Cholesterol 7α-hydroxylase activity and bile acid synthesis in hepatocytes of unwearied and weaned pigs in monolayer culture

Activity of cholesterol 7α-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7α-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7α-hydroxyease activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [¹⁴C]cholesterol. among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7α-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7α-hydroxylase activities and bile acid production rates.     

Effect of biosynthetic human epidermal growth factor on the synthesis and secretion of mucin glycoprotein from primary culture of rabbit fundal mucosa cells

Summary Biosynthetic human epidermal growth factor (Bh-EGF) induced dose-dependent synthesis and secretion of neutral mucin glycoprotein when the fundal cells isolated from rabbit stomach were cultured in serum-free medium containing Bh-EGF at concentrations as high as 10 to 100 ng/ml. At these high concentrations, Bh-EGF had no effect on the cell growth. In marked contrast, much lower concentrations from 0.1 to 1.0 ng/ml of Bh-EGF failed to stimulate mucin synthesis, but enhanced proliferation of the cells. Electrophoretic pattern of the mucin secreted from the cultured mucosal cells was very similar to that of the authentic mucin obtained from rabbit stomach. Maximal secretion of the mucin from the cells was observed at Hour 96 of the culture. Although fetal bovine serum (5%) and insulin (0.5 μg/ml) also stimulated the mucosal cells, both in growth and in mucin synthesis and release, the enhancing activity of the mucin synthesized and released by Bh-EGF at a concentration of 100 ng/ml per microgram DNA of cultured cells was far superior to that of 5% fetal bovine serum and 0.5 μg/ml insulin.     

Differentiation of lymphoid cells. A selective anti-mitogenic component of normal serum which inhibits the induction of immunoglobulin production.

Rabbit lymph node cell populations cultured in vitro in the presence of fetal calf serum are induced to produce immunoglobulin M-secreting cells. The induction of such immunoglobulin production, measured by the capacity of the cell population to secrete immunoglobulins, was inhibited when cells were cultured with sera from a variety of species despite the presence of fetal calf serum. The addition of such inhibitory serum 36 hours after initiation of the cell culture or thereafter was without effect on the extent of induction of immunoglobulin production. On the other hand, the presence of inhibitory serum in culture during only the first 24 hours yielded the same inhibition as when serum was present throughout the 72-hour culture period. Inhibitory sera also suppressed the incorporation of thymidine into DNA. The induction of immunoglobulin production and the incorporation of thymidine into DNA were essentially equally inhibited by the same range of serum concentrations. Unlike conventional inhibitors of DNA synthesis, the inhibitory sera exhibited selective specificity with regard to the kind of cells that could be affected. Thus, such sera inhibited the DNA synthesis of lymph node cells cultured in the presence of fetal calf serum but did not inhibit concanavalin A-stimulated DNA synthesis of such cultured cells and, similarly, serum did not inhibit DNA synthesis of thymus cells cultured in the presence of fetal calf serum. The sera of all species examined were inhibitory except for fetal sera. As judged from a quantitative assay, bovine and porcine serum contained the highest titer of inhibitor, whereas sera from human, rat, mouse, and rabbit were clustered in a group exhibiting less inhibitor. Ascites fluid and lymph node extracellular fluids contained less inhibitor than found in the serum of the same animal and lysates of washed lymph node cells were devoid of inhibitor. Although fetal bovine serum and newborn bovine serum did not contain the inhibitor, it was detectable within 24 hours of parturition. The inhibitor is of relatively large apparent molecular weight (about 300,000) and has been purified about 70-fold.     

Cholesterol 7 alpha-hydroxylase activity and bile acid synthesis in hepatocytes of unweaned and weaned pigs in monolayer culture

Activity of cholesterol 7 alpha-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7 alpha-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7 alpha-hydroxylase activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [14C]cholesterol, among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7 alpha-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7 alpha-hydroxylase activities and bile acid production rates.     

Modulation of albumin-like protein and lysozyme production by bovine tracheal gland serous cells. Dependence on culture conditions

Bovine tracheal submucosal gland serous cells were cultured in medium supplemented with either 10% fetal calf serum or 2% Ultroser G, a commercial serum substitute for cell culture. The proteins synthesized and secreted into the culture medium during [35S]methionine pulse, chase and isoproterenol-stimulated periods were analyzed. Marked differences in the patterns of secretory radiolabeled proteins with Mr values ranging from 15,000 to 95,000 were observed between pulse and chase media of cells cultured in fetal calf serum and Ultroser G. In the presence of Ultroser G, albumin-like protein production was inhibited 95% as compared to cultures incubated with fetal calf serum. A bovine lysozyme-type enzymatic activity was detected only in medium from stimulated cells cultured in Ultroser G. The results suggest that bovine tracheal serous cells synthesize different proteins according to the composition of culture medium and release certain proteins when adrenergically stimulated.     

Modulation of response to adenosine in vascular smooth muscle cells cultured in defined medium

Summary Cultured pig aortic smooth muscle cells maintain a viable, quiescent state in a chemically defined medium that contains 10⁻⁶ M insulin, 5μg/ml transferrin, and 0.2 mM ascorbate. DNA synthesis and DNA content were determined by measuring tritiated thymidine incorporation and DNA-binding to the fluorescent probe 4′,6-diamidino-2-phenylindole, respectively. The majority of the population of cells in defined medium cultures were diploid. Tritiated thymidine uptake in cells in defined medium was one-tenth that observed in cells in fetal bovine serum-containing medium. The study of cellular cyclic AMP level in response to extracellular adenosine stimulation in dividing cells and quiescent cells showed that cells in defined medium had a lower extent of response to adenosine compared to cells cultured in serum-containing medium. Both the cell growth index and the response to adenosine of cells cultured in defined medium were reversible after replacing the medium with 10% fetal bovine serum-containing medium, which suggests that the cells in defined medium were healthy and were capable of modulating cellular metabolism depending on culture conditions. This work was supported in part by National Institutes of Health grants HL31854, HL38130, and RR07048.     

旋毛虫肌幼虫细胞传代培养及超微结构观察

消化、分离观察旋毛虫(Trichinella spiralis)肌幼虫,获得肌幼虫细胞,用含10%胎牛血清的RPMI-1640培养液培养原代细胞,胰酶(含0.02?TA)消化法进行传代,透射电镜观察培养细胞超微结构,用多重PCR鉴定培养细胞。结果表明,在培养24～72h原代细胞开始贴壁,7～8d形成单层细胞,细胞间融合现象不明显,10～12d传一代。透射电镜显示旋毛虫细胞核为椭圆形,核膜、核仁清晰,核内染色质较丰富,胞浆含丰富的线粒体。细胞主要有两种类型:椭圆形和多角形,以椭圆形为主。多重PCR扩增培养细胞DNA,可见1条与旋毛虫肌幼虫DNA扩增产物相同的条带(173bp)。结果表明,旋毛虫肌幼虫细胞可在含10%胎牛血清的RPMI-1640培养液中传代培养。     

The effects of division rate-limiting amounts of fetal bovine serum on the proliferation and aging of cultured chick cells

Chick embryo fibroblasts serially propagated in media containing division ratelimiting amounts of fetal bovine serum underwent premature culture senescence as illustrated by accelerated declines in the number of cells incorporating ³H-thymidine, increased population doubling times, reduced cell densities at subcultivation, and reduced replicative life-spans compared to cells grown in medium containing non-rate-limiting amounts of serum. Low serum serially propagated “senescent” cultures returned to 10% serum containing medium had proliferative rates, incorporated ³H-thymidine, and attained saturation densities at confluency similar to younger cells. “Senescent” cells serially propagated in low serum and returned to 10% serum achieved life-spans similar to cells continuously grown in the presence of 10% serum. The results of these and other studies show that cells serially propagated in the presence of division rate-limiting amounts of fetal bovine serum, or at high inoculation densities, accumulate a substantial number of cells in the population during exponential growth conditions that are not senescent but are prevented from entering DNA synthesis becuase of mitogen limitations. Our results indicate that the amount of serum mitogen in the growth medium affects only the rate at which cells express their genetically predetermined replicative potential and not the replicative lifespan per se. These results are discussed in relation to the techniques that should be employed for studying cellular aging and the mechanism of senescent cell formation.     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture

A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin, transferrin, epidermal growth factor, poly-D-lysine, bovine albumin, oleic acid, and bovine alpha-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetal bovine serum (FBS), and colonies, albeit of smaller sizes, did form. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking alpha-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding alpha-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Characterization of bovine pancreatic ductal cells isolated by a perfusion-digestion technique

Summary Bovine pancreatic ductal cells isolated by perfusing an enzyme solution into the lumen of the main duct were obtained as sheets of cells. Morphologic features of these cells were those of pancreatic ductal epithelial cells. These cells also contained alcian blue/periodic acid-Schiff positive material and bound lectins, and they stained for keratin in the same manner as intact ductal epithelium. In culture, the plating efficiency was high (13.6%) as determined by DNA content before and after 24 h plating, perhaps due to the gentle isolation technique and the isolation of sheets of cells rather than a single cell. Cell doubling time was 34.4 h in Eagle's minimal essential medium with 10% heat inactivated fetal bovine serum and antibodies, and over 95% of the cell incorporated [³H]thymidine during a 6 h labeling period after 4 d in primary culture. Isolated cells grew best in medium CMRL 1066 with 10% heat inactivated fetal bovine serum as determined by measuring DNA content. The paper is Publication 1100 from the Department of Pathology, University of Maryland School of Medicine. This study was supported in part by National Cancer Institute (Bethesda, MD) Contract NO1-CP-75947 and Grant CA-19197-06 through the National Pancreatic Cancer Project.     

Effect of β-alanyl-L-histidinato zinc on differentiation of osteoblastic MC3T3-El cells: Increases in alkaline phosphatase activity and protein concentration

The effect of -alany-L-histidinato zinc (AHZ) on bone cell function was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus AHZ (10^–7–10^–5 M) or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5 M) produced a remarkable increase of alkaline phosphatase activity and protein concentration in osteoblastic cells. Thus increases were seen with the prolonged cultivation (12–21 days). With the culture of 1, 3 and 12 days, the effect of AHZ (10^–6 M) to increase alkaline phosphatase activity and protein concentration was more intensive than the effect of zinc sulfate, (10^–6 M). The AHZ effects were completely abolished by the presence of cycloheximide (10^–6 M), indicating that AHZ stimulates protein synthesis in the cells. The present study suggests that AHZ has a stimulatory effect on cell differentiation, and that this effect is partly involved on protein synthesis in osteoblastic cells.     

Effects of Fetal Bovine Serum on Ferrous Ion-Induced Oxidative Stress in Pheochromocytoma (PC12) Cells

Ferrous ion (Fe²⁺) has been considered to be a cause of neuronal oxidative injury. Since body fluids contain protein and serum is an essential component of tissue culture medium, we have examined the role of serum protein on Fe²⁺-mediated oxidative stress using PC12 cells and rat cerebral cortices. Fe²⁺ or the combination of ascorbate and Fe²⁺ increased concentrations of thiobarbituric acid reactive substances (TBARS) in PC12 cells and cerebrocortical homogenates in medium (RPMI 1640), but did not increase TBARS when the medium was supplemented with 10% fetal bovine serum. Treatment with ascorbate/Fe²⁺ in serum-free medium reduced endogenous glutathione (GSH) concentration in PC12 cells. However, the medium supplemented with serum did not reduce GSH concentrations. PC12 cell death induced by ascorbate/Fe²⁺ was alleviated by increasing serum or bovine albumin concentrations in the medium. These observations indicated that oxidative injury caused by the transition metal ion could be lessened by adding fetal bovine serum to culture medium.     

Pyruvate-Induced changes in hepatoma cell morphology and macromolecular synthesis

Cells maintained in basal growth medium with 0.2–1.0% serum often require citric acid cycle intermediates for optimal viability. We have found that pyruvate added to minimal growth medium causes cellular flattening and formation of external processes accompanied by increaded DNA synthesis in cultured hepatoma cells (HTC cells). Cells were cultured in plalstic T-flasks (0.5, 1.0, or 2.0 × 10⁶ cells/flask) containing 5 ml medium (90% Eagle's Basal Medium (BME) and 10% Swim's S-77) with various concentrations of fetal calf serum (0.2,0.25, 0.5, 1.0, 2.0, 10%) and either pyruvate (50, 100, 250,500, 1,000μg/ml), or one of: dibutyryl cAMP (DBcAMP) or dibutyryl cGMP (DBcGMP) at 10^?3, 10^?4, or 10^?5 M. At 44–48 hr cultures were pulsed with tritiated thymidine, uridine, or lecucine. Cells became attached to the plastic surface within 24hr. Cells in medium with 0.25 to 2.0% serum had a rounded appearance. With added pyruvate, cellular flattening, process formation, and an increased adherence to the substratum was absorbed. By 48 hr, culture without pyruvate grew in rounded clusters; with pyruvate, cells formed extensive interconnecting processes that appeared loosely attached to the monolayer surface. At the cell densities tested, process formation was maximal with 250 to 500 μg/ml pyruvate. Cytochalasin B blocked flattening and process formation; EDTA (1 mg/ml) caused retraction of processes within 3 min, and a slow dissolution of these structures within cells was observed. DBcAMP or DBcGMP did not induce process formation. Flattening and process foormation in pyruvate-enriched cultures were accompanied by marked stimulation of DNA synthesis and smaller increases in RNA and protein synthesis. Cell number was not affected. These pyruvate-induced changes suggest that alterations in energy metabolism, or precursors that enhance viability and macromolecular synthesis in mammalian cell cultures, may exert marked effects on cellular morphology without corresponding changes in growth of neoplastic liver cells.     

Regulatory role of endogenous regucalcin in the enhancement of nuclear deoxyribonuleic acid synthesis with proliferation of cloned rat hepatoma cells (H4-II-E)

The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.     

Effect of a serum extender containing growth factors on development of IVM and IVF bovine embryos

The present experiments were conducted to determine if supplementation of the culture medium with a serum extender containing growth factors would increase development of bovine embryos into morulae or blastocysts, following in vitro maturation (IVM) and in vitro fertilization (IVF). In Experiment 1, bovine zygotes were cultured in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 2, bovine zygotes were cultured in the presence of cumulus cells in CR1 medium supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 3, bovine oocytes were matured in Medium 199 supplemented with 0, 0.01, 0.1, 1 or 10% serum extender. In Experiment 4, oocytes were matured in Medium 199 with 10% fetal bovine serum (FBS) or 5% FBS with serum extender. Following maturation, zygotes were cultured in CR1 medium with 10% FBS or 5 % FBS and serum extender. In all 4 experiments, the embryos were cultured in vitro until Day 7 after IVF, and development to the morula or blastocyst stage was assessed. The findings of the first 2 experiments showed that the serum extender did not directly influence embryo development but did stimulate development when cumulus cells were included in the culture system. The remaining 2 experiments showed that the serum extender did influence development through its interactions with cumulus cells during maturation and/or culture. These findings suggest that although growth factors or other products do not directly stimulate bovine embryo development their effects may be mediated through secondary cell systems.     

Supplementation of Ham's F10 culture medium with three different sera in the culturing of baboon oocytes

1. Ten female baboons (Papio ursinus) were stimulated for a total of 20 cycles with 3 ovulation induction agents. 2. Oocytes obtained were randomly allocated to Ham's F10 culture medium supplemented with human fetal cord serum, primate serum or commercial fetal bovine serum respectively. 3. Fertilization occurred (38.1-45.5%) in all 3 supplements, but cleavage and embryo development was more successful in culture medium supplemented with fetal bovine serum. 4. Eight embryos were cultured and 6 (75%) of these were cultured in fetal bovine serum supplemented medium.     

Additive effect of oestradiol-17 beta and serum on synthesis of deoxyribonucleic acid in guinea-pig endometrial cells in culture.

Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [3H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [3H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) added to the basal medium. Oestradiol-17 beta added to the control medium at concentrations ranging from 10(-10) to 10(-5) mol/l not only failed to increase but even inhibited [3H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17 beta (10(-9) mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 x 10(-9) mol oestradiol-17 beta/l was combined with either EGF (100 ng/ml) or insulin (10 micrograms/ml). Oestradiol-17 beta appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.     

Obligatory Relationship Between the Sterol Biosynthetic Pathway and DNA Synthesis and Cellular Proliferation in Glial Primary Cultures

Primary cultures of newborn rat brain, which are composed predominantly of astroglia, were used to examine the relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation. Reduction of the fetal calf serum content of the culture medium from 10 to 0.1% (vol/vol) for an interval of 48 h between days 4 and 6 in culture resulted in a quiescent state characterized by inhibition of DNA synthesis and cellular proliferation. When 10% fetal calf serum was returned to the medium for these quiescent cells, within 24 h DNA synthesis increased markedly. Preceding the rise in DNA synthesis was an increase in sterol synthesis, which occurred within 12 h of the return of the quiescent cells to the 10% fetal calf serum. Exposure of the quiescent cells to mevinolin, a specific inhibitor of sterol synthesis at the 3-hydroxy-3-methylglutaryl-CoA reductase step, completely inhibited the increase in DNA synthesis that followed serum repletion. The increase in total protein synthesis that followed serum repletion was not similarly inhibited by mevinolin. When mevinolin was removed after causing the 24-h inhibition of DNA synthesis, the cultured cells underwent active DNA synthesis and proliferation. Thus, inhibition of the sterol biosynthetic pathway resulted in a specific and reversible inhibition of DNA synthesis and glial proliferation in developing glial cells. These findings establish a valuable system for the examination of glial proliferation, i.e., primary glial cultures subjected to serum depletion and subsequent repletion. Moreover, the data establish an obligatory relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation in developing glia.     

Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15 +/- 4 (X 10(6] cells per g of tracheal submucosa with 72 +/- 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1:1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelioid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS-staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post-confluent culture of BTG cells on EHS matrix, we observed numerous dome-like structures formed by differentiated cells which were joined together around luminal spaces.     

Explant culture of rat esophagus in a chemically defined medium

Summary Esophagus from adult male CDF rats was cultured for a period of 28 d in CMRL-1066 medium supplemented with pyruvic acid, HEPES buffer, β-retinyl acetate, and antibiotics. Morphological, radioautographic, and biochemical studies indicated that the survival of the tissue in serum-free medium was equivalent to that in medium containing 5% heat-inactivated fetal bovine serum. There was a relatively constant uptake of [³H]thymidine into DNA and [³H]leucine into protein of the esophageal explants during the incubation. Only the basal cells of the epithelium incorporated [³H]thymidine into their nuclei. The normal morphology of the tissue was preserved when the explants were maintained at both 37 and 30° C, and in either 50 or 20% O₂. Ninety-five percent O₂ was highly toxic to the cells of the explants. This culture system should be suitable for a variety of investigations in esophageal cell differentiation and carcinogenesis.