Immobilized Cell Membrane Research Articles

Biohydrogen Production in Semi-Continuous System Using Immobilized Cell Membrane

Biohydrogen Production in Semi-Continuous System Using Immobilized Cell Membrane

Establishment of cluster of differentiation 20 immobilized cell membrane chromatography for the screening of active antitumor components in traditional Chinese medicine

Non-Hodgkin lymphoma (NHL) is a heterogeneous group of malignant tumors occurring in B or T lymphocytes, and no small molecule-positive drugs to treat NHL have been marketed. Cluster of differentiation 20 (CD20) is an important molecule regulating signaling for the life and differentiation of B lymphocytes and possesses the characteristics of a drug target for treating NHL. 2-Methoxyestradiol induces apoptosis in lymphoma Raji cells and CD20 protein is highly expressed by Raji lymphoma cells. Therefore, in this study, a CD20-SNAP-tag/CMC model was developed to validate the interaction of 2-methoxyestradiol with CD20. 2-Methoxyestradiol was used as a small molecule control compound, and the system was validated for good applicability. The cell membrane chromatography model was combined with high-performance liquid chromatography ion trap time-of-flight mass spectroscopy (HPLC-IT-TOF-MS) in a two-dimensional system to successfully identify, analyze, and characterize the potential active compounds of Schisandra chinensis (Turcz.) Baill. extract and Lysionotus pauciflorus Maxim. extract, including Schisandrin A, Schizandrol A, Schizandrol B, Schisantherin B, and Nevadensin, which can act on CD20 receptors. The five potential active compounds were analyzed by non-linear chromatography. The thermodynamic and kinetic parameters of their interaction with CD20 were also analyzed, and the mode of interaction was simulated by molecular docking. Their inhibitory effects on lymphoma cell growth were assessed using a Cell Counting Kit-8 (CCK-8). Nevadensin and Schizandrin A were able to induce apoptosis in Raji cells within a certain concentration range. In conclusion, the present experiments provide some bases for improving NHL treatment and developing small molecule lead compounds targeting CD20 with low toxicity and high specificity.

Enhanced stability designs of cell membrane chromatography for screening drug leads

Cell membrane chromatography is an effective method for screening bioactive components acting on specific receptors in complex systems, which maintains the biological activity of the membrane receptors and improves screening efficiency. However, traditional cell membrane chromatography suffers from poor stability, resulting in a limited life span and low reproducibility, greatly limiting the application of this method. To address this problem, cyanuric chloride-decorated silica gel was used for the covalent immobilization of the cell membranes. Cyanuric chloride reacts with amino groups on the cell membranes and membrane receptors to form covalent bonds. In this way, the cell membranes are not easy to fall off. The column life of the cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography column was extended to more than 8 days, whereas the column life of the normal cell membrane chromatography column dropped sharply in the first 3 days. A cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography online HPLC-IT-TOF-MSn system was applied for screening drug leads from Trifolium pratense L. One potential drug lead, formononetin, which acts on the epidermal growth factor receptor, was screened. Our strategy of covalently immobilizing cell membrane receptors also improved the stability of cell membrane chromatography.

Covalent Design of Cell Membrane Stationary Phase with Enhanced Stability for Fast Screening P-Glycoprotein Inhibitors.

Cell membrane chromatography (CMC) has been widely used for characterizing the interaction between drugs and membrane receptors to screen target components from herbal medicines. However, the column life, stability, and the efficiency cannot meet the needs of high-throughput screening purpose. In this study, a P-glycoprotein immobilized cell membrane stationary phase (P-gp/CMSP) was prepared with a simple and mild two-step aldehyde modification, realizing the covalent bonding between cell membrane and stationary phase. The column life and stability were significantly enhanced compared with the unmodified columns. The P-gp/CMC column was equipped into a comprehensive 2D P-gp/CMC/Capcell-C18/TOFMS system, which actualizes the automated and high-throughput analytical process and rapid identification of complex chemical samples with no data loss. Five compounds with significant retention were screened out and unambiguously identified by the comprehensive 2D analytical system. Baicalin was confirmed as a P-gp inhibitor with ATP depletion inhibition ratio of 83.4%. Moreover, the reversal index of baicalin on DOX significantly increased to 11.13 when its concentration reached 25 μM, revealing that baicalin could effectively reverse the MDR cell model induced by DOX. The integrated system is a practical drug discovery platform and could be applied to other transmembrane protein models.

Kinetic evaluation of nitrification performance in an immobilized cell membrane bioreactor.

High rate membrane bioreactor (MBR) systems operated at extremely low sludge ages (superfast membrane bioreactors (SFMBRs)) are inefficient to achieve nitrogen removal, due to insufficient retention time for nitrifiers. Moreover, frequent chemical cleaning is required due to high biomass flux. This study aims to satisfy the nitrification in SFMBRs by using sponge as carriers, leading to the extension of the residence time of microorganisms. In order to test the limits of nitrification, bioreactor was run under 52, 5 and 2 days of carrier residence time (CRT), with a hydraulic retention time of 6 h. Different degrees of nitrification were obtained for different CRTs. Sponge immobilized SFMBR operation with short CRT resulted in partial nitrification indicating selective dominancy of ammonia oxidizers. At higher CRT, simultaneous nitrification-denitrification was achieved when accompanying with oxygen limitation. Process kinetics was determined through evaluation of the results by a modeling study. Nitrifier partition in the reactor was also identified by model calibration.

Biological Denitrification of Groundwater by a Composite Membrane Bioreactor

A composite membrane bioreactor (CMBR) integrating the immobilized cell technique and the membrane separation technology was developed for biological denitrification of groundwater. In CMBR the groundwater and external carbon source (ethanol solution) are separated by the composite membranes consisting of a microporous membrane facing the groundwater and a plate-like immobilized cell membrane facing the ethanol solution. Nitrate and ethanol molecules diffused from the respective frames into the plate-like immobilized cell membrane where nitrate was reduced to gaseous nitrogen by the denitrifying bacteria present there with ethanol as carbon source. The microporous membrane attached to one side of plate-like immobilized cell membrane was used to separate product water from a plate-like immobilized cell membrane for retention of the disaggregated bacteria. Using the CMBR for groundwater denitrification, the over dosed external carbon source can be reused, and its treatment performance was perfect during continuous operation up to 98 days, and almost all effluent NO3--N, NO2--N, and CODMn concentrations are below their maximum contaminant levels as the NO3--N loading was less than 4.968 g.m-2.d-1.

Preliminary study of groundwater denitrification using a composite membrane bioreactor

A composite membrane bioreactor (CMBR) integrating the immobilized cell technique and the membrane separation technology was developed for groundwater denitrification. The CMBR had two well mixed compartments with one filled with the nitrate-containing influent and the other with a dilute ethanol solution; the compartments were separated by the composite membrane consisting of a microporous membrane facing the influent and an immobilized cell membrane facing the ethanol solution. Nitrate and ethanol molecules diffused from the respective compartments into the immobilized cell membrane where nitrate was reduced to gaseous nitrogen by the denitrifying bacteria present there with ethanol as the carbon source. The microporous membrane was attached to one side of the immobilized cell membrane for retention of the disaggregated bacteria. Relative to the single dose of external ethanol, the two-dose supplementation produced better treatment results as evidenced by the lower concentrations of NO3−-N and ethanol (as measured by total organic carbon) of the effluent. The batch treatment in CMBR removed most of the nitrate in the influent and attained a stable denitrification rate of 0.1 g·m−2·h−1 for most of the 96-h cycles during the 30-cycle study. The effluent was essentially free of ethanol and nitrite nitrogen.

Development of a novel, multi-analyte biosensor system for assaying cell division: Identification of cell proliferation/death precursor events

A novel, miniaturized biosensor system was created by combining the electrophysiological response of immobilized cells with superoxide-sensing technology, optical and fluorescence microscopy. Vero cells were immobilized in a calcium alginate matrix (at a density of 1.7 x 10(6) cells ml(-1)). A 0.5 cm x 0.5 cm piece of cell-containing gel matrix was aseptically adhered on a glass microscope slide with a microfabricated gold electrode array, sealed with a cover slip and provided with Dulbecco's medium +10% (v/v) fetal calf serum every day by means of a capillary feeding tube. During a culture period of 7 days, the membrane potential of immobilized cells was continuously monitored, while cell division was assayed with an optical microscope. In addition, daily measurements of immobilized cell membrane potential, viability, RNA and calcium concentration, radical oxygen species (ROS) and glutathione accumulation, were conducted by fluorescence microscopy after provision of an appropriate dye. Superoxide accumulation was assayed by covering the electrodes with superoxide dismutase (SOD). Maximum cell membrane potential values and superoxide production were observed upon initiation of cell division. Using the novel biosensor, we were able to correlate seven different cell physiological parameters to each other and formulate a model for ROS-mediated signaling function on cell division and death. In addition, we were able to predict cell proliferation or death by comparing the relative response of the electrophysiological and superoxide sensor during the culture period.

Construction of phage display libraries from reactive lymph nodes of breast carcinoma patients and selection for specifically binding human single chain Fv on cell lines.

The display of recombinant antibody fragments on the surface of filamentous phage mimicks B cells and is therefore a technology ideal to generate antibodies against any potential target antigen in vitro. In order to obtain tumor specific, high-affinity single chain antibody fragments (scFv), it has been speculated that lymph node tissue from cancer patients infiltrated with activated B cells must be a valuable source of antibody V-genes. The aim of this study was to generate a human scFv-phage library from lymph nodes of patients with breast cancer and to develop a stringent depletion and selection protocol in order to isolate specific single chain antibodies recognizing potentially new antigens in breast cancer. The amplification of the V-genes cloned from regional lymph node tissue and their assembly to single chain variable fragments was optimized in terms of library size and diversity. A large set of degenerated primers, annealing to all known V-gene families, was designed and used under optimized PCR conditions. The amplified V-genes were genetically fused in all possible combinations and cloned into a phagemid vector. Depletion and selection on mammary epithelial and primary breast carcinoma cell lines, respectively led to the isolation of a breast cancer cell line specific scFv (BCK-1 scFv) from this patient-derived scFv-phage display library as demonstrated in polyclonal and monoclonal ELISA, using immobilized cell membrane fractions of the indicated cell lines. A new recombinant breast cancer cell line specific antibody based on V-genes derived from reactive B-lymphocyte-infiltrated lymph nodes of patients with breast cancer was isolated via phage display, performing stringent depletion and selection protocols. We believe that this combination of antibody V-gene source and elaborated phage display depletion and selection strategy will be successful for the retrieval of numerous other recombinant, tumor specific antibody fragments.

Study on the mechanism of Bioelectric Recognition Assay: evidence for immobilized cell membrane interactions with viral fragments

The Bioelectric Recognition Assay (BERA) is a whole-cell based biosensing system that detects the electric response of cultured cells, suspended in a gel matrix, to various ligands, which bind to the cell and/or affect its physiology. Previous studies have demonstrated the potential application of this method for rapid, inexpensive detection of viruses in a crude sample. However, the understanding, so far, of the fundamental processes that take place during cell–virus interactions within the probe has been rather limited. In the present study, we combined electrophysiological and fluorescence microscopical assays, so that we can prove that animal and plant cells immobilized in BERA sensors respond to different viruses primarily by changing their membrane potential. The response of immobilized cells against different viruses did not depend on the virus ability to penetrate the cell, but was modified after binding each virus to a virus-specific antibody or removal of its coat protein after treatment with a protease. Consequently, we were able to assay the presence of a virus in its complete form or fragments thereof. Combination of immunological recognition with the electrophysiological response of immobilized cells allows for a considerable increase of the specificity of the BERA biosensory assay. In addition, rather than simply detect the presence of a protein or genomic sequence, the method can help gain information on the bioactivity of a virus.

Immobilized-artificial-membrane chromatography: measurements of membrane partition coefficient and predicting drug membrane permeability

Immobilized artificial membranes (IAMs) are chromatographic surfaces prepared by covalently immobilizing cell membrane phospholipids to solid surfaces at monolayer densities. IAM surfaces mimic fluid cell membranes. For 23 structurally unrelated compounds, solute capacity factors [log ( k′ IAM)] measured on IAM columns correlate very well with the solute equilibrium partition coefficients [log ( K m)] measured in fluid liposome systems ( r = 0.907). This indicates that solute partitioning between the IAM bonded phase and the aqueous mobile phase is similar to the solute partitioning between liposomes and the aqueous phase. IAMs also predicted oral drug absorption in mice and drug permeability through Caco-2 cells. IAM chromatography is experimentally simple and large volume screening of experimental compounds for drug absorption is possible. Solute retention on IAMs was found to be dominated by a partitioning mechanism. The structural requirements for HPLC bonded to predict solute-membrane partitioning are briefly discussed.

Membrane partition coefficients chromatographically measured using immobilized artificial membrane surfaces.

Immobilized artificial membranes (IAMs) are chromatographic surfaces prepared by covalently immobilizing cell membrane phospholipids. IAM surfaces mimic fluid cell membranes. Solute capacity factors (k'IAM) measured on IAM columns correlate very well with solute equilibrium partition coefficients (Km') measured in fluid liposome systems. For 23 structurally unrelated compounds, log-(k'IAM) correlates with log(Km') with a linear correlation coefficient r = 0.907. This indicates that solute partitioning between the IAM bonded phase and the aqueous mobile phase is similar to the solute partitioning between liposomes and the aqueous phase. Although both IAM chromatography and liposome partitioning can be used as in vitro methods to predict solute partitioning into cell membranes, IAM chromatography is experimentally convenient compared to liposome systems. To study the effect of lipid structure on drug binding to IAMs, IAMs were prepared from three different phosphatidylcholine ligands: (i) a diacylated phosphatidylcholine ligand, (ii) a single chain ether phosphatidylcholine ligand, and (iii) a single chain phosphatidylcholine ligand that lacks a glycerol backbone. Solute retention data were identical for all of these IAMs, and consequently, predictions of solute binding to fluid membranes were also identical. This indicates that the structure of the phosphatidylcholine ligand that is immobilized is not critical for the binding of solutes. Since the structure is not important, the binding of solutes to membranes is a bulk phase property, i.e., it is the interface created by the ligands that determines the solute binding properties, not the ligands themselves. Solute partitioning using octanol/water systems does not correlate with k'IAM unless a homologous series of hydrophobic solutes is being evaluated.

Agar-entrapped bacteria as an in vitro model of biofilms and their susceptibility to antibiotics

A simple in vitro system was developed as a model structure of biofilms and to evaluate their susceptibility to antibiotics. Viable Escherichia coli cells were entrapped in agar gel layers and incubated for 2 days in a minimal salt medium supplemented with glucose. After subsequent culture for 3 weeks under metal ion depletion, the biomass distribution inside the gel layer was highly heterogeneous. The cell concentration reached 1011 cfu/g gel in the outer regions of the agar structure whereas the inner gel areas were less colonized (109 cfu/g gel). Immobilized cells displayed enhanced resistance to latamoxef as compared with free microorganisms. Moreover, a 3-week-old immobilized-cell membrane was less susceptible to the antibiotic than a younger (2 days old) one. The exposure for 11 h to 64 μg/cm3 latamoxef killed about 90% of the bacteria entrapped in the older agar layer, whereas the number of killed cells was 100-fold higher in the younger structure. Effective diffusivity measurements showed that the diffusion of latamoxef in the biofilm-like agar structures was moderately restricted as compared to that in water, and independent of the immobilized-cell content.

Agar-entrapped bacteria as an in vitro model of biofilms and their susceptibility to antibiotics.

A simple in vitro system was developed as a model structure of biofilms and to evaluate their susceptibility to antibiotics. Viable Escherichia coli cells were entrapped in agar gel layers and incubated for 2 days in a minimal salt medium supplemented with glucose. After subsequent culture for 3 weeks under metal ion depletion, the biomass distribution inside the gel layer was highly heterogeneous. The cell concentration reached 10(11) cfu/g gel in the outer regions of the agar structure whereas the inner gel areas were less colonized (10(9) cfu/g gel). Immobilized cells displayed enhanced resistance to latamoxef as compared with free microorganisms. Moreover, a 3-week-old immobilized-cell membrane was less susceptible to the antibiotic than a younger (2 days old) one. The exposure for 11 h to 64 micrograms/cm3 latamoxef killed about 90% of the bacteria entrapped in the older agar layer, whereas the number of killed cells was 100-fold higher in the younger structure. Effective diffusivity measurements showed that the diffusion of latamoxef in the biofilm-like agar structures was moderately restricted as compared to that in water, and independent of the immobilized-cell content.

Enzymatic biotransformations of Peritan-Na by immobilized fungal mycelium fragments ofTrametes versicolor

The mycelium ofTrametes versicolor after 5 d growing in submerged cultures was homogenized and the pellets containing cell membranes were immobilized on alkylamine glass beads. The immobilized mycelial fragments were able to modify the molecular weight of Peritan-Na after only 4 h of incubation. Besides this phenomenon, the release of six free phenolic compounds was detected. Among these there were, for example, vanillin derivatives, syringic acid, andp-hydroxybenzaldehyde. This release was dependent upon pH, temperature, and the reaction time of the supposed enzymatic mechanism of the reaction. In short term (up to 6 h) experiments, the complex polymer of Peritan-Na might be transformed into molecules approximately closer to monomers of lignin as well as new lignin-like forms. According to our observations, the biotransformation of Peritan-Na follows a series of enzymatic reactions. It is supposed that enzymes necessary for these reactions are present in immobilized cell membrane fragments ofTrametes versicolor.

Study of the chemical receptor for diphtheria toxin in cell membranes

Adsorption of diphtheria toxin on immobilized cell membrane preparations in the presence of gangliosides was studied. A mixture of gangliosides completely inhibited the adsorption of diphtheria toxin on the cell membranes, both sensitive (HeLa cells, macrophages) and resistant (L-cells) to its action. Neuraminidase-treated gangliosides were less effective. Immobilized gangliosides adsorbed diphtheria toxin effectively in the presence of a protective colloid and detergent triton X-100. The results obtained suggest gangliosides to be diphtheria toxin receptors.