Cell Cycle Progression in Serum-Free Cultures of Sf9 Insect Cells: Modulation by Conditioned Medium Factors and Implications for Proliferation and Productivity (original) (raw)
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Kinetic data for the BM-5 insect cell line in repeated-batch suspension cultures
Biotechnology and Bioengineering, 1991
The kinetics and growth characteristics of the BM-5 insect cell line of Bombyx mori (silkworm) have been experimentally investigated in order to develop optimal growth protocols when these cells are used to produce large quantities of human proteins by recombinant baculoviruses. Experiments were performed in 200-mL spinner flasks, which were operated at 80 rpm with 0.3% methyl cellulose (MCL) added to the medium to protect the cells from shear stress. Exposure of the cells to varying nutrient and metabolite concentrations was accomplished through a repeated-batch mode of feeding. The results indicate that glutamine is a limiting nutrient and that lactate has an inhibitory effect on cell growth. Ammonia depletion from the medium was accompanied by uric acid accumulation suggesting that ammonia is converted to this metabolic product by the "uricotelic" and "nucleicolytic" metabolic pathways. With the examined medium formulation, glucose, fructose, and sucrose remained at high concentrations throughout the cultivation period.
Cells, whether prokaryotic or eukaryotic, eventually reproduce or die. For prokaryotes, the mechanism of reproduction is relatively simple, since there are no internal organ-elles. The process consists of three distinct but short phases: first, a growth phase in which the mass of the cell is increased, then the chromosomal replication phase, and finally the chromosomes are separated and the cells are physically split into two independent new cells. In bacteria, these are referred to as the B, C, and D periods, respectively. Initiation of the reproductive process appears to be primarily a function of cell size. The length of the overall cell cycle is determined by the B period, as the C and D periods have relatively fixed time constraints. The length of B is determined, in part, by environmental conditions and the gain in cell mass. Generation times for bacteria can vary from under half an hour to several days, although most bacterial cultures in laboratory settings and nutrient-rich media have generation times under a day. DNA replication has already been covered in detail in chapter 7. In bacteria, the process is initiated at the origin of replication by DnaA. However, in archaea, synchronous initiation of replication at multiple sites on the chromosome as well as recognition proteins homologous to eukaryotic ORC proteins suggests that there are similarities between archaebacterial and eukaryotic DNA replication to be explored. Once the DNA is replicated and moved to opposite sides of the cell, the midcell septum forms to split the cell. At least 9 gene products are involved in this process including FtsZ, the prokaryotic tubulin homologue that forms a circumferential ring, FtsI, a pepti-doglycan synthetase involved in septum formation, FtsL, whose function is unclear but is involved in ingrowth of the cell wall at the septum, and ZipA, which anchors the FtsZ ring. The ring contracts, pulling the membrane in with it. Eventually the membrane is pinched in enough to fuse and generate two completely separate cytoplasmic compartments. Other septation enzymes make cell wall components that fill in as the septum forms simultaneously with membrane/FtsZ contraction, and the cells separate. Cell Cycle : Life cycle of the cell and Gametogenesis Using this book: This book is designed to be used in both introductory and advanced cell biology courses. The primary text is generally on the left side of the vertical divider, and printed in black. Details that are usually left to an advanced course are printed in blue and found on the right side of the divider. Finally, additional biomedically relevant information can be found in red print on either side of the divider.
Biotechnology Progress, 2000
On-line monitoring of insect cell cultures used for the production of recombinant proteins with the baculovirus expression vector system (BEVS) provides valuable tools for the optimization, operation, and control of the production process. The relative permittivity ( ′) and CO 2 evolution rates (CER) were measured on-line using the biomass monitor and the infrared CO 2 analyzer, respectively. The growth and infection phases of two different cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni (High-5), were monitored using the above measurements. These in turn were correlated to the progress of the culture by using the off-line measurements of protein produced, virus titer, and biovolume, which is the product of viable cell density and mean cell volume. The ′, CER, and the biovolume profiles were closely matched during the growth phase of cells when grown in a batch or fed batch culture. The relationship became more complex when the cultures were either in stationary phase or in the postinfection phase. The ′ profile was found to be a good indicator of the process of synchronous baculoviral infection, showing a plateau between 18 and 24 h postinfection (hpi), the period during which budded virus is produced, and a peak at approximately 48 hpi correlated to the onset of accelerated cell lysis. The CER profile continues to increase after the growth period with a peak around the 24 hpi period, after which there is a decline in the profile corresponding to release of virus as seen from virus titer determinations. This was examined for Sf-9 cultures under conditions of cell densities from 3 to 50 × 10 6 cells/mL and MOI values ranging from 0.001 to 1000. The profiles were found to be similar also in the case of the High-5 cells. Thus both measurements give reliable information regarding the physiological status of the cells as seen from their correlation to virus and protein production. A further combination of these with the off-line measured parameters such as the biovolume and metabolite concentrations can give a more detailed understanding of the process and help in the better design and automation of these processes.
Correlation between cell nutrition, cell size and division control. Part II
Biosystems, 1979
The conventional concept of division control assumes that a certain sequence of biochemical events throughout the cell cycle results in the generation of some specific mitotic trigger. An alternative approach has been examined by us on Tetrahymena pyriformis, namely, that division is started without any specific starter but by deficiency in the cell pool of nutrients. This deficiency, in its turn, is caused by various space disproportions accompanying cell growth. In accordance wi~;h the hypothesis under examination, a prompt shift-down in cell nutrition has induced a wave of division in an asynchronous culture of Tetrabymena; a shift-up in nutrition has resulted in delay of division and in a stepwise increase of mean volume of dividing cells.
Correlation between cell nutrition, cell size and division control. Part I
Bio Systems, 1979
The conventional concept of division control assumes that a certain sequence of biochemical events throughout the cell cycle results in the generation of some specific mitotic trigger. An alternative approach has been examined by us on Tetrahymena pyriformis, namely, that division is started without any specific starter but by deficiency in the cell pool of nutrients. This deficiency, in its turn, is caused by various space disproportions accompanying cell growth. In accordance wi~;h the hypothesis under examination, a prompt shift-down in cell nutrition has induced a wave of division in an asynchronous culture of Tetrabymena; a shift-up in nutrition has resulted in delay of division and in a stepwise increase of mean volume of dividing cells.
Biotechnology and Bioengineering, 2009
The cell density effect (i.e., the drop in the specific productivity in the baculovirus-insect cells expression system when cells are infected at high cell densities) has been extensively described in the literature. In this article, a model for the central metabolism of serum-free suspension cultures of Spodoptera frugiperda Sf9 cells is proposed and used to investigate the metabolic basis for this phenomenon. The main metabolic pathways (glycolysis, pentose phosphate pathway, tricarboxylic acids cycle, glutaminolysis, and amino acids metabolism), cellular growth and energetics were considered. The analysis of the stoichiometric model allowed further understanding of the interplay of the consumption of carbon and nitrogen sources in insect cells. Moreover, metabolic flux analysis revealed that Sf9 cells undergo a progressive inhibition of central metabolism when grown to high cell densities, for which the incorporation of amino acids carbon backbones into the TCA cycle (mainly glutamine) and the down-regulation of glycolysis are partially responsible. Following infection by baculovirus and cellular division arrest, central energy metabolism depended on the infection strategy chosen (cell concentration at the moment of infection and multiplicity of infection), inhibition being observed at high cell densities. Interestingly, the energetic status of the culture correlated with the decrease in cellular production of baculovirus, meaning that there is room for process optimization through the application of metabolic engineering techniques.
The rate of cell growth is governed by cell cycle stage
Genes & Development, 2009
Cell growth is an essential requirement for cell cycle progression. While it is often held that growth is independent of cell cycle position, this relationship has not been closely scrutinized. Here we show that in budding yeast, the ability of cells to grow changes during the cell cycle. We find that cell growth is faster in cells arrested in anaphase and G1 than in other cell cycle stages. We demonstrate that the establishment of a polarized actin cytoskeleton-either as a consequence of normal cell division or through activation of the mating pheromone response-potently attenuates protein synthesis and growth. We furthermore show by population and single-cell analysis that growth varies during an unperturbed cell cycle, slowing at the time of polarized growth. Our study uncovers a fundamental relationship whereby cell cycle position regulates growth.