Genomic and proteomic perspectives in cell culture engineering.

In the last few years, the number of biologics produced by mammalian cells have been steadily increasing. The advances in cell culture engineering science have contributed significantly to this increase. A common path of product and process development has emerged in the last decade and the host cell lines frequently used have converged to only a few. Selection of cell clones, their adaptation to a desired growth environment, and improving their productivity has been key to developing a new process. However, the fundamental understanding of changes during the selection and adaptation process is still lacking. Some cells may undergo irreversible alteration at the genome level, some may exhibit changes in their gene expression pattern, while others may incur neither genetic reconstruction nor gene expression changes, but only modulation of various fluxes by changing nutrient/metabolite concentrations and enzyme activities. It is likely that the selection of cell clones and their adaptation to various culture conditions may involve alterations not only in cellular machinery directly related to the selected marker or adapted behavior, but also those which may or may not be essential for selection or adaptation. The genomic and proteomic research tools enable one to globally survey the alterations at mRNA and protein levels and to unveil their regulation. Undoubtedly, a better understanding of these cellular processes at the molecular level will lead to a better strategy for 'designing' producing cells. Herein the genomic and proteomic tools are briefly reviewed and their impact on cell culture engineering is discussed.     

Molecular and allergenic characterization of recombinant tropomyosin from mud crab Scylla olivacea

Molecular Biology Reports - Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this...     

4:2:1 conduction of an AF initiating trigger

A 44 year old male with idiopathic dilated cardiomyopathy was undergoing persistent atrial fibrillation (AF) ablation. Following antral ablation, AF terminated into a regular narrow complex rhythm. Earliest activation was mapped to a focus in the superior vena cava (SVC) which was conducted in a 2:1 ratio to the atria which in turn was conducted with 2:1 ratio to the ventricles, resulting in an unusual 4:2:1 conduction of the SVC tachycardia. 1:1 conduction of the SVC tachycardia to the atrium preceded initiation of AF. During AF, SVC tachycardia continued unperturbed. Sinus rhythm was restored following catheter ablation of the focus.     

Harmonizing Energy and Power Density toward 2.7 V Asymmetric Aqueous Supercapacitor

Tremendous research efforts are devoted to developing wide potential window aqueous supercapacitors to resolve their low energy density concern. While the operational potential window is dictated by the intrinsic electrochemical stability of water (1.23 V), such a bottleneck may be surpassed by leveraging the additional overpotential of the oxygen evolution reaction and the hydrogen evolution reaction (HER). Herein, by employing an electroreduction technique, Na⁺ is adsorbed onto the carbon negative electrode which effectively acts as a physical barrier to hinder intermediate HER product formation, thereby reducing HER activity. To complement the wide potential carbon electrode, Na_0.25MnO₂ is employed as the positive electrode to take advantage of the extra energy (i.e., increased overpotential) required for Na⁺ insertion process into the structure. The asymmetric supercapacitor exhibits high energy density of 61.1 W h kg^?1 at a power density of 982 W kg^?1, and even at an ultrahigh power density of 42.9 kW kg^?1, a respectable energy density of 16.3 W h kg^?1 is attained. In addition, 93.7% capacitance retention is recorded after cycling for 10 000 cycles which further demonstrates its suitability as supercapacitor. The present success in fabricating a 2.7 V asymmetric supercapacitor will open a promising research route toward achieving high energy density and high power density.     

Exchange of ATP for ADP on high-force cross-bridges of skinned rabbit muscle fibers

The contractile properties of rabbit skinned muscle fibers were studied at 1-2 degrees C in different concentrations of MgATP and MgADP. Double-reciprocal plots of maximum velocity against MgATP concentration at different MgADP concentrations all extrapolated to the same value. This finding suggests that MgATP and MgADP compete for the same site on the cross-bridge, and that the exchange of MgATP for MgADP occurs without a detectable step intervening. The K(m) for ATP was 0.32 mM. The K(i) for MgADP was 0.33 mM. Control experiments suggested that the tortuosity of diffusion paths within the fibers reduced the radial diffusion coefficients for reactants about sixfold. Increasing MgADP from 0.18 to 2 mM at 5 mM ATP or lowering MgATP from 10 to 2 mM at 0.18 mM MgADP, respectively, increased isometric force by 25% and 23%, increased stiffness by 10% and 20%, and decreased maximum velocity by 35% and 31%. Two mechanisms appeared to be responsible. One detained bridges in high-force states, where they recovered from a length step with a slower time course. The other increased the fraction of attached bridges without altering the kinetics of their responses, possibly by an increased activation resulting from cooperative effects of the detained, high-force bridges. The rigor bridge was more effective than the ADP-bound bridge in increasing the number of attached bridges with unaltered kinetics.     

Proteomic investigation of metabolic shift in mammalian cell culture.

Mammalian cells, under typical cultivation conditions, produce large quantities of lactate and ammonia that affect cell growth adversely and result in low cell concentration. Controlled nutrient feeding to maintain low concentrations of glucose and glutamine reduces metabolite production drastically, altering the metabolism of the cells. This metabolic shift results in higher cell concentration in continuous cultures and does not affect the specific productivity of the cells. We have taken a proteomics approach to investigate the differential protein expression with metabolic shift. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we have found at least eight differentially expressed spots; two proteins were down-regulated, and the others were up-regulated with metabolic shift. These included metabolic enzymes, the brain form of phosphoglycerate mutase, which was down-regulated, and the precursor of the 23 kDa subunit of NADH-ubiquinone oxidoreductase, which was up-regulated. Another enzyme, the L1 isozyme of ubiquitin carboxyl-terminal hydrolase, which is involved in protein turnover and degradation, was also up-regulated in the metabolically altered cells. The remaining down-regulated spot had been identified as two isoforms of cytoplasmic actins, while three of the up-regulated spots were viral GAG polyproteins from various murine viruses. An unidentified protein was also up-regulated in the cells with altered metabolic state. This study shows the potential of using a proteomics approach in deciphering the intracellular changes in cells with physiological changes such as metabolism shift. The new insight into cell metabolism afforded by this analysis will greatly facilitate process optimization of continuous cell cultures.     

Generation of oxygen deficiency in cell culture using a two-enzyme system to evaluate agents targeting hypoxic tumor cells

The poor and aberrant vascularization of solid tumors makes them susceptible to localized areas of oxygen deficiency that can be considered sites of tumor vulnerability to prodrugs that are preferentially activated to cytotoxic species under conditions of low oxygenation. To readily facilitate the selection of agents targeted to oxygen-deficient cells in solid tumors, we have developed a simple and convenient two-enzyme system to generate oxygen deficiency in cell cultures. Glucose oxidase is employed to deplete oxygen from the medium by selectively oxidizing glucose and reducing molecular oxygen to hydrogen peroxide; an excess of catalase is also used to scavenge the peroxide molecules. Rapid and sustained depletion of oxygen occurs in medium or buffer, even in the presence of oxygen at the liquid/air interface. Studies using CHO/AA8 Chinese hamster cells, EMT6 murine mammary carcinoma cells, and U251 human glioma cells indicate that this system generates an oxygen deficiency that produces activation of the hypoxia-targeted prodrug KS119. This method of generating oxygen deficiency in cell culture is inexpensive, does not require cumbersome equipment, permits longer incubation times to be used without the loss of sample volume, and should be adaptable for high-throughput screening in 96-well plates.     

The air‐lift photobioreactors with flow patterning for high‐density cultures of microalgae and carbon dioxide removal

A photobioreactor containing microalgae is a highly efficient system for converting carbon dioxide (CO₂) into biomass. Using a microalgal photobioreactor as a CO₂ mitigation system is a practical approach to the problem of CO₂ emission from waste gas. In this study, a marine microalga, Chlorella sp. NCTU‐2, was applied to assess biomass production and CO₂ removal. Three types of photobioreactors were designed and used: (i) without inner column (i.e. a bubble column), (ii) with a centric‐tube column and (iii) with a porous centric‐tube column. The specific growth rates (μ) of the batch cultures in the bubble column, the centric‐tube and the porous centric‐tube photobioreactor were 0.180, 0.226 and 0.252 day^?1, respectively. The porous centric‐tube photobioreactor, operated in semicontinuous culture mode with 10% CO₂ aeration, was evaluated. The results show that the maximum biomass productivity was 0.61 g/L when one fourth of the culture broth was recovered every 2 days. The CO₂ removal efficiency was also determined by measuring the influent and effluent loads at different aeration rates and cell densities of Chlorella sp. NCTU‐2. The results show that the CO₂ removal efficiency was related to biomass concentration and aeration rate. The maximum CO₂ removal efficiency of the Chlorella sp. NCTU‐2 culture was 63% when the biomass was maintained at 5.15 g/L concentration and 0.125 vvm aeration (volume gas per volume broth per min; 10% CO₂ in the aeration gas) in the porous centric‐tube photobioreactor.     

Stochasticity of bacterial attachment and its predictability by the extended derjaguin-landau-verwey-overbeek theory

Bacterial attachment onto materials has been suggested to be stochastic by some authors but nonstochastic and based on surface properties by others. We investigated this by attaching pairwise combinations of two Salmonella enterica serovar Sofia (S. Sofia) strains (with different physicochemical and attachment properties) with one strain each of S. enterica serovar Typhimurium, S. enterica serovar Infantis, or S. enterica serovar Virchow (all with similar physicochemical and attachment abilities) in ratios of 0.428, 1, and 2.333 onto glass, stainless steel, Teflon, and polysulfone. Attached bacterial cells were recovered and counted. If the ratio of attached cells of each Salmonella serovar pair recovered was the same as the initial inoculum ratio, the attachment process was deemed stochastic. Experimental outcomes from the study were compared to those predicted by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory. Significant differences (P < 0.05) between the initial and the attached ratios for serovar pairs containing S. Sofia S1296a for all different ratios were apparent for all materials. For S. Sofia S1635-containing pairs, 7 out of 12 combinations of serovar pairs and materials had attachment ratios not significantly different (P > 0.05) from the initial ratio of 0.428. Five out of 12 and 10 out of 12 samples had attachment ratios not significantly different (P > 0.05) from the initial ratios of 1 and 2.333, respectively. These results demonstrate that bacterial attachment to different materials is likely to be nonstochastic only when the key physicochemical properties of the bacteria were significantly different (P < 0.05) from each other. XDLVO theory could successfully predict the attachment of some individual isolates to particular materials but could not be used to predict the likelihood of stochasticity in pairwise attachment experiments.     

Margination of red blood cells infected by Plasmodium falciparum in a microvessel

We investigated numerically the mechanism of margination of Plasmodium falciparum malaria-infected red blood cells (Pf-IRBCs) in micro-scale blood flow. Our model illustrates that continuous hydrodynamic interaction between a Pf-IRBC in the trophozoite stage (Pf-T-IRBC) and healthy red blood cells (HRBCs) results in the margination of the Pf-T-IRBC and, thus, a longer duration of contact with endothelial cells. The Pf-T-IRBC and HRBCs first form a "train". The volume fraction of RBCs is then locally increased, to approximately 40%, and this value is maintained for a long period of time due to the formation of a long train in high-hematocrit conditions. Even in low-hematocrit conditions, the local volume fraction is instantaneously elevated to 40% and the Pf-T-IRBC can migrate to the wall. However, the short train formed in low-hematocrit conditions does not provide continuous interaction, and the Pf-T-IRBC moves back to the center of the channel.