Suspension Culture Medium Research Articles

Extracellular polysaccharides from Pteridium aquilinum suspension cultures

Extracellular polysaccharides were isolated from the medium of liquid suspension cultures of bracken (Pteridium aquilinum) L. Kuhn var. latiusculum (Desv.) Underw. The resulting material contained very few charged pectic polysaccharides. Glycosyl composition and linkage analysis showed that the major polysaccharide components were a large arabinogalactan rich in T-arabinosyl and 3,6-galactosyl residues, and another peak rich in mixed linkages of arabinose, galactose and glucose.

Cellular and Subcellular Localization of Peroxidase Isoenzymes in Plants and Cell Suspension Cultures from Lupinus polyphyllus

Abstract , leaf protoplasts and cell suspension cultures of Lupinus polyphyllus and isolated vacuoles were studied for cellular and subcellular localization of peroxidase isoenzymes. Isoelectric focusing revealed 16 peroxidase isoenzymes. The basic peroxidase isoenzymes are predominantly localized in the vacuole and, to a minor degree, unbound in the intercellular space. The acidic isoenzymes are cell wall-bound in plants and not detectable in suspension-cultured cells. Large amounts (up to 11.0 U/ml) of a single basic isoenzyme are detectable in the spent medium of cell suspension cultures.

Effect of nerve growth factor on the elongation of neurites from axotomized rat embryonic septal‐basal forebrain neurons: An in vitro analysis

The administration of nerve growth factor (NGF) into the brain of a fornix-fimbria lesioned rat can rescue many cholinergic, septal-basal forebrain (SBF) neurons from imminent cell death. Unfortunately, it is unclear if NGF can stimulate regenerative growth from axotomized, SBF neurons. In the present study, we used an in vitro model system to determine if NGF could affect neurite outgrowth from nonaxotomized and/or axotomized, embryonic SBF neurons. Axotomized neurons were obtained by severing the neuritic fields surrounding embryonic day (E) 15 SBF explants maintained in primary culture. Acetylcholinesterase (AChE) histochemistry was used to assess the effects of NGF on cholinergic neurites. We report that 1) neurite outgrowth on type I collagen from E15 SBF neurons in primary culture (nonaxotomized neurons) was not affected by NGF. 2) NGF enhanced the outgrowth (regeneration) of axotomized, SBF neurons on a collagen substratum; however, neurons had to be treated with NGF both before and after axotomy to stimulate regeneration effectively. Application of NGF either before or after axotomy did not enhance regenerative neurite outgrowth. 3) SBF neurons had to be axotomized for NGF to facilitate neurite outgrowth. This is supported by the observation that SBF explants, initially maintained in NGF-supplemented medium in suspension culture, did not demonstrate enhanced neurite outgrowth in the presence of NGF when plated onto a substratum. 4) The regenerative growth of AChE-negative, as well as AChE-positive, neurites was facilitated by NGF treatment. In addition to data concerning neurite outgrowth, we also found that the NGF receptor, as recognized by the antibody 192-IgG, expands its distribution as time in culture progresses; i.e., staining, originally confined to cell bodies and proximal processes within the explant, later included neurites that emanated from the explant. Thus, our results demonstrate that NGF can stimulate regenerative growth from axotomized, but not nonaxotomized, embryonic SBF neurons. We hypothesize that, given the appropriate substratum for axon elongation in vivo, NGF can stimulate the regeneration of SBF neurons in the injured adult brain.

A microassay technique for sulfate determination in plant samples

Abstract A microassay for the measurement of sulfate sensitive to below 1 nmol is described. Accuracy and reproducibility were demonstrated with trichloroacetic acid clarified plant sample volumes of only 5 μl. The sensitivity of this assay enabled, for the first time, the measurement of sulfate in the medium of plant suspension cultures during its depletion down to the compensation point in a non‐disruptive manner. This was possible because very small volumes could be sampled from continuously growing cultures.

Somatic Embryogenesis and Plant Regeneration from Suspension Cultures of Red Fescue

The development of embryogenic cell suspension cultures from which whole‐viable plants could be regenerated on a reliable basis would increase the range of techniques that may be applied by somatic approaches toward genetic modification. The objective of this study was to develop such a cultural system from embryogenic callus tissue of ‘Dawson’ red fescue (Festuca rubra var trichophylla Gaud.). Embryogenic cell suspension cultures were initiated from highly embryogenic callus tissue that had been cultured for over 4 yr. The optimal basal medium for suspension cultures consisted of one‐half strength Murashige and Skoog (MS) salts supplemented with 30 g L−1 sucrose, Gamborgs B‐5 vitamins, 3 g L−1 casein hydrolysate (CH), and 4 mg L−1 of 2,4‐dichlorophenoxyacetic acid (2,4‐D). The addition of CH significantly enhanced culture growth rates as measured by packed cell volumes. Prefiltering newly initiated suspensions through a 31‐μm filter screen was critical in removing relatively faster growing non‐embryogenic (NE) cell types. The use of a discontinuous percoll gradient was as effective as filtering in removing NE cells but was more time consuming. Prolific regeneration of whole, viable plants was attained by transferring embryogenic cell clusters to a hormone‐free, half‐strength MS basal medium supplemented B‐5 vitamins and 8 g L−1 agar.

Cross-linking of the T cell antigen receptor interferes with the generation of CD4+8+ thymocytes from their immediate CD4-8+ precursors.

The majority of thymocytes are immature cells co-expressing the surface markers CD4 and CD8. About two thirds of these cells also express the T cell antigen receptor (TcR), though at a level distinctly lower than found on mature T cells. The direct precursors of these "double-positive" thymocytes are cycling cortical blast cells of the CD4-8+ phenotype. Using a new monoclonal antibody to a constant determinant of the rat TcR alpha/beta, it is shown here that (a) about 50% of these CD8 "single-positive" committed precursor cells already express the TcR alpha/beta, though at very low levels, (b) during short-term suspension culture in medium supplemented only with fetal calf serum they not only acquire CD4 but also TcR alpha/beta levels characteristic of CD4,8 "double-positive" thymocytes, and (c) cross-linking of the TcR during culture inhibits the acquisition of the CD4 antigen in the majority of these cells.

Purification and Properties of Glucoamylase from Sugar Beet Cells in Suspension Culture

Glucoamylase and alpha-amylase are present in callus and suspension cultures of sugar beets (Beta vulgaris L.) as well as in mature roots. The subcellular localization of glucoamylase differed in callus and suspension-cultured cells: in callus, glucoamylase was present together with alpha-amylase in the soluble fraction of cells, but in suspension cultures, it was present predominantly in the extracellular fraction while most of the alpha-amylase activity remained in cells. Glucoamylase activity was considerably lower in callus protoplasts relative to the activities of alpha-mannosidase and alpha-galactosidase and the suspension of callus in Murashige-Skoog liquid medium or in mannitol by brief agitation resulted in the release of glucoamylase to the medium. These findings suggest that glucoamylase in callus may be present in a soluble form in the free space in the cell wall. Both mature roots and callus contained alpha-amylase and glucoamylase in the soluble fraction. Glucoamylases in the soluble fraction of callus and in the medium of suspension cultures were purified separately to homogeneity by the same four-step purification procedure, which included fractionation with ammonium sulfate, column chromatography on carboxymethyl cellulose, gel filtration on Bio-Gel P-150, and preparative disc electrophoresis. The identity of the glucoamylases from the two sources was confirmed by a comparison of chromatographic behavior during purification, mobility during gel electrophoresis, M(r) (83,000 D by SDS PAGE), and enzymic and kinetic properties of the catalytic reaction, such as optimal pH and temperature, heat stability, and K(m) value for soluble starch. Glucoamylase from suspension cultures was one of the major proteins that were secreted into the medium. Dedifferentiation of leaves of young plants to callus was accompanied by induction of glucoamylase and repression of some alpha-amylases and the debranching enzyme.

A System for Manipulating the Membrane Fatty Acid Composition of Soybean Cell Cultures by Adding Tween-Fatty Acid Esters to Their Growth Medium

The development of a system for modifying the membrane fatty acid composition of cultured soybean cells (Glycine max [L.] Merr.) is described. Tween-fatty acid esters carrying specific fatty acids were synthesized and added to the medium of suspension cultures. Cells transferred large quantities of exogenous fatty acids from Tweens to all acylated membrane lipids; up to 50% of membrane fatty acids were exogenously derived. C15 to C20 saturated fatty acids and C16, C18, and C20 unsaturated fatty acids with either cis or trans double bonds were incorporated into lipids. Cells elongated saturated fatty acids of C16 or less, and unsaturated fatty acids with cis double bonds were further desaturated. No other types of modifications were observed. Growth ceased in cells treated with excessive concentrations of Tween-fatty acid esters, but frequently not for several days. Cessation of cell growth was correlated with changes in membrane fatty acid composition resulting from incorporation of large amounts of exogenous fatty acids into membrane lipids, although cells tolerated large variations in fatty acid composition. Maximum tolerable Tween concentrations varied widely according to the fatty acid supplied. Potential uses of this system and implications of the observed modifications on the pathway of incorporation are discussed.

Effect of culture medium composition on pheromone receptor levels in Achlya ambisexualis

Sexual reproduction in the eukaryotic fungi Achlya is controlled by two steroid pheromones. Antheridiol is the steroid released by female cells that induces male sexual differentiation. The antheridiol-induced response of male cells has been shown to be influenced by the composition of the culture medium. The present study was designed to determine if the composition of the culture media might also affect the levels of antheridiol binding protein in the cytosol of male cells. The mycelial content of cytosolic steroid pheromone binding sites in Achlya ambisexualis E87 males was measured at daily intervals during 6 days of suspension culture in media containing different nitrogen sources. Levels of binding sites increased during the first 2 days in culture to a plateau that was maintained for the next 2–3 days. During the first 3 days in culture, levels were much lower in mycelia cultured in an enriched medium containing lactalbumin hydrolysate compared to mycelia cultured in defined media containing glutamic acid as the nitrogen source. The level of binding sites increased rapidly when mycelia were transferred from an enriched medium to a nutrient-free salt solution and decreased when mycelia were transferred from a defined to an enriched medium. The relative differences in cytosolic binding measured by in vitro radioligand saturation analysis were confirmed by in vivo uptake studies. It is concluded that the mycelial content of antheridiol binding sites can be experimentally manipulated by variations in the composition of the culture medium and/or the time period of incubation in the medium.

Release and crystallization of berberine in the liquid medium of Thalictrum minus cell suspension cultures

Cell suspension cultures of Thalictrum minus L. var. hypoleucum Miq. were found to produce a large amount of berberine (400-800 mg/l) when 5-10 μM 6-benzyladenine was added to Linsmaier and Skoog's medium containing 100 μM 1-naphthaleneacetic acid. Most of the berberine produced was released continuously from the cells into the liquid medium, and an excess of berberine crystallized as its nitrate in the medium. When the cells were cultured in a modified LS medium containing 20 mM KNO3 and 40 mM NH4Cl in place of 20.6 mM NH4NO3 as nitrogen source, most of the alkaloid crystallized to form berberine chloride instead of nitrate. Minor alkaloids, thalifendine and magnoflorine, were also isolated from the medium and identified.

Changes in cell wall polysaccharides during elongation in a 2, 4‐D free medium in a carrot suspension culture

Cell elongation occurred when carrot (Daucus carota L. ev. Kurodagosun) cells subcultured through sieving (Y. Ozeki and A. Komamine, Physiol. Plant. 53: 570‐577. 1981) were transferred to a medium lacking auxin, while the cells showed no elongation in a medium containing 2, 4‐D. Changes in polysaccharides of the cell walls and in their sugar composition during elongation were investigated. All wall components, EDTA‐soluble pectic substance, 5 and 24%, KOH‐soluble hemicelluloses and cellulose increased markedly during elongation. The increase of hemicelluloses correlated especially with elongation. In the 5% KOH‐soluble hemicellulose, galactose and arabinose contents in the walls increased significantly both in amounts (per fresh weight) and relative contents (% in total neutral sugars) during elongation, while the relative contents of glucose and xylose decreased rapidly in the 5 and 24% KOH‐soluble hemicelluloses. The methylation analysis tentatively indicated that larger amounts of galactan and/or arabinogalactan and lower amount of xyloglucan were found as components of the two hemicelluloses of elongating cells than those of non‐elongating cells. The amounts of total carbohydrate and of uronic acid of extracellular polysaccharides secreted into the medium increased to a larger extent in the elongation culture than in the non‐elongation culture. The contents of galactose and arabinose in extracellular polysaccharides increased rapidly in the elongation culture. The biochemical aspects of cell elongation in the absence of auxin were discussed from the viewpoint of the results obtained here.

Establishment and characterization of cell lines from the Walker carcinoma 256 able to grow in suspension culture and deficient in thymidine kinase.

A cell line from the Walker carcinosarcoma 256 of the rat has been established in suspension culture in medium with 5% bovine calf serum for over 350 generations, with an average population doubling time of 17 h, a plating efficiency of 56%, a colony forming efficiency of 32%, and a good capacity to form colonies in soft agar. The cells are morphologically indistinguishable from those in the solid tumor and ascites as checked by transmission and scanning electron microscopy. The karyotype is characterized by a modal number of 65 chromosomes and by the presence of a marker metacentric chromosome. The cells express thymidine kinase, gamma-glutamyl transpeptidase, and alkaline phosphatase; are agglutinable by concanavalin A; and can be synchronized by the triple thymidine block. They induce primary tumors, both subcutaneously (solid) and intraperitoneally (ascitic), in the rat; are able to metastasize upon injection by the tail vein; and invade the chorioallantoic membrane of the chick embryo. Cells in suspension can be transferred to monolayers, considerably decreasing their tumorigenicity without affecting the other parameters studied, and can be switched back to suspension culture. DNA-mediated transfection showed that DNA from these cells can transform the NIH-3T3 line. Upon growth of the monolayers in a BrdUr-containing medium, a sub-line was established that was cloned into a thymidine kinase-deficient line unable to grow in HAT medium and with properties otherwise similar to those of the parental wild type cells.

Studies on ribonucleoside-diphosphate reductase in permeable animal cells: I. Reversible permeabilization of mouse L cells with dextran sulfate

The treatment of mouse L cells with sodium dextran sulfate 500 for 20 min at 4 °C rendered them selectively permeable to small molecules. The degree of permeabilization was reproducible and depended on dextran sulfate concentration: as the dextran sulfate concentration increased, the cells became more permeable to the dye erythrosin B, lost the ability to incorporate [ 3H]thymidine into DNA, and acquired the capacity for nucleotide-dependent incorporation of [ 3H]dTTP into DNA and for K +- and ATP-dependent incorporation of [ 3H]lysine into protein. The ability to engage in macromolecular synthesis indicated that the permeabilized cells had retained a considerable amount of integrity, and this was confirmed by the observation that the cells could regain viability after an appropriate repair treatment. Permeabilization was rapidly reversed by incubating the cells in suspension culture medium plus 10% calf serum or in a defined medium composed of NaCl, NaH 2PO 4, glucose, glutamine, and CaCl 2, calcium ion being the most critical component. Using this minimally disruptive permeabilization procedure, it was possible to assay the enzyme ribonucleoside-diphosphate reductase in situ. The enzymatic conversion of CDP and GDP to the corresponding deoxyribonucleotides was studied; enzyme activity was almost entirely intracellular and responded to the allosteric activators and inhibitors that are known to modulate the activity of ribonucleotide reductase in vitro.

Accumulation of 9β,19-cyclopropyl sterols in suspension cultures of bramble cells cultured with tridemorph

The addition of tridemorph, a systemic fungicide, to the medium of suspension cultures of bramble cells resulted after 4 weeks of growth in a strong accumulation of 9β,19-cyclopropyl sterols (90% of total sterols in treated cells) and in the disappearance of δ 5-sterols (98% of total sterols in control cells). Cycloeucalenol and 24-methylene pollinastanol (both together constitute 70% of total sterols) are the major sterols of treated cells. Tridemorph probably inhibits the cyclocucalenol-obtusifoliol isomerase. As the fungicide impairs only slightly the growth of the cells, the possibility that 9β,19-cyclopropyl sterols substitute for δ 5-sterols in the membranes of the treated cells is considered.

Manipulation by 25-azacycloartanol of the relative percentage of C 10, C 9 and C 8 side-chain sterols in suspension cultures of bramble cells

The addition of 25-azacycloartanol to the medium of suspension cultures of bramble cells resulted, after 6 weeks of growth, in a large decrease in the percentage of C 10 side-chain sterols, sitosterol and isofucosterol (83 % of the total in the control, 9 % in the treated cells), and in a spectacular increase in the percentage of C 8 side-chain sterols, cycloartenol, desmosterol and cholesterol (less than 1 % in the control, 53 % in the treated cells). In addition the relative percentage of C 9 side-chain sterols, mainly 24-methylene cholesterol increased significantly (from 16 to 37 %). A secondary effect of 25-azacycloartanol consisted in an increase of the percentage of Δ 24 sterols and in a decrease of the percentage of sterols with a saturated side chain. These results are in agreement with an inhibition by 25-azacycloartanol of the C-24 and C-28 methyltransferases and of the Δ 24 reductase.

Isolation of abscisic acid-resistant variants from tobacco cell cultures

The goal of this work was to begin a genetic study of the molecular mode of action of abscisic acid (ABA), by isolating variant cultured cells resistant to the hormone, or to a factor which induces ABA synthesis, namely water stress. Cell cultures of Nicotiana tabacum L. cv. Wisconsin 38 and N. silvestris Speg. and Comes were chosen as the experimental materials. Studies of the effects of the two stresses on the growth of the cultures demonstrated that ABA or water stress imposed by mannitol could completely inhibit growth. These effects arose in both cases from a constant reduction of the growth rate of the cells throughout the culture period. Mannitol also induced an increase in ABA content of the cells and media of suspension cultures, although not to the concentrations required to achieve the same degree of growth inhibition when the hormone was applied exogenously.

Einfluß von Ammonium und Sulfat auf die Glutathion-Produktion in Suspensionskulturen von Nicotiana tabacum

Summary Release and accumulation of glutathione in the medium of suspension cultures of Nicotiana tabacum var. SAMSUN grown photoheterotrophically in a modified MURASHIGE-SKOOG medium are controlled by the mineral nutrition of the cells. In batch cultures the glutathione accumulation is limited by the sulfur supply of the medium, the sulfate concentration in a range from 0.3 mM to 1.73 mM influencing the duration of accumulation but not the rate of GSH release into the medium. After the sulfate supply of the medium has been exhausted, the GSH level in the medium declines. The increase in cell protein during this period indicates that GSH is taken up by the cells and used as sulfur source. Besides high concentrations of sulfate the surplus production of glutathione is depending on the supply of the tabacco cells with ammonium. In cultures grown with 60 mM nitrate as sole nitrogen source up to 7 micromoles glutathione per liter are found in the medium in contrast to 700 micromoles accumulated in the medium of cultures supplied with 20 mM ammonium and 40 mM nitrate. There is no difference in the glutathione content of cells grown with ammonium-nitrate and with nitrate alone, and the addition of ammonia to nitrate grown cells results in a fast increase of glutathione in the medium. The present data support the idea that glutathione can function as storage and transport form of cysteine. They also pose the question how the glutathione synthesis is regulated in cells assimilating ammonium-nitrate.

Continuous Cultivation of Glucose-Limited Bean Cells (Phaseolus vulgarisL.) in a Modified Bacterial Fermentor

A commercially available bacterial fermentor was modified to allow the continuous cultivation of glucose-limited bean cells (Phaseolus vulgaris L.) at a defined pH. A steady state was reached using a dilution rate of 0-004 h_1 and was maintained for 4 months; during this period the dry weight and packed cell volume of the culture remained constant; the yield for glucose was 0-34 (dry wt./dry wt.) and for carbon 0-27 (g atom/g atom). Glucose-limited cells had lower C/N ratios both for cytoplasm and cell walls than cells in the exponential phase of growth in batch cultures. INTRODUCTION Continuous cultures of substrate-limited plant cells are useful for studying meta bolism, because the cells grow in a solution of constant composition at a constant rate. The cells can thus be kept indefinitely in particular metabolic states. However, most studies have been performed with sycamore cells and only a few substrate limitations have so far been investigated (nitrogen limitation: Young, 1973; King, 1976, 1977; phosphate limitation: Wilson, 1976; glucose limitation: King, 1977). One of the difficulties arising from this type of work is the lack of a cheap, commercially available fermentor suitable for the cultivation of plant cells. This paper describes the continuous cultivation of glucose-limited bean cells at a defined pH in a relatively cheap bacterial fermentor that we have adapted to this purpose. MATERIALS AND METHODS Cell suspension culture The culture was initiated from seedling stem expiants of Phaseolus vulgaris L., var. Prelude (Klis and Eeltink, 1979) and was maintained by subculturing every 2 weeks. Cells were grown in 500 ml Erlenmeyer flasks containing 200 ml defined medium (Table 1). The flasks were placed on a rotary shaker (125 rev. min-1) and kept at 27 °C in continuous light (2000 lx). Culture vessel The continuous culture system is shown in Fig. 1. Cells were cultured in a 2 1 glass vessel from a Biolafitte laboratory fermentor. Two glass outlet tubes (i.d. 6 mm) were inserted in the vessel This content downloaded from 207.46.13.111 on Tue, 09 Aug 2016 06:21:12 UTC All use subject to http://about.jstor.org/terms 1224 Bertola and Klis—Glucose-Limited Bean Cells Table 1. Composition of synthetic medium for bean suspension cultures o ^ o o 9 00 g> ^00° C .roo—' <N <N U .9 ^ i «i a ■§ 1 •§ «1 •3.S o « H .S •« 2 I o a .a ■£ o « .s 8 12 -a £,0 .2 4 .9 S3 h2^fcfflri^<0

The effect of membrane lipid perturbers on survival of mammalian cells to cold

Butylated hydroxytoluene (BHT), an antioxidant and common food additive, is an organic soluble molecule which modifies the properties of lipid bilayers and biological membranes. Adamantane and its derivatives, although structurally quite different, have similar effects on membranes. When Chinese hamster lung cells (V79) were pretreated with 0.1 m m BHT before exposure to +5 °C for up to 12 days in suspension culture or attached to plastic, significant protection, as assessed by colony survival, was observed compared to controls. No protection was observed when the cells were exposed to +20 °C. Use of adamantane or 2-adamantanone in serum-free medium in suspension culture at +5 °C showed protection of cells as good as, or better than, that provided by the addition of serum to control cells. Experiments with synchronized cells indicated that BHT protected cells in all phases of the cell cycle against the effects of exposure to +5 °C. However, the protection was greatest in G 1 and early S phase. Exposure of cells containing BHT to +20 °C resulted in no preferential protection in any phase of the cell cycle.

Isolation and characteristics of HeLa surface proteins

HeLa 71 and 65 cells grown in attached culture possess a coat of extracellular proteins that can be released from the cell by mild EDTA-detachment, with no significant effect on cellular integrity. This suggests that these surface proteins are weakly associated with the cell, possibly through divalent cations. The high affinity of surface proteins for critical divalent cations, shown by their high precipitability by Zn 2+, Ca 2+ and Mg 2+, supports this assumption. Since surface proteins appear to be phosphoproteins, as suggested by significant incorporation of 32Pi in vitro, it is possible that binding occurs through M 2+− PO = 4· The amount of surface protein on HeLa 65 cells grown in suspension culture is greatly reduced compared with cells grown in monolayer culture. This may be related to impaired availability of Ca 2+ in suspension culture medium. In monolayer grown HeLa cells surface proteins are predominantly distributed underneath the cells. The highest amount of these proteins is found on cells prior to growth initiation and steadily decreases as cells approach confluency. As shown by radioactive leucine protein labeling, surface proteins are primarily comprised of proteins synthesized within HeLa cells and released to the outer cell surface. The presence of serum proteins in surface protein matrix is physiologically significant.