Final Cell Density Research Articles

Cell retention by encapsulation for the cultivation of Jurkat cells in fixed and fluidized bed reactors.

Jurkat cells are accepted model cells for primary human T lymphocytes, for example, in medical research. Their growth to tissue-like cell densities (up to 100 × 10(6) cells/mLcapsule ) in semi-permeable (molecular weight cut off <10,000 Da) sodium cellulose sulfate/poly(diallyldimethylammonium chloride) polyelectrolyte capsules has previously been shown by us [Werner et al. (2013). Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells. Biotechnol Prog 29(4): 986-993]. Herein, we demonstrate that encapsulation can be used to retain the cells in continuously operated bioreactors, which opens new possibilities for research, for example, the use of Jurkat cells in pulse response experiments under steady state conditions. Two reactor concepts are presented, a fluidized and a fixed bed reactor. A direct comparison of the growth kinetics in batch and repeated batch spinner cultivations, that is, under conditions where both encapsulated and non-encapsulated cells can be cultivated under otherwise identical conditions, showed that maximum specific growth rates were higher for the encapsulated than for the non-encapsulated cells. In the subsequent batch and repeated batch bioreactor experiments (only encapsulated cells), growth rates were similar, with the exception of the fixed bed batch reactor, where growth kinetics were significantly slower. Concomitantly, a significant fraction of the cells towards the bottom of the bed were no longer metabolically active, though apparently not dead. In the repeated batch fluidized bed reactor cellular division could be maintained for more than two weeks, albeit with a specific growth rate below the maximum one, leading to final cell densities of approximately 180 × 10(6) cell/gcapsule . At the same time, the cell cycle distribution of the cells was shifted to the S and G2/M phases.

Production of the short peptide surfactant DAMP4 from glucose or sucrose in high cell density cultures of Escherichia coli BL21(DE3).

BackgroundPeptides are increasingly used in industry as highly functional materials. Bacterial production of recombinant peptides has the potential to provide large amounts of renewable and low cost peptides, however, achieving high product titers from Chemically Defined Media (CDM) supplemented with simple sugars remains challenging.ResultsIn this work, the short peptide surfactant, DAMP4, was used as a model peptide to investigate production in Escherichia coli BL21(DE3), a classical strain used for protein production. Under the same fermentation conditions, switching production of DAMP4 from rich complex media to CDM resulted in a reduction in yield that could be attributed to the reduction in final cell density more so than a significant reduction in specific productivity. To maximize product titer, cell density at induction was maximized using a fed-batch approach. In fed-batch DAMP4 product titer increased 9-fold compared to batch, while maintaining 60% specific productivity. Under the fed-batch conditions, the final product titer of DAMP4 reached more than 7 g/L which is the highest titer of DAMP4 reported to date. To investigate production from sucrose, sucrose metabolism was engineered into BL21(DE3) using a simple plasmid approach. Using this strain, growth and DAMP4 production characteristics obtained from CDM supplemented with sucrose were similar to those obtained when culturing the parent strain on CDM supplemented with glucose.ConclusionsProduction of a model peptide was increased to several grams per liter using a CDM medium with either glucose or sucrose feedstock. It is hoped that this work will contribute cost reduction for production of designer peptide surfactants to facilitate their commercial application.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0099-y) contains supplementary material, which is available to authorized users.

Effect of phosphorus limitation on coccolith morphology and element ratios in Mediterranean strains of the coccolithophore Emiliania huxleyi

Six Emiliania huxleyi strains isolated in the Mediterranean Sea were grown under phosphorus limitation and replete (control) conditions in batch culture. We tested the hypothesis that increasing phosphorus limitation, resulting from ocean warming, will affect coccolithophores and change their contribution to the PIC:POC ratios. Four strains were isolated in the oligotrophic Algerian basin (western Mediterranean), and two strains in the extremely oligotrophic and often phosphorus depleted Levantine basin (eastern Mediterranean). Samples for particulate inorganic carbon, PIC; particulate organic carbon, POC; particulate organic phosphorus, POP; and total particulate nitrogen, TPN, were taken at equal cell densities in phosphorus limited and control culture conditions (harvesting in exponential and stationary phases respectively). Different morphological features of coccoliths were analyzed. Phosphorus limitation was inferred from: 1. a decrease in growth rate between 22% and 34% compared to phosphorus replete cultures; 2. a reduction in final cell density from ca. 2×106cells/ml to ca. 8×104cells/ml, and 3. markedly lower POP quotas of the limited cells. Differences in the phosphate uptake machinery are suggested based on the fact that three western Mediterranean strains decreased the phosphate concentration in the culture media until the detection limit in the first 3 experimental days while 4–5days were necessary for the other strains. Cellular carbon quotas increased in all strains under phosphorus limitation in comparison to their respective control cultures. The increase in POC quotas ranged between 179% and 260% and in PIC quotas between 43% and 201%. TPN quotas increased under phosphorus limitation between 31% and 86% in five out of six strains. POC:TPN increased in all strains, which suggests a change in the molecular composition of the cells when exposed to phosphorus limitation. Strain specific responses were also observed in the PIC:POC ratio, which decreased in five strains and did not change in another. Phosphorus limitation did not lead to malformations. However, morphological changes of coccoliths occurred, also in a strain specific way, namely the percentage of over-calcified coccoliths was altered by P limitation, but we observed all signs in the responses among different strains. Nevertheless, this morphological feature does not reflect the changes in calcite production (PICp nor PIC:POC). Strain specific responses were not related to the isolation location, suggesting that the isolated western and eastern strains do not represent distinct eco-genotypes featuring different phosphorus usage strategies.

Elucidation of Zymomonas mobilis physiology and stress responses by quantitative proteomics and transcriptomics.

Zymomonas mobilis is an excellent ethanologenic bacterium. Biomass pretreatment and saccharification provides access to simple sugars, but also produces inhibitors such as acetate and furfural. Our previous work has identified and confirmed the genetic change of a 1.5-kb deletion in the sodium acetate tolerant Z. mobilis mutant (AcR) leading to constitutively elevated expression of a sodium proton antiporter encoding gene nhaA, which contributes to the sodium acetate tolerance of AcR mutant. In this study, we further investigated the responses of AcR and wild-type ZM4 to sodium acetate stress in minimum media using both transcriptomics and a metabolic labeling approach for quantitative proteomics the first time. Proteomic measurements at two time points identified about eight hundreds proteins, or about half of the predicted proteome. Extracellular metabolite analysis indicated AcR overcame the acetate stress quicker than ZM4 with a concomitant earlier ethanol production in AcR mutant, although the final ethanol yields and cell densities were similar between two strains. Transcriptomic samples were analyzed for four time points and revealed that the response of Z. mobilis to sodium acetate stress is dynamic, complex, and involved about one-fifth of the total predicted genes from all different functional categories. The modest correlations between proteomic and transcriptomic data may suggest the involvement of posttranscriptional control. In addition, the transcriptomic data of forty-four microarrays from four experiments for ZM4 and AcR under different conditions were combined to identify strain-specific, media-responsive, growth phase-dependent, and treatment-responsive gene expression profiles. Together this study indicates that minimal medium has the most dramatic effect on gene expression compared to rich medium followed by growth phase, inhibitor, and strain background. Genes involved in protein biosynthesis, glycolysis and fermentation as well as ATP synthesis and stress response play key roles in Z. mobilis metabolism with consistently strong expression levels under different conditions.

Foam stability and polymer phase morphology of flexible polyurethane foams synthesized from castor oil

ABSTRACTFoam stability and segmented polymeric phase morphology of polyurethane foams synthesized partially and completely from castor oil are investigated. Preliminary analysis of the impact of alterations in the polymeric phase on macroscopic stress dissipation in foams is also carried out. The stability and morphology show unique trends depending on the concentration of castor oil used in foam synthesis. While low and intermediate concentrations of castor oil does not significantly affect the foaming process; at high concentrations, the volumetrically expanding liquid matrix remains in a nonequilibrium state during the entire foaming period, resulting in significant foam decay from top. This increases the final foam cell density and decreases the plateau border thickness at bottom. In the polymeric phase of castor oil based foams, the fraction of monodentate urea increases at the cost of non‐hydrogen bonded urea. These monodentate urea domains undergo flocculation in foams synthesized completely from castor oil, thus prominently modifying the segmented morphology. The glass transition temperature of soft segments of partially substituted foams shows moderate increase, with indications of phase mixing between the polyether and castor oil generated urethane domains. Foams synthesized entirely from castor oil have significant sol fraction due to unreacted oligomers. The microscopic alterations in polymeric phase reduce the elastic recovery of partially substituted castor oil foams compared to its viscous dissipation under an applied stress. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40668.

Porphyromonas gingivalis and Treponema denticola Exhibit Metabolic Symbioses

Porphyromonas gingivalis and Treponema denticola are strongly associated with chronic periodontitis. These bacteria have been co-localized in subgingival plaque and demonstrated to exhibit symbiosis in growth in vitro and synergistic virulence upon co-infection in animal models of disease. Here we show that during continuous co-culture a P. gingivalis:T. denticola cell ratio of 6∶1 was maintained with a respective increase of 54% and 30% in cell numbers when compared with mono-culture. Co-culture caused significant changes in global gene expression in both species with altered expression of 184 T. denticola and 134 P. gingivalis genes. P. gingivalis genes encoding a predicted thiamine biosynthesis pathway were up-regulated whilst genes involved in fatty acid biosynthesis were down-regulated. T. denticola genes encoding virulence factors including dentilisin and glycine catabolic pathways were significantly up-regulated during co-culture. Metabolic labeling using 13C-glycine showed that T. denticola rapidly metabolized this amino acid resulting in the production of acetate and lactate. P. gingivalis may be an important source of free glycine for T. denticola as mono-cultures of P. gingivalis and T. denticola were found to produce and consume free glycine, respectively; free glycine production by P. gingivalis was stimulated by T. denticola conditioned medium and glycine supplementation of T. denticola medium increased final cell density 1.7-fold. Collectively these data show P. gingivalis and T. denticola respond metabolically to the presence of each other with T. denticola displaying responses that help explain enhanced virulence of co-infections.

Metabolic changes of recombinant Escherichia coli BL21 (DE3) during overexpression of recombinant human interferon beta in HCDC

The influence of the overexpression of recombinant human interferon beta (rhINF-β) during high cell density cultivation (HCDC) on metabolic characteristics of recombinant E. coli BL21 (DE3) was studied. Fed-batch culture technique was employed to grow the host cell for the production of human interferon beta in pET expression system in a 5L bench-top bioreactor. The final cell density of 42.15 g L-1 and cell growth (OD600nm) of 103.5 were obtained respectively in fed-batch culture experiment. Four hours after the induction, the specific growth rate was decreased. The initial concentration of the glucose, ammonium and phosphate in the TB culture medium were 5.0, 12.0 and 3.12 g L-1 respectively. The maximum amount of the produced rhINF-β was 2.8 g L-1 after applying the fed-batch cultivation technique. Besides, time profiles of the acetate and lactate production were similar, the acetate concentration (4.91 g L -1 ) was lower than that of lactate (10.08 g L -1 ) and the

2,3-Butanediol fermentation promotes growth of Serratia plymuthica at low pH but not survival of extreme acid challenge

The mechanisms by which Enterobacteriaceae can survive or grow at low pH are of interest because members of this family are increasingly linked to problems of spoilage and foodborne infection related to mildly acidic foods. In this work, we investigated the contribution of the 2,3-butanediol fermentation pathway in coping with specific forms of acid stress in Serratia plymuthica RVH1. This pathway consumes intracellular protons, similar to the amino acid decarboxylases which are involved in acid resistance in Enterobacteriaceae. While its role in preventing excessive acidification in media with an initial neutral pH but containing fermentable sugars has been established, we here addressed the question whether it supports survival of severe acid challenge (pH2.5–3.5) and/or enhances the ability to initiate growth at moderately low pH (pH4.0–5.0) in acidified LB medium and in tomato juice. Using a budAB::cat mutant, deficient in 2,3-butanediol fermentation, we showed that the pathway did not influence survival in simulated gastric fluid and is not involved in the acid tolerance response (ATR) in S. plymuthica RVH1. On the other hand, the pathway promoted growth at moderately low pH. In acidified LB medium, the mutant stopped growing at a lower final cell density than the wild-type strain. In tomato juice, additionally, the minimal pH at which the mutant could grow (pH4.20–4.30) was increased compared to that of the wild-type (pH4.10). Growth of the wild-type strain was often accompanied by a pH increase, in contrast to the budAB::cat mutant, where the opposite was observed. However, the differences in growth between the wild-type and budAB::cat mutant could not only be explained by external pH, suggesting that the 2,3-butanediol fermentation contributed to intracellular pH homeostasis. Based on these data, we propose the contribution to growth at low pH as a novel biological function of 2,3-butanediol fermentation in Enterobacteriaceae.

Effect of Fe3+ on the growth and lipid content of Isochrysis galbana

Inducing lipid accumulation in microalgae cells without suppressing cell growth is vital to the economical production of biodiesel from microalgae. In two experiments, we demonstrate that the cell concentration and lipid content of marine microalgae Isochrysis galbana depend upon the iron concentration in the growth media. In Experiment I, adding chelated FeCl3 to the medium at the late exponential growth phase prolonged this phase and increased the lipid content in I. galbana cells. The final cell density and lipid content of I. galbana supplemented with chelated FeCl3 was approximately 2 and 1.65 times higher than that of non-supplemented cultures, respectively. In Experiment II, I. galbana cells in the late exponential phase were collected and re-inoculated into new media containing Fe3+ at various concentrations. The final cell concentration and lipid content were maximized at the highest iron concentration (38% biomass by dry weight at 1.2x10(-5) mol/L Fe3+). In this study, intracellular neutral lipid storage was evaluated by fluorescent spectrophotometry using fluorochrome Nile red, and the measurement conditions were optimized.

Effects of additional Mg2+ on the growth, lipid production, and fatty acid composition of Monoraphidium sp. FXY-10 under different culture conditions

The effects of supplementing the culture medium with Mg2+ on the growth, lipid production, and fatty acid composition of Monoraphidium sp. FXY-10 were studied under photoautotrophic, heterotrophic, and mixotrophic conditions. Under the photoautotrophic condition, microalgae supplemented with 100 μM Mg2+ grew significantly better than the control group and exhibited a secondary growth state. The final cell density was 1.25-fold higher than that of the control group (2.98 g L−1), and the peak lipid content reached 59.8 % (control group 52.3 %). Culture under the heterotrophic condition did not significantly increase the growth rate, but the experimental group (100 μM Mg2+ supplementation) achieved a 37.03 % lipid content compared to 28.47 % by the control group. The lipid productivity of the experimental group (100 μM Mg2+ supplementation) was higher, reaching 65.93 mg L−1 day−1 compared with 56.10 mg L−1 day−1 for the group without additional Mg2+. Under the mixotrophic condition, the experimental group achieved a final density of 3.10 g L−1, which was higher than that of the control group (2.98 g L−1). There was also no variation in fatty acid composition between the experimental group and the control group. Under the heterotrophic and mixotrophic conditions, the experimental group produced more than 50% saturated fatty and mono-unsaturated fatty acids, and the degree of unsaturation was <137. This result was relatively lower than that of the control.

Utilization of glycerin byproduct derived from soybean oil biodiesel as a carbon source for heterologous protein production in Pichia pastoris

Crude glycerol, also known as glycerin, is the main byproduct of the biodiesel industry. It has been estimated that up to 40,000 tons of glycerin will be produced each year by 2020. This study evaluated the value-added use of crude glycerol derived from soybean biodiesel preparation as a carbon source for heterologous protein production using the yeast Pichia pastoris. Eleven glycerin samples were obtained by methanolysis of soybean oil using different acids or bases as catalysts. Cell growth experiments showed that crude glycerol containing either potassium or sodium hydroxide resulted in 1.5-2 times higher final cell densities when compared to glycerol P.A. Finally, crude glycerol containing sodium hydroxide was successfully utilized for constitutive heterologous α-amylase production in P. pastoris. This study demonstrated that crude glycerol without any purification steps may be directly used as carbon source for protein production in P. pastoris.

Effect of spaceflight on Pseudomonas aeruginosa final cell density is modulated by nutrient and oxygen availability

BackgroundAbundant populations of bacteria have been observed on Mir and the International Space Station. While some experiments have shown that bacteria cultured during spaceflight exhibit a range of potentially troublesome characteristics, including increases in growth, antibiotic resistance and virulence, other studies have shown minimal differences when cells were cultured during spaceflight or on Earth. Although the final cell density of bacteria grown during spaceflight has been reported for several species, we are not yet able to predict how different microorganisms will respond to the microgravity environment. In order to build our understanding of how spaceflight affects bacterial final cell densities, additional studies are needed to determine whether the observed differences are due to varied methods, experimental conditions, or organism specific responses.ResultsHere, we have explored how phosphate concentration, carbon source, oxygen availability, and motility affect the growth of Pseudomonas aeruginosa in modified artificial urine media during spaceflight. We observed that P. aeruginosa grown during spaceflight exhibited increased final cell density relative to normal gravity controls when low concentrations of phosphate in the media were combined with decreased oxygen availability. In contrast, when the availability of either phosphate or oxygen was increased, no difference in final cell density was observed between spaceflight and normal gravity. Because motility has been suggested to affect how microbes respond to microgravity, we compared the growth of wild-type P. aeruginosa to a ΔmotABCD mutant deficient in swimming motility. However, the final cell densities observed with the motility mutant were consistent with those observed with wild type for all conditions tested.ConclusionsThese results indicate that differences in bacterial final cell densities observed between spaceflight and normal gravity are due to an interplay between microgravity conditions and the availability of substrates essential for growth. Further, our results suggest that microbes grown under nutrient-limiting conditions are likely to reach higher cell densities under microgravity conditions than they would on Earth. Considering that the majority of bacteria inhabiting spacecrafts and space stations are likely to live under nutrient limitations, our findings highlight the need to explore the impact microgravity and other aspects of the spaceflight environment have on microbial growth and physiology.

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins.

Botanical pesticides as potential rotifer-control agents in microalgal mass culture

With the aim of identifying an effective and safe technique for reducing rotifer contamination in microalgal mass cultivation, the toxic effects of four botanical pesticides on the rotifer Brachionus plicatilis were studied. Acute toxicity tests showed that celangulin, matrine and toosendanin are highly toxic to B. plicatilis, with 24h LC50 values of 0.175mgL−1, 0.061mgL−1 and 2.132×10−3mgL−1, respectively. Azadirachtin was the least toxic, with a 24h LC50 value of 18.386mgL−1. Chronic toxicity tests showed that life history parameters (Ro, T, rm and λ) and population density of rotifers decreased significantly when exposed to 0.110mgL−1 celangulin, ≥0.050mgL−1 matrine or 0.380×10−3mgL−1 toosendanin. In addition, the rotifer-control effects of toosendanin and its safety in Chlorella and Nannochloropsis sp. cultivation were evaluated. Results demonstrated that 1.755−2.132×10−3mgL−1 toosendanin had no obvious toxic influence on final cell density and photosynthesis of Chlorella and Nannochloropsis sp., but effectively reduced the rotifer density and its fecundity. Based on the high toxicity to rotifers and the relative safety to microalgae, together with their low commercial price and ecological acceptability, celangulin, matrine and toosendanin are considered to be good potential botanical pesticides for controlling rotifers in microalgal mass culture.

Effect of high ferric ion concentrations on total lipids and lipid characteristics of Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis

Microalgae as sources for biodiesel production have been widely investigated. Microalgae biomass, lipid content and fatty acid profiles of microalgae are limiting factors for the cost-effective production of biodiesel. In this paper, the effects of high ferric ion concentrations on three species of microalgae (Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis) were studied. The microalgae were cultured in different concentrations (1.2 × 10−2, 1.2 × 10−1, 1.2 and 12 mmol L−1) of ferric ion. The growth, lipid content and fatty acid profiles of the three microalgae were analysed. When algae were cultured in 1.2 mmol L−1 ferric ion for 10 days, the final cell density and specific growth rates of T. subcordiformis, N. oculata and P. viridis decreased significantly (p < 0.05), and the total lipid contents of the microalgae, 33.72, 37.34 and 29.48 % (dry mass) in T. subcordiformis, N. oculata and P. viridis, respectively, were higher than those at other concentrations. The neutral lipid/total lipid ratios of the three microalgae species increased with increasing ferric ion concentration. Neutral lipids accounted for 50.75, 48.37 and 46.59 % of the total lipid in T. subcordiformis, N. oculata and P. viridis, respectively, when cultured in 12 mmol L−1 ferric ion. The proportions of saturated fatty acids in all three species cultured in 12 mmol L−1 ferric ion were significantly higher than those cultured in lower ferric ion concentrations. An optimum ferric ion concentration can improve the properties of T. subcordiformis, N. oculata and P. viridis as sources for biodiesel.

The Brucella abortus General Stress Response System Regulates Chronic Mammalian Infection and Is Controlled by Phosphorylation and Proteolysis

Virulence of pathogenic bacteria is often determined by their ability to adapt to stress. The Brucella abortus general stress response (GSR) system is required for chronic mammalian infection and is regulated by phosphorylation and proteolysis. The B. abortus GSR signaling pathway has multiple layers of post-translational control and is a determinant of chronic infection. This study provides new, molecular level insight into chronic Brucella infection. Brucella spp. are adept at establishing a chronic infection in mammals. We demonstrate that core components of the α-proteobacterial general stress response (GSR) system, PhyR and σ(E1), are required for Brucella abortus stress survival in vitro and maintenance of chronic murine infection in vivo. ΔphyR and ΔrpoE1 null mutants exhibit decreased survival under acute oxidative and acid stress but are not defective in infection of primary murine macrophages or in initial colonization of BALB/c mouse spleens. However, ΔphyR and ΔrpoE1 mutants are attenuated in spleens beginning 1 month postinfection. Thus, the B. abortus GSR system is dispensable for colonization but is required to maintain chronic infection. A genome-scale analysis of the B. abortus GSR regulon identified stress response genes previously linked to virulence and genes that affect immunomodulatory components of the cell envelope. These data support a model in which the GSR system affects both stress survival and the interface between B. abortus and the host immune system. We further demonstrate that PhyR proteolysis is a unique feature of GSR control in B. abortus. Proteolysis of PhyR provides a mechanism to avoid spurious PhyR protein interactions that inappropriately activate GSR-dependent transcription. We conclude that the B. abortus GSR system regulates acute stress adaptation and long term survival within a mammalian host and that PhyR proteolysis is a novel regulatory feature in B. abortus that ensures proper control of GSR transcription.

Preservation efficiency of new cryoprotectant used for Acidithiobacillus ferrooxidans in liquid nitrogen

The efficiency of a new cryoprotectant, GP, for the preservation of Acidithiobacillus ferrooxidans (A. ferrooxidans) strain DC in liquid nitrogen was investigated. The optimal concentration of this new cryoprotectant for the maximal viable cell recovery and the highest ferrous ion oxidation activity was determined. The results show that 30% (volume fraction) GP is optimal for the cryopreservation with 84.4% of cells surviving, completely oxidizing ferrous ions within 120 h, and growing to a final density of 5.8×107 cell/mL after 6 d in the culture. Furthermore, the optimal residual GP concentration for viable cell recovery after culture of thawed cells in 9K medium for 6 d is 0.6% (volume fraction). At this concentration, strain DC completely oxidizes ferrous ions within 108 h and grows to a final cell density of 6.8×107 mL−1. Thus, GP is a simple, effective cryoprotectant for the preservation of A. ferrooxidans strain DC in liquid nitrogen.

The Protective Effects of Erythropoietin on Rat Glomerular Podocytes in Culture are Modulated by Extracellular Matrix Proteins

Background/Aims: Podocytes are typically cultured on collagen I; however, collagen I is absent from healthy glomerular basement membranes. Erythropoietin (EPO) is thought to protect podocytes in vivo. Here, we studied how various types of extracellular matrix (ECM) proteins and EPO affect podocytes in culture. Methods: Primary rat podocytes were replated on collagen I, collagen IV, whole ECM extract, laminin, or bare plastic. Cellular adhesion (8 hours after plating), proliferation (5 days, 10 % serum), and resistance to serum deprivation (3 days, 0.5 % serum) were assessed. BrdU incorporation and expression of podocyte-specific markers were employed as measures of cellular proliferation and differentiation, respectively. qPCR was used to verify expression of EPO receptor in cultured podocytes. Results: Cellular adhesion was similar on all ECM proteins and unaffected by EPO. Proliferation was accelerated by laminin and the ECM extract, but the final cell density was similar on all ECM surfaces. Collagen IV supported the serum-deprived cells better than the other ECM proteins. EPO (2-20 ng/ml) improved viability of serum-deprived podocytes on collagen I, collagen IV, and ECM, but not on laminin or bare plastic. The cells expressed mRNA for EPO receptor. Conclusion: The physiological ECM proteins are more supportive of primary podocytic cultures compared with collagen I. The protective effects of EPO during serum deprivation are modulated by the cultivation surface.

Synergistic Action of Gentamicin and Bacteriophage in a Continuous Culture Population of Staphylococcus aureus

With the increasing frequency of antibiotic resistance and the decreasing frequency of new antibiotics entering the market, interest has returned to developing bacteriophage as a therapeutic agent. Acceptance of phage therapy, however, is limited by the unknown pharmacodynamics of a replicating agent, as well as the potential for the evolution of resistant bacteria. One way to overcome some of these limitations is to incorporate phage and antibiotics into a dual therapy regimen; however, this increases the complexity of the pharmacodynamics. The aim of this study is to develop an experimental system to evaluate the pharmacodynamics of dual phage-drug therapy. A continuous culture system for Staphylococcus aureus is used to simulate the pharmacokinetics of periodic antibiotic dosing alone and in combination with lytic phage. A computer model representation of the system allows further evaluation of the conditions governing the observed pharmacodynamics. The results of this experimental/modeling approach suggest that dual therapy can be more efficacious than single therapies, particularly if there is an overlap in the physiological pathways targeted by the individual agents. In this case, treatment with gentamicin induces a population of cells with a strong aggregation phenotype. These aggregators also have an increased ability to form biofilm, which is a well-known, non-genetic mechanism of drug resistance. However, the aggregators are also more susceptible than the parental strain to the action of the phage. Thus, dual treatment with gentamicin and phage resulted in lower final cell densities than either treatment alone. Unlike in the phage-only treatment, phage-resistant isolates were not detected in the dual treatment.

Adaptation of the Spodoptera exigua Se301 insect cell line to grow in serum-free suspended culture. Comparison of SeMNPV productivity in serum-free and serum-containing media

Spodoptera exigua Se301 cells have been successfully adapted to two different commercial serum-free media (SFM; Ex-Cell 420 and Serum-Free Insect Medium-1) by gradually reducing the 10 %-added serum-containing medium content from 100 % to 0 % (v/v) in suspended cultures. Both direct adaptation to a serum-free medium and cell growth in the absence of protective additives against fluid dynamic stress [polyvinyl pyrrolidone and polyvinyl alcohol] and disaggregation [dextran sulfate] proved impossible. Cells grew reproducibly in both SFMs once the serum had been completely removed, although the use of Ex-Cell 420 resulted in higher growth rates and cell densities. Turbulence was sufficiently high to reduce growth rates and final cell densities at the highest Reynolds number investigated, although no clear influence of agitation was observed on virus productivity. Both attached and suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Cells adapted to different conditions (attached or suspended culture, serum-containing or serum-free medium) showed different occlusion bodies productivities at the two multiplicities of infection assayed (0.1 and 0.5).