Isolation and characterization of epithelial cells from bovine pancreatic duct

Summary Epithelial cells derived from bovine pancreatic duct have been grown continuously in culture for 30 weeks (approximately 90 doublings of the cell population). The cells were grown in Eagle's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, and antibiotics. In confluent cultures, the cells are multilayered and form circular structures. When tested at various passages, the cells neither formed colonies in soft agar nor produced tumors after inoculation into athymic, nude mice. Hydrocortisone (1 and 5 μg per ml) and insulin (1,5 and 10 μg per ml) had no effect on the growth of the cells. β-Retinyl acetate inhibited growth rate and cell yield at a concentration of 5 μg per ml but was not growth-inhibitory at lower concentrations. By electron microscopy the cells have numerous mitochondria, Golgi and microvilli. Mucous droplets were observed in a small proportion of the cells. Desmosome-like structures and occluding junctions were observed more frequently between cells that had been transferred as aggregates than between cells transferred as single cells. Cytochemical studies indicated that some cells produce PAS positive granules that were not removed after treatment of the cultures with diastase. Eleven cell clones were isolated from the mass culture. The growth rates of the clones are different as well as the period of time in which the clones can be propagated in vitro. This work was supported in part by Y01 CP 60204 and N01 CP 43237.     

Stability of the carrier state in bacteriophage M13-infected cells.

Bacteriophage M13-infected carrier cells were shown to be unstable to prolonged growth under all conditions. Carrier Hfr cells were transferred in dilute culture (10(3) to 10(4)/ml), where reinfection was impossible and the physiology of the cell was minimally altered. After an initial period of about 10 generations, during which all cells in the culture remained infected, there was exponential decay in the proportion of infected cells in the culture. Uninfected cells that appeared were M13 sensitive. Hfr and F' males were also transferred serially at high cell densities (10(7) to 10(9)/ml), where high levels of phage should permit reinfection. The proportion of phage-producing cells in the cultures remained constant for 7 to 15 generations and then dropped exponentially on further growth. Non-phage-producing cells appearing in the culture were refractory to infection by M13; in some cases cells scored as non-phage producers for 20 generations were observed to produce phage on further growth in liquid culture. F'trp+ males infected with M13 lost trp+ function almost immediately; this was not regained in these experiments. Infected cells grown in dilute culture or on plates remained infected longer, produced more PFU per cell for a longer period, and retained trp+ function in F'trp+ males for over 90 generations. Non-phage-producing cells that appeared were sometimes phage resistant, sometimes phage sensitive. The existence of a phage-related material accumulating at high cell densities and affecting expression of free episomes, episomal expression in Hfr males, and phage synthesis itself is suggested.     

Role of nerve growth factor in the development of rat sympathetic neurons in vitro. III. Effect on acetylcholine production

The effect of nerve growth factor (NGF) on the development of cholinergic sympathetic neurons was studied in cultures grown either on monolayers of dissociated rat heart cells or in medium conditioned by them. In the presence of rat heart cells the absolute requirement of neurons for exogenous NGF was partially spared. The ability of heart cells to support neuronal survival was due at least in part to production of a diffusable NGF-like substance into the medium. Although some neurons survived on the heart cell monolayer without added NGF, increased levels of exogenous NGF increased neuronal survival until saturation was achieved at 0.5 microgram/ml 7S NGF. The ability of neurons to produce acetylcholine (ACh) from choline was also dependent on the level of exogenous NGF. In mixed neuron-heart cell cultures, NGF increased both ACh and catecholamine (CA) production per neuron to the same extent; saturation occurred at 1 microgram/ml 7S NGF. As cholinergic neurons developed in culture, they became less dependent on NGF for survival and ACh production, but even in older cultures approximately 40% of the neurons died when NGF was withdrawn. Thus, NGF is as necessary for survival, growth, and differentiation of sympathetic neurons when the neurons express cholinergic functions as when the neurons express adrenergic functions (4, 5).     

Role of nerve growth factor in the development of rat sympathetic neurons in vitro. II. Developmental studies

Adrenergic sympathetic neurons were grown for 4 wk in submaximal and saturating concentrations of nerve growth factor (NGF) in the virtual absence of non-neuronal cells. In 0.2 or 5 microgram/ml 7S NGF, the neurons gradually decreased in number during the first week, although fewer neurons died at the higher level. No significant change in cell number was observed thereafter. Total neuronal protein, a measure of cell growth, increased linearly with age in both concentrations of NGF. At each age, neurons in high NGF exhibited greater growth per cell than those in low NGF. The ability of neurons to produce catecholamine (CA) increased dramatically during the second and third weeks in both concentrations of NGF, and along a similar time-course, although neurons in submaximal NGF developed a lesser capacity for CA production. As neurons developed in culture, they became less dependent on NGF for survival and CA production, but even in older cultures, approximately 50% of the neurons died when NGF was withdrawn.     

Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.     

Correlations between heparan sulfate metabolism and hepatoma growth

A rat hepatoma cell line (Gershenson et al., Science, 170:859-861, 1970) contains a dynamic steady-state pool of free heparan sulfate (HS) chains in the nucleus that increases in amount when growing cells reach confluence (Fedarko and Conrad, J. Cell Biol., 102:587-599, 1986). In logarithmically growing cells labeled with 35SO4(2-) steady-state levels of [35SO4]HS in the nucleus are altered by a variety of culture conditions. Rapidly dividing cells (doubling time = 18-22 h) growing under optimized conditions had steady-state levels of nuclear HS within the range of 40-50 pmol 35SO4 in nuclear HS/10(6) cells. The steady-state levels of nuclear HS were lowered by several changes in culture conditions, including 1) additions of 1 mM p-nitrophenyl-beta-D-xyloside, 0.25-0.5 mM (+)-catechin, 0.5 ng/ml transforming growth factor beta, 20 ng/ml phorbol-12-myristate-13-acetate, 1 mM dibutyryl cAMP, or 1 mM inositol-2-PO4; 2) decreased levels of D-glucose; or 3) deletions of serum, insulin, or inositol. In all cases lowering of the nuclear HS level was accompanied by an increase in the cell doubling times, suggesting a correlation in which nuclear HS levels must be optimized for maximal growth rates. When cells cultured under optimal growth conditions reached confluence, the level of nuclear HS increased threefold and the cells stopped dividing. The same culture conditions that lowered the steady-state levels of HS in the logarithmically growing cells prevented this rise in the nuclear HS as the cells reached confluence and resulted in loss of contact inhibition and overgrowth of the confluent cultures. These observations suggest a second correlation in which elevated nuclear HS levels are found when cell growth is inhibited at confluence; prevention of this rise results in continued growth. Consistent with this correlation between elevated nuclear HS and reduced growth rates, it was observed that addition of either 0.5 microgram/ml hydrocortisone or 0.05 microgram/ml retinoic acid to the culture medium of logarithmically growing cultures resulted in increases in steady-state levels of nuclear HS that were accompanied by increased cell doubling times. The two agents that increased the levels of nuclear HS in logarithmically growing cultures had little effect on levels of nuclear HS in confluent cells or on contact inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)     

PGE2 and dibutyryl-cAMP enhance the response of rat granulosa cells to FSH

Granulosa cells isolated from immature Sprague-Dawley rat ovaries produce progesterone (31.7 pg/micrograms cell protein) in response to an acute FSH stimulus (5 micrograms/ml NIH-FSH-S11, 2 H). After culture for 48 h in the absence of hormones (control culture), progesterone production by the granulosa cells in response to FSH is significantly reduced (2.9 pg/micrograms cell protein). Cells cultured with prostaglandin E2 (PGE2, 1 microgram/ml) or dibutyryl-cAMP (dbcAMP, 1 mM) exhibited a discernibly greater steroidogenic response to FSH (12.5 and 53.4 pg/microgram cell protein, respectively) than that of control cultures. Therefore the presence of PGE2 or dbcAMP in the culture medium helps to maintain the steroidogenic capacity of granulosa cells in culture. It is probable that this capacity is maintained at a locus distal to the production of cAMP by FSH. Paradoxically, granulosa cells cultured with PGE2 produce less cAMP in response to FSH stimulation than cells in control cultures (15.9 vs. 250.3 fm/micrograms cell protein). This may be due to a suppressive effect of prior exposure to PGE2 on the subsequent activity of adenylate cyclase when the FSH is introduced and a concomitant elevation of phosphodiesterase activity.     

Factors involved in supporting the growth and steroidogenic functions of bovine adrenal cortical cells maintained on extracellular matrix and exposed to a serum-free medium

Bovine adrenal cortex cells maintained on extracellular matrix (ECM)-coated dishes will proliferate actively when serum is replaced by HDL (25 micrograms protein/ml), insulin (10 ng/ml), and FGF (100 ng/ml). The cells have an absolute requirement for HDL in order to survive and grow. The omission of insulin, FGF, or both results in a slower growth rate and lower final cell density of the cultures. A requirement for transferrin (1 microgram/ml) becomes apparent only when cells have been grown for at least four generations in the absence of serum. Early passage (P1-P3) bovine adrenal cortex cells cultured in serum-free medium responded to ACTH (10(-8)M) with increased 11-deoxycortisol production; this effect was not observed in later passage cells (P7-P15). The cells' ability to utilize LDL-derived cholesterol and to respond to db cAMP (1mM) by increased steroid release was preserved in cells cultured for over 60 generations in the serum-free medium. HDL, although also able to increase steroid production in early-passage cultures exposed to ACTH or to ACTH and dibutyryl cyclic AMP (db cAMP), was 10 fold less potent than LDL. It did not support steroidogenesis in cultures not exposed to these trophic agents. The life span of bovine adrenal cortex cells grown in the serum-free medium on fibronectin (FN)- versus ECM-coated dishes was compared. Cells seeded in serum-containing medium and grown in serum-free medium had a life span of 34 versus 60 generations when maintained on fibronectin- or ECM-coated dishes, respectively. Cells seeded in the complete absence of serum in the serum-free medium on ECM- or fibronectin-coated dishes could be passaged for 26 or 13 generations, respectively. While FGF was an absolute requirement for cells cultured on fibronectin-coated dishes, it was not required when cells were maintained on ECM. These observations demonstrate the influence of the ECM not only in promoting cell growth and differentiation but also on the life span of cultured cells.     

Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

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.     

The Mechanism of Onset of the Stationary Phase in Euglena gracilis Grown with 10 mM Succinate: Intracellular pH Values*

SYNOPSIS. When Euglena gracilis were grown with 10mM succinate at pH 3.5 the extracellular pH averaged 3.62 and the cultures had produced 6 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 30 μliters/10⁶ cells/hr. Total protein and dry weights per cell remained constant during the logarithmic phase and began to decline when the late logarithmic phase was reached. Added succinate caused the cultures in stationary phase to commence logarithmic growth once more. Onset of the stationary phase in cultures grown at pH 3.5 was due to depletion of succinate. When cultures were grown at pH 6.9 the extracellular pH averaged 7.62 and the cultures produced 3 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 20 μliters/10⁶ cells/hr during the logarithmic phase. The decline in total protein and dry weights per cell began at the beginning of the logarithmic phase and continued into the stationary phase of growth. Cultures grown at pH 3.5 should produce a larger number of cells/ml than cultures grown at pH 6.9 if the cells are responding to the unionized moiety of succinate and not the ionized moiety. At pH 3.5 83% of the succinate is unionized, whereas at pH 6.9 0.20% of the succinate is unionized. The onset of the stationary phase in cultures grown at pH 3.5 and pH 6.9 is due to lack of an adequate amount of extracellular unionized succinate. Intracellular pH values were determined in cultures grown at pH 6.9 using the weak acid DMO (5.5-dimethyl-2,4-oxazolidinedione). As the extracellular pH increased from 6.90 to 7.62, the intracellular pH increased from 5.89 to 6.89. As the extracellular pH increased from 7.62 to 8.44, the intracellular pH increased from 6.89 to 7.50.     

Characterization of the morphological, growth, and steroidogenic effect of TPA on mouse Y-1 adrenal cortical tumor cells in culture

The tumor-promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) caused a time- and dose-dependent morphological change in Y-1 adrenocortical tumor cells. The morphological alteration was apparent 2 hr following addition of 1 microgram/ml TPA to cell cultures and became more striking with longer treatment times. Smaller doses of TPA took a longer time to produce an effect. Cultures grown in the presence of TPA exhibited more rounding and piling up of cells than similar cultures maintained in medium lacking TPA. These TPA-stimulated morphological changes were reversible, and after 24 hr in TPA-free media, the cultured cells began to flatten. After 96 hr in TPA-free media they resembled the control cultures. The reversibility of the morphological change was also dose dependent: cells treated with 1 microgram/ml TPA took a longer time to resume the typical control morphology than did cultures treated with 0.01 microgram/ml TPA. In addition, TPA treatment resulted in a decrease in cell growth rate, an increase in steroid production, and an increase in the localization of free catalytic units of cAMP-dependent protein kinase in the cytoplasm. The steroidogenic effect of ACTH on the cell population was inhibited in cultures maintained in TPA. The results of this study indicate that TPA induces morphological changes in the Y-1 adrenocortical tumor cell population while increasing steroidogenesis and the activation of cAMP-dependent protein kinase and decreasing cell growth rate.     

Evaluation of anti-coccidial drugs' inhibition of Neospora caninum development in cell cultures

Eight anti-coccidial drugs were examined for their efficacies in preventing development of Neospora caninum in bovine monocyte cell cultures. Lasalocid sodium (0.05 microgram/ml), monensin sodium (0.05 microgram/ml), piritrexim (0.01 microgram/ml), pyrimethamine (0.05 microgram/ml), and trimethoprim (5.0 micrograms/ml) were effective in preventing development of intracellular N. caninum tachyzoites (P less than 0.05). No differences (P greater than 0.05) in mean numbers of infected cells compared to controls were observed in cultures treated with amprolium hydrochloride (10.0 micrograms/ml), sulfadiazine (200.0 micrograms/ml), and sulfamethoxazole (200.0 micrograms/ml).     

Mouse epidermal keratinocytes. Clonal proliferation and response to hormones and growth factors in serum-free medium

A serum-free medium (LEP-1) has been developed for mouse epidermal keratinocytes. LEP-1 consists of "Ca2+-free" Eagle's MEM with non-essential amino acids and seven added supplements (transferrin, 5 micrograms/ml; epidermal growth factor (EGF), 5 ng/ml; hydrocortisone, 0.5 microM; insulin, 5 micrograms/ml; phosphoethanolamine and ethanolamine, each 50 microM; bovine pituitary extract, 180 micrograms of protein/ml). Although serum-free the culture system was dependent for growth on bovine pituitary extract as the only still undefined supplement. LEP-1 supports sustained multiplication of mouse keratinocytes for 25 or more population doublings. A clonal growth assay was developed to investigate the action of growth factors, hormones and other supplements on keratinocytes. Cells grown in LEP-1 (calcium concentration was 0.03 mM) maintained a high proliferative rate and presented the typical morphology of basal epidermal cells. When the calcium concentration of the medium was raised to 1.0 mM, the cells were triggered to differentiate terminally. The epithelial nature of the cells was demonstrated both by electron microscopy and by immunostaining with anti-keratin antibody. The maturation stage of the keratinocytes was defined by several morphological features during the proliferative phase and in terminally differentiating cultures. This serum-free system supported a useful number of cell divisions while keratinocytes retained the capacity to undergo terminal differentiation when given the appropriate stimulus. It provides, therefore, provides a useful model for investigations on growth, differentiation and malignant transformation of epidermal cells in culture.     

Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium

Conditions have been described for the selective growth, serial cultivation, and postconfluent morphological differentiation in vitro of normal adult human uroepithelial cells (HUC) on collagen gel substrates in a serum-free medium without the deliberate addition of undefined components and without a requirement for a polypeptide growth factor. The culture medium used (F12) was the standard Ham's F12 medium (0.3 mM calcium) supplemented with 1 microgram/ml hydrocortisone, 5 micrograms/ml transferrin, 10 micrograms/ml insulin, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 2.7 mg/ml D-glucose, 10(-4) M ethanolamine or 10(-4) M phosphoethanolamine, and 5 X 10(-8) M selenium. HUC grown in F12 on Type I collagen gel substrates had a generation time of 33 hours and could be serially passed 3-5 times during log phase of growth (20-25 population doublings) before spontaneously senescing. Transmission electron microscopy showed that cultures of HUC grown entirely in serum-free F12 on collagen gel substrates morphologically differentiate postconfluence to resemble in some respects the stratified uroepithelium in vivo, although neither a basal lamina nor an asymmetric unit membrane develop. The addition of epidermal growth factor (EGF) to the F12 did not improve either the growth rate or the lifespan in vitro of HUC. In contrast, the addition of fetal bovine serum (FBS) to F12 was mitogenic to HUC in a dose-dependent manner in the concentration range 0.01-1.00% (4-400 micrograms/ml protein), but higher concentrations of FBS did not improve growth further. The generation time of HUC in 1% FBS-F12 decreased to 21 hours, and the potential population doublings in vitro increased to 31-36. Small amounts (140 micrograms/ml) of bovine pituitary extract (BPE) were similarly mitogenic to HUC in F12. Altering the calcium concentration in the standard Ham's F12 medium (0.3 mM), however, did not improve the growth of HUC in serum-containing or serum-free medium. Higher calcium concentrations (0.30-0.90 mM) were neither mitogenic nor inhibitory to HUC growth, but resulted in decreasing viability of HUC in growing cultures, suggesting an accelerating rate of cellular differentiation. In contrast HUC in low calcium, serum-free F12 (0.1 mM) failed to stratify and morphologically differentiate even in postconfluent cultures. This failure of HUC to differentiate in low calcium F12 medium did not confer a long-term growth advantage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Follicle stimulating hormone stimulation of 125-I-human chorionic gonadotropin binding in porcine granulosa cell cultures.

In order to see if FSH acts directly upon the granulosa cell to stimulate hCG binding, granulosa cells harvested from small 1-2 mm porcine follicles were grown in 250 ml flasks in chemically defined media containing 0.05 mug/ml highly purified human FSH for 2, 4, and 6 days. The defined medium consisted of culture medium 199 plus 0.4% bovine serum albumin, 0.2% lactalbumin hydrolysate and 10 munit/ml insulin. The cultures were harvested by scraping with a rubber policeman and incubated with 0.1 mug/ml 131-I- or 125-I-hCG. Binding expressed as cpm/culture or per mg protein yielded similar results. In five separate experiments addition of FSH stimulated hCG binding two- to fourfold above control cultures. In a typical experiment after 2 days of culture, the specific binding of control cultures to hCG was 962 plus or minus 45 cpm/culture (-x plus or minus SE; n = 3) and the binding in cultures grown in the presence of 0.05 mug/ml FSH was 3933 plus or minus 1787 (n = 3; P less than 0.01). Granulosa cells harvested from large (8-12 mm) follicles grown under similar conditions bound 29,669 plus or minus 948 cpm/culture (n = 4). These data demonstrate that FSH may have a direct stimulatory role upon induction of granulosa cell LH-hCG receptors in vitro.     

Attenuation of neurotoxicity following anoxia or glutamate receptor activation in EGF- and hippocampal extract-treated neuronal cultures

Neurotoxicity following anoxia or glutamate receptor activation was studied in primary neuronal cultures grown in serum-free, chemically defined CDM R12 medium. Exposure to 1 mM KCN, 0.5 mM kainic acid and 0.5 mM N-methyl-D-aspartate led to progressive neuronal degeneration. This damage was quantified by measuring lactate dehydrogenase released in the culture medium. The toxic effects were observed early during the development of the neuronal culture (from 4 days in vitro on) and seemed to be neuron-specific since astrocyte cultures were not affected. Chronic treatment of the neuronal cultures with epidermal growth factor at 10 ng/ml and hippocampal extract at dil. 1/833 (w/v) induced morphological alterations, increased beta-adrenergic receptor coupled adenylate cyclase activity, increased level of total lactate dehydrogenase activity in the case of epidermal growth factor-treated cultures, and attenuation of lactate dehydrogenase release following exposure to KCN or glutamate receptor agonists. The alterations observed are probably due to the proliferation and differentiation of glial cells in these treated cultures. This suggests that glial cells protect neurons in vitro from degeneration induced by anoxia or glutamate receptor activation.     

Requirement of heme for growth of Bacteroides fragilis.

Heme or protoporphyrin IX was required for growth of Bacteroides fragilis in a defined medium. The amount of heme necessary for half-maximal growth was 2 to 10 ng/ml (3.8 to 15 pmol/ml) among the Bacteroides species and strains tested. The growth rate, metabolic products from glucose fermentation, and cell yields were affected by the concentration of heme in the medium and by the length of time the culture was incubated. When heme was growth limiting (4 ng/ml), growth rates decreased by 50%, cultures started producing lactic and fumaric acids, and the cell yields declined. The cell yield for B. fragilis (ATCC 25285) at 24 h in medium containing 6.5 microgram of heme per ml was 69 g (dry weight) of cells per mol of glucose compared to 16 g (dry weight) of cells per mol of glucose with 4 ng of heme per ml. B. fragilis was unable to grow in defined medium when a porphyrin precursor, delta-aminolevulenic acid or porphobilinogen, was added in place of heme.     

Adaptation of BHK-21 Cells to Growth in Shaker Culture and Subsequent Challenge by Japanese Encephalitis Virus

Baby hamster kidney (BHK-21) cells were adapted to grow in shaker culture using Waymouth medium 752/1 containing 20 mM N-2-hydroxyethyl-piperazine-N'-2'-ethanesulfonic acid buffer and supplemented with 2.5% (vol/vol) calf serum, 0.002% (wt/vol) sodium oleate, and 0.2% fatty acid-free bovine serum albumin (WO2.5). Infectivity of Japanese encephalitis virus grown in the cells adapted to WO2.5 approached 2 x 10(8) plaque-forming units per ml. The culture volume of infected cells was reduced fivefold 12 h after infection. This step resulted in a 10-fold increase in infectivity over that obtained from infected cultures not subjected to volume reduction.     

Further studies of the use of cycloheximide for cultivating Mycobacterium lepraemurium in cell culture

The advantage of using cycloheximide for cultivating Mycobacterium lepraemurium in cell culture was further demonstrated. Continuous multiplication of the bacillus in successive subcultures was obtained in MFP, HEp-2 and Vero cells maintained in culture medium containing 0.1 microgram of cycloheximide per ml. Growth characteristics were comparable to those observed in the cultures of A31 cells previously reported. The procedure was simple and convenient. Comparable results, however, have not been obtained in cultures of other established cell lines, HeLa 229, L, MDCK, and Neuro-2a.