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Cell Separation (cell + separation)
Selected AbstractsThe CHAP domain of Cse functions as an endopeptidase that acts at mature septa to promote Streptococcus thermophilus cell separationMOLECULAR MICROBIOLOGY, Issue 5 2009Séverine Layec Summary Cell separation is dependent on cell wall hydrolases that cleave the peptidoglycan shared between daughter cells. In Streptococcus thermophilus, this step is performed by the Cse protein whose depletion resulted in the formation of extremely long chains of cells. Cse, a natural chimeric enzyme created by domain shuffling, carries at least two important domains for its activity: the LysM expected to be responsible for the cell wall-binding and the CHAP domain predicted to contain the active centre. Accordingly, the localization of Cse on S. thermophilus cell surface has been undertaken by immunogold electron and immunofluorescence microscopies using of antibodies raised against the N-terminal end of this protein. Immunolocalization shows the presence of the Cse protein at mature septa. Moreover, the CHAP domain of Cse exhibits a cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis and S. thermophilus. Additionally, RP-HPLC analysis of muropeptides released from B. subtilis and S. thermophilus cell wall after digestion with the CHAP domain shows that Cse is an endopeptidase. Altogether, these results suggest that Cse is a cell wall hydrolase involved in daughter cell separation of S. thermophilus. [source] Intercellular adhesion and cell separation in plantsPLANT CELL & ENVIRONMENT, Issue 7 2003M. C. JARVIS ABSTRACT Adhesion between plant cells is a fundamental feature of plant growth and development, and an essential part of the strategy by which growing plants achieve mechanical strength. Turgor pressure provides non-woody plant tissues with mechanical rigidity and the driving force for growth, but at the same time it generates large forces tending to separate cells. These are resisted by reinforcing zones located precisely at the points of maximum stress. In dicots the reinforcing zones are occupied by networks of specific pectic polymers. The mechanisms by which these networks cohere vary and are not fully understood. In the Poaceae their place is taken by phenolic cross-linking of arabinoxylans. Whatever the reinforcing polymers, a targeting mechanism is necessary to ensure that they become immobilized at the appropriate location, and there are secretory mutants that appear to have defects in this mechanism and hence are defective in cell adhesion. At the outer surface of most plant parts, the tendency of cells to cohere is blocked, apparently by the cuticle. Mutants with lesions in the biosynthesis of cuticular lipids show aberrant surface adhesion and other developmental abnormalities. When plant cells separate, the polymer networks that join them are locally dismantled with surgical precision. This occurs during the development of intercellular spaces; during the abscission of leaves and floral organs; during the release of seeds and pollen; during differentiation of root cap cells; and during fruit ripening. Each of these cell separation processes has its own distinctive features. Cell separation can also be induced during cooking or processing of fruit and vegetables, and the degree to which it occurs is a significant quality characteristic in potatoes, pulses, tomatoes, apples and other fruit. Control over these technological characteristics will be facilitated by understanding the role of cell adhesion and separation in the life of plants. [source] Continuous scalable blood filtration device using inertial microfluidicsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Albert J. Mach Abstract Cell separation is broadly useful for applications in clinical diagnostics, biological research, and potentially regenerative medicine. Recent attention has been paid to label-free size-based techniques that may avoid the costs or clogging issues associated with centrifugation and mechanical filtration. We present for the first time a massively parallel microfluidic device that passively separates pathogenic bacteria cells from diluted blood with macroscale performance. The device was designed to process large sample volumes in a high-throughput, continuous manner using 40 single microchannels placed in a radial array with one inlet and two rings of outlets. Each single channel consists of a short focusing, gradual expansion and collection region and uses unique differential transit times due to size-dependent inertial lift forces as a method of cell separation. The gradual channel expansion region is shown to manipulate cell equilibrium positions close to the microchannel walls, critical for higher efficiency collection. We demonstrate >80% removal of pathogenic bacteria from blood after two passes of the single channel system. The massively parallel device can process 240,mL/h with a throughput of 400 million cells/min. We expect that this parallelizable, robust, and label-free approach would be useful for filtration of blood as well as for other cell separation and concentration applications from large volume samples. Biotechnol. Bioeng. 2010;107: 302,311. © 2010 Wiley Periodicals, Inc. [source] Antibody-immobilized column for quick cell separation based on cell rollingBIOTECHNOLOGY PROGRESS, Issue 2 2010Atsushi Mahara Abstract Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label-free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody-immobilized cell-rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell-rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34-positive and -negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34-positive and CD34-negative cells on antibody-immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell-sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Preventing biofilm formation: promoting cell separation with terpenesFEMS MICROBIOLOGY ECOLOGY, Issue 3 2007Carla C.C.R. De Carvalho Abstract Both carveol and carvone were effective in dispersing Rhodococcus erythropolis cells that were being stimulated to aggregate by the presence of organic solvents. The two terpenes influenced the fatty acid composition of the cell membrane, decreasing the percentage of fatty acids with more than 16 carbon atoms, and thus cell hydrophobicity, and also the degree of saturation of the fatty acids. In the presence of 250 ,mol of terpene, the volume of biofilm was reduced by one third in comparison with biofilms in the absence of terpenes. The percentage of aggregated cells was also found to depend on carvone concentration during the bioconversion of carveol to carvone, in a membrane reactor. The extent of cell aggregation decreased from 90% to 10% when carvone concentration reached ca. 48 mM in the organic phase. [source] Calcineurin is implicated in the regulation of the septation initiation network in fission yeastGENES TO CELLS, Issue 10 2002Yabin Lu Background: In fission yeast, calcineurin has been implicated in cytokinesis because calcineurin-deleted cells form multiple septa and cell separation is impeded. However, this mechanism remains unclear. Results: We screened for mutations that confer syn-thetic lethality with calcineurin deletion and isolated a mutant, its10-1/cdc7-i10, a novel allele of the cdc7+ gene involved in the septation initiation network (SIN). The mutation created a termination codon, resulting in the truncation of Cdc7 by 162 amino acids, which is not localized in the spindle pole body. Following treatment with the immune suppressive drug FK506, cdc7-i10 and the original cdc7-24 mutant cells showed highly elongated multinuclear morphology with few visible septa, closely resembling the phenotype at the restrictive temperature. Other SIN mutants, cdc11, spg1, sid2 and mob1 showed similar phenotypes following FK506 treatment. Consistent with this, expression of the constitutively active calcineurin suppressed the growth defects and septum initiation deficiency of these SIN mutants at the restrictive temperature. Moreover, electron microscopy revealed that calcineurin-deleted cells had very thick multiple septa which were partially and ectopically formed. Conclusion: These results suggest that calcineurin is involved in the regulation of the SIN pathway, and is required for the proper formation and maturation of the septum in fission yeast. [source] Structural changes and shrinkage of potato during fryingINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 1 2001Rui M. Costa Summary Light microscopy was used to study changes in cell size, blister formation and crust evolution during potato frying. Frying experiments with both French fries and crisps of different thickness (1,5 mm) were performed at temperatures of 140 and 180 °C. Thickness, volume and density changes were also measured. The formation of crust in French fries starts after the potato surface reached approximately 103 °C, and then the crust thickness increased linearly with the square root of frying time, this increase being faster at 180 °C. The potato volume decreased during frying, although in the late stages the volume may increase because of oil uptake and cell separation caused by entrapped water vapour. Shrinkage was adequately described by the Weibull model with a residual value, with shrinkage rate increasing with temperature and decreasing with potato thickness. The residual volume was not affected by temperature (65% for French fries and 59,30 for crisps, depending on thickness). Volume appeared to decrease mainly as the result of water loss, except for very low water content, and thus potato density changes were very small. [source] Influence of obstetric factors on the yield of mononuclear cells, CD34+ cell count and volume of placental/umbilical cord bloodJOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 1 2010Atsuko Omori Abstract Aim:, Placental/umbilical cord blood (CB) has been used increasingly not only for transplantations, but also in the field of life science research. However, little information is available on the biological characteristics of CB units collected in rural areas because no medical facilities are affiliated with CB banks. Little attention has been paid to the collection of CB units in rural areas compared to CB collected in metropolitan areas. CB is a precious source for life science research due to the recent low birth rate in Japan. Therefore, to efficiently utilize CB units, the purpose of the present study was to investigate the optimum obstetric factors associated with a higher yield of mononuclear/CD34+ cells per CB unit. Methods:, CB units were collected at a single hospital (Hirosaki National Hospital). A total of 126 CB units from 105 vaginal deliveries and 21 cesarean section deliveries were available for cell separation within 24 h. Mononuclear low-density (LD) cells were separated using Ficoll-Paque and then processed for CD34+ cell enrichment using magnetic cell sorting. Associations between the maternal/neonatal factors and the yield of LD/CD34+ cells were analyzed. Results:, Despite the larger net weight of CB collected from cesarean section deliveries, the total number of LD cells collected from vaginal deliveries was significantly higher than that collected from cesarean section deliveries. The total number of LD cells per CB unit from primigravidae was significantly higher compared with that collected from from multigravidae. Conclusion:, CB units from vaginal deliveries of primigravidae may be more favorable because they contain a higher yield of mononuclear cells. [source] Dielectric cell separation of fine needle aspirates from tumor xenograftsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 21 2008Massimo Cristofanilli Abstract As an approach to isolating tumor cells from fine needle biopsy specimens, we investigated a dielectric cell preparation method using an in vivo xenographic tumor model. Cultured human MDA-MB-435 tumor cells were grown as solid tumors in nude mice and fine needle aspiration biopsies were conducted. Biopsied cells were suspended in sucrose medium and collected on slides patterned with microelectrode arrays (electrosmears) energized by electrical signals in the range 10 to 960 kHz. The unlabeled cells adhered to characteristic regions of the slides in accordance with their morphology as a result of dielectric forces. Tumor cells were trapped between 40 and 60 kHz and were separated according to whether they were mitotic, large and complex, or small. Damaged tumor cells were captured at between 60 and 120 kHz; granulocytes between 70 and 90 kHz; lymphocytes between 85 and 105 kHz; healthy erythrocytes between 140 and 180 kHz, and damaged erythrocytes above 180 kHz. Using intrinsic cell characteristics, the electrosmear presented cell subpopulations from fine needle aspiration biopsy specimens in a manner that is compatible with automated slide-based analysis systems. The approach has the potential to facilitate the analysis of the role of cell subpopulations in disease. [source] In vitro degradation of forage chicory (Cichorium intybus L.) by endopoly- galacturonaseJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 15 2007Xuezhao Sun Abstract BACKGROUND: Leaves of forage chicory break down rapidly in the rumen despite little or no rumination. Because chicory cell walls contain high concentrations of pectin, degradation of leaf midrib and leaf lamina tissues by pectinolytic enzymes was investigated. RESULTS: Treatment with endopolygalacturonase (endo-PG) degraded fresh intact chicory leaves to particles of less than 1 mm in length and solubilised more than 70% of the dry matter within 16 h. Uronic acids were released more extensively than neutral monosaccharides. In similar treatments, 77% of white clover leaflets and 12% of perennial ryegrass leaf blades were solubilised or broken down to particles with a size of less than 1 mm. The degradation of pectic polysaccharides in chicory midribs was monitored by immunofluorescence labelling with monoclonal antibodies JIM5 and JIM7 which target partially methyl-esterified epitopes of the homogalacturonan (HG) domain of pectin. Examination by fluorescence microscopy revealed that cell separation in the cortical parenchyma of chicory midrib following endo-PG treatment was associated with loss of HG from the middle lamella, the corners of intercellular spaces and from the tricellular junctions. CONCLUSION: The results of the current study suggest that one of the main contributions to chicory breakdown in the rumen may be cell separation caused by degradation of HG by pectinolytic enzymes from rumen bacteria. Copyright © 2007 Society of Chemical Industry [source] The CHAP domain of Cse functions as an endopeptidase that acts at mature septa to promote Streptococcus thermophilus cell separationMOLECULAR MICROBIOLOGY, Issue 5 2009Séverine Layec Summary Cell separation is dependent on cell wall hydrolases that cleave the peptidoglycan shared between daughter cells. In Streptococcus thermophilus, this step is performed by the Cse protein whose depletion resulted in the formation of extremely long chains of cells. Cse, a natural chimeric enzyme created by domain shuffling, carries at least two important domains for its activity: the LysM expected to be responsible for the cell wall-binding and the CHAP domain predicted to contain the active centre. Accordingly, the localization of Cse on S. thermophilus cell surface has been undertaken by immunogold electron and immunofluorescence microscopies using of antibodies raised against the N-terminal end of this protein. Immunolocalization shows the presence of the Cse protein at mature septa. Moreover, the CHAP domain of Cse exhibits a cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis and S. thermophilus. Additionally, RP-HPLC analysis of muropeptides released from B. subtilis and S. thermophilus cell wall after digestion with the CHAP domain shows that Cse is an endopeptidase. Altogether, these results suggest that Cse is a cell wall hydrolase involved in daughter cell separation of S. thermophilus. [source] The SUN41 and SUN42 genes are essential for cell separation in Candida albicansMOLECULAR MICROBIOLOGY, Issue 5 2007Arnaud Firon Summary Completion of the yeast cell cycle involves extensive remodelling of the cell wall upon separation of mother and daughter cells. We have studied two members of the ascomycete-specific SUN gene family in Candida albicans. Inactivation of SUN41 yields defects in cell separation and hyphal elongation while inactivation of SUN42 results in minor phenotypic alterations. Simultaneous inactivation of SUN41 and SUN42 is synthetically lethal due to lysis of mother cells after septation. Electronic microscopy reveals cell wall defects mainly localized in the region surrounding the septa. This phenotype is osmoremediable and the conditional double mutants show increased sensitivity to cell wall or cell membrane perturbing agents. The essential function shared by Sun41p and Sun42p is conserved among yeasts because UTH1, a Saccharomyces cerevisiae SUN gene, suppresses the lethality of SUN41 and SUN42 conditional mutants. Investigation of functional genomic data obtained in S. cerevisiae reveals links between members of the SUN gene family and the RAM pathway regulating cell wall-degrading enzymes specifically involved during cell separation. Thus, the main function of ascomycetous Sun proteins appears linked to cell wall remodelling, with a probable role in counter-balancing cell wall degradation to avoid cell lysis upon cell separation. [source] CPCR1, but not its interacting transcription factor AcFKH1, controls fungal arthrospore formation in Acremonium chrysogenumMOLECULAR MICROBIOLOGY, Issue 5 2005Birgit Hoff Summary Fungal morphogenesis and secondary metabolism are frequently associated; however, the molecular determinants connecting both processes remain largely undefined. Here we demonstrate that CPCR1 (cephalosporin C regulator 1 from Acremonium chrysogenum), a member of the winged helix/regulator factor X (RFX) transcription factor family that regulates cephalosporin C biosynthesis, also controls morphological development in the ,-lactam producer A. chrysogenum. The use of a disruption strain, multicopy strains as well as several recombinant control strains revealed that CPCR1 is required for hyphal fragmentation, and thus the formation of arthrospores. In a ,cpcR1 disruption strain that exhibits only hyphal growth, the wild-type cpcR1 gene was able to restore arthrospore formation; a phenomenon not observed for ,cpcR1 derivatives or non-related genes. The intracellular expression of cpcR1, and control genes (pcbC, egfp) was determined by in vivo monitoring of fluorescent protein fusions. Further, the role of the forkhead transcription factor AcFKH1, which directly interacts with CPCR1, was studied by generating an Acfkh1 knockout strain. In contrast to CPCR1, AcFKH1 is not directly involved in the fragmentation of hyphae. Instead, the presence of AcFKH1 seems to be necessary for CPCR1 function in A. chrysogenum morphogenesis, as overexpression of a functional cpcR1 gene in a ,Acfkh1 background has no effect on arthrospore formation. Moreover, strains lacking Acfkh1 exhibit defects in cell separation, indicating an involvement of the forkhead transcription factor in mycelial growth of A. chrysogenum. Our data offer the potential to control fungal growth in biotechnical processes that require defined morphological stages for optimal production yields. [source] Intercellular adhesion and cell separation in plantsPLANT CELL & ENVIRONMENT, Issue 7 2003M. C. JARVIS ABSTRACT Adhesion between plant cells is a fundamental feature of plant growth and development, and an essential part of the strategy by which growing plants achieve mechanical strength. Turgor pressure provides non-woody plant tissues with mechanical rigidity and the driving force for growth, but at the same time it generates large forces tending to separate cells. These are resisted by reinforcing zones located precisely at the points of maximum stress. In dicots the reinforcing zones are occupied by networks of specific pectic polymers. The mechanisms by which these networks cohere vary and are not fully understood. In the Poaceae their place is taken by phenolic cross-linking of arabinoxylans. Whatever the reinforcing polymers, a targeting mechanism is necessary to ensure that they become immobilized at the appropriate location, and there are secretory mutants that appear to have defects in this mechanism and hence are defective in cell adhesion. At the outer surface of most plant parts, the tendency of cells to cohere is blocked, apparently by the cuticle. Mutants with lesions in the biosynthesis of cuticular lipids show aberrant surface adhesion and other developmental abnormalities. When plant cells separate, the polymer networks that join them are locally dismantled with surgical precision. This occurs during the development of intercellular spaces; during the abscission of leaves and floral organs; during the release of seeds and pollen; during differentiation of root cap cells; and during fruit ripening. Each of these cell separation processes has its own distinctive features. Cell separation can also be induced during cooking or processing of fruit and vegetables, and the degree to which it occurs is a significant quality characteristic in potatoes, pulses, tomatoes, apples and other fruit. Control over these technological characteristics will be facilitated by understanding the role of cell adhesion and separation in the life of plants. [source] Blood cell separation using crosslinkable copolymers containing N,N -dimethylacrylamidePOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 4 2007Shizue Hayashi Natori Abstract Amphiphilic copolymers using hydrophilic N,N -dimethylacrylamide (DMA), hydrophobic methyl methacrylate (MMA) and a crosslinkable monomer, 3-methacryloyloxypropyl trimethoxysilane (MTSi), were synthesized and evaluated as coating materials for leukocyte removal filters for whole blood. When filters composed of non-woven fabrics were coated with crosslinked synthesized copolymers, the elution ratios of the copolymers to water were adequately low because of the crosslinking with trimethoxysilane groups of MTSi units in the copolymers. Filters coated with crosslinked poly(DMA- co -MTSi) having a 0.96 mole fraction of DMA units showed a 0.35,±,0.44% platelet permeation ratio and a logarithmic reduction of 4.0,±,0.68 for leukocytes. On the other hand, an increase in the content of MMA units in the DMA-containing copolymers improved the permeation ratio of the platelets dramatically. Filters coated with crosslinked poly(DMA- co -MMA- co -MTSi) containing a 0.39 mole fraction of MMA units and a 0.58 mole fraction of DMA units showed an 86,±,3.0% platelet permeation ratio and a logarithmic reduction of 2.1,±,1.2 for leukocytes. This indicates that an adequate content of hydrophobic monomer units, such as MMA units, is necessary for effective platelet permeation. Copyright © 2007 John Wiley & Sons, Ltd. [source] A microfluidics approach for the isolation of nucleated red blood cells (NRBCs) from the peripheral blood of pregnant womenPRENATAL DIAGNOSIS, Issue 10 2008R. Huang Abstract Objective Nucleated red blood cells (NRBCs) have been identified in maternal circulation and potentially provide a resource for the monitoring and diagnosis of maternal, fetal, and neonatal health and disease. Past strategies used to isolate and enrich for NRBCs are limited to complex approaches that result in low recovery and less than optimal cell purity. Here we report the development of a high-throughput and highly efficient microfluidic device for isolating rare NRBCs from maternal blood. Material and Methods NRBCs were isolated from the peripheral blood of 58 pregnant women using a microfluidic process that consists of a microfluidic chip for size-based cell separation and a magnetic device for hemoglobin-based cell isolation. Results The microfluidic,magnetic combination removes nontarget red blood cells and white blood cells at a very high efficiency (,99.99%). The device successfully identified NRBCs from the peripheral blood of 58/58 pretermination samples with a mean of 37.44 NRBC/mL (range 0.37,274.36 NRBC/mL). These results were compared with those from previous studies. Conclusion The microfluidic device results in an approximate 10- to 20-fold enrichment of NRBCs over methods described previously. The reliability of isolation and the purity of the NRBC product have the potential to enable the subsequent application of molecular diagnostic assays. Copyright © 2008 John Wiley & Sons, Ltd. [source] The role of bacteriolysis in the pathophysiology of inflammation, infection and post-infectious sequelaeAPMIS, Issue 11 2002Review article The literature dealing with the biochemical basis of bacteriolysis and its role in inflammation, infection and in post-infectious sequelae is reviewed and discussed. Bacteriolysis is an event that may occur when normal microbial multiplication is altered due to an uncontrolled activation of a series of autolytic cell-wall breaking enzymes (muramidases). While a low-level bacteriolysis sometimes occurs physiologically, due to "mistakes" in cell separation, a pronounced cell wall breakdown may occur following bacteriolysis induced either by beta-lactam antibiotics or by a large variety of bacteriolysis-inducing cationic peptides. These include spermine, spermidine, bactericidal peptides defensins, bacterial permeability increasing peptides from neutrophils, cationic proteins from eosinophils, lysozyme, myeloperoxidase, lactoferrin, the highly cationic proteinases elastase and cathepsins, PLA2, and certain synthetic polyamino acids. The cationic agents probably function by deregulating lipoteichoic acid (LTA) in Gram-positive bacteria and phospholipids in Gram-negative bacteria, the presumed regulators of the autolytic enzyme systems (muramidases). When bacteriolysis occurs in vivo, cell-wall- and -membrane-associated lipopolysaccharide (LPS (endotoxin)), lipoteichoic acid (LTA) and peptidoglycan (PPG), are released. These highly phlogistic agents can act on macrophages, either individually or in synergy, to induce the generation and release of reactive oxygen and nitrogen species, cytotoxic cytokines, hydrolases, proteinases, and also to activate the coagulation and complement cascades. All these agents and processes are involved in the pathophysiology of septic shock and multiple organ failure resulting from severe microbial infections. Bacteriolysis induced in in vitro models, either by polycations or by beta-lactams, could be effectively inhibited by sulfated polysaccharides, by D-amino acids as well as by certain anti-bacteriolytic antibiotics. However, within phagocytic cells in inflammatory sites, bacteriolysis tends to be strongly inhibited presumably due to the inactivation by oxidants and proteinases of the bacterial muramidases. This might results in a long persistence of non-biodegradable cell-wall components causing granulomatous inflammation. However, persistence of microbial cell walls in vivo may also boost innate immunity against infections and against tumor-cell proliferation. Therapeutic strategies to cope with the deleterious effects of bacteriolysis in vivo include combinations of autolysin inhibitors with combinations of certain anti-inflammatory agents. These might inhibit the synergistic tissue- and- organ-damaging "cross talks" which lead to septic shock and to additional post-infectious sequelae. [source] Continuous scalable blood filtration device using inertial microfluidicsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Albert J. Mach Abstract Cell separation is broadly useful for applications in clinical diagnostics, biological research, and potentially regenerative medicine. Recent attention has been paid to label-free size-based techniques that may avoid the costs or clogging issues associated with centrifugation and mechanical filtration. We present for the first time a massively parallel microfluidic device that passively separates pathogenic bacteria cells from diluted blood with macroscale performance. The device was designed to process large sample volumes in a high-throughput, continuous manner using 40 single microchannels placed in a radial array with one inlet and two rings of outlets. Each single channel consists of a short focusing, gradual expansion and collection region and uses unique differential transit times due to size-dependent inertial lift forces as a method of cell separation. The gradual channel expansion region is shown to manipulate cell equilibrium positions close to the microchannel walls, critical for higher efficiency collection. We demonstrate >80% removal of pathogenic bacteria from blood after two passes of the single channel system. The massively parallel device can process 240,mL/h with a throughput of 400 million cells/min. We expect that this parallelizable, robust, and label-free approach would be useful for filtration of blood as well as for other cell separation and concentration applications from large volume samples. Biotechnol. Bioeng. 2010;107: 302,311. © 2010 Wiley Periodicals, Inc. [source] Stem cell separation: A bottleneck in stem cell therapyBIOTECHNOLOGY JOURNAL, Issue 1 2010Kornelia Schriebl Dr. Abstract The substantial progress in embryonic stem cell (ESC) research could lead to new possibilities in the treatment of various diseases. Currently, applications of ESC for cell therapy are impeded by the presence of potentially teratoma-forming undifferentiated ESC. Thus, a selective and quantitative removal of undifferentiated ESC from a pool of differentiated and undifferentiated cells is essential before cell therapy. We evaluated the highly selective magnetic activated cell sorting (MACS) method for the quantitative removal of undifferentiated ESC. We found that the clearance rates for undifferentiated ESC decreased with decreasing amount of undifferentiated ESC in the cell pool. Using a simplified model calculation we could predict that, assuming an initial purity of 60%, an estimated 31 steps are required to achieve less than 10,1 cell per 109 cells. Thus, a log clearance rate of 10, which would be necessary for a therapeutically application, is hard to achieve. Our work clearly indicates that the current MACS technology is insufficient to meet the purification needs for cell therapy. [source] Antibody-immobilized column for quick cell separation based on cell rollingBIOTECHNOLOGY PROGRESS, Issue 2 2010Atsushi Mahara Abstract Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label-free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody-immobilized cell-rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell-rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34-positive and -negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34-positive and CD34-negative cells on antibody-immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell-sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] An Integrated Process for Mammalian Cell Perfusion Cultivation and Product Purification Using a Dynamic FilterBIOTECHNOLOGY PROGRESS, Issue 4 2002Leda R. Castilho In the present work, a dynamic filter was employed to develop an integrated perfusion/purification process. A recombinant CHO cell line producing a human anti-HIV IgG was employed in the experiments. In the first part of this work, the dynamic filter was fitted with conventional microfiltration membranes and tested as a new external cell retention device for perfusion cultivations. The filter was connected to a running perfusion bioreactor and operated for approximately 400 h at an average cell concentration of 10 million cells mL,1, whereby cell viability remained above 90% and no problems of sterility were experienced. In the second part of this work, the dynamic filter was employed to simultaneously carry out cell separation and product purification, using membrane adsorbers containing Protein A affinity ligands. An automated system was built, which integrated the features of an automated perfusion bioreactor and of a liquid chromatography system. The IgG was continuously adsorbed onto the affinity membranes and was periodically recovered through elution cycles. After connection of the filter, the system was operated for approximately 300 h, whereby three elution cycles were carried out. No progressive increase in transmembrane pressure was observed, indicating no membrane fouling problems, and the IgG was recovered practically free of contaminants in a 14-fold concentrated form, indicating that the integrated, one-step perfusion/purification process developed during this work is a promising alternative for the production of biologicals derived from mammalian cells. [source] Human epithelial ovarian carcinoma cell-derived cytokines cooperatively induce activated CD4+CD25,CD45RA+ naïve T cells to express forkhead box protein 3 and exhibit suppressive ability in vitroCANCER SCIENCE, Issue 11 2009Xiaofeng Zhao Regulatory T cells play an important role in tumor escape from host antitumor immunity. Increased frequencies of CD4+CD25+ regulatory T cells have been documented in the tumor sites, malignant effusions, and peripheral blood of patients with ovarian carcinoma. However, the mechanism involved remains unclear. In the present study, we collected high-purity human CD4+CD25,CD45RA+ naïve T cells by microbead cell separation. These cells did not express FOXP3 by single-cell analysis, and few cells expressed FOXP3 when they were activated with anti-CD3/CD28 dual signal. However, more cells expressed FOXP3 when the supernatant of human epithelial ovarian carcinoma cell culture was added, yet not the supernatant of normal human ovarian surface epithelia cell culture. Neutralization assays revealed that neutralizing antibody against transforming growth factor , (TGF-,), interleukin-10, and interleukin-4 did not abrogate elevated FOXP3 expression induced by carcinoma cell culture supernatant, whereas neutralizing leukemia inhibitory factor (LIF) partially abrogated FOXP3 expression, but LIF alone could not increase FOXP3 expression in activated naïve T cells. Further, an in vitro coculture suppression assay showed that these cells could suppress the proliferation of autologous CD4+CD25,CD45RA, T cells. In summary, our findings show that ovarian carcinoma cells are able to induce expression of FOXP3 and exhibit suppressive ability in activated naïve T cells by producing soluble substances, and multiple cytokines involve in the induction of FOXP3 expression. (Cancer Sci 2009) [source] Cover Picture: Electrophoresis 16'2010ELECTROPHORESIS, Issue 17 2010Article first published online: 7 SEP 2010 Issue no. 17 is a regular issue comprising 18 manuscripts distributed over 5 separate parts. Part I has 7 research articles on some aspects of proteins and cell separations. Part II has 3 research articles on nucleic acid research including cloning/amplification, gene regulation and STR analysis. Part III offers ways of measuring diffusion and binding constants in two separate articles. Concentration and detection approaches are treated in 4 research articles making up Part IV. The last two articles in this issue (Part V) are on CEC and EKC describing a mixed mode monolithic stationary phase and a cyclodextrin-modified MEEKC. Featured articles include: Lamp-based wavelength-resolved fluorescence detection for protein capillary electrophoresis: Set-up and detector performance ((doi: 10.1002/elps.201000246)) Electromigration diffusivity spectrometry: A way for simultaneous determination of diffusion coefficients from mixed samples ((doi: 10.1002/elps.201000252)) Sample stacking capillary electrophoretic microdevice for highly sensitive mini Y short tandem repeat genotyping ((doi: 10.1002/elps.201000270)) [source] | ||||||||||||||||