Rolling round bottle culture of HmLu-1 cells and the production of bovine ephemeral fever virus.

An attempt was made to cultivate HmLu-1 cells in a rolling round bottle. As a result, the optimum conditions of cultivation were found to consist in the number of cells transplanted per bottle being 1 X 10(8), the volume of growth medium per bottle being 250 ml, and the velocity of rolling being 6 revolutions per hour. It was possible to make a monolayer of cells develop all over the glass surface under these conditions. A preliminary experiment was carried out to clarify the production of virus in the tube culture. In it, the highest virus titer was obtained two days after inoculation of a 4-day-old culture with a 1:100 dilution of stock virus. On the other hand, when the 4-day-old culture cells in the rolling round bottle were inoculated with virus suspension and when 100, 500, or 800 ml of maintenance medium was added to each bottle, there was little difference in virus titer obtained among the culture bottles. Then the virus yield per cell was compared between the rolling round bottle culture method and the stationary square bottle culture method. The highest virus titer was reached two days after virus inoculation, regardless of the culture method. The virus yield was 1.9 times as high in the rolling method as in the stationary method. From the results mentioned above, it was clarified that the rolling round bottle culture method made it possible to obtain a large amount of bovine ephemeral fever virus at a high titer in a labor-saving manner.     

Growth and division of carrot cells in suspension culture

In a submerged culture of a strain of carrot cells, cellularmorphology and the mode of cell division were greatly affectedby growth factor(s) added to the medium. In the presence of2,4-D, cells showed two-dimensional growth and often formedtetrad-like structure after a set of two divisions. The sequenceof events was observed microscopically. Orientation of cellgrowth changed after the first division and the second cellplate formed at an oblique angle to the first. When IAA wasadded, instead of 2,4-D, cells showed one-dimensional growthand developed to a filamentous form. (Received June 1, 1970; )     

Growth of anchorage dependent mammalian cells on glycine-derivatized polystyrene in suspension culture

Summary Glycine-derivatized polystyrene beads were prepared and used as microcarriers to grow normal cells of human embryonic kidney, rhesus monkey kidney, and human foreskin fibroblasts in suspension cultures. Growth of the cells on polystyrene beads derivatized with other amino acids, peptides, and carboxylic acids also was investigated.     

Growth and sesquiterpenoid production by Calypogeia granulata inoue cells in suspension culture

Growth and the production of volatile sesquiterpenoids by a chlorophyllous cell suspension culture from gametophytes of C. granulata, a leafy liverwort, were examined. Glucose was more effective than 2,4-dichlorophenoxyacetic acid for callus induction, and elimination of glucose from the medium resulted in prompt redifferentiation of plantlets. The cells grew photoheterotrophically, but not in the dark. 1,4-Dimethylazulene, a trinorsesquiterpenoid, was produced as the major volatile sesquiterpenoid in the cultured cells; bicyclogermacrene, compound II, an indene-type aldehyde (a trinorsesquiterpenoid aldehyde), compound I and tetrahydro-1,4-dimethylazulene (a trinorsesquiterpenoid) followed in decreasing order. The azulene was produced both in light and the dark, and its yield was proportional to the growth in light. The yield in light was four times higher than that in the dark. The content of 1,4-dimethylazulene was 0.9–10.% and that of total essential oils was 2.0–3.3% of the dry werght of the cultured cells. The quantity, quality, and proportions of the volatile sesquiterpenoids of the cell culture were almost equal to those of intact (original) plants and redifferentiated plantlets. Previous name: Institute of Food Chemistry     

Arrest of 3T3 cells in G1 phase in suspension culture.

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.     

Growth arrest of vascular smooth muscle cells in suspension culture using low-acyl gellan gum

The proliferation of vascular smooth muscle cells (SMCs) causes restenosis in biomaterial vascular grafts. The purposes of this study were to establish a suspension culture system for SMCs by using a novel substrate, low-acyl gellan gum (GG) and to maintain SMCs in a state of growth inhibition. When SMCs were cultured in suspension with GG, their proliferation was inhibited. Their viability was 70% at day 2, which was maintained at more than 50% until day 5. In contrast, the viability of cells cultured in suspension without GG was 5.6% at day 2. By cell cycle analysis, the ratio of SMCs in the S phase when cultured in suspension with GG was lower than when cultured on plastic plates. In SMCs cultured in suspension with GG, the ratio of phosphorylated retinoblastoma (Rb) protein to Rb protein was decreased and p27^Kip1 expression was unchanged in comparison with SMCs cultured on plastic plates. In addition, SMCs could be induced to proliferate again by changing the culture condition from suspension with GG to plastic plates. These results suggest that our established culturing method for SMCs is useful to maintain SMCs in a state of growth inhibition with high viability.     

Production of plant virus inhibitor by Phytolacca americana suspension culture.

The inhibitory activity of tobacco mosaic virus (TMV) infection was assayed with the extracts of various callus tissues derived from the intact plants. Phytolacca americana callus was selected as a producer of the virus inhibitor and its cultural conditions in suspension were examined for cell growth and the inhibitor production. A modified liquid medium containing twofold concentrations of all components in that of Murashige and Skoog plus2,4-D (1.0 mg/liter) and sucrose (6%), but without any vitamins and glycine was chosen for production of higher levels of the inhibitor. TMV infections in tobacco, bean, and tomato plants were markedly inhibited by the introduction of the disrupted whole broth of suspension cultured P. americana.     

Photoautotrophic growth of Marchantia polymorpha L. cells in suspension culture

A cell line of M. polymorpha was grown photoautotrophically in liquid suspension culture using 1% CO₂ in air as sole carbon source. The growth rate in terms of cell dry-weight during the exponential phase was 0.171 and the doubling time was 1.76 d. The rate of increase in chlorophyll was 1.6 times higher than the growth rate. The highest content of chlorophyll was 24 mg g^-1 dry weight, and the photosynthetic activity of the cells in the exponential phase, as calculated from the growth rate, was at least 60 mol mg^-1 chlorophyll h^-1.     

Arrest of 3T3 cells in G1 phase in suspension culture

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.     

Liposome-cell interactions. A rapid assay for cells in suspension culture

A method has been developed for the rapid separation of cells in suspension from non-cell associated lipid vesicles in various assays for vesicle-cell interaction. Separation is achieved on a discontinuous Ficoll-Paque gradient. Cells and free vesicles are totally separated, as evidenced by both radiolabelled vesicles, and vesicles containing the fluorescent dye 6-carboxyfluorescein. The main advantages of this method are the rapidity, efficacy, and gentleness of the separation. Viability of the cells remains consistently high (greater than 96%) throughout the separation. Since this method involves a one-step centrifugation, it precludes the necessity for repeated washings of cells which have been incubated with lipid vesicles.     

Auxin requirements of sycamore cells in suspension culture

Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this `auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.

There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10⁶ cells) on day 2 followed by a decline to 45 nanograms per 10⁶ cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10⁶ cells) rises slowly to a peak of 1.4 nanograms per 10⁶ cells by day 9 then decreases rapidly to 0.2 nanograms per 10⁶ cells by day 15 (early declining phase) and 0.08 nanograms per 10⁶ cells by day 23 (early stationary phase).

     

Differentiation of Dictyostelium discoideum cells in suspension culture

Differentiation of Dictyostelium discoideum cells in suspension culture is reported, using a medium containing glucose, albumin, cyclic AMP, EDTA and streptomycin in a phosphate buffer. Production of UDPgalactose:polysaccharide transferase, an enzyme specifically present in prespore cells, and the formation of prespore-specific antigens in more than 60% of the cells, are demonstrated. Differentiation in this medium differs from that previously reported with other suspension systems in that (a) cells form only small, amorphous agglomerates, (b) there is an absolute requirement for cyclic AMP and (c) prior formation of loose cell mounds on a solid substratum is essential for subsequent differentiation in this medium. This last requirement indicates that the differentiation process, giving rise to the prespore-specific enzyme and antigen, can be resolved into two distinct stages, one requiring cell contact on a solid substratum and the other proceeding in small agglomerates incubated in the medium. This medium may be useful for elucidating the role of cyclic AMP and cell contact in slime mould development.     

Induced flocculation of animal cells in suspension culture

Recycle unit operations for suspension cell cultures may be improved by flocculation of the cells. A flocculant search was conducted, and it was found that where strongly cationic polymers were severly toxic to the cells, neutral and anionic polymers were nontoxic and ineffective at flocculating the cells. A weak and poorly soluble polycation, poly-L-histidine, was capable of flocculating cultures of CHO, HeLa, U-937, and CRL 1606 hybridoma cells with no toxicity to the majority of cells. In addition to the lowered acute toxicity, the polymer treatment left the cells in a growing state for recycle. Flocculation was found to be mediated by precipitates of the polymer. The low toxicity of poly-L-histidine is probably due to its low solubility and charge at physiological pH. Nonelectrostatic interactions may also play a role.     

Photoautotrophic growth of soybean cells in suspension culture. II. Optimization of culture medium and conditions

We have attempted to optimize the conditions under which a photoautotrophic soybean suspension culture line (SB-P; Horn et al. 1983) is grown. Magnesium, phosphate, and calcium concentrations were varied individually from one-tenth to five times the normal level found in the Murashige and Skoog (1962) recipe. After two subcultures, only phosphate at one-tenth the normal level caused the cells to show a substantial reduction in fresh and dry weight increase and chlorophyll level. Nitrate and ammonium levels were inversely varied in 20 millimolar increments of potassium nitrate and ammonium chloride. Neither N-source alone could support growth through two subcultures. A ratio of 40 millimolar potassium nitrate to 20 millimolar ammonium gave significantly better fresh and dry weight increases than did a ratio of 20:40 or 30:30 but the chlorophyll level was unchanged. The minor salts as a group resulted in a small improvement in growth when provided at twice the normal level.Indole-3-acetic acid at five milligrams per liter resulted in significantly better fresh and dry weight increases than did -naphthaleneacetic acid at any level but the final chlorophyll level was not changed. There was no correlation between growth and kinetin level and this resulted in the discovery that SB-P cells are cytokinin-autotrophic, as are heterotrophic SB cells, with regard to both growth and greening ability. Growing SB-P cells under a 14 h:10 h day:night photoperiod resulted in a slow but inevitable death. Increasing the carbon dioxide level to 10% for four weeks gave no increase in SB-P cell growth or chlorophyll level, but SB-P cells would not grow with carbon dioxide levels below 0.4%. The results clearly show that SB-P cells, despite their tenuous existence, are capable of adapting to a wide range of culture conditions. A simplified and improved culture medium for photoautotrophic cultures is given.Abbreviations SB-P photoautotrophic soybean cells - SB-M photomixotrophic soybean cells - SB-H heterotrophic soybean cells