the involvement of SR-BI in vitamin E trafficking across enterocytes. Apical uptake and efflux of the main dietary forms of vitamin E were examined using Caco-2 TC-7 cell monolayers as a model of human intestinal epithelium. (R,R,R)-gamma-tocopherol bioavailability was compared between wild-type mice and mice overexpressing SR-BI in the intestine. The effect of vitamin E on enterocyte SR-BI mRNA levels was measured by real-time quantitative reverse transcription-PCR. Concentration-dependent curves for vitamin E uptake were similar for (R,R,R)-alpha-, (R,R,R)-gamma-, and dl-alpha-tocopherol. (R,R,R)-alpha-tocopherol transport was dependent on incubation temperature, with a 60% reduction in absorption at 4 degrees C compared with 37 degrees C (p < 0.05). Vitamin E flux in enterocytes was directed from the apical to the basal side, with a relative 10-fold reduction in the transfer process when measured in the opposite direction (p < 0.05). Co-incubation with cholesterol, gamma-tocopherol, or lutein significantly impaired alpha-tocopherol absorption. Anti-human SR-BI antibodies and BLT1 (a chemical inhibitor of lipid transport via SR-BI) blocked up to 80% of vitamin E uptake and up to 30% of apical vitamin E efflux (p < 0.05), and similar results were obtained for (R,R,R)-gamma-tocopherol. SR-BI mRNA levels were not significantly modified after a 24-h incubation of Caco-2 cells with vitamin E. Finally, (R,R,R)-gamma-tocopherol bioavailability was 2.7-fold higher in mice overexpressing SR-BI than in wild-type mice (p < 0.05). The present data show for the first time that vitamin E intestinal absorption is, at least in part, mediated by SR-BI.

In Vitro Evaluation of the Antioxidant Activity of Calcium Fructoborate

Although increasing evidence shows the nutritional benefits of calcium fructoborate (CF) on animals and humans, its action mechanism has not been clearly identified. The present study aims to investigate the possible antioxidant function of CF. Based on its efficiency in skin wound healing, the authors tested whether CF possesses antioxidant properties on human keratinocytes cultures, in a complete serum-free medium (KMK-2; Sigma). The cells treated with CF (0-450 nmol/culture medium) were exposed to exogenous 100 micromol of hydrogen peroxide to mimic the oxidative stress. The changes in general cell oxidant production evaluated with dihydrorhodamine-123 showed that the intracellular reactive oxygen species (ROS) were markedly reduced by preincubation with CF. The maximum antioxidant activity was noticed at 90 nmol CF. To assess the reactivity of CF on ROS, we analyzed its ability to inhibit the superoxide- dependent auto-oxidation of pyrogallol. The CF inhibited the pyrogallol auto-oxidation depending on time and concentration, which suggests its possible role as a superoxide radical scavenger. Taken together, our results indicate that CF has antioxidant activity, which could have clinical significance in protecting cells from oxidant-induced injury. A hypothetic mechanism for the antioxidant activity of CF is proposed.

Dopamine β-Monooxygenase Signal/Anchor Sequence Alters Trafficking of Peptidylglycine α-Hydroxylating Monooxygenase

Dopamine beta-monooxygenase (DBM) and peptidylglycine alpha-hydroxylating monooxygenase (PHM) are essential for the biosynthesis of catecholamines and amidated peptides, respectively. The enzymes share a conserved catalytic core. We studied the role of the DBM signal sequence by appending it to soluble PHM (PHMs) and expressing the DBMsignal/PHMs chimera in AtT-20 and Chinese hamster ovary cells. PHMs produced as part of DBMsignal/PHMs was active. In vitro translated and cellular DBMsignal/PHMs had similar masses, indicating that the DBM signal was not removed. DBMsignal/PHMs was membrane-associated and had the properties of an intrinsic membrane protein. After in vitro translation in the presence of microsomal membranes, trypsin treatment removed 2 kDa from DBMsignal/PHMs while PHMs was entirely protected. In addition, a Cys residue in DBMsignal/PHMs was accessible to Cys-directed biotinylation. Thus the chimera adopts the topology of a type II membrane protein. Pulse-chase experiments indicate that DBMsignal/PHMs turns over rapidly after exiting the trans-Golgi network. Although PHMs is efficiently localized to secretory granules, DBMsignal/PHMs is largely localized to the endoplasmic reticulum in AtT-20 cells. On the basis of stimulated secretion, the small amount of PHMs generated is stored in secretory granules. In contrast, the expression of DBMsignal/PHMs in PC12 cells yields protein that is localized to secretory granules.

Production of retroviral vectors for gene therapy with the human packaging cell line FLYRD18.

The development of gene therapy is hampered by the difficulty of producing large stocks of retroviral vectors at high titer. This study aimed to improve culture conditions and to intensify the production of retroviruses by FLYRD18, a packaging cell line derived from the HT1080 human fibrosarcoma line. Batch virus production proved to be feasible in unsupplemented basal medium and provided significantly higher titers and productivities than medium supplemented with 10% serum. For longer-term production, however, AIM-V complete serum-free medium and basal medium supplemented with 2% serum gave superior results. Serum supplementation should nevertheless be optimized to take into account the presence of inhibitors of viral production. In monolayer cultures with 0.2 mL/cm(2), the cell concentration was increased up to 2 x 10(6) cells/mL without loss of cell productivity. A semicontinuous production process, which enables the collection of larger amounts of viruses from the same culture, has also been successfully used. Suspension culture processes were prevented by the anchorage dependency of the FLYRD18 cell line. Microcarrier cultures were able to produce viruses but will require further investigation and optimization for their performance to become competitive with monolayer cultures. In the course of this study, more than a 10-fold increase of titer has been achieved.

Growth and differentation of rat mammary epithelial cells cultured in serum-free medium

A new serum-free defined medium was developed that supports the growth of normal rat mammary epithelial cells. Mammary organoids from the glands of female F344 rats were cultured in a serum-free medium. Monolayer culture colonies developed within a week and remained viable for months in culture. Upon subculture of one-week-old primary colonies, almost the same morphology of colonies was developed. The scrape loading/dye transfer technique showed that most of colonies that developed in a serum-free medium containing EGF, human transferrin, insulin, and hydrocortisone (basal serum-free medium, BSFM) failed to show cell-cell communication. However, colonies cultured in BSFM supplemented with prolactin, E(2), and progesterone (complete hormone serum-free medium, CHSFM) showed cell-cell communication at 14 days of primary culture or of subculture. By flow cytometry with FITC-PNA and PE-anti-Thy-1.1 monoclonal antibody, we distinguished four RMEC subpopulations in cultures in both media: Thy-1.1+ cells, PNA+ cells, cells negative to both reagents and cells positive to both reagents. It is likely that combined prolactin, cortisol, and insulin in CHSFM stimulate terminal differentiation of clonogenic cells.

Insulin and hydrocortisone, but not triiodothyronine, are required for the differentiation of pig preadipocytes in primary culture.

The objective of this study was to establish optimal conditions for the primary culture of pig preadipocytes. We cultured pig preadipocytes for 10 d and studied the effects of insulin, hydrocortisone, and triiodothyronine (T3) added to serum-free basal medium on differentiation and gene expression of lipoprotein lipase an early marker, and adipsin, a late marker of preadipocyte differentiation. Insulin alone and hydrocortisone alone stimulated a low level of cell differentiation, as indicated by an increase in glycerol-3-phosphate dehydrogenase activity. When added together, insulin and hydrocortisone had a synergistic effect on cell differentiation. When combined with insulin or hydrocortisone, T3 had no effect on cell differentiation, indicating that T3 is not required in porcine preadipocyte culture. Gene expression studies also showed that removal of insulin or hydrocortisone from complete serum-free medium reduced both early and late marker mRNA. As expected, removal of T3 had no effect on the gene expression of early and late marker mRNA. We conclude that insulin and hydrocortisone, but not T3, are required for the differentiation of pig preadipocytes in primary culture.

The effect of rh-cytokines on the sensitivity of normal human CFU-GM progenitors to Ara-C and on the S-phase activity of light density human bone marrow cells

This study examines the effect of pre-incubation in liquid phase of normal human CFU-GM progenitors with recombinant human (rh) cytokines prior to exposure to cytosine arabinoside (Ara-C) in clonogenic culture. Light density marrow cells (LMDCs) were preincubated in Biorich™ (chemically defined serum-free complete medium) with G + GM-CSF or IL-3 prior to semi-solid culture with Ara-C (10 −4 M-10 −12 M). Cell cycle analysis was performed by flow cytometry pre- and post-rh-cytokine treatment. Data from 26 normal marrows studied clonogenically, demonstrated a 60% reduction in CFU-GM with 10 −4 M Ara-C. Preincubation in Biorich medium with the addition of G + GM-CSF or IL-3 caused a significant enhancement of sensitivity to Ara-C across the dose curve. Biorich medium alone had an apparent protective effect resulting in amelioration of the cytotoxic effect of Ara-C. Cell cycle analysis of eight subjects showed significant recruitment into S-phase following pre-treatment with cytokines. A reduction in S-phase activity was noted in cells pre-incubated in Biorich alone. In summary, it would appear that cytokines can enhance the sensitivity of normal CFU-GM to Ara-C in vitro possibly due to an increase in S-phase activity. Pre-incubation in nutrient medium without cytokines resulted in cell quiescence.

Critical variables controlling cell proliferation in primary cultures of rat tracheal epithelial cells

The purpose of our experiments was to examine variables affecting early events in the establishment of rat tracheal epithelial (RTE) cultures as well as factors regulating long-term RTE cell growth. The experiments showed that when RTE cells were seeded into complete serum-free medium between 13 and 30% of the seeded cells attached. Of the seeded cells, only approximately 2% entered into DNA synthesis and underwent repeated cell divisions to form colonies containing greater than 20 cells. Coating the dishes with extracellular matrix components had little effect on cell attachment or colony forming efficiency (CFE). However, coating the dishes with fetal bovine serum markedly increased CFE. The media components bovine serum albumin and bovine pituitary extract were shown to be important in promoting cell attachment as well as CFE. Cholera toxin on the other hand had no effect on cell attachment but significantly increased CFE. These and other studies showed that cell attachment and cell proliferation are independently regulated. Studies on long-term culture growth indicated that the number of progeny produced per colony forming unit (CFU) is inversely proportional to the number of CFUs seeded. Inasmuch as the cultures did not become confluent under any of the culture conditions tested and media obtained from high density cultures were shown to be growth inhibitory, these findings suggest that a diffusible growth restraining factor is being produced by the cultures limiting clonal expansion. Experiments showing growth inhibitory effects of media conditioned by high cell density cultures support this interpretation. The putative factor reaches critical concentrations earlier in cultures seeded with high numbers of CFU than in cultures seeded with low numbers of CFU. Because the cultures are known to produce transforming growth factor-beta, this growth regulator probably plays a role in controlling RTE cell proliferation. However, it is likely than other events, such as depletion of growth factors from the media, also are significant in regulating the growth of the cultures.

Plasticity of peptide biosynthesis in corticotropes: independent regulation of different steps in processing.

Compared to corticotropes in the adult rat anterior pituitary, neonatal corticotropes exhibit a significantly lower extent of conversion of precursor molecules into ACTH and a substantially greater extent of ACTH cleavage into smaller product peptides similar in size to alpha-melanotropin and corticotropin-like intermediate lobe peptide (CLIP). In the present study we examined pro-ACTH/endorphin (PAE; also called POMC) processing in corticotropes at different times during postnatal development to determine when these cells cease cleaving ACTH into smaller peptides and when they cease accumulating large amounts of intact precursor in vivo. The pattern of processing observed in the newborn is maintained through the second week after birth. A dramatic decrease in ACTH cleavage occurs between the second and third postnatal weeks. The extent of precursor cleavage increases toward the adult pattern by the fifth postnatal week. Explants of newborn anterior pituitary were previously shown to exhibit increased cleavage of ACTH into ACTH-(1-13)NH2 and CLIP, a process that was suppressed by glucocorticoids. To determine whether corticotropes from older animals maintained this plasticity and what intercellular interactions might be required, dissociated primary cultures were maintained in complete serum-free medium with or without added glucocorticoids. After 7 days in complete serum-free medium, the cellular content of both intact precursor and peptides the size of CLIP was increased compared to the corresponding in vivo pattern for animals from birth through adulthood. Although corticotropes from younger animals exhibited more pronounced changes when placed in culture, even cultures from adult animals exhibited some ACTH cleavage. For corticotrope cultures prepared from animals up to postnatal day 15, chronic treatment with dexamethasone did not suppress ACTH cleavage activity, although glucocorticoids did suppress ACTH cleavage in cultures from animals older than postnatal day 22. Biosynthetic labeling studies with cultures from 4- to 5-week-old rats demonstrated that the powerful suppressive effect of dexamethasone on the cleavage of newly synthesized ACTH-(1-39) was only evident 24 h after addition of the glucocorticoid to the culture medium. In contrast, removal of dexamethasone allowed cleavage of ACTH to commence within a few hours.

Growth requirements and long-term subcultivation of bovine granulosa cells cultured in a serum-free medium

We have developed an improved serum-free medium to optimize the cell growth of bovine granulosa cells. The cells on collagen-coated culture plates proliferated extensively in a nutrient medium supplemented with insulin, heparin binding growth factor-2 (HBGF-2), lipoprotein, and bovine serum albumin (BSA). The cell doubling time at logarithmic phase and final cell density at confluent cultures were equal to those of cultures grown in the presence of medium supplemented with optimal concentration (10%) of fetal bovine serum (FBS). Whereas HBGF-2 or insulin alone had a small mitogenic effect of granulosa cells, lipoprotein or BSA did not. When lipoprotein, BSA, or insulin was added together with HBGF-2, synergistic cell proliferation was observed in all combinations. Insulin or lipoprotein had an additive mitogenic stimulation of these cells in the presence of BSA. After granulosa cells were subcultivated in a serum-containing medium until three generations [8.5 cumulative population doubling level (CPDL)], subsequent subcultivation of the cells in a complete serum-free medium could be achieved up to six generations (14.4 CPDL). These results demonstrate that this serum-free medium can support the optimal cell growth and long-term subcultivation of bovine granulosa cells.

Differential regulation of neuropeptide Y and catecholamine production in superior cervical ganglion cultures

Primary cultures of newborn rat superior cervical ganglion (SCG) neurons grown in complete serum-free medium have been used to study the regulation of catecholamine and neuropeptide Y (NPY) production. The levels of mRNA for preproNPY and for tyrosine hydroxylase were determined, and the rates of synthesis and accumulation of NPY and catecholamines were measured. Drugs which effectively blocked accumulation of newly synthesized catecholamines (tyramine, reserpine) had no effect on NPY synthesis and accumulation. The catecholamine uptake blocker desipramine and the α 2-adrenergic receptor agonist clonidine inhibited accumulation of newly synthesized catecholamines, but only after prolonged exposure to drug; NPY synthesis and accumulation were unaltered by these treatments. The α 2-adrenergic receptor antagonist yohimbine had no effect on catecholamine or NPY synthesis or accumulation, regardless of the time of treatment. The net synthesis and accumulation of catecholamines and NPY decreased in parallel in response to treatment with phorbol esters, and increased in parallel in response to treatment with glucocorticoids; there was no change in the mRNA levels for tyrosine hydroxylase or preproNPY following either treatment. The levels of tyrosine hydroxylase or preproNPY mRNA were increased following treatment with dibutyryl cyclic AMP, with no change in the net synthesis and accumulation of catecholamines or NPY. Thus, the levels of mRNA for preproNPY and tyrosine hydroxylase are not faithful indicators of the rate of catecholamine or peptide production under several treatment conditions. The complex pattern of regulation of catecholamine and NPY production in SCG neurons grown under closely controlled conditions provides an ideal system in which to examine neuronal regulation of neurotransmitter and neuromodulator production.

Growth responses of prostatic epithelial cells from male mice neonatally exposed to diethylstilbestrol in serum-free collagen gel culture

Growth of anterior and ventral prostatic epithelial cells from mice neonatally exposed to diethylstilbestrol (DES) and from unexposed control mice was compared at different time points in serum-free collagen gel culture. A longer maintenance of the initial plating density (lag in growth) was observed in cultured DES-exposed ventral prostatic cells. Neonatal DES exposure resulted in two colony types: one similar to colonies arising from unexposed cells and one which appears to be non-growing. Keratinization was observed in some DES-exposed anterior prostatic cell colonies. Removal of epidermal growth factor from the serum-free medium significantly decreased growth in 3 of the 4 groups compared with their growth in the complete serum-free medium.

Serum-free medium conditions for steroidogenesis of bovine follicular thecal cells cultured on collagen gel matrix

Thecal cells isolated from bovine ovarian follicles were cultured with a serum-free basal medium or a serum-free complete medium in the presence or absence of collagen gel matrix, and their cellular proliferation and steroidogenesis were compared with those of cells cultured with a serum-containing medium. The cells cultured with the serum-free basal medium produced larger amounts of progesterone, androstenedione, and estradiol than the cells cultured with the serum-containing medium, but no appreciable cell proliferation was observed in the serum-free medium. Response of thecal cells to 8 bromo-cAMP, a steroidogenic agent, varied according to the type of steroid production examined and the type of culture medium used. In a cultivation period of 4 d, progesterone production was stimulated about five-fold by 8 bromo-cAMP in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium without collagen matrix, whereas androstenedione production was stimulated about three- to fourfold in the serum-free complete medium on collagen gel matrix and in the serum-free basal medium with or without collagen matrix. Estradiol production, however, was significantly suppressed by 8 bromo-cAMP in the serum-free complete medium on collagen gel matrix and also in the serum-containing medium. Thus, among the conditions examined, the most suitable primary culture media for steroidogenesis of thecal cells were the serum-free media, especially serum-free complete medium on collagen gel matrix.