Liquid Suspension Culture Research Articles

Uptake by soybean root cells of [14C] alanine over a wide concentration range

Uptake of [14C]alanine by soybean (Glycine max L. var. Mandarin) root cells growing in liquid suspension culture was examined over a concentration range of 10−6 M to 10−2 M. Below about 5 × 10−4 M, results could be explained by a single, active-uptake system with high affinity (Km = 4.6 × 10−5 M) and low capacity (Vmax = 5 nmol h−1 106 cells−1). Above 5 × 10−4 M, uptake was greater and could no longer be explained by the high-affinity system. A progressively greater uptake component appeared which was unaffected by metabolic inhibitors, 2,4-dinitrophenol and sodium azide, and by low temperature (2.5 °C). After subtraction of this so-called diffusional component from uptake kinetic data, a low-affinity (Km = 8 × 10−3 M), high-capacity (Vmax = 154 nmol h−1 106 cells−1), active-uptake system remained at high alanine concentrations.Uptake of alanine in these cells could be accounted for at low physiological concentrations by a single, monophasic system. Additional uptake at higher concentrations could be explained by other systems already demonstrated in these cells which are not specific for alanine, but which are, on the basis of competitive inhibition, capable of transporting it, and by a component which is probably diffusional in nature.

Isolation and in vitro differentiation of human erythroid precursor cells

There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24–48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha- globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.

Isolation and In Vitro Differentiation of Human Erythroid Precursor Cells

There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24-48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha-globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.

Growth and metabolism of cells and tissue of jack pine (Pinus banksiana). 3. Growth of cells in liquid suspension cultures in light and darkness

Friable, jack pine (Pinus banksiana Lamb.) callus on agar plates with a defined nutrient medium was transferred to a liquid medium of the same composition to establish cell suspension cultures. Dry weights and patterns of growth for daughter cells were estimated in continuous light or darkness, with or without conditioned media and arginine supplement. In all treatments, growth was near-exponential. Greatest final size was obtained with basal medium under continuous light.The distribution of clump sizes at the later stages of growth fits a stochastic model reflecting two types of daughter cell behavior described by the probability of one type remaining with the clump as opposed to sloughing off into the medium. In light, cells contained many more chloroplasts, thicker cell walls, and more compacted clumps than in darkness: yet cellular clumping patterns both in light and darkness were largely similar. Nearly 20% of the clumps showed polarity and symmetry and contained actively streaming suspensor-like cells. The remaining clumps were spherical and produced unorganized growth patterns. The progress of growth was eventually dominated by the tendency of daughter cells to remain attached to clumps compared with the initial tendency for clumps to fragment and cells to be released into the medium.

Biosynthesis of Abscisic Acid under Osmotic Stress: Studies Based on a Dual Labelling Technique

AbstractSterile liquid suspension cultures from grape pericarp tissue incorporate label from mevalonic acid into abscisic acid (ABA), some of which is subsequently released into the surrounding medium. The addition of mannitol to the culture medium caused plasmolysis of individual cells and increased ABA generation and release. Levels in the medium increased up to 30 fold over a period of 8 h in the presence of mannitol (720 milliosmol).A dual labelling technique using 14C and 3H mevalonic acid was developed to distinguish between labelled compounds produced by turgid tissue and those specifically associated with the substantial increase in ABA synthesis following mannitol treatment.

Type C RNA tumor virus isolated from cultured human acute myelogenous leukemia cells.

Previously, type C RNA tumor virus-related components have been described in blood leukocytes from patients with acute myelogenous leukemia. These components, for example, reverse transcriptase, have been shown to be most closely related to those from two oncogenic subhuman primate type C viruses (woolly monkey sarcoma virus and gibbon ape leukemia virus). Now, we report the continuous production of budding type C viruses with the same characteristic reverse transcriptase by three separate culturings of leukocytes from a single bleeding from a patient with acute myelogenous leukemia. These isolations were made possible by the discovery of a source of conditioned media which sustains exponential growth of human myelogenous leukemia cells in liquid suspension culture.

Functional cellular maturation in cultures of human haematopoietic cells.

Summary. The capacity of cells grown in colonies derived from human haematopoietic cells to phagocytize bacteria and to reduce nitroblue tetrazolium (NBT) was studied as a measurement of functional maturation. Marrow cells from 12 non‐leukaemic patients, fivc patients with acute non‐lymphocytic leukacmia, and cells taken from a myeloblastic cell line established from a patient with acute myelo‐genous leukaemia (AML) were studied. Cells were cultured in soft agar with normal human leucocyte feeder layers as the source of stimulating factor. While the leukaemic marrows gave rise to fewer colonies than the non‐leukaemic marrows, colony formation by the AML cell line was extensive. In all instances colony formation was completely dependent upon the presence of the leucocyte feeder layers. Suspensions of pooled colonies contained 42% mature granulocytes in non‐leukaemic cultures and 41% in the leukaemic cultures. Granulocytes from these cultures showed active phagocytosis of Staphylococcus aureus and reduction of NBT. In cells taken directly from the liquid suspension culture of the AML cell line, no mature granulocytes nor phagocytosis were noted, whereas 35% and 26% of the cells removed from the colonies formed by these cells were classified as mature granulocytes, or demonstrated phagocytosis and NBT reduction respectively. These observations indicate that granulocytes in soft agar colonies derived from human haematopoietic cells are functionally mature, and that cells derivcd from a human AML cell line have the capacity to form colonies with normal maturation, if they are provided with an appropriate stimulating factor.

Erythropoietic differentiation in colonies of cells transformed by Friend virus.

Cells transformed by Friend virus in liquid suspension culture respond to low concentrations of dimethylsulfoxide by initiating hemoglobin synthesis. The kinetics of appearance of such differentiated erythroid cells is consistent with either the induction of differentiation in a uniformly susceptible population of transformed cells or selection for the growth of a distinct erythropoietic subpopulation. The dimethylsulfoxide response of individual colonies of cells transformed by Friend virus grown in semisolid medium was studied in order to distinguish between these alternatives. The data do not support a selective effect of dimethylsulfoxide on the growth of a unique erythropoietic subpopulation; they indicate, rather, that the 745A strain of cells transformed by Friend virus consists of a relatively uniform population of dimethylsulfoxide-sensitive erythropoietic cells.

THE EFFECT OF PLASMA WITH COLONY SIMULATING ACTIVITY (CSA) IN CYCLIC NEUTROPENIA (CN)

A 2 year old M and a 10 year old F with 21-day cycles of neutropenia and apthous stomatitis were studied. Bone marrow aspirates at the cycles' nadir showed a maturation arrest at the myelocyte stage. In vitro bone marrow culture in agar documented quantitatively normal colony formation with rare colonies of neutrophils (PMN). Serial liquid suspension cultures (Marbrook chamber) yielded cell counts which were in the normal range or higher than companion control cultures. CN PMN maturation was defective early in culture; some mature PMNs appeared after 7 days and persisted for up to 25 days. Plasma, obtained from normal donors and from those given typhoid vaccine and plasmaphoresed 1 hour later, was assayed for CSA, using normal and CN bone marrow. Only plasma which stimulated normal marrow promoted PMN maturation in CN marrow in vitro. In vivo, administration of plasma containing CSA increased PMN production and ameliorated the clinical symptoms of neutropenia. It is concluded that one type of CN involves a maturation defect of PMNs, which may be altered in vitro and in vivo by plasma containing CSA. Supported by Grant 5-M01-RR00199.

Partial purification of a cyclic amp phosphodiesterase from soybean callus Isolation of a non-dialysable inhibitor

Cyclic AMP phosphodiesterase has been partially purified from soybean liquid suspension culture. It has a K m of 6.7·10 −5 M and an optimum pH of 4.2. Its activity is not affected by inorganic cations. Methyl xanthines and papaverine are not inhibitory. It is inhibited by inorganic phosphate and dibutyryl cyclic AMP. At least 35% of the total phosphodiesterase activity of the cell is associated with the plasmalemma The enzyme can be resolved into two fractions by DEAE-cellulose chromatography. These have apparently identical properties and each is associated with acid phosphatase activity. It is suggested that these two fractions may represent different aggregation states of a multienzyme complex. A non-dialysable inhibitor of the enzyme has been isolated by gel filtration on Sephadex G-75. It is heat stable and its association with phosphodiesterase is reversible.

Chemical composition of the cell walls of Lolium multiflorum endosperm

Cell walls isolated from Lolium multiflorum endosperm grown in liquid suspension culture contain 90% carbohydrate (as anhydro-glucose), 0·3 nitrogen, 1·9% lipid and 4·3% ash. The relative proportions of neutral sugars present in hydrolysates of the wall polysaccharides are glucose, 50%; arabinose, 19%; xylose, 26% and galactose, 5%. Extraction of the wall with 7 M urea solubilizes a polysaccharide representing 19% of the wall and composed of glucose and minor amounts of pentoses. This fraction has been examined by acid and enzymic hydrolysis and by periodate oxidation, and was shown to be a β-1,3; 1,4-glucan with approx. 79% 1,4-linkages. A specific β-glucan hydrolase has been used to determine the content of this mixed-linked glucan in isolated endosperm cell walls.

Studies on lolium multiflorum endosperm in tissue culture I. Nutrition

Stocks of L. multiflorum endosperm callus have been maintained in liquid suspension culture on a modified White's medium for 5 years. The mean doubling time under the conditions used is 3� 2 days. Best growth is obtained on sucrose; fructose and glucose are good carbon sources, whereas growth is only moderate on an equimolar mixture of both.

Studies on lolium multiflorum endosperm in tissue culture II. Fine structure of cells and cell walls and the development of cell walls

Log phase cells of L. multiflorum endosperm grown in liquid suspension cultures contain large nuclei, mitochondria, protein bodies, compound starch granules, small vacuoles, and multilayered membranous structures. At later stages of growth the cell organelles, protein bodies, and starch granules disappear and in senescent cultures many empty cells are seen.

Effects of environmental changes on sugars, tannins, and organized growth in cell suspension cultures of white spruce

Callus from hypocotyls of white spruce (Picea glauca [Moench] Voss) was grown on agar under defined conditions with high levels of calcium nitrate. Transfer of callus to liquid suspension cultures and maintenance of suspensions either under a regime of constant temperature and light or under alternating conditions similar to those of a late spring day, affected the content of free sugars, tannins, and aldehydes. Under the alternating conditions the levels of these substances increased greatly compared to those under the constant environment. By contrast, vascularization of cell clumps, which was comparable to the differentiation of hypocotyls in seedlings, was obtained only under constant conditions. Cells at the centre of the clumps developed secondary wall thickenings and bordered pits, and were surrounded by cambial-like initials.

Photosynthetic growth of tobacco cells in liquid suspension

Two clones of Nicotiana tabacum in liquid suspension culture were grown photosynthetically using air plus 2% CO2 as carbon source, and agitation. In general, the transfer from heterotrophic to autotrophic growth in these cells was accompanied by an increase in the concentration of photosynthetic pigments, an increase in the N, C, and H contents and in the N:C ratio, an increase in the photosynthetic oxygen evolution capacity, and a decrease in the respiratory rate. The limiting factors are discussed.Several clones were also grown photosynthetically on agar slants and the development of their photosynthetic pigments studied. Results showed that one cannot accurately predict the photosynthetic capability of tobacco cells from their appearance in heterotrophic culture conditions.

The growth of bone marrow cells in liquid culture.

Summary. Liquid suspension cultures of mouse bone marrow cells at high and low density were prepared in supplemented Eagle's medium containing 10% of a partially purified extract of mouse embryos and pregnant mouse uterus (PMU). In the low cell density cultures the number of cells decreased for 2 days; by 4 days the agar colony‐forming cells (agar CFC) had risen ten‐fold and the spleen colony‐forming units (spleen CFU) had fallen to one tenth; between 4 and 8 days the total cell count showed a four‐fold increase and the final cell number exceeded the number of the original culture. The cells produced were mainly macrophages. If PMU was not included in the culture the agar CFC disappeared after 4 days and there was no cell multiplication. In the high cell density cultures a similar pattern was observed; in the presence of PMU the agar CFC showed an increase in number, the spleen CFU decreased and an increase in cell number occurred between 4 and 8 days. However, the cells produced were predominantly granulocytes. In the absence of PMU from this cell culture, agar CFC were maintained for 6 days and the cell population remained predominantly granulocytic. These methods of growing cell enable cell recovery from the cultures to be made at any stage and provide an opportunity to study the kinetics and functional capacity of the cells produced.

Growth of mouse L cells in shaken culture and mengovirus plaque formation on L cells suspended in agar.

Procedures are described that require a minimum of equipment and maintenance for growing mouse L cells suspended in liquid medium and for plaquing mengovirus on L cells suspended in agar. Viability of L cells during storage for 1 to 2 hr at relatively high concentrations was better in media at 30 C than at 0 C, as measured by viable counts after growth for 24 hr at 35 C. The number and size of plaques increased with increasing concentration of NaHCO(3) in the agar layers, but the relative difference in plaque size was maintained. Large- and small-plaque-size variants had similar virulence as determined by the rates of viability loss of L cells in liquid suspension cultures.

Growth of Mouse L Cells in Shaken Culture and Mengovirus Plaque Formation on L Cells Suspended in Agar

Procedures are described that require a minimum of equipment and maintenance for growing mouse L cells suspended in liquid medium and for plaquing mengovirus on L cells suspended in agar. Viability of L cells during storage for 1 to 2 hr at relatively high concentrations was better in media at 30 C than at 0 C, as measured by viable counts after growth for 24 hr at 35 C. The number and size of plaques increased with increasing concentration of NaHCO(3) in the agar layers, but the relative difference in plaque size was maintained. Large- and small-plaque-size variants had similar virulence as determined by the rates of viability loss of L cells in liquid suspension cultures.

Studies on the polyphenol metabolism of tissue cultures derived from the tea pant (Camellia sinensis L.).

1. The growth characteristics on various media of solid and liquid suspension cultures derived from the stem of the tea plant are described; chlorophyll and anthocyanin synthesis occurred in the light. 2. Only the simplest catechins and leucoanthocyanins were present in callus tissue, although oligomeric and polymeric leucoanthocyanin fractions were also represented. Light caused an increase in all monomeric components analysed, but inhibited polymerization of the leucoanthocyanins. 3. The polyphenol oxidase activity of cultures was comparable with that of the apical regions of the intact plant, and was inversely correlated with growth rate. 4. Growth was stimulated by hormonal variation, and inhibited by high concentrations of sucrose and by high light-intensity; polyphenol concentrations were generally inversely correlated with growth rate. 5. From the inability of callus tissue and of cultured root apices to synthesize complex catechins, it is inferred that complex catechin formation in intact plants is associated with the process of cell vacuolation.

Tissue culture of the monocotLilium

The monocot Lilium, possessing very large nuclei and chromosomes, is a favorable material for studying problems of development, parti cularly with regard to the proteins of the nucleus (Sheridan and Stern, 1967). Since it would broaden the possible approaches to such study if successful, an attempt was made to establish Lilium in tissue culture. This communication reports (1) the successful establishment in both agar and liquid shake cultures of callus of Lilium longiflorum Thunb ; (2) the repeated subculturing of the callus on growth factor free media; and (3) the production of large numbers of plantlets from callus under certain growth conditions in liquid suspension cultures. Callus was established by placing tissue expiants from steBi apices onto the basal medium supplemented with 2 mg/1 of indoleacetic acid (IAA). The terminal 10 cm of stems which were approximately 25 cm in height were removed and all but the smallest leaves were stripped off. The apices were washed for 5 min in a solution of the detergent Heikol, 10 min in 10% Clorox, and 5 min in sterile distilled water. Each apex was then placed in a sterile petri dish and the outer portion in cluding all the leaves and primordia was removed with a sc^pel leaving a cylinder which was free of any surface tissue. The terminal 2 cm were removed and placed on the medium. The basal medium was that of Linsmaier and Skoog (1965) con taining major and minor salts, organic supplements of thiamin and inositol, and 40 g of sucrose per liter. Basal agar medium refers to the above medium solidified with 0.8% agar. Fresh weight was used for determining growth response. Within three weeks of explantation, callus appeared on the edge of the tissue block in contact with the medium. The presence of IAA in the culture medium stimulated the production of callus by the stem tissue but was not essential for it. When expiants were placed on medium lacking IAA, buds appeared on the surface of the tissue blocks and shoots and roots were formed producing new plants; on occasion callus was also formed, but usually only after the formation of roots and shoots.