Size Of Cell Aggregates Research Articles

Gradient of anthocyanin in cell aggregates of Daucus carota in suspension cultures

The size distribution of cell aggregates, and the effect of cell aggregate size on anthocyanin content of Daucus carota cells in suspension cultures, was studied. The profile of biomass distribution in various size groups of cell aggregates indicated that over 92% of biomass was present in the aggregates of 500–1500 μm in diameter. The anthocyanin content increased initially with the increase in cell aggregate diameter up to 500–850 μm, and decreased rapidly with the increase in the cell aggregate size above this critical diameter. On the other hand, the surface colour intensity showed a steady increase with the increase in cell aggregate size, indicating a steep radial gradient of anthocyanin content along the radius of the larger cell aggregates.

Three-dimensional cell aggregation enhances growth-promoting activity of NGF in adult DRG.

After removal of the connective tissues with collagenase-trypsin treatment, DRG explants without nerve fibres from mature rodents regenerated numerous neurites all around the explants in response to NGF. To observe the response in the absence of any non-neuronal cells in the ganglia, we introduced three-dimensional cell aggregates consisting of variable numbers of highly purified isolated adult DRG neurones. In this culture system, the addition of NGF to the medium increased the maximum neurite length, the number of neurites, and the cell survival rate. Furthermore, the magnitude of this effect increased with cell aggregate size. Our results suggest that the addition of NGF produces extensive neurite outgrowth in three-dimensional aggregates of matured sensory neurones in the absence of any non-neuronal cells.

Cell aggregates in wheat suspension cultures and their effects on isolation and culture of protoplasts

Fine embryogenic suspension cultures of wheat (Triticum aestivum cv Hartog and Timmo, and T. durum cv D6962) tend to grow into large cell clumps (1-3 mm), resulting in the formation of mixed suspension cultures consisting of both fine and large cell clumps. The cell clumps were separated according to their sizes and cultured as new lines to investigate their growth rate and differentiation potential and the effects of cell aggregate size on protoplast culture. The results showed that the fine clusters (<310 μm) had a higher growth rate but a lower differentiation frequency than the large cell aggregates (310-2000 μm). After 2-4 weeks incubation, all the new lines reformed mixed suspension cultures again. The large clumps (>1100 μm) released fine cell clusters into the medium so it was possible to initiate fine embryogenic suspension cultures from the large clumps. With regard to the isolation and culture of protoplasts, although the highest yield of protoplasts was obtained from the fine cell clusters, the protoplasts isolated from different sized cell aggregates all had similar potential for sustained cell division and plant regeneration.

5A11 antigen is a cell recognition molecule which is involved in neuronal-glial interactions in avian neural retina.

In continuing efforts to identify cell-surface molecules involved in cell-cell interactions in the developing avian retina, we identified a monoclonal antibody, the 5A11 antibody, which possessed the ability to interfere with contact-dependent glial cell maturation in vitro. We sought to determine the molecular and biochemical identity of the glycoprotein recognized by this antibody, and using additional criteria, establish whether the 5A11 antigen is indeed a cell-recognition molecule in the developing retina. Immunohistochemical analyses demonstrate that in the hatchling chick retina and in live cultures of embryonic retina cells, the 5A11 antigen is predominantly associated with Müller glial cells whereas little is observed on neuronal elements. Microsequencing of the major component isolated by immunoaffinity chromatography identifies the HT7 antigen (Seulberger et al.: EMBO Journal 9:2151-2158, 1990), a unique member of the immunoglobulin super gene family (IGSF), as a homologous if not identical protein to the 5A11 antigen. The HT7 antibody, furthermore, recognizes affinity purified 5A11 antigen, and both the HT7 antibody and additional probes generated against the 5A11 antigen recognize a major polypeptide of 45.5 kDa and a minor band of 69 kDa on Western blots of membrane preparations from neural retina. To verify that the 5A11 antigen mediates cell-cell recognition events in the developing neural retina, we examined the consequences of adding antibody to monolayer cultures of dissociated embryonic retina cells and to dissociated retina cells in rotation-mediated suspension culture. Addition of the 5A11 antibody to monolayer cultures results in alteration in the development of the stereotypic arrangement of neurons and glia characterized by a reduction in the number and complexity of neural extensions upon the glial-derived flat cells. Similarly, addition of antibodies generated against the 5A11 antigen to dissociated cells in rotation cultures significantly reduces retina cell reaggregation as monitored by computer-assisted image analysis of cell aggregate size. These data and the identification of the 5A11 antigen as a member of the IGSF establish a role for the 5A11 antigen as a novel recognition molecule in the developing neural retina.

Chromosomal variability and growth rate in cell suspension cultures of Stevia rebaudiana (Bert.) Bertoni

Several cell lines were obtained from a predominantly diploid ( 2n = 22) cell suspension culture of Stevia rebaudiana by colchicine treatment (10 −3M/72 h), filtration in 100–500μm sieves and plating followed by visual selection. Colchicine treatment resulted in a highly polyploid line, but with a tendency for reversion to a chromosome distribution profile (CDP) similar to the initial culture, but with a higher growth rate. Selection by filtration showed a relationship between cell aggregate size and the CDP, cells of higher ploidy having a tendency to form smaller aggregates. After plating, calli with several characteristics (shape, colour, degree of friability) were selected, and gave rise to cell lines with different CDPs; these cell lines were used to initiate predominantly diploid, tetraploid or aneuploid cell suspensions that showed characteristics growth rates and aggregate sizes.

Development of an optical method for monitoring protoplast formation from cultured plant cells

The size distribution of cell aggregates during protoplast isolation from Catharanthus roseus and Nicotiana tabacum was measured by a Coulter counter. It was observed that a gradual reduction in the size of cell aggregates occured during protoplast formation. A previously developed specialized spectrophotometer for the photometric measurement of plant cell concentration was used for continuous monitoring of the reduction in the size distribution of cell aggregates during protoplast formation. This made it possible to use changes in optical density (O.D.) to distinguish the three stages in protoplast formation—plasmolysis, maceration and cell wall digestion. During the processes of maceration and cell wall digestion, the O.D. decreased and reached a steady value at the end of each process. Consequently, changes in the O.D. could be used to determine precisely the end of each process. The cell wall digestion process was described by a simple first order reaction model and the rate of protoplast formation (cell wall digestion) was quantitatively evaluated from the rate constant ( k) of this reaction. By using the values of k, the optimal enzymatic reaction conditions for isolating protoplasts from C. roseus and N. tabacum cells were determined.

Rheological characteristics of cell suspension and cell culture of Perilla frutescens

Physical properties such as viscosity, fluid dynamic behavior of cell suspension, and size distribution of cell aggregates of a plant, Perilla frustescens, cultured in a liquid medium were studied. As a result of investigations using cells harvester after 12 days of cultivation in a flask, it was found that the apparent viscosity of the cell suspension did not change with any variation of cell concentration below 5 g dry cell/L but markedly increased when the cell concentration increased over 12.8 g dry cell/L. The cell suspension exhibited the characteristics of a Bingham plastic fluid with a small yield stress. The size of cell aggregates in the range 74 to 500 mum did not influence the rheological characteristics of the cell suspension. The rheological characteristics of cultivation mixtures of P. frutescens cultivated in a flask and in a bioreactor were also investigated. The results showed that the flow characteristics of the cell culture could be described by a Bingham plastic model. At the later stage of cultivation, the apparent viscosity increased steadily, even though the biomass concentration (by dry weight) decreased, due to the increase of individual cell size.

Kinetics of cellular growth and diosgenin biosynthesis in Dioscorea caucasica cell suspensions, cultured in batch system

By selecting small cell aggregates, several cell lines of Dioscorea with different characteristics were established: colour, size of cell aggregates, growth, accumulation of carotenoids, diosgenin and phytosterols. The white-coloured lines accumulate larger amounts of diosgenin than the unselected cell suspension and the cream-coloured cell lines, which synthesize high quantities of carotenoid pigments. There is a reverse correlation between the biosynthesis processes of the two metabolites, carotenoids and diosgenin. The cell lines consisting of small cell aggregates reveal a higher growth and a faster consumption of sucrose than the lines with larger aggregates. Intensity of diosgenin biosynthesis is correlated to cell proliferation, being maximum in the exponential phase and minimum at its beginning. In very many of the D. caucasica cell lines, more than 50% of the total steroids are phytosterols, among which the major compound is sitosterol.

Size determination of red blood cell aggregates induced by dextran using ultrasound backscattering phenomenon

Different methods are commonly used to study the red blood cell aggregation phenomenon. The major interest of the ultrasonic method presently discussed is to assess the mean size of red blood cell (RBC) aggregates by measuring ultrasonic intensity backscattered by blood. Applying Rayleigh theory of sound to blood medium, one can show that the scattered ultrasonic intensity is proportional to the 6th power of the size of the RBC aggregates. The ultrasonic method is used to evaluate the mean size of RBC aggregates induced by dextrans. RBCs are suspended at various hematocrits H, in solution of dextrans of different molecular weights M and at different weight concentrations Cw. Results are presented by using the ultrasonic backscattering coefficient chi which is a relevant quantity in a scattering experiment. For suspensions of RBCs aggregated with dextran of molecular weight 70,000 dalton (dextran 70) at concentration Cw = 40 g/l, variations of chi as a function of H are similar to those obtained for normal blood. At a fixed hematocrit, variation of chi versus Cw for dextran 70 exhibits a maximum at 40 g/l. In the case of RBCs suspended at hematocrit 20% and aggregated with dextrans of molecular weight M, 70,000 less than or equal to M less than or equal to 2,000,000, the variations of chi versus molar concentration Cm are similar to those of the microscopic aggregation index defined by Chien (1). Finally, a statistical model of the blood structure previously described (2) is applied to evaluate the mean size of the aggregates. According to this model, the mean size of aggregates is independent of hematocrit for H less than or equal to 40% and independent of the molecular weight of dextran for M greater than or equal to 150,000 dalton.

Protein synthesis stimulated in sonicated sugar beet cells and protoplasts

Sugar beet suspension cells and protoplasts were exposed to 20 kHz ultrasound and the amount of 35S-methionine incorporated into cellular protein was determined after 2 days of culture. The incorporation of 35S-methionine in cells was enhanced up to 90% after sonication for 220–770 ms at 1.2–3.5 W/cm 2 which reduced viability 5–11% and decreased the average size of cell aggregates. In protoplasts, the 35S-methionine incorporation was increased up to 31% after sonication for 220–550 ms at 0.3–1.0 W/cm 2 with a concomitant reduction in viability of 10–16%. At higher ultrasonic energies, 35S-methionine incorporation declined proportionally to the reduction in protoplast viability. At concentrations up to 30 mM, cystamine and cysteamine did not affect 35S-methionine incorporation or viability of the sonicated protoplasts. These results show that mild ultrasonic irradiation can stimulate protein synthesis in plant cells and protoplasts significantly.

Culture of photomixotrophic soybean and pine in a modified fermentor using a novel impeller

Photomixotrophic suspensions of Glycine max (soybean) and Pinus elliottii (slash pine) have been successfully cultured in a hybrid stirred tank photobioreactor using a novel cell-lift impeller. A cell-lift impeller exhibited cell viabilities over 90% and an average cell aggregate size of 1.0 mm or less. Flat-bladed turbines produced equivalent biomass to the cell-lift impeller, but cell viability was reduced (85%) and cell aggregate size increased (3-5 mm diameter). Maximum fresh weights of 82 g L(-1) (soybean) and 52 g L(-1) (slash pine) were achieved in 15 days using continuous lighting (90-100 microE m(-2) s(-1)) and supplemental 2% CO(2) inlet gas. Maximum biomass was achieved using an impeller speed of 60 rpm with air-flow rate of 0.2 vvm for the cell-lift impeller and the pair of flat bladed turbines. The lag and early exponential phases were characterized by (1) rapid hydrolysis of sucrose followed by preferential use of glucose and (2) a reduction in chlorophyll levels. Carbon dioxide (2%-5%) was an essential nutrient for photomixotrophic cell culture in the bioreactors.

Periodic cell aggregation in suspensions of Dictyostelium discoideum

Abstract Periodic activities of Dictyostelium discoideum can be observed in cell suspension as two types of oscillations in the light-scattering properties, spike-shaped and sinusoidal. Responses of suspended cells to applied chemoattractants are also reflected by transient changes in light scattering. Alterations in the light-scattering properties are due to structural changes such as changes in cell shape and/or changes in the size of cell aggregates. Therefore, changes in the aggregation state during autonomous oscillations and during attractant-induced responses were investigated. In order to be able to withdraw multiple samples and larger sample volumes from optically monitored cell suspensions, a photometer comprising glass fiber optics immersable in a cell suspension was constructed. Samples were fixed with formaldehyde and photographed. The aggregation state of the samples was quantified by counting the number of particles (cells and cell aggregates) per volume. Folic acid elicited in suspensions of undifferentiated cells a transient decrease in the number of particles per volume as did cAMP in suspensions of preaggregation cells. Periodic changes in the number of particles per volume occurred synchronously with spike-shaped and sinusoidal oscillations. The relative amplitude of the oscillations in particle number was larger during sinusoids than during spikes. Photographs showed periodic changes in the aggregate size during sinusoidal oscillations. In each cycle, the cell-aggregation phase was followed by a phase of partial disaggregation. The recurring loosening of cell-cell contacts may be relevant for sorting out the different cell types. The potential role of contact site as synchronizer and as constituent of an oscillator is discussed.

Further kinetic characterization of alginate‐entrapped cells of Mucuna pruriens L.

Alginate-entrapped cells of Mucuna pruriens L. hydroxylate L-tyrosine, tyramine, para-hydroxyphenylpropionic acid, and para-hydroxyphenylacetic acid to their corresponding catechols, which were released into the incubation medium. Michaëlis-Menten kinetics was applied for each bioconversion. The apparent affinity constants were comparable with the affinity constants obtained with a homogenate directly prepared from the cells used for entrapment and with a derived partly purified phenoloxidase. The values found for the apparent maximum rates of bioconversion of the entrapped cells were ca. 50% of the values of the maximum rates of bioconversion of the cell homogenate, indicating that the entrapped cell system was not operating optimally. The effective diffusivities of the substrates and products were measured with alginate-entrapped, inactivated cells. From the five inactivation methods tested, glutaric aldehyde treatment was chosen as the general procedure. Calculated effective diffusivities for the monophenols and catechols demonstrated that these compounds could diffuse freely into and out of the beads. For each bioconversion, the observable modulus was calculated from the initial rate of bioconversion and the effective diffusivity of the substrate. The resulting values indicated that the diffusional supply rate of the substrates was not the limiting factor, except for the conversion of tyramine for which a modulus higher than one was obtained. Analogously, the observable moduli were calculated for oxygen, which was utilized for bioconversion and cell respiration, and these values pointed towards strong oxygen limitation in all cases. The bioconversion rates of the entrapped cells increased with decreasing cell aggregate size. Therefore, it was concluded that direct cell-matrix contact determined the amount of phenoloxidase involved in the bioconversions. The bioconversion rate on a protein basis was constant with enhancement of the bead charge and thus, in spite of limitations, the mixing conditions as such were relatively optimal. In conclusion, the nonoptimal efficiency of the plant cell system studied was caused by oxygen limitation and a partial phenoloxidase participation, but not by mass transfer limitations for substrates and products with the exception of the conversion of tyramine into dopamine.

Tetrazolium staining by optical scanning overestimates colony size and number of colonies counted.

We measured the effect that staining with 2-(P-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) had on the number and size distribution of tumor colonies counted using an optical image analyzer (FAS II). Staining increased the number of tumor colonies counted. By using opaque tumor cells or pigmented melanoma cells and measuring colony growth kinetics, we demonstrated that the use of INT staining to assist in counting tumor colonies artificially increased the size of viable tumor cell aggregates by adding a red precipitate to the outside surface of the cells. Laboratories that are using the INT method for drug screening are probably measuring colonies down to and below 42 microns in diameter. These small colonies could result from as few as one or two divisions. Thus, potentially useful drugs may be missed in the screen because of the presence of abortive colonies: i.e., lethally damaged cells completing only one or two divisions.

Growth Related Accumulation of Betacyanin in Suspension Cultures of Phytolacca americana L.

Summary Maximum betacyanin accumulation was observed in the logarithmic phase of growth in suspension cultures of Phytolacca americana.The relationships between the growth, betacyanin accumulation and the initial phosphate concentration in the medium were found to be linear for phosphate concentrations in the range 0-1.25 mM.A close correlation between cell aggregate size and betacyanin accumulation was also observed.These results indicate that betacyanin in suspension cultures of Phytolacca americana shows a different accumulation pattern from most other secondary products, in being strongly affected by the growth.

Cell kinetic study on GM colonies using autoradiography and collagen gel culture with special reference to unique proliferation of eosinophils.

Using a newly developed technique of autoradiography and collagen gel culture, a kinetic study on human GM colonies was attempted. Colonies of immature cells appeared first on day 5. The number of mixed colonies (mixture of immature cells, neutrophils, and/or monocyte-macrophages) and neutrophil colonies attained a maximum on days 8 to 10 and a broad peak of monocyte-macrophage colonies was observed on days 11 to 16. Eosinophil colonies appeared first on day 12, reached a maximum on day 18, and then gradually decreased. A detailed analysis of the order of appearance of the colonies suggests that mixed, neutrophil and monocyte-macrophage colonies originate from immature cell colonies or clusters, while eosinophil colonies do not. An autoradiographic study was designed to study the proliferation characteristics of each colony. Labeling indices (LI) with 3H-TdR of the cells in immature cell colonies were always high. LI of the cells in differentiated colonies such as neutrophil, monocyte-macrophage, and mixed colonies were low throughout the observation period. In contrast, LI of the cells in eosinophil colonies were constantly high regardless of the size of cell aggregates and the duration of the culture period. Both mitotic indices and mean grain counts on the nuclei of eosinophils were similar to those of immature cells. These results suggest that eosinophil colonies develop from their own small clusters and that eosinophils retain a fairly good proliferative capacity even when differentiated to the level in which specific granules appear in the cytoplasm.

Strategies for the selection and characterization of aluminum-resistant variants from cell cultures of Nicotiana plumbaginifolia

The development of strategies for selecting and characterizing aluminum-resistant variants from Nicotiana plumbaginifolia Viv. cell cultures is described. Plated cells, smeared callus, in-vitro-grown shoots, and seedlings of wild-type N. plumbaginifolia all showed similar responses to Al, with total growth inhibition at or above 600 μM Al. The strict control of both cell density and aggregate size is important in selection experiments for total inhibition of the growth of wild-type cells. Two approaches for the selection of Al-resistant variants were used. In a direct method, cells were plated onto medium containing 600 μM Al which inhibited growth and chlorophyll synthesis in wildtype cells. A double selection strategy based on both cell growth and greening was used to isolate 29 Al-resistant variants. In the other approach, a rescue method, suspensions were cultured for 10 d in medium containing 600 μM Al, then plated onto standard medium for recovery of survivors. Using this strategy, 217 Al-resistant variants were selected. After six to twelve weeks of growth in the absence of Al, each variant was cloned and reselected from single cells. Al resistance was retained in 31% and 51% of the variants selected by the direct and rescue strategies, respectively. Seedling segregation data are presented for the progeny (selfed and backcrossed) of plants regenerated from one of the variants and are consistent with those expected for a single dominant mutation.

Accumulation and Excretion of Alkaloids by Macleaya microcarpa Cell Cultures II. Experiments in Liquid Medium

A static Macleaya microcarpa cell culture shows at most one third of its alkaloid amount in the medium (Böhm and Franke 1982). In contrast, the medium of a M. microcarpa suspension culture can contain nearly the total alkaloid quantity detectable in the culture flask. In this case most cell aggregates are very small. Their portion in the cell mass is reached under the influence of a high auxin concentration. The number of alkaloid rich cells in small cell aggregates is relatively low. Nevertheless, fine M. microcarpa suspension cultures which are poor in alkaloid accumulating cells can form substantial amounts of alkaloids. Their synthetic activity is nearly unchanged. Only their accumulating capacity is redueed. This finding decisively supports the view that in M. microcarpa cell cultures biosynthesis and accumulation of alkaloids are distributed to two types of differentiated cells. It appears not reasonable to assums that a cell specialized in both alkaloid biosynthesis and accumulation performs only the first function if the cell aggregate size decreases.

Aggregation patterns of fetal rat brain cells following exposure to X-irradiation.

In our search for a simplified in vitro test system to assess the teratogenic effects of physical factors, we studied the effects of total maternal body X-irradiation on aggregation patterns of enzymatically isolated fetal rat brain cells and on ultrastructural aggregate changes. The fetal brain cells were derived from day 14 gestation fetuses of pregnant Sprague-Dawley (CD strain) rats exposed to X-irradiation (25 - 200 R) one hour prior to sacrifice. Notable changes in the cell aggregates following X-irradiation included a reduction in cell aggregate size and an increase in number. The frequency of cell aggregates was higher in the treated than in the control group, and the mean diameter of cell aggregates was inversely related to increasing X-irradiation doses. Transmission electron microscopy revealed in isolated cells features of degenerative process which were similar to those found in intact fetal brain lesions caused by maternal X-irradiation. Furthermore, scanning electron microscopy revealed that inhibition of cell aggregation following X-irradiation could probably be attributed to inhibition of membrane filopodia development and a consequent failure fo cell aggregates to fuse into a greater cell aggregate mass. These results suggest that the membrane factors which influence cell aggregation may be a useful parameter to assess early effects of X-irradiation-induced brain deformity. Presently, the cell aggregation culture system is being further evaluated as a short term test system for environmental teratogens.

The effect of cytoskeletal disrupting agents on the morphological response of a clonedManduca sexta cell line to 20-hydroxy-ecdysone.

The embryonicManduca sexta cell line, MRRL-CH1 was cloned by dilution and single cell plating into conditioned medium, resulting in 6 cell clones with differing responses to the insect steroid hormone, 20-hydroxy-ecdysone. One of these clones, designated GV1, was studied with respect to cytoskeletal elements. Electron microscopic examination revealed that the microtubule networks became more extensive and organized increasingly parallel following exposure to the insect hormone 20-hydroxy-ecdysone, while inhibitors of microtubules were found to reverse the elongation response in the cells within 2.5 h. Agents affecting microfilaments (cytochalasins B and D) did not specifically alter the elongation response, but treatment with these agents did reveal increased cellular junctions as shown by increased cell aggregate size in hormone-treated cultures exposed to the cytochalasins.