Separation of Dictyostelium discoideum cells into density classes throughout their development and their relationship to the later cell types

This paper describes a fast, non-destructive method for the separation of large quantities of Dictyostelium discoideum cells into density classes at all stages of development. The cells were separated by low-speed centrifugation on preformed, linear Percoll density gradients. On these gradients, cells at all developmental stages showed a unimodal variation in density and this variation in density rapidly increased during the first hours of development. The density was affected by the amount of salt present in the gradient medium, which suggests that it is regulated by a permeability property of the cells. Slug cells showed a unimodal variation in density and did not form bands corresponding to the cell types. However, were able to isolate density fractions which showed a good enrichment of prespore and prestalk cells: 95% and 90%, respectively. Preaggregation cells separated on density gradients yielded fractions which contained different amounts of three developmentally regulated enzymes. Hence, cells at this stage are already heterogeneous in their enzymatic content. Sorting experiments showed a strong correlation between density and developmental fate; the least dense (light) cells preferentially became prestalk cells, and the dense (heavy) cells became prespore cells. This was found for cells at all developmental stages; even vegetative-stage cells showed considerable heterogeneity with regard to density, which was related to their developmental fate. The light cells become prestalk cells, and the heavy cells become prespore cells. Vegetative cells from the various density fractions differed in their DNA content and temporal onset of mitotic activity when resuspended in medium. Therefore, we suggest that the separation of vegetative cells on density gradients results in a separation of cells into cell-cycle phases. Hence, there appear to be cell-cycle-linked differences among vegetative cells, which bias their differentiation towards either the spore or stalk pathway.     

The reticuloplasmin calreticulun is released into the medium by carrot cell cultures

We report here the presence of a 58-kDa protein in the cells of Daucus carota L. cultivated in vitro. Two lines of carrot cells are used: wild-type line (wt) and mutant line (ts11). We describe here also presence of this protein in the media of cultured cells. Strong reaction of this intracellular and extracellular protein with an anti-calreticulin antiserum indicates that it is a major high capacity, low affinity Ca²⁺-binding reticuloplasmin–calreticulin. No differences in biochemical characterization is found between calreticulin purified from the wild-type line and the mutant line. Moreover molecular mass, type of glycosylation and the ability of extracellular protein to bind calcium is found to be indistinguishable from those of the purified intracellular calreticulin. Calreticulin release is attributed to some stress imposed on cultured cells by growth conditions. It is shown that this process can be also induced in CR-non-releasing systems such as carrot somatic embryos by applying a high-cell-density stress.     

Baculovirus production for gene therapy: the role of cell density,multiplicity of infection and medium exchange

One of the major concerns regarding the use of insect cells and baculovirus expression vectors for the production of recombinant proteins is the drop in production observed when infecting cultures at high cell densities; this work attempts to understand this so-called cell density effect in the scope of baculovirus production for gene therapy purposes. A Spodoptera frugiperda insect cell line (Sf-9) was cultured and infected in serum-free medium, and the patterns of production of a recombinant baculovirus expressing the green fluorescent protein (GFP) were analyzed at different cell concentrations at infection (CCIs) and multiplicities of infection (MOIs). The results confirm that a cell density effect on productivity occurs which is dependent on the MOI used, with a high MOI “delaying” the drop in production to higher cell densities. Medium replacement at the time of infection using a high MOI considerably improved baculovirus production, with the different production indicators, namely the titer, specific yield, amplification factor, and time of harvesting, increasing with cell concentration for the CCI range tested. Virus titers as high as 2.6 × 10¹⁰ IP.mL⁻¹ were obtained in cultures infected at 3.5 × 10⁶ cells.mL⁻¹, while the amplification factor was roughly 19 times higher than the highest value obtained without medium exchange.     

Relationship between cytoagglutination and saturation density of cell growth

The agglutination with concanavalin A and wheat germ agglutinin of the established malignant cells, HEp 2, KB, HeLa, TDB-3, HTC and RV 3T3, and of the putatively normal cells, BHK 21, 3T3 and Wi-38 was examined as a function of their saturation densities in culture. A positive correlation between the saturation density of the cell lines and the capacity to agglutinate was demonstrated. Incubation for 15 minutes with 1.25 mg/ml of trypsin converted non-agglutinating and poorly agglutinating cells into agglutinable ones, while leaving the highly agglutinating lines largely unchanged. The magnitude of change in agglutination after trypsin treatment correlated inversely with saturation density. Although the extent of agglutination varied with the saturation density, the agglutinability of a particular line remained relatively unchanged at different cell densities.     

The use of sister-chromatid exchange in Chinese hamster primary lung cell cultures to measure genotoxicity

Primary cell cultures derived from Chinese hamster lung (CHL) were established, and their response for the induction of sister-chromatid exchange (SCE) by direct- and indirect-acting mutagens was characterized. An increase in SCE frequency was induced in CHL cells by 3-methylcholanthrene (MCA), benzo[a]pyrene (BaP), and 2-aminoanthracene (2AA). The SCE frequency increased slightly after exposure to cyclophosphamide, but did not respond to the hepatocarcinogen dimethylnitrosamine (DMN). A slight increase in SCE frequency by DMN was observed in the CHL system with use of Aroclor-1254-induced rat liver homogenate fraction (S9). This response to DMN in CHL cells was lower than that seen when CHO cells were the target in the presence of S9. At low (1) and high (20) passages, the CHL cells responded with a similar dose-related increase in SCE frequency to direct- (ethyl methanesulfonate, EMS) and indirect- (MCA) acting mutagens. This response indicates that even after prolonged culturing in vitro, the cells retained the ability to metabolically activate xenobiotic promutagens. The induction of SCE by MCA occurred at concentrations that also induced macromolecular binding. SCE induction was also examined in primary lung cell cultures from animals exposed by nose-only inhalation to MCA aerosol. A significant increase in SCE frequency above controls was observed in cells from animals after a single exposure to MCA. No detectable increase in SCE frequency was observed after repeated inhalation exposures. Because CHL cells are of lung origin and showed metabolic activity, the CHL system appears to be appropriate for study of the genotoxic potential of inhaled compounds.     

Growth responses of cell cultures in a galactose medium

Under specific conditions utilized, Eagle's minimum essential medium containing “substantially glucose-free” galactose instead of glucose supported the growth of every cell culture tested with the exception of embryonic cells. Growth of various primary kidney cultures in galactose-EMEM was greater or, at least, equal to that obtained with the same medium containing glucose. Cell lines of non-human origin showed extensive growth in galactose-EMEM and were further stimulated by supplemental pyruvate. Only limited, if any, growth of human cell lines occurred in galactose-EMEM under routine conditions of culture. The growth response of these cells was greatly increased and approached that with glucose if the initial pH of the galactose medium was adjusted in the acid pH range or, with WISH cells, if the galactose medium was supplemented with pyruvate.     

A serum-free medium that supports the growth of piscine cell cultures

Summary An undefined, serum-free medium was developed for use with fish cell cultures. Lactalbumin hydrolyzate, trypticase-soy broth, Bacto-peptone, dextrose, yeastolate, and polyvinylpyrrolidone were initially combined in 100 ml of distilled H₂O, autoclaved, and added to 5% of the final volume of Medium 199. In addition, filter sterilized bovine pancreatic insulin, glutamine, and nonessential amino acids were added to the medium. The addition of insulin was observed to be unnecessary. Five fish cell lines [goldfish-derived CAR cells, fathead minnow (FHM) cells, epithelioma papillosum cyprini (EPC) cells, chinook salmon embryo (CHSE-214) cells, and a new cell line from goldfish air bladders (ABIII)] were all capable of growth in the serum-free medium at rates equivalent to cells grown in fetal bovine serum (FBS). The morphology of all cell lines, except CHSE-214 cells, was identical to cells grown in FBS. All cell lines were capable of long-term growth in the serum-free medium. The CAR, ABIII, EPC, and CHSE-214 cells in the serum-free medium supported the replication of goldfish virus-2 at levels equivalent to cells grown in FBS.     

Increase of latent HMG-CoA reductase activity with increasing density of cell cultures

The total (active latent) activity of HMG-CoA reductase declined linearly with increasing cell density in cultures of three lines of mammalian cells. The active form disappeared almost entirely under this condition, while the latent (presumably phosphorylated) form increased to some extent. The disappearance of active HMG-CoA reductase with concomitant increase in the proportion of latent HMG-CoA reductase was correlated with the decline in cellular multiplication and sterol synthesis. These results suggest that interconversion of HMG-CoA reductase between active and inactive forms through phosphorylation-dephosphorylation can be associated with changes in the rate of cellular proliferation in cell cultures. However, the decreased rate of sterol synthesis followed more closely the slower disappearance of the total HMG-CoA reductase activity than the rapid decrease of the active form of the reductase alone. Therefore, changes in the rate of cellular proliferation can affect the interconversion of HMG-CoA reductase between active and inactive forms through reversible phosphorylation. However, phosphorylation of the enzyme to the inactive form appears not to be the mechanism by which the sterol synthetic rate is regulated in confluent cell cultures. Rather, the amount of total HMG-CoA reductase determines the rate of sterol synthesis.     

On-line control of feeding of medium components to attain high cell density

On line control coupled with an expert system was constructed for the control of a fed-batch culture with the aim of achieving a high cell density. During the cultivation, the expert system could monitor the extents of sufficiencies in the amounts of chemical elements in the medium every 10 min, and suggest a modification to the feeding control policy if the amount of a particular component was inadequate for cell growth. However, we often encounter such a kind of cultivations in which some particular carbon source such as glucose and ethanol should be controlled at low concentration in many culture processes, because the excess feeding might cause the growth inhibition by its own accumulation in the culture broth, or the biproduct accumulation like as lactate or acetate, unsuitable substances for smoothed growth. In this study, we developed an online control system based on production rules which managed the glucose feed rate from DO signal. The online control was carried out by the control computer connected with the other computer for expert system, because a relatively long time (several minutes) was needed for the inference of the expert system and the influence of the starvation of carbon source on the cell growth is not negligible even in several minutes. The on-line control system with expert system was applied to the production of cell mass of E. coli W3110 and the final cell concentration reached 137 g-dry cell weight/l.     

Optimization of medium with perfusion microbioreactors for high density CHO cell cultures at very low renewal rate aided by design of experiments

A novel approach of design of experiment (DoE) is developed for the optimization of key substrates of the culture medium, amino acids, and sugars, by utilizing perfusion microbioreactors with 2 mL working volume, operated in high cell density continuous mode, to explore the design space. A mixture DoE based on a simplex-centroid is proposed to test multiple medium blends in parallel perfusion runs, where the amino acids concentrations are selected based on the culture behavior in presence of different amino acid mixtures, and using targeted specific consumption rates. An optimized medium is identified with models predicting the culture parameters and product quality attributes (G0 and G1 level N-glycans) as a function of the medium composition. It is then validated in runs performed in perfusion microbioreactor in comparison with stirred-tank bioreactors equipped with alternating tangential flow filtration (ATF) or with tangential flow filtration (TFF) for cell separation, showing overall a similar process performance and N-glycosylation profile of the produced antibody. These results demonstrate that the present development strategy generates a perfusion medium with optimized performance for stable Chinese hamster ovary (CHO) cell cultures operated with very high cell densities of 60 × 10⁶ and 120 × 10⁶ cells/mL and a low cell-specific perfusion rate of 17 pL/cell/day, which is among the lowest reported and is in line with the framework recently published by the industry.     

Commensal bacteria modulate innate immune responses of vaginal epithelial cell multilayer cultures

The human vaginal microbiome plays a critical but poorly defined role in reproductive health. Vaginal microbiome alterations are associated with increased susceptibility to sexually-transmitted infections (STI) possibly due to related changes in innate defense responses from epithelial cells. Study of the impact of commensal bacteria on the vaginal mucosal surface has been hindered by current vaginal epithelial cell (VEC) culture systems that lack an appropriate interface between the apical surface of stratified squamous epithelium and the air-filled vaginal lumen. Therefore we developed a reproducible multilayer VEC culture system with an apical (luminal) air-interface that supported colonization with selected commensal bacteria. Multilayer VEC developed tight-junctions and other hallmarks of the vaginal mucosa including predictable proinflammatory cytokine secretion following TLR stimulation. Colonization of multilayers by common vaginal commensals including Lactobacillus crispatus, L. jensenii, and L. rhamnosus led to intimate associations with the VEC exclusively on the apical surface. Vaginal commensals did not trigger cytokine secretion but Staphylococcus epidermidis, a skin commensal, was inflammatory. Lactobacilli reduced cytokine secretion in an isolate-specific fashion following TLR stimulation. This tempering of inflammation offers a potential explanation for increased susceptibility to STI in the absence of common commensals and has implications for testing of potential STI preventatives.     

Decrease of saturation density of cells of hamster cell lines after treatment with dextran sulfate

Dextran sulfates of various molecular weights were added to cultures of 3 transformed cell lines of hamster, 3T6 cells and embryonic fibroblastic cells. Dextran sulfate of high molecular weight reduced the saturation densities of all the cell lines of hamster and 3T6 cells, but those of low molecular weight did not. The mitotic rate of the treated cells decreased at stationary cell density. Dextran sulfate had no effect on the growth of normal fibroblastic cells derived from mouse and hamster embryos. Viability of treated cells was indicated by the following results. Cells of cultures seeded at different cell densities grew at almost the same rate in the presence of dextran sulfate. Treated cells remaining in the monolayer stage began to grow after removal of dextran sulfate. The colony formation rate of treated cells was the same as that of untreated cells. With the exception of one cell line, the morphology of cells treated with dextran sulfate of high molecular weight was more flattened and there was less overlapping than in untreated cells. Treated cells were less agglutinable to concanavalin A than were untreated cells. These results suggest that dextran sulfate affects the cell surface, resulting in the decrease of saturation density of cell lines.     

The effect of transferrin saturation on internal iron exchange

Radioiron was introduced into the intestinal lumen to evaluate absorption, injected as nonviable red cells to evaluate reticuloendothelial (RE) processing of iron, and injected as hemoglobin to evaluate hepatocyte iron processing. Redistribution of iron through the plasma was evaluated in control animals and animals whose transferrin was saturated by iron infusion. Radioiron introduced into the lumen of the gut as ferrous sulfate and as transferrin-bound iron was absorbed about half as well in iron-infused animals, and absorbed iron was localized in the liver. The similar absorption of transferrin-bound iron suggested that absorption of ferrous iron occurred via the mucosal cell and did not enter by diffusion. The decrease in absorption was associated with an increase in mucosal iron and ferritin content produced by the iron infusion. An inverse relationship (r = -0.895) was shown between mucosal ferritin iron and absorption. When iron was injected as nonviable red cells, it was deposited predominantly in reticuloendothelial cells of the spleen. Return of this radioiron to the plasma was only 6% of that in control animals. While there was some movement of iron from spleen to liver, this could be accounted for by intravascular hemolysis. Injected hemoglobin tagged with radioiron was for the most part taken up and held by the liver. Some 13% initially localized in the marrow in iron-infused animals was shown to be storage iron unavailable for hemoglobin synthesis. These studies demonstrate the hepatic trapping of absorbed iron and the inability of either RE cell or hepatocyte to release iron in the transferrin-saturated animal.     

The capacity of anaerobically grown bacteria to exchange the 2H+ of the cell for the K+ of the medium and to maintain a high K+ distribution between cell and medium

The N,N'-dicyclohexylcarbodiimide sensitive exchange of 2H+ of a cell for K+ of medium stable to pH, K+ activity and temperature changes has been discovered in anaerobically grown gram-negative Escherichia coli, Salmonella typhimurium. S. enteritidis, Proteus mirabilis, P. vulgaris, anaerobic gram-positive bacteria Streptococcus faecalis, Lactobacillus salivarius, L. lactis in the presence of exogenic energy source. This exchange in gram-negative bacteria is operating only at increase of medium osmolarity. The high K+ distribution between cell and medium has been reached during the exchange of 2H+ for one K+ and the corresponding potassium equilibrium potential is much more than the measured delta psi. In aerobically grown E. coli, S. typhimurium, Brevibacterium flavum and aerobic Micrococcus luteus exchange of 2H+ for K+ does not take place, the K+ distribution is lower and in good conformity with the measured delta psi. It is assumed that exchange of 2H+ for K+ in anaerobic bacteria is carried out by the H+-ATPase complex and the Trk (or Trk-like) system of K+ absorption united into the same membrane supercomplex which functions as the H+-K+-pump and supports the high K+ distribution between cell and medium.     

Improved production of ginseng polysaccharide by adding conditioned medium to Panax notoginseng cell cultures

By adding 50% (v/v) filtered culture broth to fresh MS medium, the specific growth rate of Panax notoginseng was increased from 0.046 d–1 to 0.068 d–1, and the polysaccharide production and productivity reached 1.21 g l–1 and 61 mg/(ld), respectively, which were 1.3- and 2.3-fold of the control. Further supplementation of the conditioned medium with sucrose, ammonium, nitrate and phosphate gave a cell density of 13.7 g l–1 and a specific growth rate of 0.086 d–1. Polysaccharide production was 1.65 g l–1 and the productivity was 78 mg/(ld).     

The cytogenetic effect of an X-ray contrast medium in Chinese hamster cell cultures.

Chinese hamster cell cultures were treated with different concentrations of Joduron, a water-soluble iodized X-ray contrast medium. The cytogenetic effect was analysed for a treatment period of 2 or 16 h. Jorudron primarily acts on the mitotic apparatus of the cell. The type of damage ranges from different forms of initial "C-mitosis" to unpolarized stathmokinesis. Additionally, structural chromosome damage, mainly of the chromatid type, was observed, especially after a Joduron treatment for 2 h.