Enhanced Carbofuran Degradation Using Immobilized and Free Cells of Enterobacter sp. Isolated from Soil

Extensive use of carbofuran insecticide harms the environment and human health. Carbofuran is an endocrine disruptor and has the highest acute toxicity to humans than all groups of carbamate pesticides used. Carbofuran is highly mobile in soil and soluble in water with a lengthy half-life (50 days). Therefore, it has the potential to contaminate groundwater and nearby water bodies after rainfall events. A bacterial strain BRC05 was isolated from agricultural soil characterized and presumptively identified as Enterobacter sp. The strain was immobilized using gellan gum as an entrapment material. The effect of different heavy metals and the ability of the immobilized cells to degrade carbofuran were compared with their free cell counterparts. The results showed a significant increase in the degradation of carbofuran by immobilized cells compared with freely suspended cells. Carbofuran was completely degraded within 9 h by immobilized cells at 50 mg/L, while it took 12 h for free cells to degrade carbofuran at the same concentration. Besides, the immobilized cells completely degraded carbofuran within 38 h at 100 mg/L. On the other hand, free cells degraded the compound in 68 h. The viability of the freely suspended cell and degradation efficiency was inhibited at a concentration greater than 100 mg/L. Whereas, the immobilized cells almost completely degraded carbofuran at 100 mg/L. At 250 mg/L concentration, the rate of degradation decreased significantly in free cells. The immobilized cells could also be reused for about nine cycles without losing their degradation activity. Hence, the gellan gum-immobilized cells of Enterobacter sp. could be potentially used in the bioremediation of carbofuran in contaminated soil.

In-flow measurement of cell–cell adhesion using oscillatory inertial microfluidics

Cell–cell adhesion strength of freely suspended cell clusters can be measured using an oscillatory inertial microfluidic system.

Sequential Cell-Processing System by Integrating Hydrodynamic Purification and Dielectrophoretic Trapping for Analyses of Suspended Cancer Cells

Microfluidic devices employing dielectrophoresis (DEP) have been widely studied and applied in the manipulation and analysis of single cells. However, several pre-processing steps, such as the preparation of purified target samples and buffer exchanges, are necessary to utilize DEP forces for suspended cell samples. In this paper, a sequential cell-processing device, which is composed of pre-processing modules that employ deterministic lateral displacement (DLD) and a single-cell trapping device employing an electroactive microwell array (EMA), is proposed to perform the medium exchange followed by arraying single cells sequentially using DEP. Two original microfluidic devices were efficiently integrated by using the interconnecting substrate containing rubber gaskets that tightly connect the inlet and outlet of each device. Prostate cancer cells (PC3) suspended in phosphate-buffered saline buffer mixed with microbeads were separated and then resuspended into the DEP buffer in the integrated system. Thereafter, purified PC3 cells were trapped in a microwell array by using the positive DEP force. The achieved separation and trapping efficiencies exceeded 94% and 93%, respectively, when using the integrated processing system. This study demonstrates an integrated microfluidic device by processing suspended cell samples, without the requirement of complex preparation steps.

Optimization of Vaccine Virus Accumulation in the Development of Smallpox Drugs Based on Cell Cultures

Objective. Optimization of vaccine virus cultivation in the suspended cell culture BHK-21 for infectious activity increment of virus-containing suspension as the base material for smallpox vaccine preparations. Materials and methods. We used suspended culture line of the cells BHK-21 of 72-hour age and nutrient medium of the MEM type in accordance with the guidelines on preparation in our studies. For challenging of the cells, vaccine virus (strain B-51) was used. The virus was adapted through three consequent passages on horion-allantois shell of developing chicken embryos of commercial dermovaccine series 449а at the premises of the Federal State Budgetary Institution “the 48th Central Research Institute” of the Ministry of Defense of the Russian Federation. Information on its genetic features is absent. Cultivation and precipitation of infected cells BHK-21 was carried out in bioreactor with priming volume of 1 liter at (36.5±0.5) °C and aeration with air mixture with varying content of CO2. Results and conclusions. Gas massexchange intensity was enhanced alongside simultaneous maintaining of sparing hydrodynamic conditions for mixing suspended cell cultures in bioreactor. Two-fold increase (up to (4.48±0.63)·109 cell/l) in suspended BHK-21 cell culture concentration at the end of reproduction cycle was achieved. Concentration of the vaccine virus was 3–5 times raised, from (8.1±0.3) lg PFU (plaque forming unit)/ml up to the level of infectious activity – (8.8±0.3) lg PFU/ml. Specific multiplicity of cell infection in recalculation per a cell was 1–5 PFU/cell and by virus yield – 20–100 PFU/cell. Enhanced infectious activity of the virus in concentrated suspension of infected BHK-21 cells substantiates the perspectives of the proposed method for improvement of vaccine virus accumulation phase in the development of anti-smallpox preparations based on cell cultures.

Controlling Heterogeneity and Increasing Titer from Riboswitch-RegulatedBacillus subtilisSpores for Time-Delayed Protein Expression Applications

Sporulated cells have potential as time-delayed expression chassis of proteins for applications such as ‘on-demand’ biologics production, whole cell biosensors, or oral vaccines. However, the desired attributes of high expression rates and low product variances are difficult to maintain from germinated spores. In this work we study the effect of an integratingvs.theta replicating plasmid in a wild-typeBacillus subtilisand two PolY mutants. The cells were engineered to produce a fluorescent reporter protein (RFP) under the control of a riboswitch activated by theophylline. This allowed for greater sensitivity to point mutations. The fluorescence and cell growth curves were fit with a custom kinetic model and a peak kinetic rate (LKPmax) was extracted for each clonal population (n = 30 for all cell, vector, and growth combinations). Plasmid based expression yields higher (8.7x) expression rates due to an increased copy number of the expression cassette (10x over integrated). The variance of LKPmaxvalues increased 2.07x after sporulation for the wild type strain. This increase in variance from sporulation is very similar to what is observed with UV exposure. This effect can be partially mitigated by the use of PolY knockouts observed in suspended cell growths and adherent biofilms.

Probing compression versus stretch activated recruitment of cortical actin and apical junction proteins using mechanical stimulations of suspended doublets

ABSTRACTWe report an experimental approach to study the mechanosensitivity of cellcell contact upon mechanical stimulation in suspended cell-doublets. The doublet is placed astride an hourglass aperture, and a hydrodynamic force is selectively exerted on only one of the cells. The geometry of the device concentrates the mechanical shear over the junction area. Together with mechanical shear, the system also allows confocal quantitative live imaging of the recruitment of junction proteins (e.g. E-cadherin, ZO-1, Occludin and actin). We observed the time sequence over which proteins were recruited to the stretched region of the contact. The compressed side of the contact showed no response. We demonstrated how this mechanism polarizes the stress-induced recruitment of junctional components within one single junction. Finally, we demonstrated that stabilizing the actin cortex dynamics abolishes the mechanosensitive response of the junction. Our experimental design provides an original approach to study the role of mechanical force at a cell-cell contact with unprecedented control over stress application and quantitative optical analysis.

Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method

12–14 days of culturing of bone marrow (BM) cells containing various growth factors is widely used method for generating dendritic cells (DCs) from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs’ purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P<0.05) between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.