Serum effects on the response of mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae.

The response of mammalian cells to Pseudomonas and diphtheria exotoxins was studied. A method was developed whereby the sensitivity of cells to these two toxins could be quantitated. The method is versatile and can be used to study the effects of toxins on many cellular metabolic or transport processes. The type of serum used in the culture medium significantly influenced the response of cells to the toxins. Calf, horse, and human sera protected cells while fetal calf serum did not. Precipitation with (NH4)2SO4 demonstrated the probable presence of toxin-specific antibody in the protective calf serum while none was detected in the nonprotective fetal calf serum. The level of antibody in calf serum, as titrated by hemagglutination, was sufficient to account for all the observed protection. It is suggested that fetal calf serum be used for all future cell culture studies of bacterial toxins.     

Changes in serum influence the fatty acid composition of established cell lines

Summary The fatty acid composition of different kinds of commercially available serum used to supplement cell culture media differs widely. As compared with fetal bovine serum, horse and bovine calf serum have a very high content of linoleic acid (18:2) and are low in arachidonic acid (20:4). (Fatty acids are abbreviated as number of carbon atoms: number of double bonds). Swine serum contains substantial amounts of both 18:2 and 20:4. Only fetal bovine serum contains more than 1% docosahexaenoic acid (22:6). Considerable differences in fatty acid composition occur when cells are grown in media containing any of these different serum supplements. The 18:2 and 20:4 content of 3T3 mouse fibroblast phospholipids is highest when the medium contains horse serum, intermediate with bovine calf serum, and lowest with swine or fetal bovine serum. Likewise, the highest phospholipid 18:2 content in Madin-Darby canine kidney cells (MDCK) occurs when the medium contains horse serum. With MDCK cells, however, growth in swine serum produces the highest 20:4 content. The 3T3 cell phospholipids accumulate more than 1% 22:6 only when the medium contains fetal bovine serum, whereas in no case do the MDCK cell phospholipids accumulate appreciable amounts of 22:6. The fact that the cellular fattyacid composition is likely to change should be taken into account when changes are contemplated in the serum used to grow established cell lines. These studies were supported by Arteriosclerosis Specialized Center of Research Grant HL 14,230 from the National Heart, Lung, and Blood Institute, National Institutes of Health.     

Class Specificity of Avian and Mammalian Sera in Regards to Myogenic Cell Growth in vitro

Chick myogenic cells grew in the presence of a small amount of avian serum in a culture medium composed of Eagle's minimum essential medium (MEM) and horse serum. Mammalian sera, except for fetal bovine serum at high concentrations, could not substitute for the avian serum.
Rat myogenic cells grew in the presence of a small amount of mammalian serum in a culture medium composed of MEM and chick serum: avian sera, except for dove serum at high concentrations, could not substitute for the mammalian serum.
Serum from animals of the class from which the myoblasts were obtained was needed for cell growth. It is thus concluded that there is a class specificity among sera in regards to myogenic cell growth. The only exceptions to this hypothesis found so far were fetal bovine and dove sera.     

Fetal bovine serum and other sera used in tissue culture increase epithelial permeability

Summary Fetal bovine serum (FBS) or heat-inactivated FBS (56° C for 30 min, HFBS) caused a dose-dependent decrease in the transepithelial electrical resistance of an epithelial monolayer (MDCK). A saturating concentration of HFBS (30%) caused an average fall of 25 ± 2% within 60 min. Upon removal of HFBS, the resistance returned to its starting value within 1 h. Flux studies with [³H]mannitol demonstrate that the fall in resistance is due to an increased permeability of the tight junctions. Thirty percent heat inactivated sera from goat, newborn calf, calf, bovine, and horse caused falls ranging from 26 to 47%. In contrast with the basolateral preference of human and bovine adult sera, fetal bovine and newborn calf sera elicit this response primarily by interacting with the apical surface of the epithelium. HFBS-treated monolayers show a significant increase in the condensation of F-actin at points where ≥3 cells meet. These results demonstrate that FBS and other sera used as nutritional supplements can increase the permeability of the tight junctions of cultured epithelial cells.     

Polyamine cytotoxicity in the presence of bovine serum amine oxidase

The toxicity of extracellular spermine, determined in the presence of fetal calf serum, was studied using three cell lines: FM3A, L1210, and NIH3T3 cells. Amine oxidase in fetal calf serum produces aminodialdehyde generating acrolein spontaneously, H(2)O(2), and ammonia from spermine. Spermine toxicity was prevented by aldehyde dehydrogenase, but not by catalase. Similar concentrations of spermine and acrolein were needed to produce toxicity. Other aldehydes (formaldehyde, acetaldehyde, and propionaldehyde) and hydrogen peroxide were less toxic than acrolein. Spermidine and 3-aminopropanal, which produces acrolein, also exhibited severe cytotoxicity. The degree of cytotoxicity of spermine, spermidine, and 3-aminopropanal was nearly parallel with the amount of acrolein produced from each compound. Thus, it was deduced that acrolein is a major toxic compound produced from polyamines (spermine and spermidine) by amine oxidase.     

Immunomodulating activities associated with the cytosol fraction of a 3-methylcholanthrene-induced rat fibrosarcoma--II. Association with polyamine complexes

This paper characterizes the molecular nature of the factors present in cytosol from F-344 rat McFiFi2(s) fibrosarcoma cells (FiCF) which mediate inhibition of PHA-induced lymphoproliferative responses. These are polyamines (spermine/spermidine) conjugated to different protein carriers. Interaction of these complexes with polyamine oxidase (PAO) present in fetal calf or rat serum is responsible of the suppression observed.     

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.     

Composition and enzyme activities of Spiroplasma citri membranes.

Spiroplasma citri was cultured in three different media that supplied cholesterol and fatty acids from: (i) horse serum, (ii) pleuropneumonia-like organism (PPLO) serum fraction, or (iii) bovine serum albumin-fatty acid-cholesterol. The ability of PPLO serum fraction to support growth varied by lot number. Neither PPLO serum fraction nor the bovine serum albumin medium supported growth as well as the horse serum medium. Analysis of cholesterol, lipid phosphorus, and membrane protein showed the horse serum- and PPLO-grown cells to be indistinguishable, but the bovine serum albumin-grown cells were deficient in lipid phosphorus. The three cultures did not show markedly different fatty acid compositions, but, in all cases, the cultures preferentially incorporated palmitic acid and discriminated against linoleic acid. Cultures grown for different times from logarithmic growth through a degenerative phase showed relatively constant ratios of cholesterol/protein and lipid phosphorus/protein. Fatty acid composition was also relatively constant at the different stages. Adenosine triphosphatase and p-nitrophenyl phosphatase were mainly associated with the membrane, whereas reduced nicotinamide adenine dinucleotide oxidase was either readily removed or not associated with the membrane. The reduced nicotinamide adenine dinucleotide oxidase was inactivated at temperatures above 35 degrees C.     

Effect of Serum on the Yield of Lymphocytic Choriomeningitis Virus in Baby Hamster Kidney Cells

The yields of the Armstrong and WE strains of lymphocytic choriomeningitis virus in baby hamster kidney (BHK) cells cultivated in either bovine, calf, fetal bovine, or horse serum were investigated. Lines of BHK cells were established in these sera. When the infected cell lines were observed by immunofluorescence, the per cent fluorescing cells for a given virus strain did not vary. However, for both strains, the extracellular virus yields per cell were significantly greater in the fetal bovine-cell line than in the other serum-cell lines.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk     

Effect of purine supplementation on the growth of salmonid cell lines in different mammalian sera

The Chinook salmon embryo cell line, CHSE-214, grew well in fetal bovine serum (FBS) but poorly in dialyzed (d) FBS. Purines restored most but not all growth-promoting activity to dFBS, which suggests that purines account for a large portion of the dialyzable fraction's growth-promoting activity. CHSE-214 died in newborn calf serum (NCS) but grew slightly in dNCS, which suggests that the dialyzable fraction of NCS contains a toxic component(s). Little or no proliferation occurred in calf serum (CS); some took place in horse serum (HS). Porcine serum (PS) was very toxic. In all these sera except PS and HS, the purine nucleoside, inosine, significantly enhanced growth, whereas the pyrimidine nucleoside, uridine, was without effect. The other purines, hypoxanthine, adenine, adenosine and guanosine also stimulated proliferation but not as well as inosine. Inosine also enhanced the growth of the rainbow trout gonadal cell line, RTG-2. Although their morphology underwent minor alterations in medium with inosine, CHSE-214 cells could be grown indefinitely in CS and inosine as effectively as in the more expensive FBS.     

Selective inhibition of fibroblasts by spermine in primary cultures of normal human skin epithelial cells

Summary Overgrowth with fibroblasts has been a major problem in the cultivation of normal human skin epithelium. In the present study it is shown that the addition of spermine to the culture medium in micromolar concentrations has a differential cytotoxic effect on fibroblasts allowing the cultivation of human skin epithelial cells in primary culture without fibroblastic overgrowth. Putrescine, another polyamine, is shown to be equally cytotoxic to fibroblasts and epithelial cells when added in millimolar concentrations; below this concentration range no cytotoxic effect could be demonstrated. This difference in cytotoxicity between spermine and putrescine is suggested to depend on the conversion of spermine, but not putrescine, and to highly cytotoxic products by an amine oxidase present in fetal bovine serum. This project was supported by the Novo foundation.     

Effects of fetal versus postnatal sera upon adipose tissue stromal-vascular cells in primary culture

Summary This experiment was conducted to determine if serum factors are responsible for differences in cellularity of prenatal and postnatal pig adipose tissue as determined by in vitro measurement of cellular proliferation and enzyme-histochemical metabolic development. Cellular proliferation of stromal-vascular cells derived from rat inguinal adipose tissue was measured by [³H]-thymidine incorporation. Coverslip cultures were used for analysis of histochemical differentiation. Cells were incubated in media containing 10% fetal bovine, fetal pig, mature pig, or various combinations of these sera. Fetal bovine serum promoted more [³H]-thymidine incorporation than fetal or postnatal pig sera. Fetal pig sera also stimulated more [³H]-thymidine incorporation than mature pig sera. Sera from adult pigs promoted differentiation and lipid filling of adipocytes. Fetal pig sera stimulated histochemical expression of enzymes, but did not induce lipid filling. Fetal bovine serum produced histochemically undifferentiated cells. Addition of fetal bovine serum to media containing mature pig sera reduced lipid accumulation and histochemical reactivity of cells. This effect of fetal serum was thus due to specific inhibition of lipid deposition and not substrate restriction. These experiments demonstrated that serum factors have a major influence on morphological development of fetal and postnatal adipose tissue.     

Effect of serum on organogenesis of the rat testis in vitro

Summary It was observed previously that primordia of fetal rat testes when explanted in vitro in a synthetic medium at the outset of sexual differentiation differentiate seminiferous cords during the following days, but that the addition of 15% fetal bovine serum prevents this morphogenesis. In the present study, human, horse, bovine calf, and rat sera were shown to exert the same effect. Very low concentrations of human or fetal bovine serum (0.5 or 1%) were sufficient to produce the serum effect, which was only slightly reduced when the serum was heated. The serum activity was not removed by dialysis (membrane cut-off 15 000), but it disappeared after treatment with trichloroacetic or perchloric acids or after trypsin digestion. Partial purification of the active factor(s) from human serum was achieved by successive gel filtration, affinity chromatography, and ion exchange chromatography. Analysis of the active fractions by electrofocusing and immunoelectrophoresis placed the activity within the α globulin group. Among a series of purified serum proteins tested, α₂-HS-glycoprotein was found to exhibit the serum effect, though this activity was heat labile.     

In vitro cultivation of Plasmodium falciparum: studies with modified medium supplemented with ALBUMAX II and various animal sera

RPNI, a combination of three commercially available growth media (RPMI-1640, NCTC-135 and IMDM) has been found to support long term continuous cultivation of 3D7 strain of Plasmodium falciparum in the presence of 10% bovine calf serum. During the present study, the suitability of this medium was evaluated for the development of P. falciparum in the presence of horse, goat and rabbit sera as well as various concentrations of ALBUMAX II. RPNI medium supplemented with 10% bovine calf serum (RPNI-BCS) was used as control. The cultures were maintained in candle jars protocol and parasitaemia was monitored daily up to day 7. Horse, goat and rabbit sera all supported the development of P. falciparum. Horse serum gave best results in RPNI medium and supported continuous culture up to day 100. The parasitaemia in the presence of ALBUMAX was significantly higher in RPNI than in RPMI-1640. Addition of hypoxanthine in RPMI-1640 caused an increase in parasitaemia whereas no obvious advantage could be observed in RPNI. The findings exhibited that medium RPNI has an edge over conventional RPMI-1640 medium for in vitro cultivation of P. falciparum.     

Growth in primary culture of mouse submandibular epithelial cells embedded in collagen gels

Summary Mouse submandibular glands were dissociated and the epithelial cells embedded in a collagen gel matrix. A characteristic and reproducible pattern of growth was seen resulting in three-dimensional outgrowths with ductlike structures projecting into the matrix. A sustained cell growth leading to a 5 to 10-fold increase in cell number was observed in less than 2 wk. The extent of this growth was found to be dependent on serum concentration. Of the three sera tested, swine serum was found to promote greater growth compared to fetal bovine serum or horse serum. Swine serum dose response studies have shown that a concentration of 2 to 5% in the medium elicited only a modest increase, if any, in cell number compared to the initial value within a period of 2 wk. Various hormones and growth factors were then added to this “maintenance” medium. Insulin was found to stimulate growth consistently and reproducibly in a dose-dependent manner. Ultrastructurally, the resulting outgrowths were comprised of polarized cells joined by apical tight junctions and desmosomes. These outgrowths produced epidermal growth factor in response to dihydrotestosterone, triiodothyronine, and cortisol. The present system provides a method for sustaining growth and functional differentiation in primary culture of mouse submandibular gland epithelial cells. This investigation was supported by PHS Grants CA05388 and CA09041, awarded by the National Cancer Institute, Department of Health and Human Services.     

Inhibition of cells in culture by polyamines does not depend on the presence of ruminant serum

Using T-lymphocyte (T-LC) and granulocyte colony (GC) assays with truly proliferating cells, the inhibitory dose-response relationships of spermine and spermidine in the presence of selected sera have been examined. In contrast to previous studies which used [3H]thymidine uptake as an index of proliferation, in vitro inhibition by polyamines was shown to require neither foetal calf serum (FCS) nor the addition of any exogenous polyamine oxidase. Cells grown in the absence of FCS were between 5-50% as sensitive to polyamines as in its presence. By using specific inhibitors of polyamine oxidase, it was shown that polyamine-elicited mitotic inhibition in the absence of FCS was still dependent on a polyamine oxidase, and evidence is presented to show that the source of the enzyme is the cells themselves.     

LHRH inactivation by reconstituted horse and fetal bovine sera: assessment by reduction of immunoreactivity and biological activity in pituitary cell cultures

Lyophilized horse and fetal bovine sera are commonly incorporated into the growth media used for primary pituitary cell cultures. LHRH degrading activity has been assumed to exist in these preparations but has not actually been demonstrated. During our studies with pituitary cultures, it became necessary to ascertain if LHRH inactivating activity could be demonstrated in these sera. Luteinizing hormone releasing hormone (LHRH) was preincubated in either serum-free medium or medium containing fetal bovine and horse serum. Whether LHRH was lost during these incubations was assessed by diminished immunoreactivity as indicated by radioimmunoassay (RIA) and by diminished biological activity as indicated by reduced release of LH from pituitary cell cultures. Both the RIA and bioassay results indicated LHRH inactivating activity; the loss of LHRH could be prevented by inclusion of bacitracin in the incubations.     

5-Methylthioribose. Its effects and function in mammalian cells

The growth responses of 5-deoxy-5-methylthioribose on a 5'-deoxy-5'-methylthioadenosine phosphorylase containing cell line (BW5147) and the methylthioadenosine phosphorylase-deficient cell line (L1210D) were examined. Methylthioribose was shown to dramatically affect these cells, increasing their growth rate, saturation density, and viability. It was also found that methylthioribose could satisfy the methylthio dependence of the enzyme-deficient cell line, L1210D. A model is proposed to explain the selective growth of methylthioadenosine phosphorylase-deficient cells in medium lacking a methylthio donor but containing fetal calf serum. It is hypothesized that cellularly exported methylthioadenosine is degraded to methylthioribose in the presence of medium containing serum of high methylthioadenosine phosphorylase activity (i.e. fetal calf serum). The resultant methylthioribose can then be used to satisfy the methylthio requirement of these cells. To test this theory, various purified preparations of bovine liver methylthioadenosine phosphorylase were used to artificially increase the specific activity of methylthioadenosine phosphorylase in horse serum. In each case, it was demonstrated that only medium containing serum of enzyme activity nearly equal to that of the glutathione-stimulated fetal calf serum activity, supported the growth of methylthio-dependent cells in the absence of methylthio compounds. The data suggest that the degradation of methylthioadenosine and subsequent formation of methylthioribose represents an essential process in the growth of mammalian cells.     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.