Repeated-batch cultures of Baby Hamster Kidney cell aggregates in stirred vessels

Natural aggregates of Baby Hamster Kidney cells were grown in stirred vessels operated as repeated-batch cultures during more than 600 hours. Different protocols were applied to passaging different fractions of the initial culture: single cells, large size distributed aggregates and large aggregates. When single cells or aggregates with the same size distribution found in culture are used as inoculum, it is possible to maintain semi-continuous cultures during more than 600 hours while keeping cell growth and viability. These results suggest that aggregate culture in large scale might be feasible, since a small scale culture can easily be used as inoculum for larger vessels without noticeable modification of the aggregate chacteristics. However, when only the large aggregates are used as inoculum, it was shown that much lower cell concentrations are obtained, cell viability in aggregates dropping to less than 60%. Under this selection procedure, aggregates maintain a constant size, larger than under batch experiments, up to approximately 400 hours; after this time, aggregate size increases to almost twice the size expected from batch cultures.     

Frequency, size, and localization of bacterial aggregates on bean leaf surfaces

Using epifluorescence microscopy and image analysis, we have quantitatively described the frequency, size, and spatial distribution of bacterial aggregates on leaf surfaces of greenhouse-grown bean plants inoculated with the plant-pathogenic bacterium Pseudomonas syringae pv. syringae strain B728a. Bacterial cells were not randomly distributed on the leaf surface but occurred in a wide range of cluster sizes, ranging from single cells to over 10(4) cells per aggregate. The average cluster size increased through time, and aggregates were more numerous and larger when plants were maintained under conditions of high relative humidity levels than under dry conditions. The large majority of aggregates observed were small (less than 100 cells), and aggregate sizes exhibited a strong right-hand-skewed frequency distribution. While large aggregates are not frequent on a given leaf, they often accounted for the majority of cells present. We observed that up to 50% of cells present on a leaf were located in aggregates containing 10(3) cells or more. Aggregates were associated with several different anatomical features of the leaf surface but not with stomates. Aggregates were preferentially associated with glandular trichomes and veins. The biological and ecological significance of aggregate formation by epiphytic bacteria is discussed.     

Frequency, Size, and Localization of Bacterial Aggregates on Bean Leaf Surfaces

Using epifluorescence microscopy and image analysis, we have quantitatively described the frequency, size, and spatial distribution of bacterial aggregates on leaf surfaces of greenhouse-grown bean plants inoculated with the plant-pathogenic bacterium Pseudomonas syringae pv. syringae strain B728a. Bacterial cells were not randomly distributed on the leaf surface but occurred in a wide range of cluster sizes, ranging from single cells to over 10⁴ cells per aggregate. The average cluster size increased through time, and aggregates were more numerous and larger when plants were maintained under conditions of high relative humidity levels than under dry conditions. The large majority of aggregates observed were small (less than 100 cells), and aggregate sizes exhibited a strong right-hand-skewed frequency distribution. While large aggregates are not frequent on a given leaf, they often accounted for the majority of cells present. We observed that up to 50% of cells present on a leaf were located in aggregates containing 10³ cells or more. Aggregates were associated with several different anatomical features of the leaf surface but not with stomates. Aggregates were preferentially associated with glandular trichomes and veins. The biological and ecological significance of aggregate formation by epiphytic bacteria is discussed.     

Use of preformed cell aggregates and layers to measure tissue-specific differences in intercellular adhesion

The binding of aggregates formed from various 7-day chick embryo tissues to cultured cell layers was analyzed 24 hr following trypsin dissociation of the tissues. The proprotion of aggregates binding is independent of the number of aggregates added, and changes with time over 60 min in a manner consistent with a first-order process. The adhesive parameter measured, the percentage of aggregates binding to cell layers per unit time, varies slightly with aggregate size but is not dependent upon the probability of collision of the aggregate with the layer. The rate of binding and the effect of modifiers of binding (temperature, inhibitors of oxidative metabolism and glutaraldehyde) are substantially different for neural retina interactions than for liver or heart interactions, suggesting that retina cells may form intercellular bonds via a mechanism distinct from that of liver or heart cells. The rate of binding between like tissue types is, with one exception, greater than between unlike types. Glutaraldehyde treatment of only one of the reactants abolishes this adhesive specificity. Aggregate binding provides a means of quantitatively assessing intercellular adhesion which has the advantage of reducing the effects of trypsinization on measurements of adhesion, and therefore lends itself to the investigation of cellular consequences of adhesion.     

Behavior of cell aggregate of Carthamus tinctorius L. cultured cells and correlation with red pigment formation

The formation processes of Carthamus tinctorius cell aggregates in a growth medium and the correlation of red pigment formation with cell aggregate sizes were investigated. About 80% of cell aggregates in the growth medium were > 1.00 mm in size. The growth rate of large cell aggregates was more rapid than that of small cell aggregates. Most cell aggregates > 0.50 mm in size became larger or smaller than their original sizes during the culture. A high level of red pigment formation was observed when cell aggregates obtained by the preculture using cell aggregates < 1.00 mm in size were cultured in the production medium.     

Modeling aggregation and growth processes in an algal population model: analysis and computations

 Aggregation, the formation of large particles through multiple collision of smaller ones is a highly visible phenomena in oceanic waters which can control material flux to the deep sea. Oceanic aggregates more than 1 cm in diameter have been observed and are frequently described to consist of phytoplankton cells as well as other organic matter such as fecel pellets and mucus nets from pteropods. Division of live phytoplankton cells within an aggregate can also increase the size of aggregate (assuming some daughter cells stay in the aggregate) and hence could be a significant factor in speeding up the formation process of larger aggregate. Due to the difficulty of modeling cell division within aggregates, few efforts have been made in this direction. In this paper, we propose a size structured approach that includes growth of aggregate size due to both cell division and aggregation. We first examine some basic mathematical issues associated with the development of a numerical simulation of the resulting algal aggregation model. The numerical algorithm is then used to examine the basic model behavior and present a comparison between aggregate distribution with and without division in aggregates. Results indicate that the inclusion of a growth term in aggregates, due to cell division, results in higher densities of larger aggregates; hence it has the impact to speed clearance of organic matter from the surface layer of the ocean. Received 1 July 1994; received in revised form 23 February 1996     

A population balance equation model of aggregation dynamics in Taxus suspension cell cultures

The nature of plant cells to grow as multicellular aggregates in suspension culture has profound effects on bioprocess performance. Recent advances in the measurement of plant cell aggregate size allow for routine process monitoring of this property. We have exploited this capability to develop a conceptual model to describe changes in the aggregate size distribution that are observed over the course of a Taxus cell suspension batch culture. We utilized the population balance equation framework to describe plant cell aggregates as a particulate system, accounting for the relevant phenomenological processes underlying aggregation, such as growth and breakage. We compared model predictions to experimental data to select appropriate kernel functions, and found that larger aggregates had a higher breakage rate, biomass was partitioned asymmetrically following a breakage event, and aggregates grew exponentially. Our model was then validated against several datasets with different initial aggregate size distributions and was able to quantitatively predict changes in total biomass and mean aggregate size, as well as actual size distributions. We proposed a breakage mechanism where a fraction of biomass was lost upon each breakage event, and demonstrated that even though smaller aggregates have been shown to produce more paclitaxel, an optimum breakage rate was predicted for maximum paclitaxel accumulation. We believe this is the first model to use a segregated, corpuscular approach to describe changes in the size distribution of plant cell aggregates, and represents an important first step in the design of rational strategies to control aggregation and optimize process performance.     

Role for Hsp70 chaperone in Saccharomyces cerevisiae prion seed replication

The Saccharomyces cerevisiae [PSI+] prion is a misfolded form of Sup35p that propagates as self-replicating cytoplasmic aggregates. Replication is believed to occur through breakage of transmissible [PSI+] prion particles, or seeds, into more numerous pieces. In [PSI+] cells, large Sup35p aggregates are formed by coalescence of smaller sodium dodecyl sulfate-insoluble polymers. It is uncertain if polymers or higher-order aggregates or both act as prion seeds. A mutant Hsp70 chaperone, Ssa1-21p, reduces the number of transmissible [PSI+] seeds per cell by 10-fold but the overall amount of aggregated Sup35p by only two- to threefold. This discrepancy could be explained if, in SSA1-21 cells, [PSI+] seeds are larger or more of the aggregated Sup35p does not function as a seed. To visualize differences in aggregate size, we constructed a Sup35-green fluorescent protein (GFP) fusion (NGMC) that has normal Sup35p function and can propagate like [PSI+]. Unlike GFP fusions lacking Sup35p's essential C-terminal domain, NGMC did not form fluorescent foci in log-phase [PSI+] cells. However, using fluorescence recovery after photobleaching and size fractionation techniques, we find evidence that NGMC is aggregated in these cells. Furthermore, the aggregates were larger in SSA1-21 cells, but the size of NGMC polymers was unchanged. Possibly, NGMC aggregates are bigger in SSA1-21 cells because they contain more polymers. Our data suggest that Ssa1-21p interferes with disruption of large Sup35p aggregates, which lack or have limited capacity to function as seed, into polymers that function more efficiently as [PSI+] seeds.     

The factors that promote the development of symmetry properties in aggregates from dissociated echinoid embryos

Summary Embryos of Hemicentrotus pulcherrimus at the 16 cell, 400 cell or mesenchyme blastula stage of development were dissociated into single cells. The cells were reaggregated, and the development of individual aggregates was monitored. Only aggregates from 16 cell embryos developed into pluteus-like larvae with radial or bilateral symmetry. When embryos at these three developmental stages were incompletely dissociated so that there were mixtures of single cells and groups of undissociated cells, the percentage of aggregates from 16 cell embryos that developed in a pluteus-like manner was greater than in aggregates from completely dissociated 16 cell embryos. Also a small percentage of aggregates from 400 cell embryos now developed into pluteus-like larvae. In both of these experiments small aggregates tend to develop in a more normal manner than larger aggregates.In order to test the role of undissociated cells in promoting pluteus-like development in aggregates from incompletely dissociated blastula stage embryos, pieces of intact animal, lateral, or vegetal blastula wall were grafted to aggregates formed from completely dissociated embryos. While each kind of graft improved the ability of the aggregate to develop in a pluteus-like manner, grafts of vegetal blastula wall were most effective. In an aggregate, a graft differentiates according to its presumptive fate and influences the cells of the aggregate to differentiate in an appropriate manner. The ability of the graft to influence the development of the other cells in the aggregate depends on the developmental stage of the cells that make up the aggregate and the size of the aggregate.     

Hydrodynamic effects on BHK cells grown as suspended natural aggregates

Baby hamster kidney (BHK) cell aggregates grown in stirred vessels with different working volumes and impeller sizes were characterized. Using batch cultures, the range of agitation rates studied (25-100 rpm) led to aggregates with maximum sizes of 150 mum. Necrotic centers were not observed and cell specific productivity was independent of aggregate size. High cell viability was found for both single and adherent cells without an increase in cell death when agitation rate was increased. The increase in agitation rate affected aggregates by reducing their size and increasing their concentration and cell concentration in aggregates, while increasing the fraction of free cells in suspension. The experimental relationship between aggregate size and power dissipation rate per unit of mass was close to -1/4, suggesting a correlation with a critical turbulence microscale; this was independent of vessel scale and impeller geometry over the range investigated. Viscous stresses in the viscous dissipation subrange (below Kolmogoroff eddies) appear to be responsible for aggregate breakage. Under intense agitation BHK cells grown in the absence of microcarriers existed as aggregates without cell damage, whereas cells grown on the surface of microcarriers were largely reduced. This is a clear advantage for scaleup purposes if aggregates are used as a natural immobilization system in stirred vessels. (c) 1995 John Wiley & Sons, Inc.     

Application of imaging tools to plant cell culture: Relationship between plant cell aggregation and flavonoid production

Summary Image analysis tools were developed to measure biomass concentration, aggregate size and distribution, and pigmentation from anthocyanin-producing cell suspension cultures of ohelo (Vaccinium pahalae). The ex situ imaging system could image cell aggregates from 30 μm to 2 mm in diameter. The image analysis algorithm was based on extracted geometric features and morphological methods for biomass volume estimates, and hue, saturation, and intensity color characteristics for pigmentation estimates. Detailed information available from sampled cell culture images was validated by comparison to standard destructive manual measurements. Image analysis measurements revealed that pigment accumulation was negatively correlated with aggregate size. Although a substantial proportion of small aggregates remained colorless, the highly-pigmented small aggregates, 18 to 238 μm in breadth, contributed over 70% of the culture anthocyanin production (mg L⁻¹), despite their minor contribution to the overall biomass. The relative frequency of pigmented aggregates was higher in large-size aggregate classes; however, the pigmented sectors were mostly confined to only the periphery of the aggregates. As a result, large aggregate classes had only a minor contribution to overall culture anthocyanin yield.     

Cultivation of mammalian cells as aggregates in bioreactors: effect of calcium concentration of spatial distribution of viability

Recombinant human kidney epithelial 293 cells were cultivated as aggregates in suspension. The concentration calcium ion, in the range of 100 muM to 1mM, affected the rate of aggregate formation. During the course of cultivation the size distribution of aggregates shifted and the fraction of larger aggregates increased. This effect was more profound in cultures with a high calcium concentration. Scanning and transmission microscopic examination of the aggregates revealed that cell packing was greater in the high calcium cultures and that ultrastructural integrity was retained in aggregates from both low and high calcium cultures. Confocal microscopy was applied to examine the viability of cells in the interior of the aggregates. High viability was observed in the aggregates obtained from exponentially growing cultures. Aggregates from the high calcium culture in the stationary phase exhibited lower viability in the interior. With its ease of retention in a perfusion bioreactor, aggregate cultures offer an alternative choice for large-scale operation. (c) 1993 John Wiley & Sons, Inc.     

Cryopreservation of pluripotent stem cell aggregates in defined protein‐free formulation

Cultivation of undifferentiated pluripotent stem cells (PSCs) as aggregates has emerged as an efficient culture configuration, enabling rapid and controlled large scale expansion. Aggregate‐based PSC cryopreservation facilitates the integrated process of cell expansion and cryopreservation, but its feasibility has not been demonstrated. The goals of current study are to assess the suitability of cryopreserving intact mouse embryonic stem cell (mESC) aggregates and investigate the effects of aggregate size and the formulation of cryopreservation solution on mESC survival and recovery. The results demonstrated the size‐dependent cell survival and recovery of intact aggregates. In particular, the generation of reactive oxygen species (ROS) and caspase activation were reduced for small aggregates (109 ± 55 μm) compared to medium (245 ± 77 μm) and large (365 ± 141 μm) ones, leading to the improved cell recovery. In addition, a defined protein‐free formulation was tested and found to promote the aggregate survival, eliminating the cell exposure to animal serum. The cryopreserved aggregates also maintained the pluripotent markers and the differentiation capacity into three‐germ layers after thawing. In summary, the cryopreservation of small PSC aggregates in a defined protein‐free formulation was shown to be a suitable approach toward a fully integrated expansion and cryopreservation process at large scale. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Characterization of cytoplasmic alpha-synuclein aggregates. Fibril formation is tightly linked to the inclusion-forming process in cells

The alpha-synuclein fibrillation process has been associated with the pathogenesis of several neurodegenerative diseases. Here, we have characterized the cytoplasmic alpha-synuclein aggregates using a fractionation procedure with which different aggregate species can be separated. Overexpression of alpha-synuclein in cells produce two distinct types of aggregates: large juxtanuclear inclusion bodies and small punctate aggregates scattered throughout the cytoplasm. Biochemical fractionation results in an inclusion-enriched fraction and two small aggregate fractions. Electron microscopy and thioflavin S reactivity of the fractions show that the juxtanuclear inclusion bodies are filled with amyloid-like alpha-synuclein fibrils, whereas both the small aggregate fractions contain non-fibrillar spherical aggregates with distinct size distributions. These aggregates appear sequentially, with the smallest population appearing the earliest and the fibrillar inclusions the latest. Based on the structural and kinetic properties, we suggest that the small spherical aggregates are the cellular equivalents of the protofibrils. The proteins that co-exist in the Lewy bodies, such as proteasome subunit, ubiquitin, and hsp70 chaperone, are present in the fibrillar inclusions but absent in the protofibrils, suggesting that these proteins may not be directly involved in the early aggregation stage. As predicted in the aggresome model, disruption of microtubules with nocodazole reduced the number of inclusions and increased the size of the protofibrils. Despite the increased size, the protofibrils remained non-fibrillar, suggesting that the deposition of the protofibrils in the juxtanuclear region is important in fibril formation. This study provides evidence that the cellular fibrillation also involves non-fibrillar intermediate species, and the microtubule-dependent inclusion-forming process is required for the protofibril-to-fibril conversion in cells.     

Aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. for triptolide, wilforgine, and wilforine production

The relationships between aggregate cell types, cell growth, and the triptolide, wilforgine, and wilforine content in aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. were examined. Aggregate cells larger than 2?mm grew quickly and constituted the majority of the white aggregates. The accumulation of triptolide was strongly correlated with the size of the aggregates and the length of the culture period. The aggregates 0.5?C2?mm in diameter accumulated higher triptolide content than those with other sizes throughout the culture. However, the size of the aggregate cells did not significantly affect on the wilforgine and wilforine content. Two other kinds of aggregate cells, the brown and green aggregate cells, also formed in the suspension cultures. The smallest aggregates (0.1?C0.5?mm) had a lower biomass and growth rate and had more chloroplasts and higher alkaloid content. The results of this study can be used to improve the selection process for the mass production of triptolide, wilforgine, and wilforine from cell suspension cultures.     

Botulinum hemagglutinin‐mediated in situ break‐up of human induced pluripotent stem cell aggregates for high‐density suspension culture

Large numbers of human induced pluripotent stem cells (hiPSCs) are required for making stable cell bank. Although suspension culture yields high cell numbers, there remain unresolved challenges for obtaining high‐density of hiPSCs because large size aggregates exhibit low growth rates. Here, we established a simple method for hiPSC aggregate break‐up using botulinum hemagglutinin (HA), which specifically bound with E‐cadherin and disrupted cell–cell connections in hiPSC aggregates. HA showed temporary activity for disrupting the E‐cadherin‐mediated cell–cell connections to facilitate the break‐up of aggregates into small sizes only 9 hr after HA addition. The transportation of HA into the aggregates was mediated by transcellular and paracellular way after HA addition to the culture medium. hiPSC aggregates broken up by HA showed a higher number of live cells, higher cell density, and higher expansion fold compared to those of aggregates dissociated with enzymatic digestion. Moreover, a maximum cell density of 4.5 ± 0.2 × 10⁶ cells ml^?1 was obtained by aggregate break‐up into small ones, which was three times higher than that with the conventional culture without aggregate break‐up. Therefore, the temporary activity of HA for disrupting E‐cadherin‐mediated cell–cell connection was key to establishing a simple in situ method for hiPSC aggregate break‐up in bioreactors, leading to high cell density in suspension culture.     

Mating aggregates in Escherichia coli conjugation.

Mating mixtures of Escherichia coli cells were shown to contain mating aggregates of two to 20 cells each rather than only mating pairs of two cells each. The mating aggregate size distribution shows two broad peaks, at two to four cells and at eight to 13 cells. The quantitative mating aggregate size distribution and the proportion of male cells in mating aggregates are dependent on the input ratio of male to female cells. At an input ratio of one to one, the average mating aggregate contains equal proportions of male and female cells and most of the cells involved in mating are in large aggregates of seven or more cells each. The deoxyribonucleic acid (DNA) transfer efficiency per mating aggregate cell was constant regardless of average aggregate size or of the ratio of male to female cells in the aggregate. Under optimal conditions essentially every male cell or every female cell in a mating aggregate can be involved in DNA transfer. A comparison of light microscopy, sucrose gradient centrifugation, and analysis with a modified Coulter counter indicated that the number of cells in mating aggregates is best equantitated using a modified Coulter counter.     

Characterization of spatial growth and distribution of chondrocyte cells embedded in collagen gels through a stereoscopic cell imaging system

The stereoscopic image analysis of fluorescence-labeled chondrocyte cells for cytoplasm and nucleus was performed for the quantitative determination of spatial cell distribution as well as cell aggregate size in the collagen-embedded culture. The three-dimensional histomorphometric data indicated that the cells in the gels formed aggregates by cell division, and the size of aggregates increased with elapsed culture time. In the culture seeded at 2.0 x 10(6) cells/cm(3), the cells showed a semilunar shape that is a typical chondrocytic morphology, and formed the dense cell aggregates producing collagen type II. From the quantitative analysis of aggregate size, in addition, it was found that the cell division caused the aggregate growth with an increase of cell number in respective aggregates at 7 days, and some of aggregates made coalescence at 14 days. In the gel surface region, further coalescence of aggregates accompanied with cell division produced larger cell clusters, creating cell layers on the gel surface at the end of culture (21 days). In the culture seeded at 2.0 x 10(5) cells/cm(3), the different manner of aggregation was observed. At 14 days, the loose clusters of spindle-shaped cells emerged in the deeper region of gels, suggesting that the cell migration and gathering occurred in the gels. This loose-clustered aggregates did not produce collagen type II. Our results suggest that the seeding density is a factor to cause different mechanisms of cell distribution accompanied with the formation of aggregates as well as collagen type II.     

Serum proteins enhance aggregate formation of dissociated fetal rat brain cells in an aggregating culture

Summary Dissociated fetal rat brain cells (Day 14.5 of gestation) reaggregated into small cell clusters and formed large aggregates in a medium supplemented with serum or dialyzed serum in an aggregating culture. In contrast, only small aggregates were produced in a serum-free medium. The present results indicated that albumin, fetuin, transferrin, and {ie1031-1}-antitrypsin enhanced the aggregate formation. Small aggregates produced in a serum-free medium elongated neurites when they were cultured within a collagen gel matrix. Total DNA per flask was almost the same in small and large aggregates. Thus, these serum proteins may well play an important role in the adhesion of small cell clusters and cause the formation of large aggregates in this short-term aggregating culture.     

Effect of soil aggregate size on methanogenesis and archaeal community structure in anoxic rice field soil

In anoxically incubated slurries of Italian rice field soil, CH(4) production is initiated after a lag phase during which ferric iron and sulfate are reduced. The production of CH(4) was affected by the size of soil aggregates used for the preparation of the soil slurry. Rates of CH(4) production were lowest with small aggregates (<50 and 50-100 μm), were highest with aggregates of 200-2000 μm size and were intermediate with aggregates of 2000-15000 μm size. The different amounts of CH(4) accumulated were positively correlated to the concentrations of acetate, propionate and caproate that transiently accumulated in the slurries prepared from different aggregate sizes and also to the organic carbon content. The addition of organic debris that was collected from large-size aggregates to the aggregate size fractions <200 and <50 μm resulted in an increase of CH(4) production to amounts that were comparable to those measured in unamended aggregates of 200-2000 μm size, indicating that CH(4) production in the different aggregate size fractions was limited by substrate. The distribution of archaeal small-subunit rRNA genes in the different soil aggregate fractions was analyzed by terminal restriction fragment length polymorphism which allowed seven different archaeal ribotypes to be distinguished. Ribotype-182 (consisting of members of the Methanosarcinaceae and rice cluster VI), ribotype-389 (rice cluster I and II) and ribotype-820 (undigested DNA, rice cluster IV and members of the Methanosarcinaceae) accounted for >20, >30 and >10% of the total, respectively. The other ribotypes accounted for <10% of the total. The relative quantity of the individual ribotypes changed only slightly with incubation time and was almost the same among the different soil aggregate fractions. Ribotype-389, for example, slightly decreased with time, whereas ribotype-182 slightly increased. At the end of incubation, the relative quantity of ribotype-182 seemed to be slightly higher in soil fractions with larger than with smaller aggregates, whereas it was the opposite with ribotype-80 (Methanomicrobiaceae) and ribotype-88 (Methanobacteriaceae). Ribotype-280 (Methanosaetaceae and rice cluster V), ribotype-375 (rice cluster III), ribotype-389 and ribotype-820, on the other hand, were not much different among the different soil aggregate size fractions. However, the differences were not significant relative to the errors encountered during the extraction of polymerase chain reaction (PCR)-amplifiable DNA from soil. In conclusion, soil aggregate size and incubation time showed a strong effect on the function but only a small effect on the structure of the methanogenic microbial community.