Optimization of reovirus production from mouse L-929 cells in suspension culture

Reovirus serotype 3 Dearing (T3D) has shown potential as a novel cancer therapy. To support the increasing demand for reovirus, a two-stage perfusion mode scheme is proposed for cell growth and reovirus production. Mouse L-929 cells were used as the host for reovirus infection due to their ability to grow well in suspension culture. Several L-929 cell growth and reovirus infection characteristics were investigated and optimized in spinner flask batch cultures. For the growth of L-929 cells, a balanced nutrient-fortification of SMEM medium increased the maximum cell density by 30%, compared to normal SMEM; however, ammonia and lactate accumulations were found to inhibit further cell growth. For the production of reovirus, approximately 90% increase in viral yield resulted when the infection temperature was reduced from 37 to 33 degrees C. Infectious reovirus particles were shown to be stable in conditioned medium at 37 and 33 degrees C. The final virus titer was dependent on the multiplicity of infection (MOI) and the host cell density at the time of infection. A combination of an MOI of 0.1 pfu/cell and an initial host cell density of 1.0 x 10(6) cells/mL in fortified medium resulted in a maximum virus titer of (4.59 +/- 0.16) x 10(9) pfu/mL and a specific yield of (2.34 +/- 0.08) x 10(3) pfu/cell. At an optimal harvest time of the infection process, 99% of the virus was associated with the cellular debris. Finally, the presence of 5.0 mM ammonia in the culture medium was shown to seriously inhibit the reovirus yield, whereas lactate concentrations up to 20 mM had no effect.     

Respiration of mengovirus-infected L-929 cells

Iglewski, W. J. (The Pennsylvania State University, University Park), and E. H. Ludwig. Respiration of mengovirus-infected L-929 cells. J. Bacteriol. 92:733-738. 1966.-Polarographic techniques were employed to study the oxidative metabolism of L-929 cells during a one-step mengovirus growth cycle. Virus maturation began 3.5 hr after infection and was complete with 7 hr. Virus maturation was accompanied by a decreased rate of endogenous respiration and an increased rate of oxidation of succinate and alpha-glycerophosphate by L-929 cells. The rate of glucose uptake was the same for mengovirus-infected and control L-929 cells. However, there was a decreased oxidation of glucose to carbon dioxide and a decreased production of lactic acid by L cells infected with mengovirus under aerobic conditions. Mengovirus was produced equally well under aerobic and anaerobic conditions. The implications of the alterations in metabolism with respect to virus synthesis are discussed.     

Anti-steroid action in cultured L-929 mouse fibroblasts

L-929 mouse fibroblast growth is arrested by the glucocorticosteroid dexamethasone (dex), which also decreases adhesiveness to culture plates. Both dex effects were abolished when RU 486, a new synthetic anti-hormonal steroid, was added to the culture medium. Using [3H]RU 486, binding studies revealed that RU 486 bound approximately 25,000 sites/cell of the glucocorticosteroid receptor (GR) with affinity higher than that of dex and translocated GR to the nucleus. However, the distribution of steroid-receptor complexes between cytosol and nuclei was different for the agonist and the antagonist, with more nuclear accumulation in the case of dex. Estradiol increases L-929 cell growth and adhesiveness to culture plates, but not if the anti-estrogen tamoxifen (tam) was added. These observations initially made in serum containing medium, were confirmed in serum-free, chemically defined culture medium (SF). In SF medium, tam (1 microM) provoked death of most L-929 cells after 10 days of culture, leading to the selection of a variant clone LB of tam-resistant cells. Tam binds to the estrogen receptor (ER), but with less affinity than estradiol. ER concentration, estimated by the binding of 4-hydroxytamoxifen (OH-tam) and of estradiol was lower in LB cells than in original tam-sensitive L-929 cells. The concentration of specific anti-estrogen binding sites in the particulate fraction of the cells, measured by OH-tam binding, non competed by estradiol, was also significantly diminished in tam-resistant LB cells.     

Glycolysis in permeabilized L-929 cells.

Mouse L-929 cells permeabilized with dextran sulphate (DSP cells) carry out glycolysis when supplemented with glucose, ATP and NAD+ in a suitable incubation buffer. Glycolytic rates were linear and generally independent of cell density over the range examined (1 x 10(6)-10 x 10(6) cells/ml). Electron microscopy revealed characteristic changes in DSP cell ultrastructure, notably for nuclei and mitochondria. Some cells lacked plasma membranes, while others appeared intact. In the latter case, estimates of the lesion size in plasma membranes were obtained from volume of distribution studies using 14C-labelled proteins, and infiltration of fluorescein isothiocyanate dextran. The results indicated the presence of lesions large enough to allow globular proteins of about 400 kDa to cross the cell surface. In spite of that, only about 10% of total cell protein exited from DSP cells during a 30 min incubation period. We propose that none of the glycolytic enzymes in DSP cells can exist completely in solution in the 'cytosol', suggesting extensive enzyme organization. The results are interpreted within the broader picture of metabolic organization in animal cells and the nature of the 'cytosol'.     

L-929 cells under hyperosmotic conditions

Changes in cell water content resulting from sorbitol addition to the environment of L-929 cells were evaluated gravimetrically using¹⁴C-labeled polyethylene glycol as a probe of extracellular space. Reductions in cell water were proportional to sorbitol supplements up to 0.6 molal, above which no further measurable decrease occurred. No volume regulation occurred for at least 1 h but the percentage of cell water lost was quickly regained when physiological conditions were restored. The amount of cell water lost because of a given hyperosmotic exposure was found to exceed the loss of cell volume. That discrepancy could be the result of an overestimation of extracellular space and/or an underestimation of cell volume reduction as a result of infolding of the cell surface. Na⁺ and K⁺ were also measured in cells of variable water content and volume: no significant change occurred in the amounts of these ions per cell, but large increases in total cell concentration resulted from hyperosmotic exposure. The sum of Na⁺ and K⁺ concentrations exceeds the total osmotic pressure of the medium indicating that an appreciable fraction of Na⁺ and K⁺ must be bound to fixed charges within the cells. The results are evaluated in the context of intracellular organization.     

The establishment and characterization of mouse L-929 cells in protein-free Eagle's Minimal Essential Medium

The mouse cell line L-929 was established in protein-free Eagle's Minimal Essential Medium. The cells have been 'adapted' to continuous growth in the medium using stepwise reductions in the concentration of fetal bovine serum. The cells designated L-929-WS have now been propagated in protein-free Eagle's Minimal Essential Medium for two years. The population-doubling time was about 37 h. The addition of serum stimulated cell growth only slightly, but the saturation density was significantly increased. Morphological examination, a study of the secretion of colony stimulating activity and cytochemical investigations for acid phosphatase and alkaline phosphatase showed that L-929-WS cells, grown in protein-free Eagle's Minimal Essential Medium, did not differ markedly from cells propagated in medium containing serum. The cells provided a simple model for the study of cell growth in the absence of serum or the other macromolecular substances usually added to cell cultures. The general application of the cells for purposes in which the addition of serum or growth factors might interfere, is suggested.     

Characterization of the lysosomal cystine transport system in mouse L-929 fibroblasts

We characterize here a lysosomal cystine transporter in mouse L-929 fibroblasts. Granular fractions from cells preloaded with cystine demonstrated countertransport that showed no dependence on Na+ or K+. The Michaelis constant for infinite-trans influx was 0.27 +/- 0.06 mM (n = 3), and a nonsaturable component of cystine entry was observed with Kd = 0.8-1.8 nmol of cystine.min-1.unit of hexosaminidase-1.mM-1. We found no evidence that cystine was also carried on any of the other known lysosomal amino acid transporters. Over 50 analogs were tested for their ability to inhibit countertransport. The inhibition constants are reported for selenocystine, cystathionine, selenomethionine, and leucine. Significant requirements for recognition by the transporter were the presence of amino groups, L configuration, and a chain length not greater than eight atoms. A net positive or negative charge was not required. Some di- as well as tetrapolar amino acids were recognized. We have surmised that the binding site has polar and apolar domains, the latter being large enough to accommodate branching on C-3 and the substitution of selenium or carbon in place of sulfur.     

Dexamethasone induces gelsolin synthesis and altered morphology in L929 cells

When L929 cells are exposed to 5 μg/ml dexamethasone, synthesis of a 90,000 M(r) polypeptide is induced within 12 h. Flattening of the cells begins at about this time and progresses to become quite prominent after 48 h of exposure. Two-dimensional PAGE and partial proteolytic fingerprints identify the 90,000 M(r) polypeptide as gelsolin, a Ca(++)-dependent inhibitor of actin polymerization. Thus, this system provides evidence that gelsolin may have a role in regulating cell shape in response to physiological agents such as glucocorticoids.     

Dynamics of spreading of cells of L-929 line after the mitosis

Using time-lapse microscopy, the changes in L-929 cells shape were analyzed during a cell cycle. During this time the cells were established to pass through three spreading stages. The highest rate of the cell spreading was observed during the first 1.5 h of mitosis. In this period, the cell area increases approximately 3-3.5 times following sigmoid dependence. After a short plateau the augmentation of the cell area starts also as a sigmoid dependence. This period is longer (up to 6 h after the beginning of cell division) with an additional 1.5-fold augmentation of the cells size. Next, the augmentation of the cells area goes linearly up to the beginning of the following mitosis. After the mother L-929 cell division, the daughter cells remained to be bridged together in the fission furrow site almost in 100% cases. The structure known as an intercellular bridge is related to a late telophase. In this connected state the L-cells are spreading and migrating up to 2.13 +/- 0.06 h where upon they are separated. Transition of the daughter cells from a round shape to the spread one occurring with the simultaneous maintenance of the intercellular bridge during a strictly determined time allows us to consider this phenomenon as independent and not relating to mitosis. We suggest naming this junction between the daughter cells as the "posttelophase intercellular bridge".     

Evidence for intermediate channelling in the glycolytic pathway of permeabilized L-929 cells

L-929 cells permeabilized by dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Unlabeled 3-phosphoglycerate (PGA) added to DSP cells reduces the specific activity of lactate coming from [14C]glucose but the extent of this reduction can not be accounted for on the basis of free diffusion of PGA coming from [14C]glucose. Studies on other glycolytic intermediates, although preliminary, yield similar results. PGA also inhibits the production of lactate from glucose; however, this effect, like that of the reduction of lactate specific activity, becomes apparent only at concentrations of PGA well in excess of those considered to be physiological. We conclude that channelling of PGA, and probably other intermediates, occurs but is of the "leaky" type.     

Glucose metabolism and the channeling of glycolytic intermediates in permeabilized L-929 cells

L-929 cells (mouse fibroblasts) permeabilized with dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Glycolysis in DSP cells is pH dependent, being strongly inhibited at pH 6.5. Compared to their nonpermeabilized counterparts, DSP cells exhibit faster glycolytic rates, but tend to convert a smaller proportion of the glucose utilized to lactate. [14C]Glucose is converted to lactate by DSP cells without dilution from endogenous substrates. When exogenous 12C-labeled glycolytic intermediates (12C-I) are added to glycolyzing DSP cells the [14C]lactate produced from [14C]glucose is diluted to varying extents, depending on the intermediate. However, the extent of that dilution (reduced specific activity) is not that expected from the complete mixing of exogenous 12C-I with their corresponding 14C-labeled intermediates coming from [14C]-glucose. DSP cells also respire and convert glucose to CO2. The amount of 14CO2 produced from [14C]glucose is also reduced by addition of most 12C-I, an interesting exception being pyruvate, which had no measurable effect on 14CO2 production and caused only a modest stimulation of respiration in glycolyzing DSP cells. These results suggest that channeling, or some other form of coupling, takes place between the glycolytic production of pyruvate and its further oxidation. These observations confirm previously published data and add further support to the proposition that channeling of glycolytic intermediates occurs in DSP cells but is of the "leaky" type. Although abundant evidence in the literature indicates that various glycolytic enzymes associate with F-actin, as well as other elements of the cytomatrix, we observed no effect of cytochalasin D on lactate production even at very high concentrations of this compound. Our results are compared with those from other laboratories and discussed in the context of metabolic organization.