A new principle of cell cultivation: No air-liquid interface but gas exchange across a synthetic membrane

Summary A completely liquid-filled growth chamber for axenic cultures ofTetrahymena pyriformis is described; gas exchange is ensured across a synthetic membrane. The chamber may be incorporated into a continuous flow system with inoculation and removal of cell samples under sterile conditions. Initially, the generation time of the cells was slightly prolonged, about 10%, but after some cell doublings decreased to 5%. The capacity of the cells to form food vacuoles (endocytosis) was unaltered during growth in the chamber. The synthetic membrane was highly permeable to O₂ and CO₂; however, cells grown in the chamber contained small refractive granules. The culture chamber permits the culture volume to be varied and it may be used for other protozoa, bacteria, and even tissue culture cells.     

Automatic Cell Counting in Continuous Flow Cultures of Tetrahymena pyriformis*

This report describes an electronic cell counter constructed for determining cell number in cultures of the ciliate, Tetrahymena pyriformis. The culture chamber has been equipped with a device which determines the number of cells per unit volume and records the number automatically. As cell multiplication is unaffected by the counting procedure the cells are returned to the culture. Furthermore, keeping the culture volume constant we have arranged a continuous flow of fresh nutrient medium through the culture chamber and thus established conditions under which cell multiplication has continued for months while determinations of cell concentrations have been recorded every 10 min. Since the culture volume has been small, ～25 ml, growth studies utilizing this method require less than one liter of fresh medium per week in spite of the fast multiplication (9 generations per 24 hr) occurring in cultures of Tetrahymena pyriformis under optimal conditions.     

Primary culture of antvenom gland cells

Summary Venom from the antPseudomyrmex triplarinus reduces the symptoms and swelling of rheumatoid arthritis. The cells that produce the venom were dissected from larval and pupal ants and culture conditions studied. Cell dissociation, with minimal amount of damage, was done with 0.25% trypsin at 4°C with subsequent use of soybean trypsin inhibitor. A new medium was formulated and epidermal growth factor, fibroblast growth factor, insulin, cAMP, cGMP, and isoproterenol were beneficial. The optimum osmotic pressure was a relatively high 500 mOsm. Conditioning the medium with an established insect cell line was essential for long-term cell survival. Under these culture conditions the structural and metabolic integrity of the cells were maintained for up 12 mo.     

Optimization of in vitro multiple shoot clump induction and plantlet regeneration of Kentucky bluegrass (Poa pratensis)

An efficient protocol for Kentucky bluegrass (Poa pratensis L.) in vitro culture was established using shoot apices of seedlings as explants. The optimal procedure of this protocol for majority of the genotypes was that meristematic cell clumps and small calluses were firstly induced from the bases of explants on initial culture medium supplemented with 0.9 μM 2,4-d and 8.9 μM 6-BA for 20 d, then were separated and transferred to shoot clumps induction medium containing 8.9 μM 6-BA for the formation of multiple shoot clumps. The percentage of multiple shoot clumps and numbers of shoots per clump were deeply related with the combinations of different plant growth regulators, duration of initial culture, the intensity of illumination and genotypes. Histological observation of the induced explants revealed that the meristematic cell clumps were produced from repeated division of the cortical cells and original meristematic primodium cells of explants, and the multiple shoots were formed via organogenesis pathway in the meristematic cell regions of cultures on shoot clumps induction medium. In this study, plantlets were efficiently regenerated on large scale from seven cultivars of Kentucky bluegrass. Hence the meristematic cell clumps and small calluses in this protocol could be considered good targets for genetic transformation of Kentucky bluegrass.     

Improvement of somatic embryogenesis in highland-papaya cell suspensions

Axillary buds (2 mm) from 3-year-old Carica pubescens Lenné et Koch (highland papaya) fruit-bearing plants grown in the greenhouse were cultivated in NN-medium supplemented with different growth regulators naphthaleneacetic acid and indoleacetic acid in combination with Zeatin, benzyladenine, Kinetin and thidiazuron. Several responses were observed within 2–3 months; namely, sprouting of the preformed axillary buds, bud branching into multiple shoots, callus formation at the basal end of the explant and somatic embryogenesis in the preformed callus. Somatic embryogenesis was frequent in most of the tested growth regulator combinations, with the exception of thidiazuron which showed no effect. A much higher yield of somatic embryos could be obtained in suspensions. Somatic embryogenesis was enhanced by the occurence of adventive embryogenesis on single embryos as globular embryo clusters. This was observed in cell suspensions initially grown in a WPM-medium with 2,4-dichlorophenoxyacetic acid, or in combination with benzyladenine or zeatin, for 6 days, then maintained in a growth regulator-free medium under continuous agitation (50 RPM) on an orbital shaker for 3 months. Single cells grown in the absence of 2,4-dichlorophenoxyacetic acid did not initiate embryogenesis and de-differentiated into callus. Plantlets were recovered after transfer of mature embryos from cell suspensions into Magenta flasks. In a second subculture, adventitious embryogenesis occurred spontaneously in clusters at the globular embryo stage under the same growth conditions, yielding a high number of embryos. The culture conditions described above allowed initiation of a large number of somatic embryos directly from cell suspensions through adventive somatic embryogenesis and indirectly from callus on axillary buds.Abbreviations 2,4-d dichlorophenoxyacetic acid - CH casein enzymatic hydrolysate - BA benzyladenine - FAA formalin:acetic acid:alcohol - Glu l-glutamine - IAA indoleacetic acid - NAA naphthaleneacetic acid - NN Nitsch and Nitsch-medium (1969) - TDZ thidiazuron - SD standard deviation     

Improvement of the cell volume ofCandida blankii through protoplast fusion

Summary The yeastCandida blankii ESP-94, capable of utilizing xylose as substrate, was isolated for the production of single-cell protein (SCP) on bagasse hydrolysates. However, the small cell volume of strain ESP-94 would complicate harvesting of the cells during a continuous fermentation process. Auxotrophic mutants of strain ESP-94 were generated and intraspecific protoplast fusion experiments performed in an attempt to increase the cell volume of strain ESP-94. The fusion products were characterised with respect to cell volume, DNA content and genetic stability. Six genetically stable fusants with bigger cell volumes and higher DNA contents were obtained. One such fusant, fusant F17, had a cell volume 3-times that of strain ESP-94, while exhibiting similar growth rates to strain ESP-94 ond-xylose as carbon source.     

Plant regeneration through somatic embryogenesis in protoplast cultures of sandalwood (Santalum album L.)

Summary Protoplasts were isolated from embryogenic cell suspension cultures derived from proliferating shoot segments of a 20-year-old sandalwood tree (Santalum album Linn.). Under appropriate conditions, isolated protoplasts divided in liquid culture medium and produced embryogenic cell aggregates and globular embryos. Plating of cell aggregates on a fresh medium facilitated the differentiation of somatic emryos which further developed into plantlets.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA indoleacetic acid - IBA indolebutrytic acid - MES 2-(N-morpholino)ethane sulfonic acid - MS Murashige and Skoog's medium as modified in the text     

A method for the regulation of microbial population density during continuous culture at high growth rates

A method for the continuous culture of microorganisms is described which employs growth-dependent pH changes to control the rate of addition of fresh medium to a culture vessel. The apparatus (the phauxostat) supports, at constant pH, longterm continuous culture at rates near or at the maximum of which the organisms are capable. The buffering capacity of the inflowing medium determines the steady-state population density of the culture, but the rate of growth is independent of the buffering capacity. The fundamental theory of operation is tested and some basic parameters of growth are estimated using Escherichia coli B growing continuously in media containing glucose, glycerol or DL-lactate.Joseph C. Wilson Scholar.     

Changes in peroxidases in the suspension culture of Rubus fruticosus during growth

The growth parameters of a cell suspension culture of Rubus fruticosus L. were determined over a culture period including exponential growth, stationary phase and a glucose starvation period at the end of the normal culture cycle. Peroxidase activities were measured in the cytoplasm, in the cell wall, and in the culture medium by the guaiacol assay. There is a relationship between the activity found in the spent medium and the dry matter mass of the cells during the exponential growth. In the three compartments a bimodal repartition of peroxidase activities was observed, with the two peaks at day 4 and day 26, respectively. This suggests that the first peak corresponds to actively dividing cells whereas the second is associated with senescence, or stress due to starvation. Fractionation of the peroxidases from the culture mediuim revealed the presence of two sets of cationic isoenzymes, with minor amount of anionic peroxidases. Interestingly, the second peak of cationic enzymes which was of weak intensity at day 10 of the culture, becameprevalent at day 26. This indicates that not only the total amount of peroxidases varies as a function of culture time, but also that the nature of the peroxidases secreted into the medium changes during growth.Abbreviations DW dry weight - FW fresh weight - MV medium volume - SV suspension volume - BSA bovine serum albumin     

Monitoring of haploid maize cell suspension culture conditions in bioreactors

Maize (Zea mays L.) haploid cells were cultivated in a 1500 ml aerated and stirred batch bioreactor using modified BM medium. Cell growth was highly affected by pH and dissolved oxygen, and we observed two fairly distinct growth phases. During the first two days after inoculation at pH 5.8, oxygen consumption was high and the cells lowered the pH to a value around 4.3. After this period the pH stabilized at 4.5 and the dissolved oxygen reached a steady level. Decreasing dissolved oxygen concentration leads to lower growth rate and to higher pH. Both events mean stress conditions for the cell culture and probably result in increased genetic variability, and the loss of regeneration capacity. The stress condition during the adaptation phase can be eliminated by decreasing the pH of the medium to 4.7 before inoculation and by keeping dissolved oxygen above 40%. These conditions provide prolonged exponential growth dynamics and the cell suspensions could be the basis of large scale cultures also.Abbreviations 2,4-d 2,4-dichlorophenoxyacetitc acid - NAA naphthalene acetic acid     

Continuous culture of Candida utilis: influence of medium nitrogen concentration

When Candida utilis was grown in continuous culture, decreasing the concentration of N in the medium affected cell composition, biomass yield, biomass productivity, maximal growth rate and cell morphology. When the dilution rate was low (0.1 h^-1), reducing N from 1100 to 100 mg/l led to a 40% decrease in RNA content of the cells. Nitrogen-limited growth, which occurred when N<420 mg/l, was associated with significant changes in cell-wall carbohydrates and a significant reduction in the glycogen content of the cells. A set of culture conditions was established which permitted maximal consumption of the main nutrients in the medium and the production of yeast biomass suitable as a source of single-cell protein.     

A repeated batch process for cultivation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

A repeated batch process was performed to culture Bifidobacterium longum CCRC 14634. An on-line device, oxidation-reduction potential (ORP), was used to monitor cell growth and uptake of nutrients in the culture. The ORP of the culture medium decreased substantially during fermentation until nutrients were depleted. Six cycles of batch fermentation using ORP as a control parameter were successfully carried out. As soon as ORP remained constant or increased, three-quarters of the broth was removed, and the same volume of fresh medium was fed to the fermenter for a new cycle of cultivation. Average cell concentrations of 1.9×10⁹ and 3.4×10⁹ cfu ml^–1 for repeated batch fermentation in MRS (Lactobacilli MRS broth) and WY (containing whey hydrolyzates, yeast extract, l-cysteine) medium, respectively, were achieved. Cell mass productivities for batch, fed-batch and repeated batch fermentation using MRS medium were 0.51, 0.41, and 0.64 g l^–1 h^–1, respectively, and those for batch and repeated batch using WY medium were 0.76, 0.99 g l^–1 h^–1, respectively. The results indicate a possible industrial process to culture Bifidobacteria sp.     

Somatic embryogenesis in Ranunculus asiaticus L. hybr. thalamus cultivated in vitro

Calli derived from in vitro cultivated thalamus of Ranunculus asiaticus L. were initiated and maintained for 75 days on Murashige & Skoog's medium containing five concentrations of 2,4-d (0.1, 0.2, 0.4, 0.8, 1.6 mg l^-1). Embryoid differentiation occurred on calli initiated on 1.6 mg l^-1 2,4-d 75 days after subculture onto hormone-free medium. Calli which were initiated and maintained for 75 days on lower 2,4-d concentrations, then transferred to medium without hormones for 75 days, showed the first embryoids one month after further subculture on medium containing 0.05 mg l^-1 2,4-d. All the somatic embryos developed into plants, and 96% survived transplantation to in vivo growth conditions.     

PEG-tolerant cell clones of chili pepper: Growth,osmotic potentials and solute accumulation

Cell cultures of chili pepper (Capsicum annuum L.) were established from callus tissue inoculated in MS liquid medium supplemented with 6.25 M 2,4-d and 0.44 M BA. Cell clones were isolated by plating the cell suspension on filter paper discs supported by polyurethane foam that were bathed with culture medium containing 15% PEG. The cell clones T6 and T7 were chosen based on their characteristics of growth and friability. These cell clones were established as cell suspensions in the presence of 15% PEG and subsequently subcultured in increasing concentrations of osmoticum. By this approach the cell clones T7 and T6 were capable of growing in the presence of 20 and 25% PEG, respectively. The cell clone T7 was found to grow better in the presence of 5–10% PEG after a period of subculturing in the absence of osmoticum indicating that the tolerance trait was stable. The tolerant cell clones exhibited a 3 to 3.5-fold decrease in the osmotic potentials in comparison with the nonselected cells suggesting that osmotic adjustment occurred. K⁺ was the major contributing solute to the osmotic potential in all the cell cultures among those tested and was found to be higher in concentration in the PEG-tolerant clones (1.3–3 times higher than nonselected cells). Proline and glycine betaine levels showed a positive correlation with the degree of tolerance to water deficit in the PEG-tolerant cell clones. The levels of proline in the cell clone T7 subcultured in the absence of PEG in the culture medium decreased to values similar to those of nonselected cells, whereas the contents of glycine betaine in the same conditions were maintained at high levels.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - PEG polyethylene glycol     

Bioreactor culture of Picea mariana Mill. (black spruce) and the species complex Picea glauca-engelmannii (interior spruce) somatic embryos. Growth parameters

Somatic embryo cultures of Picea mariana and the species complex P. glauca-engelmannii were each grown in 7.5-l-capacity mechanically-stirred bioreactors containing 61 medium (LP, von Arnold and Eriksson) with 30 mm sucrose. Growth of both species occurred with no observable signs of shear stress due to mechanical agitation. Growth kinetics were analysed using an array of parameters (settled culture volume, packed culture volume, osmolarity, conductivity, pH). These were compared with fresh weight, dry weight, and somatic embryo number in order to determine what parameters were highly correlated with growth and embryo number. Increasing the sucrose concentration from 30 mm to 60 mm resulted in an increase in biomass and total number of somatic embryos. For P. mariana a maximum dry weight of 6.3 gl^–1 and 3076 embryos ml^–1 occurred in LP medium with 60 mm sucrose after 10–12 days of culture. For P. glauca-engelmannii a maximum dry weight of 4.3 gl^–1 and 2278 embryos ml^–1 occurred in LP medium with 60 mm sucrose after 6–8 days culture. For all sucrose concentrations, fresh weight, dry weight and embryo number were closely correlated with packed culture volume and conductivity for P. mariana, and settled culture volume, packed culture volume and conductivity for P. glauca-engelmannii.Correspondence to: D. I. Dunstan     

Continuous insect cell (Sf-9) culture with aeration through sparging

The continuous growth of Spodoptera frugiperda Sf-9 cells in a 250-ml blown-glass jacketed spinner flask under a direct air sparging environment was investigated. Even at 220 ml working volume (about 90% of total volume), this spinner flask provided good mixing and oxygenation as demonstrated by a higher cell density compared with fermentor cultures. This eliminates a common limitation of the traditional spinner flask, namely much lower cell density at high working volume. Furthermore, this spinner flask has been run with Sf-9 cell culture at five different dilution rates and two different air sparging rates at steady state, demonstrating its utility in research applications where cell size, metabolic activity and environmental conditions can be constantly maintained. In addition to demonstrating the utility of the reactor, three novel points are made in this report. First, cell density in continuous cultures is increased significantly due to a high agitation rate and, especially, air sparging rate, which is seldom used in animal cell or insect cell culture. Second, there is no apparent difference in the specific death rate at two different sparging rates (0.0093 vvm and 0.0125 vvm). Finally, we have maintained Sf-9 cells for more than 4 months in a continuous culture using a serum-free medium without loss of recombinant protein expression in infected cells.     

High-frequency somatic embryogenesis from small suspension-cultured clusters of cells of an interspecific hybrid of Oryza

Suspension cultures in which cell clusters were small and had a high capacity for somatic embryogenesis (about 60%) were established from immature panicles of F₁ plants from a cross between Oryza sativa and Oryza latifolia The cells were subcultured at seven-day intervals in a modified N6 medium. The cell clusters were quite small (approximately 30–200m in diameter) after culture for two months in this medium. When small clusters of cells were transferred to N6 medium, that had been diluted with an equal volume of water and supplemented with -naphthalenacetic acid (53 nM), 4-pyridylurea (2.2 M) and sucrose (30 gl^-1), somatic embryogenesis occurred at high frequency (about 60%). The system established in the present work is useful for biochemical and molecular biological research of the somatic embryogenesis in the Gramineae.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthalenacetic acid - 4-PU 4-pyridylurea - MS Murashige and Skoog (1962)     

Alexandrium ostenfeldii growth and spirolide production in batch culture and photobioreactor

Growth and spirolide production of the toxic dinoflagellate Alexandrium ostenfeldii (Danish strain CCMP1773) were studied in batch culture and a photobioreactor (continuous cultures). First, batch cultures were grown in 450 mL flasks without aeration and under varying conditions of temperature (16 and 22 °C) and culture medium (L1, f/2 and L1 with addition of soil extract). Second, cultures were grown at 16 °C in 8 L aerated flat-bottomed vessels using L1 with soil extract as culture medium. Finally, continuous cultures in a photobioreactor were conducted at 18 °C in L1 with soil extract; pH was maintained at 8.5 and continuous stirring was applied.This study showed that A. ostenfeldii growth was significantly affected by temperature. At the end of the exponential phase, maximum cell concentration and cell diameter were significantly higher at 16 °C than at 22 °C. In batch culture, maximum spirolide quota per cell (approx. 5 pg SPX 13-desMeC eq cell⁻¹) was detected during lag phase for all conditions used. Spirolide quota per cell was negatively and significantly correlated to cell concentration according to the following equation: y = 4013.9x^−0.858. Temperature and culture medium affected the spirolide profile which was characterized by the dominance of 13,19-didesMeC (29–46%), followed by SPX-D (21–28%), 13-desMeC (21–23%), and 13-desMeD (17–21%).Stable growth of A. ostenfeldii was maintained in a photobioreactor over two months, with maximum cell concentration of 7 × 10⁴ cells mL⁻¹. As in batch culture, maximum spirolide cell quota was found in lag phase and then decreased significantly throughout the exponential phase. Spirolide cell quota was negatively and significantly correlated to cell concentration according to the equation: y = 12,858x^−0.8986. In photobioreactor, spirolide profile was characterized by higher proportion of 13,19-didesMeC (60–87%) and lower proportions of SPX-D (3–12%) and 13-desMeD (1.6–10%) as compared to batch culture.     

Primary culture of normal rat adrenocortical cells

Summary Rat adrenocortical cells retiained their differentiated characteristics over 2 wk in culture without a specific requirement for additives other than inorganic salts, amino acids, vitamins, and fetal bovine serum. The cells were maintained free from fibroblast overgrowth by substitution ofd-valine in place ofl-valine in the medium. Corticotropin (ACTH) inhibited the growth of adrenocortical cells in this medium and the effect was reversible. The adrenocortical cells had a limited capacity for growth as reflected by total cell counts and [³H]thymidine uptake with cells from young animals demonstrated a greater potential for DNA synthesis than cells obtained from mature animals. A very sensitive assay for ACTH using a small number of cells in primary culture also is described. This work was supported by Grant CA-16417 from the National Cancer Institute.     

Ultrastructural study of different types of callus from leaf expiants ofAesculus hippocastanum L.

Summary The cell ultrastructure in three types of callus obtained from leaf explants ofAesculus hippocastanum L. has been studied. Remarkable differences have been shown between the cells of the forerunner E₁ callus and those of the callus arising from it, according to the culture conditions.The peculiar characteristics of E₁ are the scarcity of intercellular spaces and the occurrence of autophagic vacuoles in the cells.An embryogenic friable callus (E₂) is formed in time when E₁ is maintained on solid culture medium. The E₂ cells show cytological features typical of a higher metabolic level and contain starch. Diffused middle lamella digestion leads to the detachment of small embryogenic cell aggregates consisting of vacuolated parenchymatous-like cells and small meristematic cells which may be regarded as embryoids initials.Shaking E₁ in the same liquid medium and subsequent culture on solid medium lead to the differentiation of a non-embryogenic callus (NE), whose cells are very large and highly vacuolated, devoid of starch and with organelle-rich cytoplasm. The NE callus shows a high degree of growth, but does not attain embryogenic competence in time.Abbreviations c cell - cr crystal - cw cell wall - d dictyosome - er endoplasmic reticulum - m mitochondrion - mb microbody - n nucleus - p plastid - s starch - v vacuole