Liquid Suspension Culture Research Articles

RECOMBINANT HUMAN INTERLEUKIN-6 ENHANCES THE ANTIPROLIFERATION AND DIFFERENTIATION INDUCING EFFECTS OF RETINOIC ACID IN HL-60 HUMAN MYELOID LEUKAEMIC CELLS

1. The effect of recombinant human interleukin-6 on the antiproliferation and differentiation inducing actions of all-trans retinoic acid in HL-60 human myeloid leukaemia cells was studied in short-term liquid suspension culture. 2. Interleukin-6 alone showed no significant effect on HL-60 human myeloid leukaemia cells. 3. The addition of interleukin-6 to all-trans retinoic acid-treated cultures of HL-60 human myeloid leukaemia cells significantly enhanced the desired antiproliferation effect of all-trans retinoic acid. 4. The combination of interleukin-6 with all-trans retinoic acid reduced the doses of all-trans retinoic acid required to induce the same differentiation of HL-60 cells as single agent by between 1.7- and 4.8-fold; that is, the efficacy of all-trans retinoic acid in inducing the differentiation of human myeloid leukaemia HL-60 cells was increased up to 4.8 times by its combination with interleukin-6. 5. The combination of all-trans retinoic acid and interleukin-6 could provide an effective alternative therapy for elderly myeloid leukaemia patients.

In vitro propagation of Lilium testaceum and structural investigation of the storage β-1,4-glucomannan

Bulblet and callus cultures of Lilium testaceum were initiated in vitro from bulbscales. Continous propagation of the bulblet cultures was achieved on a modified Murashige and Skoog agar medium containing 1-naphthalene acetic acid (0.1 mg/l) and kinetin (0.1 mg/l) as phytohormones. The in vitro grown bulbs synthesized large quantities of storage ß-1,4-glucomannans (mannose: glucose = 7∶3; molecular weight = 200 kd) with an identical structure to the glucomannans from the in vivo grown bulbs. Higher 1-naphthalene acetic acid concentrations (1 mg/l) resulted in increased callus formation. Liquid suspension cultures derived from callus exhibited only small amounts of reserve glucomannans.

Role of interleukin-6 in the proliferation of human multiple myeloma cell lines OCI-My 1 to 7 established from patients with advanced stage of the disease

Interleukin-6 (IL-6) has been shown to stimulate the proliferation of multiple myeloma cells purified to a high degree from human bone marrow. IL-6 production in multiple myeloma has been attributed to cells belonging to the myeloma clone, thus supporting a mechanism of autostimulation. In addition, it has been shown that IL-6 may be produced by auxiliary cell populations of the bone marrow that are not part of the myeloma clone. A definitive separation of both putative sources for IL-6 may be difficult to achieve in fresh patient IL-6 growth requirement and production by pure myeloma cell populations using seven human myeloma cell lines (OCI-My 1 to 7) that were established from patients with advanced disease. The proliferative response of each line to recombinant IL-6 was measured in a clonogenic assay providing human plasma and methylcellulose as a viscous support and by 3H-thymidine uptake in liquid suspension culture. We observed marked heterogeneity, ranging from IL-6-dependent colony formation by OCI-My 4, to IL-6-independent growth. All lines expressed mRNA for the IL-6 receptor. Expression of IL-6 mRNA was analyzed after amplification by polymerase chain reaction and was present in five of seven lines. IL- 6 protein was detected by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of two lines (OCI-My 3 and 2). Its functional activity was confirmed in a bioassay using the IL-6-dependent murine hybridoma line B 13.29. This activity was neutralized by anti-IL-6 antibody. Two lines did not express mRNA for IL-6. The remaining three lines expressed mRNA for IL-6, but did not secrete IL-6 protein. Immunoprecipitation experiments with lysates of one of these three lines did not detect the presence of IL-6 protein. These results suggest that autocrine stimulation by IL-6 may occur in some cell lines derived from patients with multiple myeloma. However, it does not represent a universal mechanism in myeloma cell growth.

Role of Interleukin-6 in the Proliferation of Human Multiple Myeloma Cell Lines OCI-My 1 to 7 Established From Patients With Advanced Stage of the Disease

Interleukin-6 (IL-6) has been shown to stimulate the proliferation of multiple myeloma cells purified to a high degree from human bone marrow. IL-6 production in multiple myeloma has been attributed to cells belonging to the myeloma clone, thus supporting a mechanism of autostimulation. In addition, it has been shown that IL-6 may be produced by auxiliary cell populations of the bone marrow that are not part of the myeloma clone. A definitive separation of both putative sources for IL-6 may be difficult to achieve in fresh patient IL-6 growth requirement and production by pure myeloma cell populations using seven human myeloma cell lines (OCI-My 1 to 7) that were established from patients with advanced disease. The proliferative response of each line to recombinant IL-6 was measured in a clonogenic assay providing human plasma and methylcellulose as a viscous support and by 3H-thymidine uptake in liquid suspension culture. We observed marked heterogeneity, ranging from IL-6-dependent colony formation by OCI-My 4, to IL-6-independent growth. All lines expressed mRNA for the IL-6 receptor. Expression of IL-6 mRNA was analyzed after amplification by polymerase chain reaction and was present in five of seven lines. IL-6 protein was detected by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of two lines (OCI-My 3 and 2). Its functional activity was confirmed in a bioassay using the IL-6-dependent murine hybridoma line B 13.29. This activity was neutralized by anti-IL-6 antibody. Two lines did not express mRNA for IL-6. The remaining three lines expressed mRNA for IL-6, but did not secrete IL-6 protein. Immunoprecipitation experiments with lysates of one of these three lines did not detect the presence of IL-6 protein. These results suggest that autocrine stimulation by IL-6 may occur in some cell lines derived from patients with multiple myeloma. However, it does not represent a universal mechanism in myeloma cell growth.

Effects of hematopoietic growth factors on the survival of primitive stem cells in liquid suspension culture

We have examined the effects of 10 different growth factors either alone or in combination on colony-forming unit-spleen (CFU-S) and repopulating stem cell survival in vitro. Either interleukin-3 (IL-3), granulocyte-colony-stimulating factor (G-CSF), or IL-4 alone support CFU-S in vitro. The effects of IL-3 or G-CSF could be neutralized by adding antibodies against IL-3 or G-CSF, respectively. However, the effects of IL-4 could be neutralized with antibodies to IL-4 as well as with antibodies to IL-3 and G-CSF. The combinations of IL-3 and IL-6, IL-3 and G-CSF, IL-3 and IL-1 alpha, IL-3 and granulocyte-macrophage CSF (GM-CSF), and IL-4 and IL-6 acted synergistically to increase CFU-S number. Addition of macrophage inflammatory protein-1 alpha (MIP-1 alpha) to IL-3 and IL-6 inhibited the increase in CFU-S number. Repopulating stem cell function was measured in a competitive repopulation assay. Either IL-3 or IL-4 alone could preserve stem cell function in vitro. The combinations of IL-3 and IL-6, and IL-3 and G- CSF increased stem cell function approximately twofold. The combinations of IL-3 + G-CSF + IL-6, and IL-4 and IL-6 (both of which increased CFU-S number fivefold to 10-fold) decreased stem cell function approximately fourfold. These results demonstrate that certain combinations of growth factors can increase CFU-S number at the expense of stem cell function.

Effects of Hematopoietic Growth Factors on the Survival of Primitive Stem Cells in Liquid Suspension Culture

We have examined the effects of 10 different growth factors either alone or in combination on colony-forming unit-spleen (CFU-S) and repopulating stem cell survival in vitro. Either interleukin-3 (IL-3), granulocyte-colony-stimulating factor (G-CSF), or IL-4 alone support CFU-S in vitro. The effects of IL-3 or G-CSF could be neutralized by adding antibodies against IL-3 or G-CSF, respectively. However, the effects of IL-4 could be neutralized with antibodies to IL-4 as well as with antibodies to IL-3 and G-CSF. The combinations of IL-3 and IL-6, IL-3 and G-CSF, IL-3 and IL-1 alpha, IL-3 and granulocyte-macrophage CSF (GM-CSF), and IL-4 and IL-6 acted synergistically to increase CFU-S number. Addition of macrophage inflammatory protein-1 alpha (MIP-1 alpha) to IL-3 and IL-6 inhibited the increase in CFU-S number. Repopulating stem cell function was measured in a competitive repopulation assay. Either IL-3 or IL-4 alone could preserve stem cell function in vitro. The combinations of IL-3 and IL-6, and IL-3 and G-CSF increased stem cell function approximately twofold. The combinations of IL-3 + G-CSF + IL-6, and IL-4 and IL-6 (both of which increased CFU-S number fivefold to 10-fold) decreased stem cell function approximately fourfold. These results demonstrate that certain combinations of growth factors can increase CFU-S number at the expense of stem cell function.

The effect of temperature, water activity and sorbic acid on ketone rancidity produced by Penicillium crustosum Thom in coconut and palm kernel oils

In a model system medium‐chain fatty acids (MCFA) C6–C12 in coconut and palm kernel oil are converted to methyl ketones, one carbon atom less than the parent fatty acids, by two strains of Penicillium crustosum Thom. Conversion rates of up to 32% were seen for decanoic acid at 25°C. The optimum temperature for ketone production was 25°C in liquid suspension culture. Coconut oil contains 3.23 mmol/g MCFAs compared with 2.34 mmol/g for palm kernel oil. Coconut oil is more prone to fungal spoilage (growth and ketone production) than palm kernel oil. The main end product of fermentation was 2‐undecanone reflecting the high concentration of dodecanoic acid in the substrates. Ketonic rancidity is fungal engendered. The reaction can be controlled by reducing the temperature (4°C), reducing the water activity (0.91) or by addition of sorbic acid (20 mmol/l).

Toxicity of medium chain fatty acids to Penicillium crustosum Thom and their detoxification to methyl ketones

Conversion of medium chain length fatty acids into methyl ketones by some filamentous fungi is important in the development of flavour in the blue mould‐ripened cheeses and in the promotion of ketonic rancidity in the lauric acid oils. The effect of fatty acid composition of single acid glycerides, pH, temperature and medium chain length fatty acids on conversion of fatty acids into methyl ketones has been studied with two strains of Penicillium crustosum Thom. Medium chain length fatty acids were fungitoxic. The minimum inhibitory concentration was 3776, 2884, 1723 and 3066 mg/l for the C6:0, 8:0, 10:0 and 12:0 acids, respectively, at pH 7·0 in liquid suspension culture. Evidence is provided to suggest that the cellular site for this conversion is the microbody. Growth of P. crustosum on single acid glycerides involves two distinct physiological processes; the first, a general inhibition of growth, and the second, a detoxification. The fatty acid composition and the slip point of the substrate are of paramount importance in these two processes.

Effects of Recombinant Human Cytokines on Cytarabine Activity in K562 Human Myeloid Leukaemia Cells

The K562 cell line provides a unique population of primitive human myeloid leukaemia cells which can be induced to differentiate along the erythroid, granulocytic, macrophage and megakaryocytic lineages in response to several agents. Cytarabine is not only the most widely used drug in the treatment of myeloid leukaemia but also the most effective agent in K562 cells. The effects of five recombinant human cytokines - interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-alpha, interferon-beta and interferon-gamma on cytarabine-induced growth inhibition and differentiation of K562 cells was studied in liquid suspension cultures. GM-CSF and to a lesser extent IL-3 enhanced the antiproliferative effect of cytarabine in K562 cells, whereas the three interferons reduced it. The efficacy of cytarabine in inhibiting the growth of K562 cells was doubled by its combination with GM-CSF or IL-3 but was halved by its combination with interferons. The five cytokines did not significantly affect cytarabine-induced erythroid differentiation of K562 cells. The present results appear to favour the use of GM-CSF and IL-3 but not of interferons in future treatment strategies based on a combined cytokine and chemotherapy approach for myeloid leukaemia.

Phytoalexin aurone induced in Cephalocereus senilis liquid suspension culture

A new aurone, 4,5-methylenedioxy-6-hydoxyaurone, isolated from chitin-treated liquid suspension cultures of Cephalocereus senilis showed antibacterial activity. The structure of the aurone, cephalocerone, was elucidated by chemical and spectroscopic methods.

Stimulation of macrophage growth and multinucleated cell formation in rat bone marrow cultures by insulin-like growth factor I

In this study the effects of rhIGF-I on macrophage differentiation and growth have been studied using liquid suspension cultures of rat bone marrow cells. IGF-I stimulated macrophage growth in a dose-dependent manner, a maximum response was found at a concentration of 20 ng/ml. IGF-I effects could be ascribed to stimulation of both postmitotic and proliferating cells. A remarkable finding was that IGF-I induced formation of multinucleated cells (MNC). The MNC resembled macrophage-like cells (AcP, NSE positive). A monoclonal antibody to rhIGF-I significantly inhibited IGF-stimulated macrophage growth and MNC formation. A specific antibody to mouse CSF-1 reduced IGF-stimulated macrophage growth in mouse bone marrow cultures indicating that IGF-I effects could, at least in part, be ascribed to endogenous production of CSF-1. These findings indicate that IGF-I in concert with locally induced CSF-1 can influence the differentiation and growth of bone marrow-derived macrophages.

Interleukin 6 is a differentiation factor for human megakaryocytes in vitro

The response of cells of the megakaryocytic lineage to interleukin 6 (IL6), a cytokine with multiple biological activities, was studied by adding the factor to human bone marrow (BM) cultures. IL6 alone had no effect on megakaryocytic colony formation in methylcellulose; however, in the presence of maximally stimulating concentrations of IL 3, almost a twofold increment in colony formation was observed. Tritiated thymidine suicide studies of BM incubated for 2 h with growth factors showed that almost one-half of megakaryocytic progenitors (CFU-Mk) preincubated with IL3 or IL3 plus IL6 were in S phase, whereas BM incubated with IL6 alone was similar to control (approximately 24% of CFU-Mk in S phase). When greater than or equal to 1 ng/ml of IL6 was added to liquid suspension cultures of BM, the size of individual megakaryocytes was significantly augmented compared with that seen in control cultures. Moreover, the DNA content of megakaryocytes grown in the presence of IL6 was increased (modal ploidy 16N) compared with cultures grown with IL3 (modal ploidy 8N). To determine if the effect of IL6 could be direct rather than mediated via accessory BM cells, the factor was added to cultures of isolated single megakaryocytes. Seventy-one percent of cells increased in diameter in the presence of IL6, while only 27% increased in size in the absence of the factor. The data show that IL6 is a direct maturation factor for human megakaryocytes, promoting increments in size and ploidy of these cells.

Ontogeny of Somatic Embryos of Vigna aconitifolia, Vigna mungo and Vigna radiata

Callus cultures were initiated from immature cotyledons of Vigna aconitifolia, V. mungo and V. radiata on MS medium supplemented with NAA, picloram or 2, 4-D. On transfer to L-6 liquid medium supplemented with low concentrations of picloram, GA3 and cytokinins, large number of somatic embryos differentiated from the callus. The cells destined to become somatic embryos divided to form spherical or filamentous proembryos. From the filamentous proembryo, the embryo proper developed either at single or multiple sites. Development of somatic embryos from multiple sites resulted in several embryos connected by a common suspensor at the radicle end. Continued divisions of the proembryos led to globular, heart shaped and dicotyledonary stages of somatic embryogenesis. The somatic embryos of V. mungo and V. aconitifolia differentiated into tiny plantlets at low frequency (1%) in liquid suspension cultures supplemented with zeatin, picloram and GA3.

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.

Expression of a cell surface antigen from Rhizobium leguminosarum 3841 is regulated by oxygen and pH.

Rhizobium leguminosarum bv. viciae 3841 was grown in liquid suspension culture to investigate how culture conditions could affect the expression of a developmentally regulated cell surface antigen associated with lipopolysaccharide. The antigen, which is recognized by monoclonal antibody AFRC MAC 203, was expressed when cultures were grown at neutral pH under low-oxygen conditions (less than 7.5% [vol/vol] O2 in the gas phase). Antigen was also expressed in aerobically grown cultures at pH values below 5.3. The nature of the nitrogen and the carbon sources had no effect on antigen expression except by indirect changes on the pH of the culture medium; similarly, growth in 0.3 M NaCl did not result in antigen expression. The induction of MAC 203 antigen by low-oxygen or low-pH culture conditions is discussed in the context of tissue-specific expression within the legume root nodule.

Production of growth factors by malignant lymphoma cell lines

Fourteen Epstein-Barr virus (EBV)-negative cell lines were raised from bone marrow (BM), peripheral blood (PB), or lymph node samples of patients with intermediate- or high-grade malignant lymphoma. The cell lines were propagated in liquid suspension culture. They contain clonogenic progenitors capable of forming lymphoma colonies in semi- solid culture medium. Cells of these lines were used to examine the growth factor requirements of their clonogenic progenitors and to assess their ability to produce their own growth factors. Two of the cell lines (OCI-Ly9 and OCI-Ly13.1) required addition of exogenous factors for colony growth. These factors were routinely provided by media conditioned by phytohemagglutinin-stimulated leukocytes (PHA- LCM). Three lines formed some and nine lines gave rise to optimal numbers of colonies without addition of growth factors. Eight of these factor-independent lines were able to function as feeder cells and promoted colony formation by both factor-dependent lines. Cell lines that displayed feeder cell function released activities into supernatants able to replace their cellular source. Some of these endogenously produced growth-promoting activities could be replaced by known hematopoietic growth factors. Both factor-dependent cell lines were cultured with recombinant IL-1 alpha, IL-2, IL-3, IL-6, and GM colony-stimulating factor (CSF) and semipurified B-cell growth factor (BCGF) interleukin-4 (IL-4). A heterogeneous response pattern was observed. Both lines formed colonies with IL-4. The colonies were comparable in frequency and size with colonies observed with (PHA-LCM). OCI-Ly9 responded to IL-6 but showed no growth with IL-2. In contrast, the TAC-positive line OCI-Ly13.1 gave rise to colonies with IL-2 while remaining unresponsive to IL-6. A moderate number of colonies was observed when cells of this line were cultured with GM-CSF. Colony formation of both lines was uninfluenced by IL1 alpha or IL-3.

Production of Growth Factors by Malignant Lymphoma Cell Lines

Fourteen Epstein-Barr virus (EBV)-negative cell lines were raised from bone marrow (BM), peripheral blood (PB), or lymph node samples of patients with intermediate- or high-grade malignant lymphoma. The cell lines were propagated in liquid suspension culture. They contain clonogenic progenitors capable of forming lymphoma colonies in semi-solid culture medium. Cells of these lines were used to examine the growth factor requirements of their clonogenic progenitors and to assess their ability to produce their own growth factors. Two of the cell lines (OCI-Ly9 and OCI-Ly13.1) required addition of exogenous factors for colony growth. These factors were routinely provided by media conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM). Three lines formed some and nine lines gave rise to optimal numbers of colonies without addition of growth factors. Eight of these factor-independent lines were able to function as feeder cells and promoted colony formation by both factor-dependent lines. Cell lines that displayed feeder cell function released activities into supernatants able to replace their cellular source. Some of these endogenously produced growth-promoting activities could be replaced by known hematopoietic growth factors. Both factor-dependent cell lines were cultured with recombinant IL-1 alpha, IL-2, IL-3, IL-6, and GM colony-stimulating factor (CSF) and semipurified B-cell growth factor (BCGF) interleukin-4 (IL-4). A heterogeneous response pattern was observed. Both lines formed colonies with IL-4. The colonies were comparable in frequency and size with colonies observed with (PHA-LCM). OCI-Ly9 responded to IL-6 but showed no growth with IL-2. In contrast, the TAC-positive line OCI-Ly13.1 gave rise to colonies with IL-2 while remaining unresponsive to IL-6. A moderate number of colonies was observed when cells of this line were cultured with GM-CSF. Colony formation of both lines was uninfluenced by IL1 alpha or IL-3.

Cooperative effects of gamma-interferon and 1?, 25-dihydroxyvitamin D3 on in vitro differentiation of the blast cells of RAEB and RAEB-T

We added recombinant human gamma-interferon (gamma-IFN) and 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25 (OH)2D3) to the bone marrow cells from six patients with RAEB or RAEB-T in liquid suspension cultures. After cultivation for 7 to 9 days, numerical, morphological and functional changes of the cells were assessed. gamma-IFN and 1 alpha, 25 (OH)2D3 additively suppressed cell growth, especially the number of blast cells decreased. The expression of alpha-naphthylbutyrate esterase (NBE) activity appeared to be promoted but that of naphthol AS-D chloroacetate esterase (NAE) activity was apparently suppressed by the addition of gamma-IFN and/or 1 alpha, 25 (OH)2D3. The percentage of NBT reduction-positive cells and latex-phagocytizing cells was only slightly increased by both agents. These results indicate that gamma-IFN and 1 alpha, 25 (OH)2D3 cooperate to induce monocytoid differentiation of the patients' blast cells. Combination therapy with both agents merits further study.

Development of A Tissue Culture System from Immature Strawberry Fruits

ABSTRACTThe potential for production of strawberry flavor from cells grown in plant tissue culture was investigated. Immature strawberry fruits from strawberry cultivars (Fragaria x ananassa Brighton, Earliglow, and Aptos) were cultured on modified Murashige and Skoog (MS) basal medium supplemented with kinetin and 2,4‐dichlorophenoxyacetic acid. Parameters that influence callus formation and growth rate including medium composition, phytohormone concentration and photoperiod were studied. Cultivars varied in their ability to initiate callus formation and of the three types identified, only the friable type II callus was capable of growth in liquid suspension culture. Medium composition, types and levels of phytohormones, and inoculum size influenced cell growth in liquid suspension culture.

Pivotal roles of picloram and gelrite in banana callus culture

Vigorous development of callus was achieved only when the nutrient medium was supplemented with picloram and solidified with gelrite. Gelrite also eliminated tissue and medium discoloration. The callus resulted from intensified activity of the primary thickening meristem of shoot-tip explants. After continous culture for 1.5 years, only a small proportion of the callus cells displayed the normal chromosome number, 3 N = 33, the majority being variable aneuploids. Liquid suspension cultures were established by transfering the relatively friable callus to a constantly shaken liquid medium.