Liquid Suspension Culture Research Articles

Cocoa bean cell and embryo culture

AbstractCallus culture of cocoa bean was initiated from immature cotyledons on agar medium. By dispersing these callus cells, a liquid suspension culture was established. The lipid composition of cocoa suspension culture was investigated and compared with those of cocoa beans of different maturities. Factors affecting fatty acid and triglyceride synthesis in cocoa suspension cultures also were studied. In parallel studies, asexual embryos were proliferated from immature zygotic embryos cultured in semisolid or liquid medium. The factors affecting the lipid composition in cultured embryos are discussed.

Tghe Presence of Common Precursor for Basophils and Eosinophils

AbstractWhen human bone marrow cells were incubated in the presence of medium conditioned by PHA‐stimulated leukocytes (PHALCM) in methylcellulose culture, small compact colonies grew. The compact colonies were identified to consist of eosinophils, using Wright and Giemsa staining. However, among som of the eosinophilic colonies, several mixed granule leukocytes containing both basophilic and eosinophilic granules (but not basophils) were identified using astra blue‐biebrich scarlet staining. When some of the eosinophilic colonies were transferred into liquid suspension culture containing PHALCM, the growth of mixed granule leukocytes and basophils was observed in the culture. These results show that basophils and eosinophils may have a common precursor.

The effects of tumor-promoting phorbol esters on human granulopoiesis in vitro

In order to determine whether the tumor-promoting phorbol esters are capable of inducing normal human committed granulocytic-monocytic progenitor cells (CFUc) to proliferate and differentiate in the absence of granulocyte-monocyte colony-stimulating activity (CSA), we studied the effects of these compounds on human granulopoiesis in vitro. We found that when light-density human marrow cells or peripheral blood leukocytes were depleted of adherent cells and then incubated in semisolid tissue culture medium under conditions optimal for CFUc growth, phorbol myristate acetate (PMA) and its congeners produced no measurable stimulatory effect on the proliferation of CFUc in the absence of added CSA. Likewise, when light-density marrow cells that had not been depleted of adherent cells were plated in the cultures, no stimulation of CFUc colony growth resulted from the addition of PMA. However, when light-density peripheral blood leukocytes were used as a target source of CFUc without first subjecting them to adherence separation, enhanced proliferation and differentiation of CFUc were noted in cultures that contained PMA. To investigate the possibility that CSA production by monocytes in these cultures in response to activation by PMA might account for the enhanced colony formation that we observed, we incubated isolated peripheral blood monocytes in short-term liquid suspension cultures and found that in the presence of PMA, large quantities of CSA were secreted into the surrounding medium. Finally, we noted that when marrow cell suspensions were suboptimally stimulated by low concentrations of CSA added to the cultures, the effects of PMA on CFUc proliferation were unpredictable, enhancing colony formation in some cases and inhibiting it in others. Our data indicate that although the tumor-promoting phorbol esters do not appear capable of directly stimulating the proliferation or differentiation of human CFUc in the absence of CSA, they may do so indirectly by causing auxiliary cells such as monocytes to secrete CSA.

The Effects of Tumor-Promoting Phorbol Esters on Human Granulopoiesis In Vitro

Abstract In order to determine whether the tumor-promoting phorbol esters are capable of inducing normal human committed granulocytic-monocytic progenitor cells (CFUc) to proliferate and differentiate in the absence of granulocyte-monocyte colony-stimulating activity (CSA), we studied the effects of these compounds on human granulopoiesis in vitro. We found that when light-density human marrow cells or peripheral blood leukocytes were depleted of adherent cells and then incubated in semisolid tissue culture medium under conditions optimal for CFUc growth, phorbol myristate acetate (PMA) and its congeners produced no measurable stimulatory effect on the proliferation of CFUc in the absence of added CSA. Likewise, when light-density marrow cells that had not been depleted of adherent cells were plated in the cultures, no stimulation of CFUc colony growth resulted from the addition of PMA. However, when light-density peripheral blood leukocytes were used as a target source of CFUc without first subjecting them to adherence separation, enhanced proliferation and differentiation of CFUc were noted in cultures that contained PMA. To investigate the possibility that CSA production by monocytes in these cultures in response to activation by PMA might account for the enhanced colony formation that we observed, we incubated isolated peripheral blood monocytes in short- term liquid suspension cultures and found that in the presence of PMA, large quantities of CSA were secreted into the surrounding medium. Finally, we noted that when marrow cell suspensions were suboptimally stimulated by low concentrations of CSA added to the cultures, the effects of PMA on CFUc proliferation were unpredictable, enhancing colony formation in some cases and inhibiting it in others. Our data indicate that although the tumor-promoting phorbol esters do not appear capable of directly stimulating the proliferation or differentiation of human CFUc in the absence of CSA, they may do so indirectly by causing auxiliary cells such as monocytes to secrete CSA.

G6PD Isoenzyme Analysis of Myeloid and Lymphoid Cells in Human Multilineage Colonies

Some multilineage hemopoietic colonies contain, in addition to myeloid cells, T lymphocytes. These proliferate extensively in liquid suspension culture under the influence of a T cell growth factor provided by phytohemagglutinin-T cell-conditioned medium (PHA-TCM). The clonal origin of these myeloid and lymphoid components was investigated by determining the glucose-6-phosphate dehydrogenase (G6PD) isoenzyme types of multilineage colonies grown from peripheral blood of 4 G6PD heterozygous normal volunteers. The G6PD assay is sufficiently sensitive to detect enzyme concentrations contributed by as few as 30 granulocytes and erythroblasts, 4-6 megakaryocytes, 2-3 macrophages, and 50-100 T cells. T cell components can be detected even if myeloid cells are present in 10-20-fold excess. A small number of multilineage colonies with T cells produced a single G6PD isoenzyme on direct analysis and after expansion in liquid culture. This observation supports the view of a common progenitor for myeloid and lymphoid cells in the peripheral blood of normal adults.

Photoautotrophic potato cells: Transition from heterotrophic to autotrophic growth

Cells of potato (Solanum tuberosum L.) were obtained which were capable of photoautotrophic growth in liquid suspension culture under a photon flux density of 90–110 μmol m−2 s−1 PAR and in an atmosphere enriched with 2% CO2. These photoautotrophic cells contained between 100 to 200 μg Chl (g fresh weight)−1 and fixed CO2 at a maximum rate of 16 μmol CO2 (g fresh weight)−1h−1. In order to obtain cells capable of photoautotrophic growth it was necessary to adapt highly chlorophyllous heterotrophic cells (>50 μg Chl (g fresh weight)−1) for growth in medium with 2.5 g sucrose 1−1 (photomixotrophic cells). The photomixotropic cells had a Chl content of ca 100 μg Chl (g fresh weight)−1 and were capable of photosynthetic activity which allowed them to survive after sugars had been depleted from the medium. It was from the photomixotrophic cells that cells capable of photoautotrophic growth were obtained.Heterotrophic cells initially established in liquid medium with 25 g sucrose I−1 from chlorophyllous callus contained about 50 to 150 μg Chl (g fresh weight)−1. However, after 5 to 10 passages the Chl content decreased to a maximum of 15 μg Chl (g fresh weight)−1. These cells could not be adapted to photomixotrophic or photoautotrophic growth. These cells also were not able to regain Chl or initiate high rates of CO2 fixation during the stationary phase of growth as did photomixotrophic cells or chlorophyllous heterotrophic cells. The loss of Chl exhibited by the cells during adaption to heterotrophic growth could be attributed at least in part to unbalanced growth (when cell division and growth exceeds Chl accumulation). Sucrose appeared to have an inhibitory effect directly on photosynthesis independent of Chl accumulation.

G6PD isoenzyme analysis of myeloid and lymphoid cells in human multilineage colonies

Some multilineage hemopoietic colonies contain, in addition to myeloid cells, T lymphocytes. These proliferate extensively in liquid suspension culture under the influence of a T cell growth factor provided by phytohemagglutinin-T cell-conditioned medium (PHA-TCM). The clonal origin of these myeloid and lymphoid components was investigated by determining the glucose-6-phosphate dehydrogenase (G6PD) isoenzyme types of multilineage colonies grown from peripheral blood of 4 G6PD heterozygous normal volunteers. The G6PD assay is sufficiently sensitive to detect enzyme concentrations contributed by as few as 30 granulocytes and erythroblasts, 4–6 megakaryocytes, 2–3 macrophages, and 50–100 T cells. T cell components can be detected even if myeloid cells are present in 10–20-fold excess. A small number of multilineage colonies with T cells produced a single G6PD isoenzyme on direct analysis and after expansion in liquid culture. This observation supports the view of a common progenitor for myeloid and lymphoid cells in the peripheral blood of normal adults.

Maintenance of granulocyte-monocyte progenitor cells in liquid cultures of human foetal liver.

These studies describe a liquid suspension culture system for normal myeloid cells derived from human foetal liver. A simple one-step fractionation procedure was employed to obtain a cell population capable of expanding into all stages of myeloid differentiation, including committed myeloid progenitor cells (GM-CFC). Cell proliferation in these cultures resulted in the maintenance of early myeloid populations for up to a month. In order to extend myeloid cell maintenance, a specific factor in the form of media conditioned by human endothelial cells (endo C.M.) was used. Addition of endo C.M. to foetal liver cultures resulted in increased myeloid proliferation coupled to extensive myeloid differentiation. Clonally derived foetal liver culture cells proliferated for up to 2 months in the presence of endo C.M. before maturing into macrophages. These results show that endo C.M. exert an extensive proliferative effect on early myeloid cells as well as inducing their differentiation. The large quantity of cells in early stages of myeloid differentiation provided by foetal liver cultures may be useful for biochemical and molecular biology studies of myelopoiesis. In addition, these cultures are a potential source from which to derive normal myeloid lines. The separation of the potent proliferative activity present in endo C.M. may yield an effector which maintains human myeloid cell proliferation.

Rhizobium free-living nitrogen fixation occurs in specialized nongrowing cells

A model for free-living N(2) fixation by Rhizobium sp. RC3200 is presented that asserts that this process occurs in nongrowing cells. Cultures containing mixed populations of cell types, N(2)-fixing and vegetative, grow cooperatively. In nitrogen-limited liquid suspension cultures, cooperative growth occurs by means of ammonium that is produced and exported by nongrowing, N(2)-fixing cells and transported to vegetative cells. This model implies prokaryotic differentiation: the creation of metabolically specialized cells, terminally nonviable, that functionally cooperate in a higher cell order. Here, the switch to a Rhizobium N(2)-fixing cell state is regulated by both O(2) and utilizable nitrogen.

Variation in culture, isoenzyme patterns and plastid DNA in the genusDaucus

To identify markers for fusion and transformation studies, cell suspension cultures of four members of theDaucus genus were examined to determine differences in culture conditions, isoenzyme patterns, and plastid DNA. The four were:D. carota subsp.sativus cv. Danvers,D. carota subsp.gummifer, D. capillifolius, andD. pusillus. Under appropriate conditions, all four grew well as liquid cell suspension cultures and regenerated from protoplasts into plants. Enzyme activities of homoserine dehydrogenase (HSDH) and alcohol dehydrogenase from cell culture extracts were analyzed on electrophoretic gels. Although only one form of HSDH was present in eachDaucus line, the rate of migration of HSDH from cv. Danvers was different from that of the other cell lines. Multiple isoenzymic forms of ADH were present in eachDaucus cultivar.

Single cell cloning of Leishmania parasites in purine-defined medium: isolation of drug-resistant variants.

A simple technique for the isolation of Leishmania donovani and Leishmania tropica promastigote clones derived from a single cell involves the use of semi-solid agar. Both species of Leishmania form discrete colonies at high efficiency in completely defined medium lacking serum. Visible colonies appear between eight and fourteen days. Viability of colonies transferred from semi-solid agar to liquid suspension culture is 100%. Using these techniques, we have isolated clonal populations of cells resistant to tubercidin and formycin b.

Effect of mouse interferon on growth and differentiation of mouse bone marrow cells stimulated by two different types of colony-stimulating factor

The effects of mouse L-cell interferon (IFN) on growth of mouse bone marrow cells and their differentiation into macrophages and granulocytes were investigated in a liquid suspension culture system with two different types of colony-stimulating factor (CSF). Within 7 days, most bone marrow cells differentiated into macrophages in the presence of macrophage colony-stimulating factor (M-CSF) derived from mouse fibroblast L929 cells, but into both granulocytes (40%) and macrophages (23%) in the presence of a granulocyte-macrophage colony- stimulating factor (GM-CSF) from mouse lung tissue. IFN inhibited growth of bone marrow cells with both M-CSF and GM-CSF, but had 20 times more effect on bone marrow cells stimulated with M-CSF than on those stimulated with GM-CSF. A low concentration of IFN (50 IU/ml) stimulated production of macrophages by GM-CSF in liquid culture medium, whereas it selectively inhibited colony formation of macrophages in semisolid agar culture. IFN caused no detectable block of late stages of differentiation; mature macrophages and granulocytes were produced even when cell proliferation was inhibited by IFN. These results indicate that IFN preferentially affects growth and differentiation of the cell lineage of macrophages among mouse bone marrow cells.

Effect of Mouse Interferon on Growth and Differentiation of Mouse Bone Marrow Cells Stimulated by Two Different Types of Colony-Stimulating Factor

The effects of mouse L-cell interferon (IFN) on growth of mouse bone marrow cells and their differentiation into macrophages and granulocytes were investigated in a liquid suspension culture system with two different types of colony-stimulating factor (CSF). Within 7 days, most bone marrow cells differentiated into macrophages in the presence of macrophage colony-stimulating factor (M-CSF) derived from mouse fibroblast L929 cells, but into both granulocytes (40%) and macrophages (23%) in the presence of a granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse lung tissue. IFN inhibited growth of bone marrow cells with both M-CSF and GM-CSF, but had 20 times more effect on bone marrow cells stimulated with M-CSF than on those stimulated with GM-CSF. A low concentration of IFN (50 IU/ml) stimulated production of macrophages by GM-CSF in liquid culture medium, whereas it selectively inhibited colony formation of macrophages in semisolid agar culture. IFN caused no detectable block of late stages of differentiation; mature macrophages and granulocytes were produced even when cell proliferation was inhibited by IFN. These results indicate that IFN preferentially affects growth and differentiation of the cell lineage of macrophages among mouse bone marrow cells.

Induction and in vitro culture of soybean crown gall tumors

Induction of crown galls on 4-6 week old soybean (Glycine max (L.) Merr.) plants cultured in growth chambers was obtained with Agrobacterium tumefaciens strains C58, T37 and ACH5. The crown galls were isolated and cultured in vitro as sterile callus and liquid suspension cultures. Transformation was tested by opine tests and by molecular hybridization of restricted cell DNA with T-DNA fragments. Protoplasts were isolated from suspension cultures. Transformed protoplasts regenerate cell walls, divide and form calli without an exogenous supply of hormones.

High Efficiency Plating Method for Leishmania Promastigotes in Semidefined or Completely-Defined Medium

A simple technique, developed for the isolation of clones derived from single, promastigote cells of Leishmania donovani and Leishmania tropica, involved the use of semisolid agar. Both species of Leishmania promastigotes formed discrete colonies at high efficiency either in semidefined medium containing 10% fetal calf serum or in completely-defined medium lacking serum. Visible colonies appeared between 8 and 14 days in growth medium containing 10% fetal calf serum. Replacement of the fetal calf serum with bovine serum albumin and Tween-80 increased the time of colony formation by 50% but did not affect the cloning efficiency. Viability of colonies transferred from semisolid agar to liquid suspension culture was 100%.

Callus formation and organogenesis by explants of six Lycopersicon species

Stem and hypocotyl explants of six Lycopersicon species, L. esculentum var. cerasiformae, L. cheesmanii, L. hirsutum, L. pimpinellifolium, L. peruvianum, and L. glandulosum, were examined for ability to form callus, adventitious shoots, and adventitious roots on media containing varying levels of indole acetic acid (IAA) and kinetin. Callus growth of L. peruvianum and L. glandulosum explants was better than that of the other species tested. Stem explants tended to form more callus than hypocotyl explants. Shoot formation by hypocotyl explants was superior to stem explants for all species and at nearly all hormone levels. Hypocotyl explants of L. peruvianum and L. glandulosum showed the highest levels of shoot formation, and L. cheesmanii and L. esculentum var. cerasiformae hypocotyl explants showed good shoot formation on media containing 3 mg/L kinetin and 0.3 or 1 mg/L indole acetic acid. No shoot formation was observed by L. hirsutum explants, and L. pimpinellifolium explants gave only a poor response. Shoot formation by stem explants was obtained only with L.. esculentum var. cerasiformae and L. glandulosum. When callus of the six species, subcultured for 63 days on a medium optimal for callus growth, was subcultured on shoot-forming media, only callus of L. peruvianum and L. glandulosum subsequently formed shoots. Callus of L. glandulosum has retained its morphogenic potential for 1 year in culture and can be used to initiate liquid suspension cultures.

The synthesis and secretion of granulocyte-monocyte colony-stimulating activity (CSA) by isolated human monocytes: kinetics of the response to bacterial endotoxin.

We studied the kinetics of the synthesis and secretion of granulocyte-monocyte colony-stimulating activity (CSA) by human monocytes stimulated by S. typhi endotoxin. We found that these cells initially secrete copious quantities of CSA when exposed to endotoxin but rapidly become refractory to its stimulatory effect. When monocytes were incubated in liquid suspension cultures, large amounts of CSA were generated during the first 24 hr of culture after the addition of as little as 10 ng/ml of endotoxin to previously unstimulated monocytes. After the addition of endotoxin, CSA secretion abruptly took place after an initial 1 to 2 hr lag phase, and occurred primarily within the first 6 to 12 hr of culture. The addition of puromycin or cycloheximide to the cultures significantly inhibited CSA secretion in response to endotoxin, suggesting that CSA production by stimulated monocytes requires de novo protein synthesis and does not solely result from the release of preformed active CSA. After initial exposure to endotoxin, CSA production by monocytes steadily decreased after 24 hr and ceased after 72 hr. Subsequent reexposure to the same concentration of endotoxin resulted in little CSA production, whereas the refractory state could be overcome by increasing the quantity of endotoxin added. Additional studies demonstrated that refractoriness of monocytes to further CSA production after initial response to endotoxin was not due to a loss of cell viability, degradation of endotoxin, or inhibition of CSA synthesis by soluble metabolites generated by stimulated monocytes. Our data suggest that the capacity of human monocytes to synthesize CSA in response to endotoxin rapidly becomes blunted after initial exposure of the cells to the lipopolysaccharide, but that the refractory state of these cells can be overcome by increasing the concentration of endotoxin. Acquired hyporesponsiveness of cells that produce CSA may in part account for the phenomenon of immediate endotoxin tolerance observed in vivo.

Effect of granulocytic chalone on the growth rate of continuous cell lines propagated in vitro: a new assay system.

The progress of research on granulocytic chalone has been hampered by a lack of reliable assay systems. This paper reports the use of continuous cell lines growing in liquid suspension culture as a new method for assessing the effect of granulocytic chalone on cell proliferation. Partially purified granulocytic chalone (PPGC) was added to cultures of a rat myeloid cell line (W25), a human myeloid cell line (HL60), a human B lymphocytic cell line (8392), and a human T lymphocytic cell line (8402). The growth rate of the target cell populations was directly assessed by serial cell counts. PPGC pretreatment resulted in a significant inhibition of cell proliferation of the 2 myeloid permanent cell lines, while the growth rate of the lymphocytic cell lines was not affected. Moreover, inhibition of cell proliferation was spontaneously reversed when PPGC treatment was discontinued. Since PPGC from ox leucocytes displayed an inhibitory effect on cell proliferation which was specific to the myeloid cells, spontaneously reversible and not species-specific, it appears to have all the essential properties of a granulocytic chalone.

Establishment of long-term monocyte suspension cultures from normal human peripheral blood.

The long-term suspension growth of normal, immature myeloid cells from fresh human cord blood was recently reported and required cells separated on supplemented discontinuous Percoll gradients, growth in media containing hydrocortisone and vitamins D3, and gentle, continuous agitation (13). When normal adult bone marrow (six donors) or blood from Epstein-Barr virus (EBV)-seropositive donors (nine donors) was used as a source of fresh human leukocytes, only short-term proliferation of myeloid cells was achieved with the same techniques. However, when leukocytes prepared from EBV seronegative normal adult peripheral blood were used, pure populations of monocytes and macrophages that replicate slowly in liquid suspension culture for greater than 5 mo were repeatedly obtained from three independent donors. These cultures consists of several morphologically distinguishable monocytic cell types, including an approximately 20% adherent macrophage population. The monocytic nature of these cultures was confirmed by cytochemical, immunological, and functional criteria. These monocytes retain a normal chromosome pattern and can be induced to differentiate to phagocytic cells by treatment with tetradecanylphorbal acetate. Eventually, the cultures terminate as nonreplicating mature macrophages. These liquid suspension cultures should be a valuable resource for morphological, biochemical, and functional studies of developing monocyte-macrophages and their interaction with other cell types in normal and various pathological situations.

Cyanide-Resistant Respiration in Suspension Cultured Cells of Nicotiana glutinosa L

The respiration of dark-grown Nicotiana glutinosa L. cells in liquid suspension culture was found to be highly cyanide resistant and salicylhydroxamic acid (SHAM) sensitive, indicative of an active alternative respiratory pathway. This was especially true during the lag and logarithmic phases of the 14-day growth cycle. Mitochondria isolated from logarithmically growing cells exhibited active oxidation of malate, succinate, and exogenous NADH. Oxidation of all three substrates had an optimum pH of 6.5 and all were highly resistant to inhibited by cyanide and sensitive to SHAM. Respiratory control was exhibited by all three substrates but only if SHAM was present to block the alternative pathway and divert electrons to the phosphorylating cytochrome pathway. The cyanide-resistant oxidation of exogenous NADH has previously only been associated with Arum spadix mitochondria. Coemergence during evolution of the alternative respiratory pathway and the exogenous NADH dehydrogenase in plant mitochondria as a possible mechanism for removal of cytoplasmic NADH is proposed. Evidence is presented which suggests that mitochondrial assays should be performed at pH 6.5.