Comparison of different particles and methods for magnetic isolation of circulating tumor cells

NASA Astrophysics Data System (ADS)

Sieben, S.; Bergemann, C.; Lübbe, A.; Brockmann, B.; Rescheleit, D.

2001-01-01

A more effective method for tumor cell separation from peripheral blood was established. The results of optimized magnetic particles verified by analyzing yield, purity and viability of isolated epithelial tumor cells were compared with a commercial kit for immunomagnetic cell separation. Porous silica particles of 230 nm were found to give best recovery rates and high viability of extracted cells.

Separation of cancer cells from white blood cells by pinched flow fractionation.

PubMed

Pødenphant, Marie; Ashley, Neil; Koprowska, Kamila; Mir, Kalim U; Zalkovskij, Maksim; Bilenberg, Brian; Bodmer, Walter; Kristensen, Anders; Marie, Rodolphe

2015-12-21

In this paper, the microfluidic size-separation technique pinched flow fractionation (PFF) is used to separate cancer cells from white blood cells (WBCs). The cells are separated at efficiencies above 90% for both cell types. Circulating tumor cells (CTCs) are found in the blood of cancer patients and can form new tumors. CTCs are rare cells in blood, but they are important for the understanding of metastasis. There is therefore a high interest in developing a method for the enrichment of CTCs from blood samples, which also enables further analysis of the separated cells. The separation is challenged by the size overlap between cancer cells and the 10(6) times more abundant WBCs. The size overlap prevents high efficiency separation, however we demonstrate that cell deformability can be exploited in PFF devices to gain higher efficiencies than expected from the size distribution of the cells.

Biocompatible and label-free separation of cancer cells from cell culture lines from white blood cells in ferrofluids.

PubMed

Zhao, Wujun; Cheng, Rui; Lim, So Hyun; Miller, Joshua R; Zhang, Weizhong; Tang, Wei; Xie, Jin; Mao, Leidong

2017-06-27

This paper reports a biocompatible and label-free cell separation method using ferrofluids that can separate a variety of low-concentration cancer cells from cell culture lines (∼100 cancer cells per mL) from undiluted white blood cells, with a throughput of 1.2 mL h -1 and an average separation efficiency of 82.2%. The separation is based on the size difference of the cancer cells and white blood cells, and is conducted in a custom-made biocompatible ferrofluid that retains not only excellent short-term viabilities but also normal proliferations of 7 commonly used cancer cell lines. A microfluidic device is designed and optimized specifically to shorten the time of live cells' exposure to ferrofluids from hours to seconds, by eliminating time-consuming off-chip sample preparation and extraction steps and integrating them on-chip to achieve a one-step process. As a proof-of-concept demonstration, a ferrofluid with 0.26% volume fraction was used in this microfluidic device to separate spiked cancer cells from cell lines at a concentration of ∼100 cells per mL from white blood cells with a throughput of 1.2 mL h -1 . The separation efficiencies were 80 ± 3%, 81 ± 5%, 82 ± 5%, 82 ± 4%, and 86 ± 6% for A549 lung cancer, H1299 lung cancer, MCF-7 breast cancer, MDA-MB-231 breast cancer, and PC-3 prostate cancer cell lines, respectively. The separated cancer cells' purity was between 25.3% and 28.8%. In addition, the separated cancer cells from this strategy showed an average short-term viability of 94.4 ± 1.3%, and these separated cells were cultured and demonstrated normal proliferation to confluence even after the separation process. Owing to its excellent biocompatibility and label-free operation and its ability to recover low concentrations of cancer cells from white blood cells, this method could lead to a promising tool for rare cell separation.

Characterization and Separation of Cancer Cells with a Wicking Fiber Device.

PubMed

Tabbaa, Suzanne M; Sharp, Julia L; Burg, Karen J L

2017-12-01

Current cancer diagnostic methods lack the ability to quickly, simply, efficiently, and inexpensively screen cancer cells from a mixed population of cancer and normal cells. Methods based on biomarkers are unreliable due to complexity of cancer cells, plasticity of markers, and lack of common tumorigenic markers. Diagnostics are time intensive, require multiple tests, and provide limited information. In this study, we developed a novel wicking fiber device that separates cancer and normal cell types. To the best of our knowledge, no previous work has used vertical wicking of cells through fibers to identify and isolate cancer cells. The device separated mouse mammary tumor cells from a cellular mixture containing normal mouse mammary cells. Further investigation showed the device separated and isolated human cancer cells from a heterogeneous mixture of normal and cancerous human cells. We report a simple, inexpensive, and rapid technique that has potential to identify and isolate cancer cells from large volumes of liquid samples that can be translated to on-site clinic diagnosis.

A method for a separator for cells

NASA Technical Reports Server (NTRS)

Takakaki, T.; Tsujino, Y.

1982-01-01

A method is presented for manufacturing a separator for cells which is characterized by the fact that the spaces or small holes in the porous body are made even smaller, and therefore the porous body is made physically stronger.

Breast cancer cells synchronous labeling and separation based on aptamer and fluorescence-magnetic silica nanoparticles

NASA Astrophysics Data System (ADS)

Wang, Qiu-Yue; Huang, Wei; Jiang, Xing-Lin; Kang, Yan-Jun

2018-01-01

In this work, an efficient method based on biotin-labeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) has been established for human breast carcinoma MCF-7 cells synchronous labeling and separation. Carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH) were first synthesized using the Stöber method. Streptavidin (SA) was then conjugated to the surface of FITC@Fe3O4@SiNPs-COOH. The MCF-7 cell suspension was incubated with biotin-labeled MUC-1 aptamer. After centrifugation and washing, the cells were then treated with FITC@Fe3O4@SiNPs-SA. Afterwards, the mixtures were separated by a magnet. The cell-probe conjugates were then imaged using fluorescent microscopy. The results show that the MUC-1 aptamer could recognize and bind to the targeted cells with high affinity and specificity, indicating the prepared FITC@Fe3O4@SiNPs-SA with great photostability and superparamagnetism could be applied effectively in labeling and separation for MCF-7 cell in suspension synchronously. In addition, the feasibility of MCF-7 cells detection in peripheral blood was assessed. The results indicate that the method above is also applicable for cancer cells synchronous labeling and separation in complex biological system.

Hybrid microfluidics combined with active and passive approaches for continuous cell separation.

PubMed

Yan, Sheng; Zhang, Jun; Yuan, Dan; Li, Weihua

2017-01-01

Microfluidics, which is classified as either active or passive, is capable of separating cells of interest from a complex and heterogeneous sample. Active methods utilise external fields such as electric, magnetic, acoustic, and optical to drive cells for separation, while passive methods utilise channel structures, intrinsic hydrodynamic forces, and steric hindrances to manipulate cells. However, when processing complex biological samples such as whole blood with rare cells, separation with a single module microfluidic device is difficult. Hybrid microfluidics is an emerging technique, which utilises active and passive methods whilst fulfilling higher requirements for stable performance, versatility, and convenience, including (i) the ability to process multi-target cells, (ii) enhanced ability for multiplexed separation, (iii) higher sensitivity, and (iv) tunability for a wider operational range. This review introduces the fundamental physics and typical formats for subclasses of hybrid microfluidic devices based on their different physical fields; presents current examples of cell sorting to highlight the advantage and usefulness of hybrid microfluidics on biomedicine, and then discusses the challenges and perspective of future development and the promising direction of research in this field. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous separation and quantitation of amino acids and polyamines of forest tree tissues and cell cultures within a single high-performance liquid chromatography run using dansyl derivatization

Treesearch

Rakesh Minocha; Stephanie Long

2004-01-01

The objective of the present study was to develop a rapid HPLC method for simultaneous separation and quantitation of dansylated amino acids and common polyamines in the same matrix for analyzing forest tree tissues and cell cultures. The major modifications incorporated into this method as compared to previously published HPLC methods for separation of only dansyl...

Design and simulation of a microfluidic device for acoustic cell separation.

PubMed

Shamloo, Amir; Boodaghi, Miad

2018-03-01

Experimental acoustic cell separation methods have been widely used to perform separation for different types of blood cells. However, numerical simulation of acoustic cell separation has not gained enough attention and needs further investigation since by using numerical methods, it is possible to optimize different parameters involved in the design of an acoustic device and calculate particle trajectories in a simple and low cost manner before spending time and effort for fabricating these devices. In this study, we present a comprehensive finite element-based simulation of acoustic separation of platelets, red blood cells and white blood cells, using standing surface acoustic waves (SSAWs). A microfluidic channel with three inlets, including the middle inlet for sheath flow and two symmetrical tilted angle inlets for the cells were used to drive the cells through the channel. Two interdigital transducers were also considered in this device and by implementing an alternating voltage to the transducers, an acoustic field was created which can exert the acoustic radiation force to the cells. Since this force is dependent to the size of the cells, the cells are pushed towards the midline of the channel with different path lines. Particle trajectories for different cells were obtained and compared with a theoretical equation. Two types of separations were observed as a result of varying the amplitude of the acoustic field. In the first mode of separation, white blood cells were sorted out through the middle outlet and in the second mode of separation, platelets were sorted out through the side outlets. Depending on the clinical needs and by using the studied microfluidic device, each of these modes can be applied to separate the desired cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Microfluidic Blood Cell Preparation: Now and Beyond

PubMed Central

Yu, Zeta Tak For; Yong, Koh Meng Aw; Fu, Jianping

2014-01-01

Blood plays an important role in homeostatic regulation with each of its cellular components having important therapeutic and diagnostic uses. Therefore, separation and sorting of blood cells has been of a great interest to clinicians and researchers. However, while conventional methods of processing blood have been successful in generating relatively pure fractions, they are time consuming, labor intensive, and are not optimal for processing small volume blood samples. In recent years, microfluidics has garnered great interest from clinicians and researchers as a powerful technology for separating blood into different cell fractions. As microfluidics involves fluid manipulation at the microscale level, it has the potential for achieving high-resolution separation and sorting of blood cells down to a single-cell level, with an added benefit of integrating physical and biological methods for blood cell separation and analysis on the same single chip platform. This paper will first review the conventional methods of processing and sorting blood cells, followed by a discussion on how microfluidics is emerging as an efficient tool to rapidly change the field of blood cell sorting for blood-based therapeutic and diagnostic applications. PMID:24515899

Label-free density difference amplification-based cell sorting.

PubMed

Song, Jihwan; Song, Minsun; Kang, Taewook; Kim, Dongchoul; Lee, Luke P

2014-11-01

The selective cell separation is a critical step in fundamental life sciences, translational medicine, biotechnology, and energy harvesting. Conventional cell separation methods are fluorescent activated cell sorting and magnetic-activated cell sorting based on fluorescent probes and magnetic particles on cell surfaces. Label-free cell separation methods such as Raman-activated cell sorting, electro-physiologically activated cell sorting, dielectric-activated cell sorting, or inertial microfluidic cell sorting are, however, limited when separating cells of the same kind or cells with similar sizes and dielectric properties, as well as similar electrophysiological phenotypes. Here we report a label-free density difference amplification-based cell sorting (dDACS) without using any external optical, magnetic, electrical forces, or fluidic activations. The conceptual microfluidic design consists of an inlet, hydraulic jump cavity, and multiple outlets. Incoming particles experience gravity, buoyancy, and drag forces in the separation chamber. The height and distance that each particle can reach in the chamber are different and depend on its density, thus allowing for the separation of particles into multiple outlets. The separation behavior of the particles, based on the ratio of the channel heights of the inlet and chamber and Reynolds number has been systematically studied. Numerical simulation reveals that the difference between the heights of only lighter particles with densities close to that of water increases with increasing the ratio of the channel heights, while decreasing Reynolds number can amplify the difference in the heights between the particles considered irrespective of their densities.

The rotating spectrometer: Biotechnology for cell separations

NASA Technical Reports Server (NTRS)

Noever, David A.

1991-01-01

An instrument for biochemical studies, called the rotating spectrometer, separates previously inseparable cell cultures. The rotating spectrometer is intended for use in pharmacological studies which require fractional splitting of heterogeneous cell cultures based on cell morphology and swimming behavior. As a method to separate and concentrate cells in free solution, the rotating method requires active organism participation and can effectively split the large class of organisms known to form spontaneous patterns. Examples include the biochemical star, an organism called Tetrahymena pyriformis. Following focusing in a rotating frame, the separation is accomplished using different radial dependencies of concentrated algal and protozoan species. The focusing itself appears as concentric rings and arises from the coupling between swimming direction and Coriolis forces. A dense cut is taken at varying radii, and extraction is replenished at an inlet. Unlike standard separation and concentrating techniques such as filtration or centrifugation, the instrument is able to separate motile from immotile fractions. For a single pass, typical split efficiencies can reach 200 to 300 percent compared to the inlet concentration.

The rotating spectrometer: New biotechnology for cell separations

NASA Technical Reports Server (NTRS)

Noever, David A.; Matsos, Helen C.

1990-01-01

An instrument for biochemical studies, called the rotating spectrometer, separates previously inseparable cell cultures. The rotating spectrometer is intended for use in pharmacological studies which require fractional splitting of heterogeneous cell cultures based on cell morphology and swimming behavior. As a method to separate and concentrate cells in free solution, the rotating method requires active organism participation and can effectively split the large class of organisms known to form spontaneous patterns. Examples include the biochemical star, an organism called Tetrahymena pyriformis. Following focusing in a rotated frame, the separation is accomplished using different radial dependencies of concentrated algal and protozoan species. The focusing itself appears as concentric rings and arises from the coupling between swimming direction and Coriolis forces. A dense cut is taken at varying radii and extraction is replenished at an inlet. Unlike standard separation and concentrating techniques such as filtration or centrifugation, the instrument is able to separate motile from immotile fractions. For a single pass, typical split efficiencies can reach 200 to 300 percent compared to the inlet concentration.

A Self-Directed Method for Cell-Type Identification and Separation of Gene Expression Microarrays

PubMed Central

Zuckerman, Neta S.; Noam, Yair; Goldsmith, Andrea J.; Lee, Peter P.

2013-01-01

Gene expression analysis is generally performed on heterogeneous tissue samples consisting of multiple cell types. Current methods developed to separate heterogeneous gene expression rely on prior knowledge of the cell-type composition and/or signatures - these are not available in most public datasets. We present a novel method to identify the cell-type composition, signatures and proportions per sample without need for a-priori information. The method was successfully tested on controlled and semi-controlled datasets and performed as accurately as current methods that do require additional information. As such, this method enables the analysis of cell-type specific gene expression using existing large pools of publically available microarray datasets. PMID:23990767

Fresenius AS.TEC204 blood cell separator.

PubMed

Sugai, Mikiya

2003-02-01

Fresenius AS.TEC204 is a third-generation blood cell separator that incorporates the continuous centrifugal separation method and automatic control of the cell separation process. Continuous centrifugation separates cell components according to their specific gravity, and different cell components are either harvested or eliminated as needed. The interface between the red blood cell and plasma is optically detected, and the Interface Control (IFC) cooperates with different pumps, monitors and detectors to harvest required components automatically. The system is composed of three major sections; the Front Panel Unit; the Pump Unit, and the Centrifuge Unit. This unit can be used for a wide variety of clinical applications including collection of platelets, peripheral blood stem cells, bone marrow stem cells, granulocytes, mononuclear cells, and exchange of plasma or red cells, and for plasma treatment.

Size-amplified acoustofluidic separation of circulating tumor cells with removable microbeads

NASA Astrophysics Data System (ADS)

Liu, Huiqin; Ao, Zheng; Cai, Bo; Shu, Xi; Chen, Keke; Rao, Lang; Luo, Changliang; Wang, Fu-Bin; Liu, Wei; Bondesson, Maria; Guo, Shishang; Guo, Feng

2018-06-01

Isolation and analysis of rare circulating tumor cells (CTCs) is of great interest in cancer diagnosis, prognosis, and treatment efficacy evaluation. Acoustofluidic cell separation becomes an attractive method due to its contactless, noninvasive, simple, and versatile features. However, the indistinctive physical difference between CTCs and normal blood cells limits the purity of CTCs using current acoustic methods. Herein, we demonstrate a size-amplified acoustic separation and release of CTCs with removable microbeads. CTCs selectively bound to size-amplifiers (40 μm-diameter anti-EpCAM/gelatin-coated SiO2 microbeads) have significant physical differences (size and mechanics) compared to normal blood cells, resulting in an amplification of acoustic radiation force approximately a hundredfold over that of bare CTCs or normal blood cells. Therefore, CTCs can be efficiently sorted out with size-amplifiers in a traveling surface acoustic wave microfluidic device and released from size-amplifiers by enzymatic degradation for further purification or downstream analysis. We demonstrate a cell separation from blood samples with a total efficiency (E total) of ∼ 77%, purity (P) of ∼ 96%, and viability (V) of ∼83% after releasing cells from size-amplifiers. Our method substantially improves the emerging application of rare cell purification for translational medicine.

Method for forming a cell separator for use in bipolar-stack energy storage devices

DOEpatents

Mayer, Steven T.; Feikert, John H.; Kaschmitter, James L.; Pekala, Richard W.

1994-01-01

An improved multi-cell electrochemical energy storage device, such as a battery, fuel cell, or double layer capacitor using a cell separator which allows cells to be stacked and interconnected with low electrical resistance and high reliability while maximizing packaging efficiency. By adding repeating cells, higher voltages can be obtained. The cell separator is formed by applying an organic adhesive on opposing surfaces of adjacent carbon electrodes or surfaces of aerogel electrodes of a pair of adjacent cells prior to or after pyrolysis thereof to form carbon aerogel electrodes. The cell separator is electronically conductive, but ionically isolating, preventing an electrolytic conduction path between adjacent cells in the stack.

Method for forming a cell separator for use in bipolar-stack energy storage devices

DOEpatents

Mayer, S.T.; Feikert, J.H.; Kaschmitter, J.L.; Pekala, R.W.

1994-08-09

An improved multi-cell electrochemical energy storage device, such as a battery, fuel cell, or double layer capacitor using a cell separator which allows cells to be stacked and interconnected with low electrical resistance and high reliability while maximizing packaging efficiency. By adding repeating cells, higher voltages can be obtained. The cell separator is formed by applying an organic adhesive on opposing surfaces of adjacent carbon electrodes or surfaces of aerogel electrodes of a pair of adjacent cells prior to or after pyrolysis thereof to form carbon aerogel electrodes. The cell separator is electronically conductive, but ionically isolating, preventing an electrolytic conduction path between adjacent cells in the stack. 2 figs.

Advanced Method for Isolation of Mouse Hepatocytes, Liver Sinusoidal Endothelial Cells, and Kupffer Cells.

PubMed

Liu, Jia; Huang, Xuan; Werner, Melanie; Broering, Ruth; Yang, Dongliang; Lu, Mengji

2017-01-01

Separation of pure cell populations from the liver is a prerequisite to study the role of hepatic parenchymal and non-parenchymal cells in liver physiology, pathophysiology, and immunology. Traditional methods for hepatic cell separation usually purify only single cell types from liver specimens. Here, we describe an efficient method that can simultaneously purify populations of hepatocytes (HCs), liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs) from a single mouse liver specimen. A liberase-based perfusion technique in combination with a low-speed centrifugation and magnetic-activated cell sorting (MACS) led to the isolation and purification of HCs, KCs, and LSECs with high yields and purity.

Platelet-rich plasma differs according to preparation method and human variability.

PubMed

Mazzocca, Augustus D; McCarthy, Mary Beth R; Chowaniec, David M; Cote, Mark P; Romeo, Anthony A; Bradley, James P; Arciero, Robert A; Beitzel, Knut

2012-02-15

Varying concentrations of blood components in platelet-rich plasma preparations may contribute to the variable results seen in recently published clinical studies. The purposes of this investigation were (1) to quantify the level of platelets, growth factors, red blood cells, and white blood cells in so-called one-step (clinically used commercial devices) and two-step separation systems and (2) to determine the influence of three separate blood draws on the resulting components of platelet-rich plasma. Three different platelet-rich plasma (PRP) separation methods (on blood samples from eight subjects with a mean age [and standard deviation] of 31.6 ± 10.9 years) were used: two single-spin processes (PRPLP and PRPHP) and a double-spin process (PRPDS) were evaluated for concentrations of platelets, red and white blood cells, and growth factors. Additionally, the effect of three repetitive blood draws on platelet-rich plasma components was evaluated. The content and concentrations of platelets, white blood cells, and growth factors for each method of separation differed significantly. All separation techniques resulted in a significant increase in platelet concentration compared with native blood. Platelet and white blood-cell concentrations of the PRPHP procedure were significantly higher than platelet and white blood-cell concentrations produced by the so-called single-step PRPLP and the so-called two-step PRPDS procedures, although significant differences between PRPLP and PRPDS were not observed. Comparing the results of the three blood draws with regard to the reliability of platelet number and cell counts, wide variations of intra-individual numbers were observed. Single-step procedures are capable of producing sufficient amounts of platelets for clinical usage. Within the evaluated procedures, platelet numbers and numbers of white blood cells differ significantly. The intra-individual results of platelet-rich plasma separations showed wide variations in platelet and cell numbers as well as levels of growth factors regardless of separation method.

Immunomagnetic separation can enrich fixed solid tumors for epithelial cells.

PubMed

Yaremko, M L; Kelemen, P R; Kutza, C; Barker, D; Westbrook, C A

1996-01-01

Immunomagnetic separation is a highly specific technique for the enrichment or isolation of cells from a variety of fresh tissues and microorganisms or molecules from suspensions. Because new techniques for molecular analysis of solid tumors are now applicable to fixed tissue but sometimes require or benefit from enrichment for tumor cells, we tested the efficacy of immunomagnetic separation for enriching fixed solid tumors for malignant epithelial cells. We applied it to two different tumors and fixation methods to separate neoplastic from non-neoplastic cells in primary colorectal cancers and metastatic breast cancers, and were able to enrich to a high degree of purity. Immunomagnetic separation was effective in unembedded fixed tissue as well as fixed paraffin-embedded tissue. The magnetically separated cells were amenable to fluorescence in situ hybridization and polymerase chain reaction amplification of their DNA with minimal additional manipulation. The high degree of enrichment achieved before amplification contributed to interpretation of loss of heterozygosity in metastatic breast cancers, and simplified fluorescence in situ hybridization analysis because only neoplastic cells were hybridized and counted. Immunomagnetic separation is effective for the enrichment of fixed solid tumors, can be performed with widely available commercial antibodies, and requires little specialized instrumentation. It can contribute to interpretation of results in situations where enrichment by other methods is difficult or not possible.

Paramagnetic capture mode magnetophoretic microseparator for high efficiency blood cell separations.

PubMed

Han, Ki-Ho; Frazier, A Bruno

2006-02-01

This paper presents the characterization of continuous single-stage and three-stage cascade paramagnetic capture (PMC) mode magnetophoretic microseparators for high efficiency separation of red and white blood cells from diluted whole blood based on their native magnetic properties. The separation mechanism for both PMC microseparators is based on a high gradient magnetic separation (HGMS) method. This approach enables separation of blood cells without the use of additives such as magnetic beads. Experimental results for the single-stage PMC microseparator show that 91.1% of red blood cells were continuously separated from the sample at a volumetric flow rate of 5 microl h-1. In addition, the three-stage cascade PMC microseparator continuously separated 93.5% of red blood cells and 97.4% of white blood cells from whole blood at a volumetric flow rate of 5 microl h-1.

Asymmetrical Deterministic Lateral Displacement Gaps for Dual Functions of Enhanced Separation and Throughput of Red Blood Cells

PubMed Central

Zeming, Kerwin Kwek; Salafi, Thoriq; Chen, Chia-Hung; Zhang, Yong

2016-01-01

Deterministic lateral displacement (DLD) method for particle separation in microfluidic devices has been extensively used for particle separation in recent years due to its high resolution and robust separation. DLD has shown versatility for a wide spectrum of applications for sorting of micro particles such as parasites, blood cells to bacteria and DNA. DLD model is designed for spherical particles and efficient separation of blood cells is challenging due to non-uniform shape and size. Moreover, separation in sub-micron regime requires the gap size of DLD systems to be reduced which exponentially increases the device resistance, resulting in greatly reduced throughput. This paper shows how simple application of asymmetrical DLD gap-size by changing the ratio of lateral-gap (GL) to downstream-gap (GD) enables efficient separation of RBCs without greatly restricting throughput. This method reduces the need for challenging fabrication of DLD pillars and provides new insight to the current DLD model. The separation shows an increase in DLD critical diameter resolution (separate smaller particles) and increase selectivity for non-spherical RBCs. The RBCs separate better as compared to standard DLD model with symmetrical gap sizes. This method can be applied to separate non-spherical bacteria or sub-micron particles to enhance throughput and DLD resolution. PMID:26961061

Asymmetrical Deterministic Lateral Displacement Gaps for Dual Functions of Enhanced Separation and Throughput of Red Blood Cells.

PubMed

Zeming, Kerwin Kwek; Salafi, Thoriq; Chen, Chia-Hung; Zhang, Yong

2016-03-10

Deterministic lateral displacement (DLD) method for particle separation in microfluidic devices has been extensively used for particle separation in recent years due to its high resolution and robust separation. DLD has shown versatility for a wide spectrum of applications for sorting of micro particles such as parasites, blood cells to bacteria and DNA. DLD model is designed for spherical particles and efficient separation of blood cells is challenging due to non-uniform shape and size. Moreover, separation in sub-micron regime requires the gap size of DLD systems to be reduced which exponentially increases the device resistance, resulting in greatly reduced throughput. This paper shows how simple application of asymmetrical DLD gap-size by changing the ratio of lateral-gap (GL) to downstream-gap (GD) enables efficient separation of RBCs without greatly restricting throughput. This method reduces the need for challenging fabrication of DLD pillars and provides new insight to the current DLD model. The separation shows an increase in DLD critical diameter resolution (separate smaller particles) and increase selectivity for non-spherical RBCs. The RBCs separate better as compared to standard DLD model with symmetrical gap sizes. This method can be applied to separate non-spherical bacteria or sub-micron particles to enhance throughput and DLD resolution.

Cell separation using electric fields

NASA Technical Reports Server (NTRS)

Eppich, Henry M. (Inventor); Mangano, Joseph A. (Inventor)

2003-01-01

The present invention involves methods and devices which enable discrete objects having a conducting inner core, surrounded by a dielectric membrane to be selectively inactivated by electric fields via irreversible breakdown of their dielectric membrane. One important application of the invention is in the selection, purification, and/or purging of desired or undesired biological cells from cell suspensions. According to the invention, electric fields can be utilized to selectively inactivate and render non-viable particular subpopulations of cells in a suspension, while not adversely affecting other desired subpopulations. According to the inventive methods, the cells can be selected on the basis of intrinsic or induced differences in a characteristic electroporation threshold, which can depend, for example, on a difference in cell size and/or critical dielectric membrane breakdown voltage. The invention enables effective cell separation without the need to employ undesirable exogenous agents, such as toxins or antibodies. The inventive method also enables relatively rapid cell separation involving a relatively low degree of trauma or modification to the selected, desired cells. The inventive method has a variety of potential applications in clinical medicine, research, etc., with two of the more important foreseeable applications being stem cell enrichment/isolation, and cancer cell purging.

Cell separation using electric fields

NASA Technical Reports Server (NTRS)

Mangano, Joseph (Inventor); Eppich, Henry (Inventor)

2009-01-01

The present invention involves methods and devices which enable discrete objects having a conducting inner core, surrounded by a dielectric membrane to be selectively inactivated by electric fields via irreversible breakdown of their dielectric membrane. One important application of the invention is in the selection, purification, and/or purging of desired or undesired biological cells from cell suspensions. According to the invention, electric fields can be utilized to selectively inactivate and render non-viable particular subpopulations of cells in a suspension, while not adversely affecting other desired subpopulations. According to the inventive methods, the cells can be selected on the basis of intrinsic or induced differences in a characteristic electroporation threshold, which can depend, for example, on a difference in cell size and/or critical dielectric membrane breakdown voltage. The invention enables effective cell separation without the need to employ undesirable exogenous agents, such as toxins or antibodies. The inventive method also enables relatively rapid cell separation involving a relatively low degree of trauma or modification to the selected, desired cells. The inventive method has a variety of potential applications in clinical medicine, research, etc., with two of the more important foreseeable applications being stem cell enrichment/isolation, and cancer cell purging.

Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation

PubMed Central

Vykoukal, Jody; Vykoukal, Daynene M.; Freyberg, Susanne; Alt, Eckhard U.; Gascoyne, Peter R. C.

2009-01-01

We have applied the microfluidic cell separation method of dielectrophoretic field-flow fractionation (DEP-FFF) to the enrichment of a putative stem cell population from an enzyme-digested adipose tissue derived cell suspension. A DEP-FFF separator device was constructed using a novel microfluidic-microelectronic hybrid flex-circuit fabrication approach that is scaleable and anticipates future low-cost volume manufacturing. We report the separation of a nucleated cell fraction from cell debris and the bulk of the erythrocyte population, with the relatively rare (<2% starting concentration) NG2-positive cell population (pericytes and/or putative progenitor cells) being enriched up to 14-fold. This work demonstrates a potential clinical application for DEP-FFF and further establishes the utility of the method for achieving label-free fractionation of cell subpopulations. PMID:18651083

Method of sealing an ultracapacitor substantially free of water

DOEpatents

Chapman-Irwin, Patricia; Feist, Thomas Paul

2002-04-02

A method of sealing an ultracapacitor substantially free of water is disclosed. The method includes providing a multilayer cell comprising two solid, non porous current collectors, separated by two porous electrodes with a separator between the two electrodes, sealing the cell with a reclosable hermetic closure. Water inside the closure is dissociated by an applied voltage to the cell and escapes in the form of hydrogen and oxygen when the closure is unmated, the closure is then mated to hermetically seal the cell which is substantially free of water.

Novel Automated Blood Separations Validate Whole Cell Biomarkers

PubMed Central

Burger, Douglas E.; Wang, Limei; Ban, Liqin; Okubo, Yoshiaki; Kühtreiber, Willem M.; Leichliter, Ashley K.; Faustman, Denise L.

2011-01-01

Background Progress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs). Successful biomarkers could help to track drug effects at early time points in clinical trials to prevent costly trial failures late in development. One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs) of fresh blood samples. Methods and Findings To replace the Ficoll method, we developed and studied a novel blood-based magnetic separation method. The magnetic method strikingly surpassed Ficoll in viability, purity and yield of PBLs. To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations. These more accurate and labor-saving magnet configurations allowed the lymphocytes to be tested in bioassays for rare antigen-specific T cells. The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficoll's uniform failure. We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes. Conclusions Improving, automating and standardizing lymphocyte detections from whole blood may facilitate development of new cell-based biomarkers for human diseases. Improved upfront blood processes may lead to broad improvements in monitoring early trial outcome measurements in human clinical trials. PMID:21799852

Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics.

PubMed

Sarkar, Aniruddh; Hou, Han Wei; Mahan, Alison E; Han, Jongyoon; Alter, Galit

2016-03-30

Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary 'bind-elute' separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets-cells or proteins-bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients.

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system

PubMed Central

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-01-01

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice. PMID:28425472

Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system.

PubMed

Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori; Fujii, Teruo; Laurell, Thomas

2017-04-20

The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and improved methods for CTC enumeration are asked for. Microfluidics has been extensively used for this purpose but most methods require several post-separation processing steps including concentration of the sample before analysis. This induces a high risk of sample loss of the collected rare cells. Here, an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood concentration of the peripheral blood mononuclear cell (PBMC) fraction were efficiently separated and trapped at a recovery of 76.2 ± 5.9% of the cancer cells and a minute contamination of 0.12 ± 0.04% PBMCs while simultaneously enabling a 20x volumetric concentration. This constitutes a first step towards a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice.

Label-free cell separation and sorting in microfluidic systems

PubMed Central

Gossett, Daniel R.; Weaver, Westbrook M.; Mach, Albert J.; Hur, Soojung Claire; Tse, Henry Tat Kwong; Lee, Wonhee; Amini, Hamed

2010-01-01

Cell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. Figure A wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties PMID:20419490

Comparative study of different procedures for the separation of peripheral blood mononuclear cells in cytokine-induced killer cell immunotherapy for hepatocarcinoma.

PubMed

Liu, Hui; Li, Jianyu; Wang, Fengmei; Gao, Yingtang; Luo, Ying; Wang, Peng; Li, Chenglong; Zhu, Zhengyan

2015-04-01

Cytokine-induced killer (CIK) cell immunotherapy exhibits significant advantages in the clinical treatment of tumors. This study was designed to compare the biological characteristics of autologous CIK cells from patients with hepatocarcinoma following different procedures for the separation of peripheral blood mononuclear cells (PBMCs). Forty-four hepatocarcinoma patients were enrolled and distributed into two groups. PBMCs were isolated either using a blood cell separator (apheresis method) or Ficoll lymphocyte separation medium (Ficoll method). The total amount, collection efficacy, and cell status of PBMCs in the two groups were determined. According to the number and status of collected PBMCs, different cultivation procedures were used for their amplification and activation and the proliferation ability, phenotype, and killing activity of CIK cells in the two groups were evaluated. Our results indicated that the number of collected PBMCs in the apheresis group was far more than that in the Ficoll group. However, the isolation rate was lower, and more cellular debris was observed in the apheresis group, which may be the cause of some untoward effects. Following in vitro culture, the enrichment time of CIK cells was longer in the Ficoll group, and the percentages of CD3(+)CD4(+) (Th) and CD4(+)CD25(+) (Treg) cells were higher. In the apheresis group, the percentages of CD3(-)CD56(+) (NK) and CD3(+)CD56(+) (NKT) cells were higher, and the CIK cells exhibited a higher cytolytic activity against HepG2 hepatoma cells. In conclusion, different procedures for PBMCs separation can influence the biological activities of CIK cells, and the apheresis method is more effective at enhancing the antitumor efficacy of CIK cells. However, significant attention should be paid to the possibility of adverse reactions in apheresis donors.

Bioprocessing development: Immune/cellular applications: Anti-Ig autoantibody and complement-mediated destruction of neoplastic cells

NASA Technical Reports Server (NTRS)

Twomey, J. J.

1976-01-01

This space bioprocessing contract effort was comprised of four general objectives. These were: (1) the evaluation of current separation processes, (2) the identification of problems relevant to the separation of important biologicals, (3) the identification of ground-based assay methods needed for pre- and postflight analysis of space bioprocessing separation technology; and (4) the establishment of methods to determine the efficiency of space bioprocessing separation procedures. Immunology was deemed advantageous to study the diversity of cells and cell products involved and the extensive interest being given to their separation. Upon recognition of a cellular or molecular agent as foreign to the body, the immune system becomes activated to produce cells whose function is to destroy that agent and cell products whose function is to inactivate the agent and assist in its destruction. Long after the agent is removed from the body, some cells remain in a state of readiness to continue these destructive actions specifically against that agent should further exposure to it occur. This is the basis of acquired immunity to disease.

Cell separation in immunoaffinity partition in aqueous polymer two-phase systems

NASA Technical Reports Server (NTRS)

Karr, Laurel J.; Van Alstine, James M.; Snyder, Robert S.; Shafer, Steven G.; Harris, J. Milton

1989-01-01

Two methods for immunoaffinity partitioning are described. One technique involves the covalent coupling of poly (ethylene glycol) (PEG) to immunoglobulin G antibody preparations. In the second method PEG-modified Protein A is used to complex with cells and unmodified antibody. The effects of PEG molecular weight, the degree of modification, and varying phase system composition on antibody activity and its affinity for the upper phase are studied. It is observed that both methods resulted in effective cell separation.

Immunomagnetic separation can enrich fixed solid tumors for epithelial cells.

PubMed Central

Yaremko, M. L.; Kelemen, P. R.; Kutza, C.; Barker, D.; Westbrook, C. A.

1996-01-01

Immunomagnetic separation is a highly specific technique for the enrichment or isolation of cells from a variety of fresh tissues and microorganisms or molecules from suspensions. Because new techniques for molecular analysis of solid tumors are now applicable to fixed tissue but sometimes require or benefit from enrichment for tumor cells, we tested the efficacy of immunomagnetic separation for enriching fixed solid tumors for malignant epithelial cells. We applied it to two different tumors and fixation methods to separate neoplastic from non-neoplastic cells in primary colorectal cancers and metastatic breast cancers, and were able to enrich to a high degree of purity. Immunomagnetic separation was effective in unembedded fixed tissue as well as fixed paraffin-embedded tissue. The magnetically separated cells were amenable to fluorescence in situ hybridization and polymerase chain reaction amplification of their DNA with minimal additional manipulation. The high degree of enrichment achieved before amplification contributed to interpretation of loss of heterozygosity in metastatic breast cancers, and simplified fluorescence in situ hybridization analysis because only neoplastic cells were hybridized and counted. Immunomagnetic separation is effective for the enrichment of fixed solid tumors, can be performed with widely available commercial antibodies, and requires little specialized instrumentation. It can contribute to interpretation of results in situations where enrichment by other methods is difficult or not possible. Images Figure 1 Figure 2 Figure 3 PMID:8546231

A New Cell Separation Method Based on Antibody-Immobilized Nanoneedle Arrays for the Detection of Intracellular Markers.

PubMed

Kawamura, Ryuzo; Miyazaki, Minami; Shimizu, Keita; Matsumoto, Yuta; Silberberg, Yaron R; Sathuluri, Ramachandra Rao; Iijima, Masumi; Kuroda, Shun'ichi; Iwata, Futoshi; Kobayashi, Takeshi; Nakamura, Chikashi

2017-11-08

Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift ("fish") individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.

Fundamentals and Application of Magnetic Particles in Cell Isolation and Enrichment

PubMed Central

Plouffe, Brian D.; Murthy, Shashi K.; Lewis, Laura H.

2014-01-01

Magnetic sorting using magnetic beads has become a routine methodology for the separation of key cell populations from biological suspensions. Due to the inherent ability of magnets to provide forces at a distance, magnetic cell manipulation is now a standardized process step in numerous processes in tissue engineering, medicine, and in fundamental biological research. Herein we review the current status of magnetic particles to enable isolation and separation of cells, with a strong focus on the fundamental governing physical phenomena, properties and syntheses of magnetic particles and on current applications of magnet-based cell separation in laboratory and clinical settings. We highlight the contribution of cell separation to biomedical research and medicine and detail modern cell separation methods (both magnetic and non-magnetic). In addition to a review of the current state-of-the-art in magnet-based cell sorting, we discuss current challenges and available opportunities for further research, development and commercialization of magnetic particle-based cell separation systems. PMID:25471081

Numerical simulation of dielectrophoretic separation of live/dead cells using a three-dimensional nonuniform AC electric field in micro-fabricated devices.

PubMed

Tada, Shigeru

2015-01-01

The analysis of cell separation has many important biological and medical applications. Dielectrophoresis (DEP) is one of the most effective and widely used techniques for separating and identifying biological species. In the present study, a DEP flow channel, a device that exploits the differences in the dielectric properties of cells in cell separation, was numerically simulated and its cell-separation performance examined. The samples of cells used in the simulation were modeled as human leukocyte (B cell) live and dead cells. The cell-separation analysis was carried out for a flow channel equipped with a planar electrode on the channel's top face and a pair of interdigitated counter electrodes on the bottom. This yielded a three-dimensional (3D) nonuniform AC electric field in the entire space of the flow channel. To investigate the optimal separation conditions for mixtures of live and dead cells, the strength of the applied electric field was varied. With appropriately selected conditions, the device was predicted to be very effective at separating dead cells from live cells. The major advantage of the proposed method is that a large volume of sample can be processed rapidly because of a large spacing of the channel height.

Safety shutdown separators

DOEpatents

Carlson, Steven Allen; Anakor, Ifenna Kingsley; Farrell, Greg Robert

2015-06-30

The present invention pertains to electrochemical cells which comprise (a) an anode; (b) a cathode; (c) a solid porous separator, such as a polyolefin, xerogel, or inorganic oxide separator; and (d) a nonaqueous electrolyte, wherein the separator comprises a porous membrane having a microporous coating comprising polymer particles which have not coalesced to form a continuous film. This microporous coating on the separator acts as a safety shutdown layer that rapidly increases the internal resistivity and shuts the cell down upon heating to an elevated temperature, such as 110.degree. C. Also provided are methods for increasing the safety of an electrochemical cell by utilizing such separators with a safety shutdown layer.

Recycling of high purity selenium from CIGS solar cell waste materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gustafsson, Anna M.K., E-mail: anna.gustafsson@chalmers.se; Foreman, Mark R.StJ.; Ekberg, Christian

Highlights: • A new method for recycling of selenium from CIGS solar cell materials is presented. • Separation of selenium as selenium dioxide after heating in oxygen atmosphere. • Complete selenium separation after oxidation of <63 μm particles at 800 °C for 1 h. • After reduction of selenium dioxide the selenium purity was higher than 99.999 wt%. - Abstract: Copper indium gallium diselenide (CIGS) is a promising material in thin film solar cell production. To make CIGS solar cells more competitive, both economically and environmentally, in comparison to other energy sources, methods for recycling are needed. In addition tomore » the generally high price of the material, significant amounts of the metals are lost in the manufacturing process. The feasibility of recycling selenium from CIGS through oxidation at elevated temperatures was therefore examined. During oxidation gaseous selenium dioxide was formed and could be separated from the other elements, which remained in solid state. Upon cooling, the selenium dioxide sublimes and can be collected as crystals. After oxidation for 1 h at 800 °C all of the selenium was separated from the CIGS material. Two different reduction methods for reduction of the selenium dioxide to selenium were tested. In the first reduction method an organic molecule was used as the reducing agent in a Riley reaction. In the second reduction method sulphur dioxide gas was used. Both methods resulted in high purity selenium. This proves that the studied selenium separation method could be the first step in a recycling process aimed at the complete separation and recovery of high purity elements from CIGS.« less

Collection, Storage, and Preparation of Human Blood Cells

PubMed Central

Dagur, Pradeep K.; McCoy, J. Philip

2015-01-01

Human peripheral blood is often studied by flow cytometry in both the research and clinical laboratories. The methods for collection, storage, and preparation of peripheral blood will vary depending on the cell lineage to be examined as well as the type of assay to be performed. This unit presents protocols for collection of blood, separation of leukocytes from whole blood by lysis of erythrocytes, isolating mononuclear cells by density gradient separation, and assorted non-flow sorting methods, such as magnetic bead separations, for enriching specific cell populations, including monocytes, T lymphocytes, B lymphocytes, neutrophils,, , and platelets prior to flow cytometric analysis. A protocol is also offered for cryopreservation of cells since clinical research often involves retrospective flow cytometric analysis of samples stored over a period of months or years. PMID:26132177

Cell separation technique in dilectrophoretic chip with bulk electrode

NASA Astrophysics Data System (ADS)

Iliescu, Ciprian; Tay, Francis E. H.; Xu, Guolin; Yu, Liming

2006-01-01

This paper presents a new technique for separation of two cell populations in a dielectrophoretic chip with bulk silicon electrode. A characteristic of the dielectrophoretic chip is its "sandwich" structure: glass/silicon/glass that generates a unique definition of the microfluidic channel with conductive walls (silicon) and isolating floor and ceiling (glass). The structure confers the opportunity to use the electrodes not only to generate a gradient of the electric field but also to generate a gradient of velocity of the fluid inside the channel. This interesting combination gives rise to a new solution for dielectrophoretic separation of two cell populations. The separation method consists of four steps. First, the microchannel is field with the cells mixture. Second, the cells are trapped in different locations of the microfluidic channel, the cell population which exhibits positive dielectrophoresis is trapped in the area where the distance between the electrodes is the minimum whilst, the other population that exhibit negative dielectrophoresis is trapped where the distance between electrodes is the maximum. In the next step, increasing the flow in the microchannel will result in an increased hydrodynamic force that sweeps the cells trapped by positive dielectrophoresis out of the chip. In the last step, the electric field is removed and the second population is sweep out and collected at the outlet. The device was tested for separation of dead yeast cells from live yeast cells. The paper presents analytical aspects of the separation method a comparative study between different electrode profiles and experimental results.

Method for solubilization of low-rank coal using low molecular weight cell-free filtrates derived from cultures of Coriolus versicolor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, D.L.; Fredrickson, J.K.; Campbell, J.A.

1992-01-28

This patent describes a method for isolating an extracellular product derived from a broth of Coriolus versicolor. It comprises separating the cells from a broth of C. versicolor to obtain a cell-free filtrate; separating from the cell-free filtrate a fraction containing molecules of molecular weight in the range of about 500 to 1000 daltons. This patent also describes a method for degrading low-rank coal to a water-soluble material. It comprises contacting the low-rank coal with a cell-free fraction from the broth of Coriolus versicolor containing molecules in the molecular weight range of about 500 to 1000 daltons.

Cell separation using tilted-angle standing surface acoustic waves

PubMed Central

Ding, Xiaoyun; Peng, Zhangli; Lin, Sz-Chin Steven; Geri, Michela; Li, Sixing; Li, Peng; Chen, Yuchao; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2014-01-01

Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼97%. We illustrate that taSSAW is capable of effectively separating particles–cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological–biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice. PMID:25157150

Cell separation using tilted-angle standing surface acoustic waves.

PubMed

Ding, Xiaoyun; Peng, Zhangli; Lin, Sz-Chin Steven; Geri, Michela; Li, Sixing; Li, Peng; Chen, Yuchao; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2014-09-09

Separation of cells is a critical process for studying cell properties, disease diagnostics, and therapeutics. Cell sorting by acoustic waves offers a means to separate cells on the basis of their size and physical properties in a label-free, contactless, and biocompatible manner. The separation sensitivity and efficiency of currently available acoustic-based approaches, however, are limited, thereby restricting their widespread application in research and health diagnostics. In this work, we introduce a unique configuration of tilted-angle standing surface acoustic waves (taSSAW), which are oriented at an optimally designed inclination to the flow direction in the microfluidic channel. We demonstrate that this design significantly improves the efficiency and sensitivity of acoustic separation techniques. To optimize our device design, we carried out systematic simulations of cell trajectories, matching closely with experimental results. Using numerically optimized design of taSSAW, we successfully separated 2- and 10-µm-diameter polystyrene beads with a separation efficiency of ∼ 99%, and separated 7.3- and 9.9-µm-polystyrene beads with an efficiency of ∼ 97%. We illustrate that taSSAW is capable of effectively separating particles-cells of approximately the same size and density but different compressibility. Finally, we demonstrate the effectiveness of the present technique for biological-biomedical applications by sorting MCF-7 human breast cancer cells from nonmalignant leukocytes, while preserving the integrity of the separated cells. The method introduced here thus offers a unique route for separating circulating tumor cells, and for label-free cell separation with potential applications in biological research, disease diagnostics, and clinical practice.

Cell-Specific Multifunctional Processing of Heterogeneous Cell Systems in a Single Laser Pulse Treatment

PubMed Central

Lukianova-Hleb, Ekaterina Y.; Mutonga, Martin B. G.; Lapotko, Dmitri O.

2012-01-01

Current methods of cell processing for gene and cell therapies use several separate procedures for gene transfer and cell separation or elimination, because no current technology can offer simultaneous multi-functional processing of specific cell sub-sets in highly heterogeneous cell systems. Using the cell-specific generation of plasmonic nanobubbles of different sizes around cell-targeted gold nanoshells and nanospheres, we achieved simultaneous multifunctional cell-specific processing in a rapid single 70 ps laser pulse bulk treatment of heterogeneous cell suspension. This method supported the detection of cells, delivery of external molecular cargo to one type of cells and the concomitant destruction of another type of cells without damaging other cells in suspension, and real-time guidance of the two above cellular effects. PMID:23167546

Manufacturing of Proteins and Antibodies: Chapter Downstream Processing Technologies : Harvest Operations.

PubMed

Turner, Richard; Joseph, Adrian; Titchener-Hooker, Nigel; Bender, Jean

2017-08-04

Cell harvesting is the separation or retention of cells and cellular debris from the supernatant containing the target molecule Selection of harvest method strongly depends on the type of cells, mode of bioreactor operation, process scale, and characteristics of the product and cell culture fluid. Most traditional harvesting methods use some form of filtration, centrifugation, or a combination of both for cell separation and/or retention. Filtration methods include normal flow depth filtration and tangential flow microfiltration. The ability to scale down predictably the selected harvest method helps to ensure successful production and is critical for conducting small-scale characterization studies for confirming parameter targets and ranges. In this chapter we describe centrifugation and depth filtration harvesting methods, share strategies for harvest optimization, present recent developments in centrifugation scale-down models, and review alternative harvesting technologies.

Separation of active and inactive fractions from starved culture of Vibrio parahaemolyticus by density dependent cell sorting.

PubMed

Nayak, Binaya Bhusan; Kamiya, Eriko; Nishino, Tomohiko; Wada, Minoru; Nishimura, Masahiko; Kogure, Kazuhiro

2005-01-01

The co-existence of physiologically different cells in bacterial cultures is a general phenomenon. We have examined the applicability of the density dependent cell sorting (DDCS) method to separate subpopulations from a long-term starvation culture of Vibrio parahaemolyticus. The cells were subjected to Percoll density gradient and separated into 12 fractions of different buoyant densities, followed by measuring the cell numbers, culturability, respiratory activity and leucine incorporation activity. While more than 78% of cells were in lighter fractions, about 95% of culturable cells were present in heavier fractions. The high-density subpopulations also had high proportion of cells capable of forming formazan granules. Although this was accompanied by the cell specific INT-reduction rate, both leucine incorporation rates and INT-reduction rates per cell had a peak at mid-density fraction. The present results indicated that DDCS could be used to separate subpopulations of different physiological conditions.

Deformability and size-based cancer cell separation using an integrated microfluidic device.

PubMed

Pang, Long; Shen, Shaofei; Ma, Chao; Ma, Tongtong; Zhang, Rui; Tian, Chang; Zhao, Lei; Liu, Wenming; Wang, Jinyi

2015-11-07

Cell sorting by filtration techniques offers a label-free approach for cell separation on the basis of size and deformability. However, filtration is always limited by the unpredictable variation of the filter hydrodynamic resistance due to cell accumulation and clogging in the microstructures. In this study, we present a new integrated microfluidic device for cell separation based on the cell size and deformability by combining the microstructure-constricted filtration and pneumatic microvalves. Using this device, the cell populations sorted by the microstructures can be easily released in real time for subsequent analysis. Moreover, the periodical sort and release of cells greatly avoided cell accumulation and clogging and improved the selectivity. Separation of cancer cells (MCF-7, MDA-MB-231 and MDA231-LM2) with different deformability showed that the mixture of the less flexible cells (MCF-7) and the flexible cells (MDA-MB-231 and MDA231-LM2) can be well separated with more than 75% purity. Moreover, the device can be used to separate cancer cells from the blood samples with more than 90% cell recovery and more than 80% purity. Compared with the current filtration methods, the device provides a new approach for cancer cell separation with high collection recovery and purity, and also, possesses practical potential to be applied as a sample preparation platform for fundamental studies and clinical applications.

Size-Based Separation of Particles and Cells Utilizing Viscoelastic Effects in Straight Microchannels.

PubMed

Liu, Chao; Xue, Chundong; Chen, Xiaodong; Shan, Lei; Tian, Yu; Hu, Guoqing

2015-06-16

Viscoelasticity-induced particle migration has recently received increasing attention due to its ability to obtain high-quality focusing over a wide range of flow rates. However, its application is limited to low throughput regime since the particles can defocus as flow rate increases. Using an engineered carrier medium with constant and low viscosity and strong elasticity, the sample flow rates are improved to be 1 order of magnitude higher than those in existing studies. Utilizing differential focusing of particles of different sizes, here, we present sheathless particle/cell separation in simple straight microchannels that possess excellent parallelizability for further throughput enhancement. The present method can be implemented over a wide range of particle/cell sizes and flow rates. We successfully separate small particles from larger particles, MCF-7 cells from red blood cells (RBCs), and Escherichia coli (E. coli) bacteria from RBCs in different straight microchannels. The proposed method could broaden the applications of viscoelastic microfluidic devices to particle/cell separation due to the enhanced sample throughput and simple channel design.

Reducing WBC background in cancer cell separation products by negative acoustic contrast particle immuno-acoustophoresis.

PubMed

Cushing, Kevin; Undvall, Eva; Ceder, Yvonne; Lilja, Hans; Laurell, Thomas

2018-02-13

Cancer cells display acoustic properties enabling acoustophoretic separation from white blood cells (WBCs) with 2-3 log suppression of the WBC background. However, a subset of WBCs has overlapping acoustic properties with cancer cells, which is why label-free acoustophoretic cancer cell isolation needs additional purification prior to analysis. This paper reports for the first time a proof of concept for continuous flow acoustophoretic negative selection of WBCs from cancer cells using negative acoustic contrast elastomeric particles (EPs) activated with CD45-antibodies that specifically bind to WBCs. The EP/WBC complexes align at the acoustic pressure anti-nodes along the channel walls while unbound cancer cells focus to the pressure node in the channel center, enabling continuous flow based depletion of WBC background in a cancer cell product. The method does not provide a single process solution for the CTC separation challenge, but provides an elegant part to a multi-step process by further reducing the WBC background in cancer cell separation products derived from an initial step of label-free acoustophoresis. We report the recorded performance of the negative selection immuno-acoustophoretic WBC depletion and cancer cell recovery. To eliminate the negative impact of the separation due to the known problems of aggregation of negative acoustic contrast particles along the sidewalls of the acoustophoresis channel and to enable continuous separation of EP/WBC complexes from cancer cells, a new acoustic actuation method has been implemented where the ultrasound frequency is scanned (1.991MHz ± 100 kHz, scan rate 200 kHz ms -1 ). Using this frequency scanning strategy EP/WBC complexes were acoustophoretically separated from mixtures of WBCs spiked with breast and prostate cancer cells (DU145 and MCF-7). An 86-fold (MCF-7) and 52-fold (DU145) reduction of WBCs in the cancer cell fractions were recorded with separation efficiencies of 98.6% (MCF-7) and 99.7% (DU145) and cancer cell recoveries of 89.8% (MCF-7) and 85.0% (DU145). Copyright © 2017 Elsevier B.V. All rights reserved.

HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations.

PubMed

Drabbels, Jos J M; van de Keur, Carin; Kemps, Berit M; Mulder, Arend; Scherjon, Sicco A; Claas, Frans H J; Eikmans, Michael

2011-11-10

Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

Cell separation and electrofusion in space

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Hofmann, G. A.

1990-01-01

In microgravity, free-fluid electrophoretic methods for separating living cells and proteins are improved significantly by the absence of gravity-driven phenomena. Cell fusion, culture, and other bioprocessing steps are being investigated to understand the limits of earth-based processing. A multistep space bioprocess is described that includes electrophoretic separation of human target cells, single-cell manipulations using receptor-specific antibodies, electrofusion to produce immortal hybridomas, gentle suspension culture, and monoclonal antibody recovery using continuous-flow electrophoresis or recirculating isoelectric focusing. Improvements in several key steps already have been demonstrated by space experiments, and others will be studied on Space Station Freedom.

Acoustic separation of circulating tumor cells

PubMed Central

Li, Peng; Mao, Zhangming; Peng, Zhangli; Zhou, Lanlan; Chen, Yuchao; Huang, Po-Hsun; Truica, Cristina I.; Drabick, Joseph J.; El-Deiry, Wafik S.; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2015-01-01

Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acoustic-based methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contact-free sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (∼100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state. PMID:25848039

Acoustic separation of circulating tumor cells.

PubMed

Li, Peng; Mao, Zhangming; Peng, Zhangli; Zhou, Lanlan; Chen, Yuchao; Huang, Po-Hsun; Truica, Cristina I; Drabick, Joseph J; El-Deiry, Wafik S; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2015-04-21

Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acoustic-based methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contact-free sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (∼100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state.

Ion transport restriction in mechanically strained separator membranes

NASA Astrophysics Data System (ADS)

Cannarella, John; Arnold, Craig B.

2013-03-01

We use AC impedance methods to investigate the effect of mechanical deformation on ion transport in commercial separator membranes and lithium-ion cells as a whole. A Bruggeman type power law relationship is found to provide an accurate correlation between porosity and tortuosity of deformed separators, which allows the impedance of a separator membrane to be predicted as a function of deformation. By using mechanical compression to vary the porosity of the separator membranes during impedance measurements it is possible to determine both the α and γ parameters from the modified Bruggeman relation for individual separator membranes. From impedance testing of compressed pouch cells it is found that separator deformation accounts for the majority of the transport restrictions arising from compressive stress in a lithium-ion cell. Finally, a charge state dependent increase in the impedance associated with charge transfer is observed with increasing cell compression.

Solar cell with doped groove regions separated by ridges

DOEpatents

Molesa, Steven Edward; Pass, Thomas; Kraft, Steve

2017-01-31

Solar cells with doped groove regions separated by ridges and methods of fabricating solar cells are described. In an example, a solar cell includes a substrate having a surface with a plurality of grooves and ridges. A first doped region of a first conductivity type is disposed in a first of the grooves. A second doped region of a second conductivity type, opposite the first conductivity type, is disposed in a second of the grooves. The first and second grooves are separated by one of the ridges.

Method of preparing porous, rigid ceramic separators for an electrochemical cell

DOEpatents

Bandyopadhyay, Gautam; Dusek, Joseph T.

1981-01-01

Porous, rigid separators for electrochemical cells are prepared by first calcining particles of ceramic material at temperatures above about 1200.degree. C. for a sufficient period of time to reduce the sinterability of the particles. A ceramic powder that has not been calcined is blended with the original powder to control the porosity of the completed separator. The ceramic blend is then pressed into a sheet of the desired shape and sintered at a temperature somewhat lower than the calcination temperature. Separator sheets of about 1 to 2.5 mm thickness and 30 to 70% porosity can be prepared by this technique. Ceramics such as yttria, magnesium oxide and magnesium-aluminum oxide have advantageously been used to form separators by this method.

Detection of E. coli O157:H7 from ground beef using Fourier transform infrared (FT-IR) spectroscopy and chemometrics.

PubMed

Davis, Reeta; Irudayaraj, Joseph; Reuhs, Bradley L; Mauer, Lisa J

2010-08-01

FT-IR spectroscopy methods for detection, differentiation, and quantification of E. coli O157:H7 strains separated from ground beef were developed. Filtration and immunomagnetic separation (IMS) were used to extract live and dead E. coli O157:H7 cells from contaminated ground beef prior to spectral acquisition. Spectra were analyzed using chemometric techniques in OPUS, TQ Analyst, and WinDAS software programs. Standard plate counts were used for development and validation of spectral analyses. The detection limit based on a selectivity value using the OPUS ident test was 10(5) CFU/g for both Filtration-FT-IR and IMS-FT-IR methods. Experiments using ground beef inoculated with fewer cells (10(1) to 10(2) CFU/g) reached the detection limit at 6 h incubation. Partial least squares (PLS) models with cross validation were used to establish relationships between plate counts and FT-IR spectra. Better PLS predictions were obtained for quantifying live E. coli O157:H7 strains (R(2)> or = 0.9955, RMSEE < or = 0.17, RPD > or = 14) and different ratios of live and dead E. coli O157:H7 cells (R(2)= 0.9945, RMSEE = 2.75, RPD = 13.43) from ground beef using Filtration-FT-IR than IMS-FT-IR methods. Discriminant analysis and canonical variate analysis (CVA) of the spectra differentiated various strains of E. coli O157:H7 from an apathogenic control strain. CVA also separated spectra of 100% dead cells separated from ground beef from spectra of 0.5% live cells in the presence of 99.5% dead cells of E. coli O157:H7. These combined separation and FT-IR methods could be useful for rapid detection and differentiation of pathogens in complex foods.

Urokinase production by electrophoretically separated cultured human embryonic kidney cells

NASA Technical Reports Server (NTRS)

Kunze, M. E.; Plank, L. D.; Giranda, V.; Sedor, K.; Todd, P. W.

1985-01-01

Urokinase is a plasminogen activator found in urine. Relatively pure preparations have been tested in Europe, Japan and the United States for the treatment of deep vein thrombosis and other dangerous blood clots. Human embryonic kidney cell cultures have been found to produce urokinase at much higher concentrations, but less than 5% of the cells in typical cultures are producers. Since human diploid cells become senescent in culture the selection of clones derived from single cells will not provide enough material to be useful, so a bulk purification method is needed for the isolation of urokinase producing cell populations. Preparative cell electrophoresis was chosen as the method, since evidence exists that human embryonic cell cultures are richly heterogeneous with respect to electrophoretic mobility, and preliminary electrophoretic separations on the Apollo-Soyuz space flight produced cell populations that were rich in urokinase production. Similarly, erythropoietin is useful in the treatment of certain anemias and is a kidney cell duct, and electrophoretically enriched cell populations producing this product have been reported. Thus, there is a clear need for diploid human cells that produce these products, and there is evidence that such cells should be separable by free-flow cell electrophoresis.

Wall effects in continuous microfluidic magneto-affinity cell separation.

PubMed

Wu, Liqun; Zhang, Yong; Palaniapan, Moorthi; Roy, Partha

2010-05-01

Continuous microfluidic magneto-affinity cell separator combines unique microscale flow phenomenon with advantageous nanobead properties, to isolate cells with high specificity. Owing to the comparable size of the cell-bead complexes and the microchannels, the walls of the microchannel exert a strong influence on the separation of cells by this method. We present a theoretical and experimental study that provides a quantitative description of hydrodynamic wall interactions and wall rolling velocity of cells. A transient convection model describes the transport of cells in two-phase microfluidic flow under the influence of an external magnetic field. Transport of cells along the microchannel walls is also considered via an additional equation. Results show the variation of cell flux in the fluid phases and the wall as a function of a dimensionless parameter arising in the equations. Our results suggest that conditions may be optimized to maximize cell separation while minimizing contact with the wall surfaces. Experimentally measured cell rolling velocities on the wall indicate the presence of other near-wall forces in addition to fluid shear forces. Separation of a human colon carcinoma cell line from a mixture of red blood cells, with folic acid conjugated 1 microm and 200 nm beads, is reported.

Separation of malignant human breast cancer epithelial cells from healthy epithelial cells using an advanced dielectrophoresis-activated cell sorter (DACS).

PubMed

An, Jaemin; Lee, Jangwon; Lee, Sang Ho; Park, Jungyul; Kim, Byungkyu

2009-06-01

In this paper, we successfully separated malignant human breast cancer epithelial cells (MCF 7) from healthy breast cells (MCF 10A) and analyzed the main parameters that influence the separation efficiency with an advanced dielectrophoresis (DEP)-activated cell sorter (DACS). Using the efficient DACS, the malignant cancer cells (MCF 7) were isolated successfully by noninvasive methods from normal cells with similar cell size distributions (MCF 10A), depending on differences between their material properties such as conductivity and permittivity, because our system was able to discern the subtle differences in the properties by generating continuously changed electrical field gradients. In order to evaluate the separation performance without considering size variations, the cells collected from each outlet were divided into size-dependent groups and counted statistically. Following that, the quantitative relative ratio of numbers between MCF 7 and MCF 10A cells in each size-dependent group separated by the DEP were compared according to applied frequencies in the range 48, 51, and 53 MHz with an applied amplitude of 8 V(pp). Finally, under the applied voltage of 48 MHz-8 V(pp) and a flow rate of 290 microm/s, MCF 7 and MCF 10A cells were separated with a maximum efficiency of 86.67% and 98.73% respectively. Therefore, our suggested system shows it can be used for detection and separation of cancerous epithelial cells from noncancerous cells in clinical applications.

Method of preparing porous, rigid ceramic separators for an electrochemical cell. [Patent application

DOEpatents

Bandyopadhyay, G.; Dusek, J.T.

Porous, rigid separators for electrochemical cells are prepared by first calcining particles of ceramic material at temperatures above about 1200/sup 0/C for a sufficient period of time to reduce the sinterability of the particles. A ceramic powder that has not been calcined is blended with the original powder to control the porosity of the completed separator. The ceramic blend is then pressed into a sheet of the desired shape and sintered at a temperature somewhat lower than the calcination temperature. Separator sheets of about 1 to 2.5 mm thickness and 30 to 70% porosity can be prepared by this technique. Ceramics such as yttria, magnesium oxide, and magnesium-aluminium oxide have advantageously been used to form separators by this method.

Controlled method of reducing electrophoretic mobility of various substances

NASA Technical Reports Server (NTRS)

Vanalstine, James M. (Inventor)

1989-01-01

A method of reducing electrophoretic mobility of macromolecules, particles, cells, and the like is provided. The method comprises interacting the particles or cells with a polymer-linked affinity compound composed of: a hydrophilic neutral polymer such as polyethylene glycol, and an affinity component consisting of a hydrophobic compound such as a fatty acid ester, an immunocompound such as an antibody or active fragment thereof or simular macromolecule, or other ligands. The reduction of electrophoretic mobility achieved is directly proportional to the concentration of the polymer-linked affinity compound employed, and the mobility reduction obtainable is up to 100 percent for particular particles and cells. The present invention is advantageous in that analytical electrophoretic separation can not be achieved for macromolecules, particles, and cells whose native surface charge structure had prevented them from being separated by normal electrophoretic means. Depending on the affinity component utilized, separation can be achieved on the basis of specific/irreversible, specific/reversible, semi-specific/reversible, relatively nonspecific/reversible, or relatively nonspecific/irreversible ligand-substance interactions. The present method is also advantageous in that it can be used in a variety of standard laboratory electrophoresis equipment.

Microfluidic Separation of Circulating Tumor Cells Based on Size and Deformability.

PubMed

Park, Emily S; Duffy, Simon P; Ma, Hongshen

2017-01-01

Circulating tumor cells (CTCs) have been implicated as the seeds of cancer metastasis and therefore have the potential to provide significant prognostic and diagnostic values. Here, we describe a procedure for separating CTCs from whole blood based on size and deformability using the microfluidic ratchet device. This device leverages the ratcheting motion of single cells created as they are deformed through funnel-shaped constrictions using oscillatory flow in order to divert cells based on differences in size and deformability. Subsequent methods for CTC identification and enumeration using immunofluorescence after separation are also described.

Microfluidics separation reveals the stem-cell-like deformability of tumor-initiating cells.

PubMed

Zhang, Weijia; Kai, Kazuharu; Choi, Dong Soon; Iwamoto, Takayuki; Nguyen, Yen H; Wong, Helen; Landis, Melissa D; Ueno, Naoto T; Chang, Jenny; Qin, Lidong

2012-11-13

Here we report a microfluidics method to enrich physically deformable cells by mechanical manipulation through artificial microbarriers. Driven by hydrodynamic forces, flexible cells or cells with high metastatic propensity change shape to pass through the microbarriers and exit the separation device, whereas stiff cells remain trapped. We demonstrate the separation of (i) a mixture of two breast cancer cell types (MDA-MB-436 and MCF-7) with distinct deformabilities and metastatic potentials, and (ii) a heterogeneous breast cancer cell line (SUM149), into enriched flexible and stiff subpopulations. We show that the flexible phenotype is associated with overexpression of multiple genes involved in cancer cell motility and metastasis, and greater mammosphere formation efficiency. Our observations support the relationship between tumor-initiating capacity and cell deformability, and demonstrate that tumor-initiating cells are less differentiated in terms of cell biomechanics.

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.

PubMed

Macaulay, Iain C; Teng, Mabel J; Haerty, Wilfried; Kumar, Parveen; Ponting, Chris P; Voet, Thierry

2016-11-01

Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ∼3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.

Aluminum oxyhydroxide based separator/electrolyte and battery system, and a method making the same

DOEpatents

Gerald, II, Rex E.; Klingler, Robert J [Glenview, IL; Rathke, Jerome W [Homer Glen, IL

2011-03-08

The instant invention relates a solid-state electrochemical cell and a novel separator/electrolyte incorporated therein. A preferred embodiment of the invented electrochemical cell generally comprises a unique metal oxyhydroxide based (i.e. AlOOH) separator/electrolyte membrane sandwiched between a first electrode and a second electrode. A preferred novel separator/electrolyte comprises a nanoparticulate metal oxyhydroxide, preferably AlOOH and a salt which are mixed and then pressed together to form a monolithic metal oxyhydroxide-salt membrane.

Aluminum oxyhydroxide based separator/electrolyte and battery system, and a method of making the same

DOEpatents

Gerald, II; Rex, E [Brookfield, IL; Klingler, Robert J [Glenview, IL; Rathke, Jerome W [Homer Glen, IL

2011-02-15

The instant invention relates a solid-state electrochemical cell and a novel separator/electrolyte incorporated therein. The invented electrochemical cell generally comprising: a unique metal oxyhydroxide based (i.e. AlOOH) separator/electrolyte membrane sandwiched between a first electrode and a second electrode. The novel separator/electrolyte comprises a nanoparticulate metal oxyhydroxide, preferably AlOOH and a salt which are mixed and then pressed together to form a monolithic metal oxyhydroxide-salt membrane.

Spray-on polyvinyl alcohol separators and impact on power production in air-cathode microbial fuel cells with different solution conductivities.

PubMed

Hoskins, Daniel L; Zhang, Xiaoyuan; Hickner, Michael A; Logan, Bruce E

2014-11-01

Separators are used to protect cathodes from biofouling and to avoid electrode short-circuiting, but they can adversely affect microbial fuel cell (MFC) performance. A spray method was used to apply a polyvinyl alcohol (PVA) separator to the cathode. Power densities were unaffected by the PVA separator (339±29mW/m(2)), compared to a control lacking a separator in a low conductivity solution (1mS/cm) similar to wastewater. Power was reduced with separators in solutions typical of laboratory tests (7-13mS/cm), compared to separatorless controls. The PVA separator produced more power in a separator assembly (SEA) configuration (444±8mW/m(2)) in the 1mS/cm solution, but power was reduced if a PVA or wipe separator was used in higher conductivity solutions with either Pt or activated carbon catalysts. Spray and cast PVA separators performed similarly, but the spray method is preferred as it was easier to apply and use. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Removal of Human Breast Cancer Cells from Hematopoietic CD34+ Stem Cells by Dielectrophoretic Field-Flow-Fractionation

PubMed Central

HUANG, YING; YANG, JUN; WANG, XIAO-BO; BECKER, FREDERICK F.; GASCOYNE, PETER R.C.

2009-01-01

Dielectrophoretic field-flow-fractionation (DEP-FFF) was used to purge human breast cancer MDA-435 cells from hematopoietic CD34+ stem cells. An array of interdigitated microelectrodes lining the bottom surface of a thin chamber was used to generate dielectrophoretic forces that levitated the cell mixture in a fluid flow profile. CD34+ stem cells were levitated higher, were carried faster by the fluid flow, and exited the separation chamber earlier than the cancer cells. Using on-line flow cytometry, efficient separation of the cell mixture was observed in less than 12 min, and CD34+ stem cell fractions with a purity >99.2% were obtained. The method of DEP-FFF is potentially applicable to many biomedical cell separation problems, including microfluidic-scale diagnosis and preparative-scale purification of cell subpopulations. PMID:10791899

Charge-based separation of particles and cells with similar sizes via the wall-induced electrical lift.

PubMed

Thomas, Cory; Lu, Xinyu; Todd, Andrew; Raval, Yash; Tzeng, Tzuen-Rong; Song, Yongxin; Wang, Junsheng; Li, Dongqing; Xuan, Xiangchun

2017-01-01

The separation of particles and cells in a uniform mixture has been extensively studied as a necessity in many chemical and biomedical engineering and research fields. This work demonstrates a continuous charge-based separation of fluorescent and plain spherical polystyrene particles with comparable sizes in a ψ-shaped microchannel via the wall-induced electrical lift. The effects of both the direct current electric field in the main-branch and the electric field ratio in between the inlet branches for sheath fluid and particle mixture are investigated on this electrokinetic particle separation. A Lagrangian tracking method based theoretical model is also developed to understand the particle transport in the microchannel and simulate the parametric effects on particle separation. Moreover, the demonstrated charge-based separation is applied to a mixture of yeast cells and polystyrene particles with similar sizes. Good separation efficiency and purity are achieved for both the cells and the particles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing.

PubMed

Xu, Chun-Xiu; Yin, Xue-Feng

2011-02-04

A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance. Copyright © 2010 Elsevier B.V. All rights reserved.

SERS-fluorescence joint spectral encoded magnetic nanoprobes for multiplex cancer cell separation.

PubMed

Wang, Zhuyuan; Zong, Shenfei; Chen, Hui; Wang, Chunlei; Xu, Shuhong; Cui, Yiping

2014-11-01

A new kind of cancer cell separation method is demonstrated, using surface-enhanced Raman scattering (SERS) and fluorescence dual-encoded magnetic nanoprobes. The designed nanoprobes can realize SERS-fluorescence joint spectral encoding (SFJSE) and greatly improve the multiplexing ability. The nanoprobes have four main components, that is, the magnetic core, SERS generator, fluorescent agent, and targeting antibody. These components are assembled with a multi-layered structure to form the nanoprobes. Specifically, silica-coated magnetic nanobeads (MBs) are used as the inner core. Au core-Ag shell nanorods (Au@Ag NRs) are employed as the SERS generators and attached on the silica-coated MBs. After burying these Au@Ag NRs with another silica layer, CdTe quantum dots (QDs), that is, the fluorescent agent, are anchored onto the silica layer. Finally, antibodies are covalently linked to CdTe QDs. SFJSE is fulfilled by using different Raman molecules and QDs with different emission wavelengths. By utilizing four human cancer cell lines and one normal cell line as the model cells, the nanoprobes can specifically and simultaneously separate target cancer cells from the normal ones. This SFJSE-based method greatly facilitates the multiplex, rapid, and accurate cancer cell separation, and has a prosperous potential in high-throughput analysis and cancer diagnosis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-free ferrohydrodynamic cell separation of circulating tumor cells.

PubMed

Zhao, Wujun; Cheng, Rui; Jenkins, Brittany D; Zhu, Taotao; Okonkwo, Nneoma E; Jones, Courtney E; Davis, Melissa B; Kavuri, Sravan K; Hao, Zhonglin; Schroeder, Carsten; Mao, Leidong

2017-09-12

Circulating tumor cells (CTCs) have significant implications in both basic cancer research and clinical applications. To address the limited availability of viable CTCs for fundamental and clinical investigations, effective separation of extremely rare CTCs from blood is critical. Ferrohydrodynamic cell separation (FCS), a label-free method that conducted cell sorting based on cell size difference in biocompatible ferrofluids, has thus far not been able to enrich low-concentration CTCs from cancer patients' blood because of technical challenges associated with processing clinical samples. In this study, we demonstrated the development of a laminar-flow microfluidic FCS device that was capable of enriching rare CTCs from patients' blood in a biocompatible manner with a high throughput (6 mL h -1 ) and a high rate of recovery (92.9%). Systematic optimization of the FCS devices through a validated analytical model was performed to determine optimal magnetic field and its gradient, ferrofluid properties, and cell throughput that could process clinically relevant amount of blood. We first validated the capability of the FCS devices by successfully separating low-concentration (∼100 cells per mL) cancer cells using six cultured cell lines from undiluted white blood cells (WBCs), with an average 92.9% cancer cell recovery rate and an average 11.7% purity of separated cancer cells, at a throughput of 6 mL per hour. Specifically, at ∼100 cancer cells per mL spike ratio, the recovery rates of cancer cells were 92.3 ± 3.6% (H1299 lung cancer), 88.3 ± 5.5% (A549 lung cancer), 93.7 ± 5.5% (H3122 lung cancer), 95.3 ± 6.0% (PC-3 prostate cancer), 94.7 ± 4.0% (MCF-7 breast cancer), and 93.0 ± 5.3% (HCC1806 breast cancer), and the corresponding purities of separated cancer cells were 11.1 ± 1.2% (H1299 lung cancer), 10.1 ± 1.7% (A549 lung cancer), 12.1 ± 2.1% (H3122 lung cancer), 12.8 ± 1.6% (PC-3 prostate cancer), 11.9 ± 1.8% (MCF-7 breast cancer), and 12.2 ± 1.6% (HCC1806 breast cancer). Biocompatibility study on H1299 cell line and HCC1806 cell line showed that separated cancer cells had excellent short-term viability, normal proliferation and unaffected key biomarker expressions. We then demonstrated the enrichment of CTCs in blood samples obtained from two patients with newly diagnosed advanced non-small cell lung cancer (NSCLC). While still at its early stage of development, FCS could become a complementary tool for CTC separation for its high recovery rate and excellent biocompatibility, as well as its potential for further optimization and integration with other separation methods.

High-rate overcharge-protection separators for rechargeable lithium-ion batteries and the method of making the same

DOEpatents

Chen, Guoying; Richardson, Thomas J.

2016-12-20

This invention relates to low-cost, electroactive-polymer incorporated fine-fiber composite membranes for use as overcharge and/or overdischarge protection separators in non-aqueous electrochemical cells and the methods for making such membranes.

Methods for fabricating thin film III-V compound solar cell

DOEpatents

Pan, Noren; Hillier, Glen; Vu, Duy Phach; Tatavarti, Rao; Youtsey, Christopher; McCallum, David; Martin, Genevieve

2011-08-09

The present invention utilizes epitaxial lift-off in which a sacrificial layer is included in the epitaxial growth between the substrate and a thin film III-V compound solar cell. To provide support for the thin film III-V compound solar cell in absence of the substrate, a backing layer is applied to a surface of the thin film III-V compound solar cell before it is separated from the substrate. To separate the thin film III-V compound solar cell from the substrate, the sacrificial layer is removed as part of the epitaxial lift-off. Once the substrate is separated from the thin film III-V compound solar cell, the substrate may then be reused in the formation of another thin film III-V compound solar cell.

Cross-linked polyvinyl alcohol films as alkaline battery separators

NASA Technical Reports Server (NTRS)

Sheibley, D. W.; Manzo, M. A.; Gonzalez-Sanabria, O. D.

1983-01-01

Cross-linking methods have been investigated to determine their effect on the performance of polyvinyl alcohol (PVA) films as alkaline battery separators. The following types of cross-linked PVA films are discussed: (1) PVA-dialdehyde blends post-treated with an acid or acid periodate solution (two-step method) and (2) PVA-dialdehyde blends cross-linked during film formation (drying) by using a reagent with both aldehyde and acid functionality (one-step method). Laboratory samples of each cross-linked type of film were prepared and evaluated in standard separator screening tests. Then pilot-plant batches of films were prepared and compared to measure differences due to the cross-linking method. The pilot-plant materials were then tested in nickel oxide-zinc cells to compare the two methods with respect to performance characteristics and cycle life. Cell test results are compared with those from tests with Celgard.

Cross-linked polyvinyl alcohol films as alkaline battery separators

NASA Technical Reports Server (NTRS)

Sheibley, D. W.; Manzo, M. A.; Gonzalez-Sanabria, O. D.

1982-01-01

Cross-linking methods were investigated to determine their effect on the performance of polyvinyl alcohol (PVA) films as alkaline battery separators. The following types of cross-linked PVA films are discussed: (1) PVA-dialdehyde blends post-treated with an acid or acid periodate solution (two-step method) and (2) PVA-dialdehyde blends cross-linked during film formation (drying) by using a reagent with both aldehyde and acid functionality (one-step method). Laboratory samples of each cross-linked type of film were prepared and evaluated in standard separator screening tests. The pilot-plant batches of films were prepared and compared to measure differences due to the cross-linking method. The pilot-plant materials were then tested in nickel oxide - zinc cells to compare the two methods with respect to performance characteristics and cycle life. Cell test results are compared with those from tests with Celgard.

FogBank: a single cell segmentation across multiple cell lines and image modalities.

PubMed

Chalfoun, Joe; Majurski, Michael; Dima, Alden; Stuelten, Christina; Peskin, Adele; Brady, Mary

2014-12-30

Many cell lines currently used in medical research, such as cancer cells or stem cells, grow in confluent sheets or colonies. The biology of individual cells provide valuable information, thus the separation of touching cells in these microscopy images is critical for counting, identification and measurement of individual cells. Over-segmentation of single cells continues to be a major problem for methods based on morphological watershed due to the high level of noise in microscopy cell images. There is a need for a new segmentation method that is robust over a wide variety of biological images and can accurately separate individual cells even in challenging datasets such as confluent sheets or colonies. We present a new automated segmentation method called FogBank that accurately separates cells when confluent and touching each other. This technique is successfully applied to phase contrast, bright field, fluorescence microscopy and binary images. The method is based on morphological watershed principles with two new features to improve accuracy and minimize over-segmentation. First, FogBank uses histogram binning to quantize pixel intensities which minimizes the image noise that causes over-segmentation. Second, FogBank uses a geodesic distance mask derived from raw images to detect the shapes of individual cells, in contrast to the more linear cell edges that other watershed-like algorithms produce. We evaluated the segmentation accuracy against manually segmented datasets using two metrics. FogBank achieved segmentation accuracy on the order of 0.75 (1 being a perfect match). We compared our method with other available segmentation techniques in term of achieved performance over the reference data sets. FogBank outperformed all related algorithms. The accuracy has also been visually verified on data sets with 14 cell lines across 3 imaging modalities leading to 876 segmentation evaluation images. FogBank produces single cell segmentation from confluent cell sheets with high accuracy. It can be applied to microscopy images of multiple cell lines and a variety of imaging modalities. The code for the segmentation method is available as open-source and includes a Graphical User Interface for user friendly execution.

Rapid Characterization of Magnetic Moment of Cells for Magnetic Separation

PubMed Central

Ooi, Chinchun; Earhart, Christopher M.; Wilson, Robert J.; Wang, Shan X.

2014-01-01

NCI-H1650 lung cancer cell lines labeled with magnetic nanoparticles via the Epithelial Cell Adhesion Molecule (EpCAM) antigen were previously shown to be captured at high efficiencies by a microfabricated magnetic sifter. If fine control and optimization of the magnetic separation process is to be achieved, it is vital to be able to characterize the labeled cells’ magnetic moment rapidly. We have thus adapted a rapid prototyping method to obtain the saturation magnetic moment of these cells. This method utilizes a cross-correlation algorithm to analyze the cells’ motion in a simple fluidic channel to obtain their magnetophoretic velocity, and is effective even when the magnetic moments of cells are small. This rapid characterization is proven useful in optimizing our microfabricated magnetic sifter procedures for magnetic cell capture. PMID:24771946

Method and apparatus for electrostatically sorting biological cells

DOEpatents

Merrill, John T.

1982-01-01

An improved method of sorting biological cells in a conventional cell sorter apparatus includes generating a fluid jet containing cells to be sorted, measuring the distance between the centers of adjacent droplets in a zone thereof defined at the point where the fluid jet separates into descrete droplets, setting the distance between the center of a droplet in said separation zone and the position along said fluid jet at which the cell is optically sensed for specific characteristics to be an integral multiple of said center-to-center distance, and disabling a charger from electrically charging a specific droplet if a cell is detected by the optical sensor in a position wherein it will be in the neck area between droplets during droplet formation rather than within a predetermined distance from the droplet center.

An Overview of NASA Biotechnology

NASA Technical Reports Server (NTRS)

Pusey, Marc L.

1997-01-01

Biotechnology research at NASA has comprised three separate areas; cell science and tissue culture, separations methods, and macromolecular crystal growth. This presentation will primarily focus on the macromolecular crystal growth.

Lipid-Based Immuno-Magnetic Separation of Archaea from a Mixed Community

NASA Astrophysics Data System (ADS)

Frickle, C. M.; Bailey, J.; Lloyd, K. G.; Shumaker, A.; Flood, B.

2014-12-01

Despite advancing techniques in microbiology, an estimated 98% of all microbial species on Earth have yet to be isolated in pure culture. Natural samples, once transferred to the lab, are commonly overgrown by "weed" species whose metabolic advantages enable them to monopolize available resources. Developing new methods for the isolation of thus-far uncultivable microorganisms would allow us to better understand their ecology, physiology and genetic potential. Physically separating target organisms from a mixed community is one approach that may allow enrichment and growth of the desired strain. Here we report on a novel method that uses known physiological variations between taxa, in this case membrane lipids, to segregate the desired organisms while keeping them alive and viable for reproduction. Magnetic antibodies bound to the molecule squalene, which is found in the cell membranes of certain archaea, but not bacteria, enable separation of archaea from bacteria in mixed samples. Viability of cells was tested by growing the separated fractions in batch culture. Efficacy and optimization of the antibody separation technique are being evaluated using qPCR and cell counts. Future work will apply this new separation technique to natural samples.

DAPNe with micro-capillary separatory chemistry-coupled to MALDI-MS for the analysis of polar and non-polar lipid metabolism in one cell

NASA Astrophysics Data System (ADS)

Hamilton, Jason S.; Aguilar, Roberto; Petros, Robby A.; Verbeck, Guido F.

2017-05-01

The cellular metabolome is considered to be a representation of cellular phenotype and cellular response to changes to internal or external events. Methods to expand the coverage of the expansive physiochemical properties that makeup the metabolome currently utilize multi-step extractions and chromatographic separations prior to chemical detection, leading to lengthy analysis times. In this study, a single-step procedure for the extraction and separation of a sample using a micro-capillary as a separatory funnel to achieve analyte partitioning within an organic/aqueous immiscible solvent system is described. The separated analytes are then spotted for MALDI-MS imaging and distribution ratios are calculated. Initially, the method is applied to standard mixtures for proof of partitioning. The extraction of an individual cell is non-reproducible; therefore, a broad chemical analysis of metabolites is necessary and will be illustrated with the one-cell analysis of a single Snu-5 gastric cancer cell taken from a cellular suspension. The method presented here shows a broad partitioning dynamic range as a single-step method for lipid analysis demonstrating a decrease in ion suppression often present in MALDI analysis of lipids.

Streptavidin-coated magnetic beads for DNA strand separation implicate a multitude of problems during cell-SELEX.

PubMed

Paul, Angela; Avci-Adali, Meltem; Ziemer, Gerhard; Wendel, Hans P

2009-09-01

Using whole living cells as a target for SELEX (systematic evolution of ligands by exponential enrichment) experiments represents a promising method to generate cell receptor-specific aptamers. These aptamers have a huge potential in diagnostics, therapeutics, imaging, regenerative medicine, and target validation. During the SELEX for selecting DNA aptamers, one important step is the separation of 2 DNA strands to yield one of the 2 strands as single-stranded DNA aptamer. This is being done routinely by biotin labeling of the complementary DNA strand to the desired aptamer and then separating the DNA strand by using streptavidin-coated magnetic beads. After immobilization of the double-stranded DNA on these magnetic beads and alkaline denaturation, the non-biotinylated strand is being eluted and the biotinylated strand is retarded. Using Western blot analysis, we demonstrated the detachment of covalent-bonded streptavidin from the bead surface after alkaline treatment. The eluates were also contaminated with undesired biotinylated strands. Furthermore, a streptavidin-induced aggregation of target cells was demonstrated by flow cytometry and microscopic methods. Cell-specific enrichment of aptamers was not possible due to clustering and patching effects triggered by streptavidin. Therefore, the use of streptavidin-coated magnetic beads for DNA strand separation should be examined thoroughly, especially for cell-SELEX applications.

Free zone electrophoresis simulation of static column electrophoresis in microgravity on shuttle flight STS-3

NASA Technical Reports Server (NTRS)

Todd, P. W.; Hjerten, S.

1985-01-01

Experiments were designed to replicate, as closely as possible in 1-G, the conditions of the STS-3 red blood cell (RBC) experiments. Free zone electrophoresis was the method of choice, since it minimizes the role of gravity in cell migration. The physical conditions of the STS-3 experiments were used, and human and rabbit RBC's fixed by the same method were the test particles. The effects of cell concentration, electroosmotic mobility, and sample composition were tested in order to seek explanations for the STS-3 results and to provide data on cell concentration effects for future zero-G separation on the continuous-flow zero-G electrophoretics separator.

Method of producing purified carotenoid compounds

NASA Technical Reports Server (NTRS)

Eggink, Laura (Inventor)

2007-01-01

A method of producing a carotenoid in solid form includes culturing a strain of Chlorophyta algae cells in a minimal inorganic medium and separating the algae comprising a solid form of carotenoid. In one embodiment f the invention, the strain of Chlorophyta algae cells includes a strain f Chlamydomonas algae cells.

An integrated system for synchronous culture of animal cells under controlled conditions.

PubMed

Mendoza-Pérez, Elena; Hernández, Vanessa; Palomares, Laura A; Serrato, José A

2016-01-01

The cell cycle has fundamental effects on cell cultures and their products. Tools to synchronize cultured cells allow the study of cellular physiology and metabolism at particular cell cycle phases. However, cells are most often arrested by methods that alter their homeostasis and are then cultivated in poorly controlled environments. Cell behavior could then be affected by the synchronization method and culture conditions used, and not just by the particular cell cycle phase under study. Moreover, only a few viable cells are recovered. Here, we designed an integrated system where a large number of cells from a controlled bioreactor culture is separated by centrifugal elutriation at high viabilities. In contrast to current elutriation methods, cells are injected directly from a bioreactor into an injection loop, allowing the introduction of a large number of cells into the separation chamber without stressful centrifugation. A low pulsation peristaltic pump increases the stability of the elutriation chamber. Using this approach, a large number of healthy cells at each cell cycle phase were obtained, allowing their direct inoculation into fully instrumented bioreactors. Hybridoma cells synchronized and cultured in this system behaved as expected for a synchronous culture.

Ferromagnetic nickel silicide nanowires for isolating primary CD4+ T lymphocytes

NASA Astrophysics Data System (ADS)

Kim, Dong-Joo; Seol, Jin-Kyeong; Lee, Mi-Ri; Hyung, Jung-Hwan; Kim, Gil-Sung; Ohgai, Takeshi; Lee, Sang-Kwon

2012-04-01

Direct CD4+ T lymphocytes were separated from whole mouse splenocytes using 1-dimensional ferromagnetic nickel silicide nanowires (NiSi NWs). NiSi NWs were prepared by silver-assisted wet chemical etching of silicon and subsequent deposition and annealing of Ni. This method exhibits a separation efficiency of ˜93.5%, which is comparable to that of the state-of-the-art superparamagnetic bead-based cell capture (˜96.8%). Furthermore, this research shows potential for separation of other lymphocytes, B, natural killer and natural killer T cells, and even rare tumor cells simply by changing the biotin-conjugated antibodies.

Method of preparing a powdered, electrically insulative separator for use in an electrochemical cell

DOEpatents

Cooper, Tom O.; Miller, William E.

1978-01-01

A secondary electrochemical cell includes electrodes separated by a layer of electrically insulative powder. The powder includes refractory materials selected from the oxides and nitrides of metals and metaloids. The powdered refractory material, blended with electrolyte particles, is compacted as layers onto an electrode to form an integral electrode structure and assembled into the cell. The assembled cell is heated to its operating temperature leaving porous layers of electrically insulative, refractory particles, containing molten electrolyte between the electrodes.

Optimized signal detection and analysis methods for in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Wang, Qiyan; Zhou, Quanyu; Yang, Ping; Wang, Xiaoling; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Gao, Wenyuan; Tang, Shuo; Wei, Xunbin

2017-02-01

Melanoma is known as a malignant tumor of melanocytes, which usually appear in the blood circulation at the metastasis stage of cancer. Thus the detection of circulating melanoma cells is useful for early diagnosis and therapy of cancer. Here we have developed an in vivo photoacoustic flow cytometry (PAFC) based on the photoacoustic effect to detect melanoma cells. However, the raw signals we obtain from the target cells contain noises such as environmental sonic noises and electronic noises. Therefore we apply correlation comparison and feature separation methods to the detection and verification of the in vivo signals. Due to similar shape and structure of cells, the photoacoustic signals usually have similar vibration mode. By analyzing the correlations and the signal features in time domain and frequency domain, we are able to provide a method for separating photoacoustic signals generated by target cells from background noises. The method introduced here has proved to optimize the signal acquisition and signal processing, which can improve the detection accuracy in PAFC.

Controlled method of reducing electrophoretic mobility of macromolecules, particles, or cells

NASA Technical Reports Server (NTRS)

Vanalstine, James M. (Inventor)

1992-01-01

A method of reducing electrophoretic mobility of macromolecules, particles, cells, and other substances is provided which comprises interacting in a conventional electrophoretic separating procedure, the substances with a polymer-linked affinity compound comprised of a hydrophilic neutral polymer such as polyethylene glycol bound to a second component such as a hydrophobic compound, an immunocompound such as an antibody or antibody active fragment, or a ligand such as a hormone, drug, antigen, or a hapten. The reduction of electrophoretic mobility achieved is directly proportional to the concentration of the polymer-linked affinity compound employed, and such reduction can comprise up to 100 percent for particular particles and cells. The present invention is advantageous in that electrophoretic separation can now be achieved for substances whose native surface charge structure had prevented them from being separated by normal electrophoretic means. Depending on the affinity component utilized, separation can be achieved on the basis of the specific/irreversible, specific/reversible, semi-specific/reversible, relatively nonspecific/reversible, or relatively nonspecific/irreversible ligand-substance interactions.

Extending metabolome coverage for untargeted metabolite profiling of adherent cultured hepatic cells.

PubMed

García-Cañaveras, Juan Carlos; López, Silvia; Castell, José Vicente; Donato, M Teresa; Lahoz, Agustín

2016-02-01

MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (-) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids.

A simple method for the investigation of cell separation effects of blood with physiological hematocrit values.

PubMed

Gester, Kathrin; Jansen, Sebastian V; Stahl, Marion; Steinseifer, Ulrich

2015-05-01

Even though the separation of blood into erythrocyte-rich and erythrocyte-poor areas is well known in physiological setups such as small vessels, it has recently come into focus in small gaps in cardiovascular applications. Studies show that separation effects occur, for example, in gaps in hydrodynamic bearings, where they can have a positive effect on hemolysis. Separation effects depend on the hematocrit value, but due to visualization issues, studies in small gaps used very low hematocrit values. In this study, a test setup and an evaluation method for the investigation of separation effects of blood with hematocrit values of 30, 45, and 60% were developed. The erythrocyte distribution was evaluated by means of gray scale value distribution. This principle is based on the fact that an erythrocyte-rich region is more opaque than an erythrocyte-poor region. The experimental setup is designed in a way that no further processes (e.g., fluorescence labeling) need to be carried out which might change the properties of the membrane of the erythrocytes, and therefore their flow properties. Additionally, the method is executable with basic laboratory equipment, which makes it applicable for many laboratories. To validate the feasibility of the method, the influence of the diameter and the flow rate on the migration of erythrocytes were studied in micro channels for three different physiological hematocrit values. Even though no individual cells were traced, plasma layer and areas of high erythrocyte concentration could be identified. Dependencies of the erythrocyte distribution on flow rate and channel diameter were validated. The influence of the hematocrit value was demonstrated as well and showed the hematocrit value to be a crucial factor when investigating cell separation. The experimental results were consistent with findings in the literature. As the developed method is suitable for physiological hematocrit values and easy to handle, it provides an optimal basis for cell separation studies in gap models with whole blood, for example, hydrodynamic bearings, where it can be used to optimize these devices. Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Rare Cell Capture in Microfluidic Devices

PubMed Central

Pratt, Erica D.; Huang, Chao; Hawkins, Benjamin G.; Gleghorn, Jason P.; Kirby, Brian J.

2010-01-01

This article reviews existing methods for the isolation, fractionation, or capture of rare cells in microfluidic devices. Rare cell capture devices face the challenge of maintaining the efficiency standard of traditional bulk separation methods such as flow cytometers and immunomagnetic separators while requiring very high purity of the target cell population, which is typically already at very low starting concentrations. Two major classifications of rare cell capture approaches are covered: (1) non-electrokinetic methods (e.g., immobilization via antibody or aptamer chemistry, size-based sorting, and sheath flow and streamline sorting) are discussed for applications using blood cells, cancer cells, and other mammalian cells, and (2) electrokinetic (primarily dielectrophoretic) methods using both electrode-based and insulative geometries are presented with a view towards pathogen detection, blood fractionation, and cancer cell isolation. The included methods were evaluated based on performance criteria including cell type modeled and used, number of steps/stages, cell viability, and enrichment, efficiency, and/or purity. Major areas for improvement are increasing viability and capture efficiency/purity of directly processed biological samples, as a majority of current studies only process spiked cell lines or pre-diluted/lysed samples. Despite these current challenges, multiple advances have been made in the development of devices for rare cell capture and the subsequent elucidation of new biological phenomena; this article serves to highlight this progress as well as the electrokinetic and non-electrokinetic methods that can potentially be combined to improve performance in future studies. PMID:21532971

Microfluidic immunomagnetic cell separation from whole blood.

PubMed

Bhuvanendran Nair Gourikutty, Sajay; Chang, Chia-Pin; Puiu, Poenar Daniel

2016-02-01

Immunomagnetic-based separation has become a viable technique for the separation of cells and biomolecules. Here we report on the design and analysis of a simple and efficient microfluidic device for high throughput and high efficiency capture of cells tagged with magnetic particles. This is made possible by using a microfluidic chip integrated with customized arrays of permanent magnets capable of creating large magnetic field gradients, which determine the effective capturing of the tagged cells. This method is based on manipulating the cells which are under the influence of a combination of magnetic and fluid dynamic forces in a fluid under laminar flow through a microfluidic chip. A finite element analysis (FEA) model is developed to analyze the cell separation process and predict its behavior, which is validated subsequently by the experimental results. The magnetic field gradients created by various arrangements of magnetic arrays have been simulated using FEA and the influence of these field gradients on cell separation has been studied with the design of our microfluidic chip. The proof-of-concept for the proposed technique is demonstrated by capturing white blood cells (WBCs) from whole human blood. CD45-conjugated magnetic particles were added into whole blood samples to label WBCs and the mixture was flown through our microfluidic device to separate the labeled cells. After the separation process, the remaining WBCs in the elute were counted to determine the capture efficiency, and it was found that more than 99.9% WBCs have been successfully separated from whole blood. The proposed design can be used for positive selection as well as for negative enrichment of rare cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and evaluation of polyvinyl-alcohol blend polymer films as battery separators

NASA Technical Reports Server (NTRS)

Manzo, M. A.

1982-01-01

Several dialdehydes and epoxies were evaluated for their suitability as cross-linkers. Optium concentrations of several cross-linking reagents were determined. A two-step method of cross-linking, which involves treatment of the film in an acid or acid periodate bath, was investigated and dropped in favor of a one-step method in which the acid catalyst, which initiates cross-linking, is added to the PVA - cross-linker solution before casting. The cross-linking was thus achieved during the drying step. This one-step method was much more adaptable to commercial processing. Cross-linked films were characterized as alkaline battery separators. Films were prepared in the lab and tested in cells in order to evaluate the effect of film composition and a number of processing parameters on cell performance. These tests were conducted in order to provide a broader data base from which to select optimum processing parameters. Results of the separator screening tests and the cell tests are discussed.

Method and apparatus for biological material separation

DOEpatents

Robinson, Donna L.

2005-05-10

There has been invented an apparatus comprising a separation barrier for excluding denser cell materials from less dense cell materials after centrifuging of the cells so that selected materials can be withdrawn from the less dense cell materials without inclusion of the denser cell materials or clogging of sampling equipment with denser cell materials. Cells from which selected material is to be withdrawn are centrifuged, either as cells or cells in media. Once the denser cell materials are isolated in a layer by centrifugal force, an invention screen or seive is submerged in the less dense cell material to a level above the layer of denser cell materials to isolate the denser cell materials from the less dense cell materials, preventing mixing of the denser cell materials back into the less dense cell materials when the cells or the cells in media are no longer being centrifuged and to prevent clogging of sampling equipment with denser cell materials. In a particularly useful application of the invention method and apparatus, plasmid DNA can be withdrawn from less dense cell materials without contamination or interference with denser cell materials.

Separation of distinct adhesion complexes and associated cytoskeleton by a micro-stencil-printing method.

PubMed

Caballero, David; Osmani, Naël; Georges-Labouesse, Elisabeth; Labouesse, Michel; Riveline, Daniel

2012-01-01

Adhesion between cells and the extracellular matrix is mediated by different types of transmembraneous proteins. Their associations to specific partners lead to the assembly of contacts such as focal adhesions and hemidesmosomes. The spatial overlap between both contacts within cells has however limited the study of each type of contact. Here we show that with "stampcils" focal contacts and hemidesmosomes can be spatially separated: cells are plated within the cavities of a stencil and the grids of the stencil serve as stamps for grafting an extracellular matrix protein-fibronectin. Cells engage new contacts on stamped zones leading to the segregation of adhesions and their associated cytoskeletons, i.e., actin and intermediate filaments of keratins. This new method should provide new insights into cell contacts compositions and dynamics.

Cup-Shaped Superparamagnetic Hemispheres for Size-Selective Cell Filtration

PubMed Central

Kim, Hyonchol; Terazono, Hideyuki; Takei, Hiroyuki; Yasuda, Kenji

2014-01-01

We propose a new method of size separation of cells exploiting precisely size-controlled hemispherical superparamagnetic microparticles. A three-layered structure of a 2-nm nickel layer inserted between 15-nm silicon dioxide layers was formed on polystyrene cast spheres by vapor deposition. The polystyrene was then removed by burning and the hemispherical superparamagnetic microparticles, “magcups”, were obtained. The standard target cells (CCRF-CEM, 12 ± 2 μm) were mixed with a set of different sizes of the fabricated magcups, and we confirmed that the cells were captured in the magcups having cavities larger than 15 μm in diameter, and then gathered by magnetic force. The collected cells were grown in a culture medium without any damage. The results suggest that this method is quick, simple and non-invasive size separation of target cells. PMID:25219418

Improved method and apparatus for electrostatically sorting biological cells. [DOE patent application

DOEpatents

Merrill, J.T.

An improved method of sorting biological cells in a conventional cell sorter apparatus includes generating a fluid jet containing cells to be sorted, measuring the distance between the centers of adjacent droplets in a zone thereof defined at the point where the fluid jet separates into descrete droplets, setting the distance between the center of a droplet in said separation zone and the position along said fluid jet at which the cell is optically sensed for specific characteristics to be an integral multiple of said center-to-center distance, and disabling a charger from electrically charging a specific droplet if a cell is detected by the optical sensor in a position wherein it will be in the neck area between droplets during droplet formation rather than within a predetermined distance from the droplet center.

Separation and quantification of monothiols and phytochelatins from a wide variety of cell cultures and tissues of trees and other plants using high performance liquid chromatography

Treesearch

Rakesh Minocha; P. Thangavel; Om Parkash Dhankher; Stephanie Long

2008-01-01

The HPLC method presented here for the quantification of metal-binding thiols is considerably shorter than most previously published methods. It is a sensitive and highly reproducible method that separates monobromobimane tagged monothiols (cysteine, glutathione, γ-glutamylcysteine) along with polythiols (PC2, PC3...

Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics.

PubMed

Liang, Wenfeng; Zhao, Yuliang; Liu, Lianqing; Wang, Yuechao; Dong, Zaili; Li, Wen Jung; Lee, Gwo-Bin; Xiao, Xiubin; Zhang, Weijing

2014-01-01

Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP) force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell) sample from red blood cells (RBCs) with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK) chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for efficient and effective purification of Raji cells from RBCs.

IB-LBM simulation on blood cell sorting with a micro-fence structure.

PubMed

Wei, Qiang; Xu, Yuan-Qing; Tian, Fang-bao; Gao, Tian-xin; Tang, Xiao-ying; Zu, Wen-Hong

2014-01-01

A size-based blood cell sorting model with a micro-fence structure is proposed in the frame of immersed boundary and lattice Boltzmann method (IB-LBM). The fluid dynamics is obtained by solving the discrete lattice Boltzmann equation, and the cells motion and deformation are handled by the immersed boundary method. A micro-fence consists of two parallel slope post rows which are adopted to separate red blood cells (RBCs) from white blood cells (WBCs), in which the cells to be separated are transported one after another by the flow into the passageway between the two post rows. Effected by the cross flow, RBCs are schemed to get through the pores of the nether post row since they are smaller and more deformable compared with WBCs. WBCs are required to move along the nether post row till they get out the micro-fence. Simulation results indicate that for a fix width of pores, the slope angle of the post row plays an important role in cell sorting. The cells mixture can not be separated properly in a small slope angle, while obvious blockages by WBCs will take place to disturb the continuous cell sorting in a big slope angle. As an optimal result, an adaptive slope angle is found to sort RBCs form WBCs correctly and continuously.

Dissolved air flotation and centrifugation as methods for oil recovery from ruptured microalgal cells.

PubMed

Ghasemi Naghdi, Forough; Schenk, Peer M

2016-10-01

Solvent-free microalgal lipid recovery is highly desirable for safer, more sustainable and more economical microalgal oil production. Dispersed air flotation and centrifugation were evaluated for the ability to separate oil and debris from a slurry mixture of osmotically fractured Chaetoceros muelleri cells with and without utilizing collectors. Microalgal oil partially phase-separated as a top layer and partially formed an oil-in-water emulsion. Although collectors, such as sodium dodecyl sulphate enhanced selective flotation, by just adjusting the pH and cell concentration of the mixture, up to 78% of the lipids were recovered in the froth. Using centrifugation of fractured microalgal slurry resulted in removal of 60% cell debris and up to 68.5% of microalgal oil was present in the supernatant. Both methods, centrifugation and flotation provided options for separation of microalgal oil from C. muelleri slurry with similar fatty acid recoveries of 57% and 60%, respectively. Copyright © 2016. Published by Elsevier Ltd.

Acoustic impedance matched buffers enable separation of bacteria from blood cells at high cell concentrations.

PubMed

Ohlsson, Pelle; Petersson, Klara; Augustsson, Per; Laurell, Thomas

2018-06-14

Sepsis is a common and often deadly systemic response to an infection, usually caused by bacteria. The gold standard for finding the causing pathogen in a blood sample is blood culture, which may take hours to days. Shortening the time to diagnosis would significantly reduce mortality. To replace the time-consuming blood culture we are developing a method to directly separate bacteria from red and white blood cells to enable faster bacteria identification. The blood cells are moved from the sample flow into a parallel stream using acoustophoresis. Due to their smaller size, the bacteria are not affected by the acoustic field and therefore remain in the blood plasma flow and can be directed to a separate outlet. When optimizing for sample throughput, 1 ml of undiluted whole blood equivalent can be processed within 12.5 min, while maintaining the bacteria recovery at 90% and the blood cell removal above 99%. That makes this the fastest label-free microfluidic continuous flow method per channel to separate bacteria from blood with high bacteria recovery (>80%). The high throughput was achieved by matching the acoustic impedance of the parallel stream to that of the blood sample, to avoid that acoustic forces relocate the fluid streams.

Determination of Amino Acids in Cell Culture and Fermentation Broth Media Using Anion-Exchange Chromatography with Integrated Pulsed Amperometric Detection

PubMed Central

Hanko, Valoran P.; Heckenberg, Andrea; Rohrer, Jeffrey S.

2004-01-01

Anion-exchange chromatography with integrated pulsed amperometric detection (AE-IPAD) separates and directly detects amino acids, carbohydrates, alditols, and glycols in the same injection without pre- or post-column derivatization. These separations use a combination of NaOH and NaOH/sodium acetate eluents. We previously published the successful use of this technique, also known as AAA-Direct, to determine free amino acids in cell culture and fermentation broth media. We showed that retention of carbohydrates varies with eluent NaOH concentration differently than amino acids, and thus separations can be optimized by varying the initial NaOH concentration and its duration. Unfortunately, some amino acids eluting in the acetate gradient portion of the method were not completely resolved from system-related peaks and from unknown peaks in complex cell culture and fermentation media. In this article, we present changes in method that improve amino acid resolution and system ruggedness. The success of these changes and their compatibility with the separations previously designed for fermentation and cell culture are demonstrated with yeast extract-peptone-dextrose broth, M199, Dulbecco’s modified Eagle’s (with F-12), L-15 (Leibovitz), and McCoy’s 5A cell culture media. PMID:15585828

Colon Stem Cell and Crypt Dynamics Exposed by Cell Lineage Reconstruction

PubMed Central

Itzkovitz, Shalev; Elbaz, Judith; Maruvka, Yosef E.; Segev, Elad; Shlush, Liran I.; Dekel, Nava; Shapiro, Ehud

2011-01-01

Stem cell dynamics in vivo are often being studied by lineage tracing methods. Our laboratory has previously developed a retrospective method for reconstructing cell lineage trees from somatic mutations accumulated in microsatellites. This method was applied here to explore different aspects of stem cell dynamics in the mouse colon without the use of stem cell markers. We first demonstrated the reliability of our method for the study of stem cells by confirming previously established facts, and then we addressed open questions. Our findings confirmed that colon crypts are monoclonal and that, throughout adulthood, the process of monoclonal conversion plays a major role in the maintenance of crypts. The absence of immortal strand mechanism in crypts stem cells was validated by the age-dependent accumulation of microsatellite mutations. In addition, we confirmed the positive correlation between physical and lineage proximity of crypts, by showing that the colon is separated into small domains that share a common ancestor. We gained new data demonstrating that colon epithelium is clustered separately from hematopoietic and other cell types, indicating that the colon is constituted of few progenitors and ruling out significant renewal of colonic epithelium from hematopoietic cells during adulthood. Overall, our study demonstrates the reliability of cell lineage reconstruction for the study of stem cell dynamics, and it further addresses open questions in colon stem cells. In addition, this method can be applied to study stem cell dynamics in other systems. PMID:21829376

Continuous inertial microparticle and blood cell separation in straight channels with local microstructures.

PubMed

Wu, Zhenlong; Chen, Yu; Wang, Moran; Chung, Aram J

2016-02-07

Fluid inertia which has conventionally been neglected in microfluidics has been gaining much attention for particle and cell manipulation because inertia-based methods inherently provide simple, passive, precise and high-throughput characteristics. Particularly, the inertial approach has been applied to blood separation for various biomedical research studies mainly using spiral microchannels. For higher throughput, parallelization is essential; however, it is difficult to realize using spiral channels because of their large two dimensional layouts. In this work, we present a novel inertial platform for continuous sheathless particle and blood cell separation in straight microchannels containing microstructures. Microstructures within straight channels exert secondary flows to manipulate particle positions similar to Dean flow in curved channels but with higher controllability. Through a balance between inertial lift force and microstructure-induced secondary flow, we deterministically position microspheres and cells based on their sizes to be separated downstream. Using our inertial platform, we successfully sorted microparticles and fractionized blood cells with high separation efficiencies, high purities and high throughputs. The inertial separation platform developed here can be operated to process diluted blood with a throughput of 10.8 mL min(-1)via radially arrayed single channels with one inlet and two rings of outlets.

Sample detection and analysis techniques for electrophoretic separation

NASA Technical Reports Server (NTRS)

Falb, R. D.; Hughes, K. E.; Powell, T. R.

1975-01-01

Methods for detecting and analyzing biological agents suitable for space flight operations were studied primarily by literature searches which were conducted of cell separation techniques. Detection methods discussed include: photometrometric, electric, radiometric, micrometry, ultrasonic, microscopic, and photographic. A bibliography, and a directory of vendors are included along with an index of commercial hardware.

Electrochemical cell method

DOEpatents

Kaun, T.D.; Eshman, P.F.

1980-05-09

A secondary electrochemical cell is prepared by providing positive and negative electrodes having outer enclosures of rigid perforated electrically conductive material defining an internal compartment containing the electrode material in porous solid form. The electrodes are each immersed in molten electrolyte salt prior to cell assembly to incorporate the cell electrolyte. Following solidification of the electrolyte substantially throughout the porous volume of the electrode material, the electrodes are arranged in an alternating positive-negative array with interelectrode separators of porous frangible electrically insulative material. The completed array is assembled into the cell housing and sealed such that on heating the solidified electrolyte flows into the interelectrode separator.

Analysis and enhancement of astaxanthin accumulation in Haematococcus pluvialis.

PubMed

Orosa, M; Franqueira, D; Cid, A; Abalde, J

2005-02-01

The green microalga Haematococcus pluvialis was cultured with different concentrations of NaNO(3) to determine the effect on cell growth and astaxanthin accumulation. The optimum nitrate concentration to obtain astaxanthin and to avoid the cessation of cell division was 0.15 g/l NaNO(3). The ratio chlorophyll a/total carotenoids proved a good physiological indicator of nitrogen deficiency in the cell. The effect of different carbon sources, malonate and acetate, on astaxanthin accumulation was also studied; up to 13 times more carotenoids per cell were accumulated in cultures with malonate than in cultures without this compound. The pigment analysis was performed by a new low toxicity HPLC method capable of separating chlorophylls a and b, carotenes and xanthophylls in a short-period of time, using low volumes of solvents and with an economical price. With this method even echinenone was separated, which had been unsuccessful by any other method.

High-throughput rare cell separation from blood samples using steric hindrance and inertial microfluidics.

PubMed

Shen, Shaofei; Ma, Chao; Zhao, Lei; Wang, Yaolei; Wang, Jian-Chun; Xu, Juan; Li, Tianbao; Pang, Long; Wang, Jinyi

2014-07-21

The presence and quantity of rare cells in the bloodstream of cancer patients provide a potentially accessible source for the early detection of invasive cancer and for monitoring the treatment of advanced diseases. The separation of rare cells from peripheral blood, as a "virtual and real-time liquid biopsy", is expected to replace conventional tissue biopsies of metastatic tumors for therapy guidance. However, technical obstacles, similar to looking for a needle in a haystack, have hindered the broad clinical utility of this method. In this study, we developed a multistage microfluidic device for continuous label-free separation and enrichment of rare cells from blood samples based on cell size and deformability. We successfully separated tumor cells (MCF-7 and HeLa cells) and leukemic (K562) cells spiked in diluted whole blood using a unique complementary combination of inertial microfluidics and steric hindrance in a microfluidic system. The processing parameters of the inertial focusing and steric hindrance regions were optimized to achieve high-throughput and high-efficiency separation, significant advantages compared with existing rare cell isolation technologies. The results from experiments with rare cells spiked in 1% hematocrit blood indicated >90% cell recovery at a throughput of 2.24 × 10(7) cells min(-1). The enrichment of rare cells was >2.02 × 10(5)-fold. Thus, this microfluidic system driven by purely hydrodynamic forces has practical potential to be applied either alone or as a sample preparation platform for fundamental studies and clinical applications.

Acoustic Microfluidics for Bioanalytical Application

NASA Astrophysics Data System (ADS)

Lopez, Gabriel

2013-03-01

This talk will present new methods the use of ultrasonic standing waves in microfluidic systems to manipulate microparticles for the purpose of bioassays and bioseparations. We have recently developed multi-node acoustic focusing flow cells that can position particles into many parallel flow streams and have demonstrated the potential of such flow cells in the development of high throughput, parallel flow cytometers. These experiments show the potential for the creation of high throughput flow cytometers in applications requiring high flow rates and rapid detection of rare cells. This talk will also present the development of elastomeric capture microparticles and their use in acoustophoretic separations. We have developed simple methods to form elastomeric particles that are surface functionalized with biomolecular recognition reagents. These compressible particles exhibit negative acoustic contrast in ultrasound when suspended in aqueous media, blood serum or diluted blood. These particles can be continuously separated from cells by flowing them through a microfluidic device that uses an ultrasonic standing wave to align the blood cells, which exhibit positive acoustic contrast, at a node in the acoustic pressure distribution while aligning the negative acoustic contrast elastomeric particles at the antinodes. Laminar flow of the separated particles to downstream collection ports allows for collection of the separated negative contrast particles and cells. Separated elastomeric particles were analyzed via flow cytometry to demonstrate nanomolar detection for prostate specific antigen in aqueous buffer and picomolar detection for IgG in plasma and diluted blood samples. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers (including biomolecules and cells) in a number of biological sample types. We acknowledge support through the NSF Research Triangle MRSEC.

Alkaline electrochemical cells and method of making

NASA Technical Reports Server (NTRS)

Hoyt, H. E.; Pfluger, H. L. (Inventor)

1970-01-01

Equilibrated cellulose ether membranes of increased electrolytic conductivity for use as separators in concentrated alkaline electrochemical cells are investigated. The method of making such membranes by equilibration to the degree desired in an aqueous alkali solution mantained at a temperature below about 10 C is described.

Estimation of ovular fiber production in cotton

DOEpatents

Van't Hof, Jack

1998-09-01

The present invention is a method for rendering cotton fiber cells that are post-anthesis and pre-harvest available for analysis of their physical properties. The method includes the steps of hydrolyzing cotton fiber cells and separating cotton fiber cells from cotton ovules thereby rendering the cells available for analysis. The analysis of the fiber cells is through any suitable means, e.g., visual inspection. Visual inspection of the cells can be accomplished by placing the cells under an instrument for detection, such as microscope or other means.

Benchtop Technologies for Circulating Tumor Cells Separation Based on Biophysical Properties

PubMed Central

Low, Wan Shi; Wan Abas, Wan Abu Bakar

2015-01-01

Circulating tumor cells (CTCs) are tumor cells that have detached from primary tumor site and are transported via the circulation system. The importance of CTCs as prognostic biomarker is leveraged when multiple studies found that patient with cutoff of 5 CTCs per 7.5 mL blood has poor survival rate. Despite its clinical relevance, the isolation and characterization of CTCs can be quite challenging due to their large morphological variability and the rare presence of CTCs within the blood. Numerous methods have been employed and discussed in the literature for CTCs separation. In this paper, we will focus on label free CTCs isolation methods, in which the biophysical and biomechanical properties of cells (e.g., size, deformability, and electricity) are exploited for CTCs detection. To assess the present state of various isolation methods, key performance metrics such as capture efficiency, cell viability, and throughput will be reported. Finally, we discuss the challenges and future perspectives of CTC isolation technologies. PMID:25977918

Benchtop technologies for circulating tumor cells separation based on biophysical properties.

PubMed

Low, Wan Shi; Wan Abas, Wan Abu Bakar

2015-01-01

Circulating tumor cells (CTCs) are tumor cells that have detached from primary tumor site and are transported via the circulation system. The importance of CTCs as prognostic biomarker is leveraged when multiple studies found that patient with cutoff of 5 CTCs per 7.5 mL blood has poor survival rate. Despite its clinical relevance, the isolation and characterization of CTCs can be quite challenging due to their large morphological variability and the rare presence of CTCs within the blood. Numerous methods have been employed and discussed in the literature for CTCs separation. In this paper, we will focus on label free CTCs isolation methods, in which the biophysical and biomechanical properties of cells (e.g., size, deformability, and electricity) are exploited for CTCs detection. To assess the present state of various isolation methods, key performance metrics such as capture efficiency, cell viability, and throughput will be reported. Finally, we discuss the challenges and future perspectives of CTC isolation technologies.

Method of automating of the separation of blasts and lymphocytes in the diagnosis of acute myeloid leukemia

NASA Astrophysics Data System (ADS)

Blindar, V. N.; Nikitaev, V. G.; Polyakov, E. V.; Matveeva, I. I.

2017-01-01

The work deals with the separation of the lymphocytes of healthy patients from blasts of patients with acute myeloblastic leukemia (different variants of the disease). In this study the evaluation of textural characteristics has been done for nuclei of blood cells for cells classification and for the determination of a variant of acute myeloblastic leukemia.

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

NASA Technical Reports Server (NTRS)

Mozdziak, P. E.; Pulvermacher, P. M.; Schultz, E.; Schell, K.

2000-01-01

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. Copyright 2000 Wiley-Liss, Inc.

Cell structure for electrochemical devices and method of making same

DOEpatents

Kaun, Thomas D.

1993-01-01

An electrochemical device comprises a plurality of cells, each cell including a laminate cell membrane, made up of a separator/electrolyte means interposed between alternating positive and negative electrodes, each type of electrode being respectively in common contact to a single current collector.

Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

PubMed

Dores, C; Rancourt, D; Dobrinski, I

2015-05-01

To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density, or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here, we report the use of stirred suspension bioreactors (SSB) to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: SSB followed by differential plating. After 66 h of culture, germ cell enrichment in SSBs provided 7.3 ± 1.0-fold (n = 9), differential plating 9.8 ± 2.4-fold (n = 6) and combination of both methods resulted in 9.1 ± 0.3-fold enrichment of germ cells from the initial germ cell population (n = 3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the SSB allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability owing to handling. © 2015 American Society of Andrology and European Academy of Andrology.

Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis

PubMed Central

Dores, Camila; Rancourt, Derrick; Dobrinski, Ina

2015-01-01

To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here we report the use of stirred suspension bioreactors to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: stirred suspension bioreactor followed by differential plating. After 66 hours of culture, germ cell enrichment in stirred suspension bioreactors provided 7.3±1.0 fold (n=9), differential plating 9.8±2.4 fold (n=6) and combination of both methods resulted in 9.1±0.3 fold enrichment of germ cells from the initial germ cell population (n=3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the stirred suspension bioreactor allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability due to handling. PMID:25877677

A lysozyme and magnetic bead based method of separating intact bacteria.

PubMed

Diler, Ebru; Obst, Ursula; Schmitz, Katja; Schwartz, Thomas

2011-07-01

As a response to environmental stress, bacterial cells can enter a physiological state called viable but noncultivable (VBNC). In this state, bacteria fail to grow on routine bacteriological media. Consequently, standard methods of contamination detection based on bacteria cultivation fail. Although they are not growing, the cells are still alive and are able to reactivate their metabolism. The VBNC state and low bacterial densities are big challenges for cultivation-based pathogen detection in drinking water and the food industry, for example. In this context, a new molecular-biological separation method for bacteria using point-mutated lysozymes immobilised on magnetic beads for separating bacteria is described. The immobilised mutated lysozymes on magnetic beads serve as bait for the specific capture of bacteria from complex matrices or water due to their remaining affinity for bacterial cell wall components. Beads with bacteria can be separated using magnetic racks. To avoid bacterial cell lysis by the lysozymes, the protein was mutated at amino acid position 35, leading to the exchange of the catalytic glutamate for alanine (LysE35A) and glutamine (LysE35Q). As proved by turbidity assay with reference bacteria, the muramidase activity was knocked out. The mutated constructs were expressed by the yeast Pichia pastoris and secreted into expression medium. Protein enrichment and purification were carried out by SO(3)-functionalised nanoscale cationic exchanger particles. For a proof of principle, the proteins were biotinylated and immobilised on streptavidin-functionalised, fluorescence dye-labelled magnetic beads. These constructs were used for the successful capture of Syto9-marked Microccocus luteus cells from cell suspension, as visualised by fluorescence microscopy, which confirmed the success of the strategy.

Continuous Flow Deformability-Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets.

PubMed

Park, Emily S; Jin, Chao; Guo, Quan; Ang, Richard R; Duffy, Simon P; Matthews, Kerryn; Azad, Arun; Abdi, Hamidreza; Todenhöfer, Tilman; Bazov, Jenny; Chi, Kim N; Black, Peter C; Ma, Hongshen

2016-04-13

Circulating tumor cells (CTCs) offer tremendous potential for the detection and characterization of cancer. A key challenge for their isolation and subsequent analysis is the extreme rarity of these cells in circulation. Here, a novel label-free method is described to enrich viable CTCs directly from whole blood based on their distinct deformability relative to hematological cells. This mechanism leverages the deformation of single cells through tapered micrometer scale constrictions using oscillatory flow in order to generate a ratcheting effect that produces distinct flow paths for CTCs, leukocytes, and erythrocytes. A label-free separation of circulating tumor cells from whole blood is demonstrated, where target cells can be separated from background cells based on deformability despite their nearly identical size. In doping experiments, this microfluidic device is able to capture >90% of cancer cells from unprocessed whole blood to achieve 10(4) -fold enrichment of target cells relative to leukocytes. In patients with metastatic castration-resistant prostate cancer, where CTCs are not significantly larger than leukocytes, CTCs can be captured based on deformability at 25× greater yield than with the conventional CellSearch system. Finally, the CTCs separated using this approach are collected in suspension and are available for downstream molecular characterization. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Method and apparatus for iterative lysis and extraction of algae

DOEpatents

Chew, Geoffrey; Boggs, Tabitha; Dykes, Jr., H. Waite H.; Doherty, Stephen J.

2015-12-01

A method and system for processing algae involves the use of an ionic liquid-containing clarified cell lysate to lyse algae cells. The resulting crude cell lysate may be clarified and subsequently used to lyse algae cells. The process may be repeated a number of times before a clarified lysate is separated into lipid and aqueous phases for further processing and/or purification of desired products.

Multipotent Mesenchymal Stromal Stem Cell Expansion by Plating Whole Bone Marrow at a Low Cellular Density: A More Advantageous Method for Clinical Use

PubMed Central

Mareschi, Katia; Rustichelli, Deborah; Calabrese, Roberto; Gunetti, Monica; Sanavio, Fiorella; Castiglia, Sara; Risso, Alessandra; Ferrero, Ivana; Tarella, Corrado; Fagioli, Franca

2012-01-01

Mesenchymal stem cells (MSCs) are a promising source for cell therapy due to their pluripotency and immunomodulant proprieties. As the identification of “optimal” conditions is important to identify a standard procedure for clinical use. Percoll, Ficoll and whole bone marrow directly plated were tested from the same sample as separation methods. The cells were seeded at the following densities: 100 000, 10 000, 1000, 100, 10 cells/cm2. After reaching confluence, the cells were detached, pooled and re-plated at 1000, 500, 100, and 10 cells/cm2. Statistical analyses were performed. Cumulative Population Doublings (PD) did not show significant differences for the separation methods and seeding densities but only for the plating density. Some small quantity samples plated in T25 flasks at plating densities of 10 and 100 cells/cm2 did not produce any expansion. However, directly plated whole bone marrow resulted in a more advantageous method in terms of CFU-F number, cellular growth and minimal manipulation. No differences were observed in terms of gross morphology, differentiation potential or immunophenotype. These data suggest that plating whole bone marrow at a low cellular density may represent a good procedure for MSC expansion for clinical use. PMID:23715383

Basic features of boron isotope separation by SILARC method in the two-step iterative static model

NASA Astrophysics Data System (ADS)

Lyakhov, K. A.; Lee, H. J.

2013-05-01

In this paper we develop a new static model for boron isotope separation by the laser assisted retardation of condensation method (SILARC) on the basis of model proposed by Jeff Eerkens. Our model is thought to be adequate to so-called two-step iterative scheme for isotope separation. This rather simple model helps to understand combined action on boron separation by SILARC method of all important parameters and relations between them. These parameters include carrier gas, molar fraction of BCl3 molecules in carrier gas, laser pulse intensity, gas pulse duration, gas pressure and temperature in reservoir and irradiation cells, optimal irradiation cell and skimmer chamber volumes, and optimal nozzle throughput. A method for finding optimal values of these parameters based on some objective function global minimum search was suggested. It turns out that minimum of this objective function is directly related to the minimum of total energy consumed, and total setup volume. Relations between nozzle throat area, IC volume, laser intensity, number of nozzles, number of vacuum pumps, and required isotope production rate were derived. Two types of industrial scale irradiation cells are compared. The first one has one large throughput slit nozzle, while the second one has numerous small nozzles arranged in parallel arrays for better overlap with laser beam. It is shown that the last one outperforms the former one significantly. It is argued that NO2 is the best carrier gas for boron isotope separation from the point of view of energy efficiency and Ar from the point of view of setup compactness.

Method of forming emitters for a back-contact solar cell

DOEpatents

Li, Bo; Cousins, Peter J.; Smith, David D.

2015-09-29

Methods of forming emitters for back-contact solar cells are described. In one embodiment, a method includes forming a first solid-state dopant source above a substrate. The first solid-state dopant source includes a plurality of regions separated by gaps. Regions of a second solid-state dopant source are formed above the substrate by printing.

Method of forming emitters for a back-contact solar cell

DOEpatents

Li, Bo; Cousins, Peter J; Smith, David D

2014-12-16

Methods of forming emitters for back-contact solar cells are described. In one embodiment, a method includes forming a first solid-state dopant source above a substrate. The first solid-state dopant source includes a plurality of regions separated by gaps. Regions of a second solid-state dopant source are formed above the substrate by printing.

Method or forming emitters for a back-contact solar cell

DOEpatents

Li, Bo; Cousins, Peter J.; Smith, David D.

2014-08-12

Methods of forming emitters for back-contact solar cells are described. In one embodiment, a method includes forming a first solid-state dopant source above a substrate. The first solid-state dopant source includes a plurality of regions separated by gaps. Regions of a second solid-state dopant source are formed above the substrate by printing.

Multiplex coherent raman spectroscopy detector and method

NASA Technical Reports Server (NTRS)

Joyner, Candace C. (Inventor); Patrick, Sheena T. (Inventor); Chen, Peter (Inventor); Guyer, Dean R. (Inventor)

2004-01-01

A multiplex coherent Raman spectrometer (10) and spectroscopy method rapidly detects and identifies individual components of a chemical mixture separated by a separation technique, such as gas chromatography. The spectrometer (10) and method accurately identify a variety of compounds because they produce the entire gas phase vibrational Raman spectrum of the unknown gas. This is accomplished by tilting a Raman cell (20) to produce a high-intensity, backward-stimulated, coherent Raman beam of 683 nm, which drives a degenerate optical parametric oscillator (28) to produce a broadband beam of 1100-1700 nm covering a range of more than 3000 wavenumber. This broadband beam is combined with a narrowband beam of 532 nm having a bandwidth of 0.003 wavenumbers and focused into a heated windowless cell (38) that receives gases separated by a gas chromatograph (40). The Raman radiation scattered from these gases is filtered and sent to a monochromator (50) with multichannel detection.

Multiplex coherent raman spectroscopy detector and method

DOEpatents

Chen, Peter; Joyner, Candace C.; Patrick, Sheena T.; Guyer, Dean R.

2004-06-08

A multiplex coherent Raman spectrometer (10) and spectroscopy method rapidly detects and identifies individual components of a chemical mixture separated by a separation technique, such as gas chromatography. The spectrometer (10) and method accurately identify a variety of compounds because they produce the entire gas phase vibrational Raman spectrum of the unknown gas. This is accomplished by tilting a Raman cell (20) to produce a high-intensity, backward-stimulated, coherent Raman beam of 683 nm, which drives a degenerate optical parametric oscillator (28) to produce a broadband beam of 1100-1700 nm covering a range of more than 3000 wavenumber. This broadband beam is combined with a narrowband beam of 532 nm having a bandwidth of 0.003 wavenumbers and focused into a heated windowless cell (38) that receives gases separated by a gas chromatograph (40). The Raman radiation scattered from these gases is filtered and sent to a monochromator (50) with multichannel detection.

On-chip Magnetic Separation and Cell Encapsulation in Droplets

NASA Astrophysics Data System (ADS)

Chen, A.; Byvank, T.; Bharde, A.; Miller, B. L.; Chalmers, J. J.; Sooryakumar, R.; Chang, W.-J.; Bashir, R.

2012-02-01

The demand for high-throughput single cell assays is gaining importance because of the heterogeneity of many cell suspensions, even after significant initial sorting. These suspensions may display cell-to-cell variability at the gene expression level that could impact single cell functional genomics, cancer, stem-cell research and drug screening. The on-chip monitoring of individual cells in an isolated environment could prevent cross-contamination, provide high recovery yield and ability to study biological traits at a single cell level These advantages of on-chip biological experiments contrast to conventional methods, which require bulk samples that provide only averaged information on cell metabolism. We report on a device that integrates microfluidic technology with a magnetic tweezers array to combine the functionality of separation and encapsulation of objects such as immunomagnetically labeled cells or magnetic beads into pico-liter droplets on the same chip. The ability to control the separation throughput that is independent of the hydrodynamic droplet generation rate allows the encapsulation efficiency to be optimized. The device can potentially be integrated with on-chip labeling and/or bio-detection to become a powerful single-cell analysis device.

Thermally assisted acoustophoresis as a new stiffness-based separation method

NASA Astrophysics Data System (ADS)

Dolatmoradi, Ata; El-Zahab, Bilal

2017-02-01

The use of acoustophoretic separation devices provides a feasible means in biomedical diagnostics for label-free separation of diseased cells. Separation via acoustophoresis, however, has been restricted mainly to size contrast. Thermally-assisted acoustophoresis, as a newly-developed approach, integrates acoustic and thermal actuators on the same platform, enabling a stiffness-based separation when adjusted properly. Using this method, we have demonstrated the possibility of separating cell-mimicking liposomes based on their membrane stiffness. In a temperature-tuned microchannel with an overlaid ultrasonic standing wave, the acoustic contrast factor of a liposome is mainly determined according to its compressibility compared to that of medium. The sign of this factor was observed to flip to a negative value at a specific temperature, unique to the composition of the liposome. This sign switch was hypothesized to be due to the thermotropic phase transitions in the liposome's membrane upon which an apparent effect on the compressibility is experienced by the liposome. By choosing the midpoint of the existing temperature window for two different compositions, within which liposomes were mechanically distinct enough to become differentiable in the acoustic radiation field, we examined the separation efficiency under different flow rate conditions.

Cell structure for electrochemical devices and method of making same

DOEpatents

Kaun, Thomas D.

2007-03-27

An electrochemical device comprising alternating layers of positive and negative electrodes separated from each other by separator layers. The electrode layers extend beyond the periphery of the separator layers providing superior contact between the electrodes and battery terminals, eliminating the need for welding the electrode to the terminal. Electrical resistance within the battery is decreased and thermal conductivity of the cell is increased allowing for superior heat removal from the battery and increased efficiency. Increased internal pressure within the battery can be alleviated without damaging or removing the battery from service while keeping the contents of the battery sealed off from the atmosphere by a pressure release system. Nonoperative cells within a battery assembly can also be removed from service by shorting the nonoperative cell thus decreasing battery life.

[Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by magnetic beads separating B cells and single cell RT-PCR cloning].

PubMed

Huang, Xiang-Ying; Yu, Shuang-Qing; Cheng, Zhan; Ye, Jing-Rong; Xu, Ke; Feng, Xia; Zeng, Yi

2013-04-01

To establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals. Human B cells were purified by negative sorting from PBMCs and memory B cells were further enriched using anti-CD27 microbeads. Gp120 antigen labbled with biotin was incubated with memory B cells to specifically bind IgG on cells membrane. The memory B cells expressing the Env-specific antibody were harvested by magnetic beads separating, counted and diluted to the level of single cell in each PCR well that loading with catch buffer containing RNase inhibitor to get RNAs. The antibody genes were amplified by single cell RT-PCR and nested PCR, cloned into eukaryotic expression vectors and transfected into 293T cells. The binding activity of recombinant antibodies to Env were tested by ELISA. Three monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual. We can obtain Env-specific antibody by biotin labbled antigen, magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.

Method for manufacturing an electrochemical cell

DOEpatents

Kaun, Thomas D.; Eshman, Paul F.

1982-01-01

A secondary electrochemical cell is prepared by providing positive and negative electrodes having outer enclosures of rigid perforated electrically conductive material defining an internal compartment containing the electrode material in porous solid form. The electrodes are each immersed in molten electrolyte salt prior to cell assembly to incorporate the cell electrolyte. Following solidification of the electrolyte substantially throughout the porous volume of the electrode material, the electrodes are arranged in an alternating positive-negative array with interelectrode separators of porous frangible electrically insulative material. The completed array is assembled into the cell housing and sealed such that on heating the solidified electrolyte flows into the interelectrode separator.

The positive effects of different platelet-rich plasma methods on human muscle, bone, and tendon cells.

PubMed

Mazzocca, Augustus D; McCarthy, Mary Beth R; Chowaniec, David M; Dugdale, Evan M; Hansen, Derek; Cote, Mark P; Bradley, James P; Romeo, Anthony A; Arciero, Robert A; Beitzel, Knut

2012-08-01

Clinical application of platelet-rich plasma (PRP) in the realm of orthopaedic sports medicine has yielded variable results. Differences in separation methods and variability of the individual may contribute to these variable results. To compare the effects of different PRP separation methods on human bone, muscle, and tendon cells in an in vitro model. Controlled laboratory study. Blood collected from 8 participants (mean ± SD age 31.6 ± 10.9 years) was used to obtain PRP preparations. Three different PRP separation methods were used: a single-spin process yielding a lower platelet concentration (PRP(LP)), a single-spin process yielding high platelet and white blood cell concentrations (PRP(HP)), and a double-spin that produces a higher platelet concentration and lower white blood cell concentration (PRP(DS)). Human bone, muscle, and tendon cells obtained from discarded tissue samples during shoulder surgery were placed into culture and treated with the 3 PRP preparations, control media (2% fetal bovine serum [FBS] and 10% FBS), and native blood. Radioactive thymidine assays were obtained to examine cell proliferation, and testing with enzyme-linked immunosorbent assay was used to determine growth factor concentrations. Addition of PRP(LP) to osteocytes, myocytes, and tenocytes significantly increased cell proliferation (P ≤ .05) compared with the controls. Adding PRP(DS) to osteoblasts and tenocytes increased cell proliferation significantly (P ≤ .05), but no significance was shown for its addition to myocytes. The addition of PRP(HP) significantly increased cell proliferation compared with the controls only when added to tenocytes (P ≤ .05). Osteoblasts: Proliferation was significantly increased by addition of PRP(LP) compared with all controls (2% FBS, 10% FBS, native blood) (P ≤ .05). Addition of PRP(DS) led to significantly increased proliferation compared with all controls, native blood, and PRP(HP) (P ≤ .05). Proliferation was significantly less when PRP(HP) was added compared with PRP(DS) (P ≤ .05). Myocytes: Proliferation was significantly increased by addition of PRP(LP) compared with native blood (P ≤ .05). Adding PRP(HP) or PRP(DS) to myocytes showed no significant increase in proliferation compared with the controls or the other separations. Tenocytes: Proliferation was significantly increased by addition of PRP(LP) compared with all controls (2% FBS, 10% FBS, native blood) (P ≤ .05). Addition of PRP(DS) showed a significant increase compared with the controls and native blood. For tenocytes, there was a significant increase (P ≤ .05) seen when PRP(HP) was added compared with the controls and native blood but not compared with the other separations. The primary findings of this study suggest the application of different PRP separations may result in a potential beneficial effect on the clinically relevant target cells in vitro. However, it is unclear which platelet concentration or PRP preparation may be optimal for the treatment of various cell types. In addition, a "more is better" theory for the use of higher platelet concentrations cannot be supported. This study was not intended to prove efficacy but to provide a platform for future research to be built upon. The utilization of different PRP separations may result in a potentially beneficial effect on the clinically relevant target cells in vitro, but it is unclear which platelet concentration or PRP preparation may be optimal for the treatment of various cell types.

Droplet electric separator microfluidic device for cell sorting

NASA Astrophysics Data System (ADS)

Guo, Feng; Ji, Xing-Hu; Liu, Kan; He, Rong-Xiang; Zhao, Li-Bo; Guo, Zhi-Xiao; Liu, Wei; Guo, Shi-Shang; Zhao, Xing-Zhong

2010-05-01

A simple and effective droplet electric separator microfluidic device was developed for cell sorting. The aqueous droplet without precharging operation was influenced to move a distance in the channel along the electric field direction by applying dc voltage on the electrodes beside the channel, which made the target droplet flowing to the collector. Single droplet can be isolated in a sorting rate of ˜100 Hz with microelectrodes under a required pulse. Single or multiple mammalian cell (HePG2) encapsulated in the surfactant free alginate droplet could be sorted out respectively. This method may be used for single cell operation or analysis.

Filtration device for rapid separation of biological particles from complex matrices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sangil; Naraghi-Arani, Pejman; Liou, Megan

2018-01-09

Methods and systems for filtering of biological particles are disclosed. Filtering membranes separate adjacent chambers. Through osmotic or electrokinetic processes, flow of particles is carried out through the filtering membranes. Cells, viruses and cell waste can be filtered depending on the size of the pores of the membrane. A polymer brush can be applied to a surface of the membrane to enhance filtering and prevent fouling.

Thermally assisted acoustofluidic separation of extracellular vesicles from cells

NASA Astrophysics Data System (ADS)

Mirtaheri, Elnaz; Dolatmoradi, Ata; Pimentel, Krystine; Bhansali, Shekhar; El-Zahab, Bilal

2018-02-01

Extracellular vesicles (EVs) have been gaining increasing attention given their role in communicating information between cells. Composition-based isolation of EVs is particularly of high significance as the proteomic and lipidomic characterization of their cargo could provide valuable clues to the role of EVs in mediating the biology of various conditions. This has, however, proved to be challenging as EVs, despite their abundance, are very small and difficult to be differentiated from the other constituents of host media. In addition, currently available methods like ultracentrifugation and filtration are cumbersome and capable of achieving mostly size-based separations. In this work, we demonstrate the possibility of separating submicron EV-like vesicles from cancer cells using a thermally-assisted acoustophoretic device. In a system composed of MCF-7 breast cancer cells spiked with two different types of same-size vesicles, composition-based isolation of vesicles was shown to be realizable through opposite focusing of the system's components at the node and antinodes of the overlaid ultrasonic standing wave. By proper choice of temperature in the microchannel, we were able to achieve separations with purities exceeding 93%. Furthermore, cells recovered from the channel were shown to be viable after the separation.

Simultaneous screening of four epidermal growth factor receptor antagonists from Curcuma longa via cell membrane chromatography online coupled with HPLC-MS.

PubMed

Sun, Meng; Ma, Wei-na; Guo, Ying; Hu, Zhi-gang; He, Lang-chong

2013-07-01

The epidermal growth factor receptors (EGFRs) are significant targets for screening active compounds. In this work, an analytical method was established for rapid screening, separation, and identification of EGFRs antagonists from Curcuma longa. Human embryonic kidney 293 cells with a steadily high expression of EGFRs were used to prepare the cell membrane stationary phase in a cell membrane chromatography model for screening active compounds. Separation and identification of the retention chromatographic peaks was achieved by HPLC-MS. The active sites, docking extents and inhibitory effects of the active compounds were also demonstrated. The screening result found that ar-turmerone, curcumin, demethoxycurcumin, and bisdemethoxycurcumin from Curcuma longa could be active components in a similar manner to gefitinib. Biological trials showed that all of four compounds can inhibit EGFRs protein secretion and cell growth in a dose-dependent manner, and downregulate the phosphorylation of EGFRs. This analytical method demonstrated fast and effective characteristics for screening, separation and identification of the active compounds from a complex system and should be useful for drug discovery with natural medicinal herbs. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical compositions, methods of making the chemical compositions, and structures made from the chemical compositions

DOEpatents

Yang, Lei; Cheng, Zhe; Liu, Ze; Liu, Meilin

2015-01-13

Embodiments of the present disclosure include chemical compositions, structures, anodes, cathodes, electrolytes for solid oxide fuel cells, solid oxide fuel cells, fuel cells, fuel cell membranes, separation membranes, catalytic membranes, sensors, coatings for electrolytes, electrodes, membranes, and catalysts, and the like, are disclosed.

Method of fabricating a monolithic solid oxide fuel cell

DOEpatents

Minh, N.Q.; Horne, C.R.

1994-03-01

In a two-step densifying process of making a monolithic solid oxide fuel cell, a limited number of anode-electrolyte-cathode cells separated by an interconnect layer are formed and partially densified. Subsequently, the partially densified cells are stacked and further densified to form a monolithic array. 10 figures.

Method of fabricating a monolithic solid oxide fuel cell

DOEpatents

Minh, Nguyen Q.; Horne, Craig R.

1994-01-01

In a two-step densifying process of making a monolithic solid oxide fuel cell, a limited number of anode-electrolyte-cathode cells separated by an interconnect layer are formed and partially densified. Subsequently, the partially densified cells are stacked and further densified to form a monolithic array.

Separation of whole blood into plasma and red cells by using a hollow-fibre filtration system.

PubMed

Hornsey, V S; McColl, K; Drummond, O; Prowse, C V

2005-08-01

The aim of this study was to assess the separation of whole blood into red cells and plasma by using the Sangofer device, which is a gravity-fed, hollow-fibre system. The components would then be compared with those produced by the use of more elaborate technical equipment. Ten whole-blood units were leucoreduced by using a WBF2 filter and immediately separated into red cells and plasma by using the Sangofer blood-separation device. Red cells were stored in additive solution and tested on days 1 and 42. The plasma was assayed for levels of various coagulation factors and for markers of both coagulation and complement activation. The red-cell parameters were similar to those obtained when routine centrifugation methods were used. The filter did not cause haemolysis. Levels of plasma factor VIII and factor XI were lower than those seen in routinely produced leucoreduced plasma units but there was no evidence of activation of the coagulation and complement systems. The Sangofer device is simple and straightforward to use and eliminates the need for both centrifugation and automated separation steps during the processing of whole blood into red cells and plasma components. Minor changes are required to make the procedure easier to incorporate into routine use.

Separation of neural stem cells by whole cell membrane capacitance using dielectrophoresis.

PubMed

Adams, Tayloria N G; Jiang, Alan Y L; Vyas, Prema D; Flanagan, Lisa A

2018-01-15

Whole cell membrane capacitance is an electrophysiological property of the plasma membrane that serves as a biomarker for stem cell fate potential. Neural stem and progenitor cells (NSPCs) that differ in ability to form neurons or astrocytes are distinguished by membrane capacitance measured by dielectrophoresis (DEP). Differences in membrane capacitance are sufficient to enable the enrichment of neuron- or astrocyte-forming cells by DEP, showing the separation of stem cells on the basis of fate potential by membrane capacitance. NSPCs sorted by DEP need not be labeled and do not experience toxic effects from the sorting procedure. Other stem cell populations also display shifts in membrane capacitance as cells differentiate to a particular fate, clarifying the value of sorting a variety of stem cell types by capacitance. Here, we describe methods developed by our lab for separating NSPCs on the basis of capacitance using several types of DEP microfluidic devices, providing basic information on the sorting procedure as well as specific advantages and disadvantages of each device. Copyright © 2017 Elsevier Inc. All rights reserved.

Estimation of ovular fiber production in cotton

DOEpatents

Van`t Hof, J.

1998-09-01

The present invention is a method for rendering cotton fiber cells that are post-anthesis and pre-harvest available for analysis of their physical properties. The method includes the steps of hydrolyzing cotton fiber cells and separating cotton fiber cells from cotton ovules thereby rendering the cells available for analysis. The analysis of the fiber cells is through any suitable means, e.g., visual inspection. Visual inspection of the cells can be accomplished by placing the cells under an instrument for detection, such as microscope or other means. 4 figs.

Estimation of ovular fiber production in cotton

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van`t Hof, J.

The present invention is a method for rendering cotton fiber cells that are post-anthesis and pre-harvest available for analysis of their physical properties. The method includes the steps of hydrolyzing cotton fiber cells and separating cotton fiber cells from cotton ovules thereby rendering the cells available for analysis. The analysis of the fiber cells is through any suitable means, e.g., visual inspection. Visual inspection of the cells can be accomplished by placing the cells under an instrument for detection, such as microscope or other means. 4 figs.

ISOLATION OF CHICKEN FOLLICULAR DENDRITIC CELLS

USDA-ARS?s Scientific Manuscript database

The aim of the present study was to isolate chicken follicular dendritic cells (FDC). A combination of methods involving panning, iodixanol density gradient centrifugation, and magnetic cell separation technology made it possible to obtain functional FDC from the cecal tonsils from chickens, which h...

Microfluidic devices for label-free separation of cells through transient interaction with asymmetric receptor patterns

NASA Astrophysics Data System (ADS)

Bose, S.; Singh, R.; Hollatz, M. H.; Lee, C.-H.; Karp, J.; Karnik, R.

2012-02-01

Cell sorting serves an important role in clinical diagnosis and biological research. Most of the existing microscale sorting techniques are either non-specific to antigen type or rely on capturing cells making sample recovery difficult. We demonstrate a simple; yet effective technique for isolating cells in an antigen specific manner by using transient interactions of the cell surface antigens with asymmetric receptor patterned surface. Using microfluidic devices incorporating P-selectin patterns we demonstrate separation of HL60 cells from K562 cells. We achieved a sorting purity above 90% and efficiency greater than 85% with this system. We also present a mathematical model incorporating flow mediated and adhesion mediated transport of cells in the microchannel that can be used to predict the performance of these devices. Lastly, we demonstrate the clinical significance of the method by demonstrating single step separation of neutrophils from whole blood. When whole blood is introduced in the device, the granulocyte population gets separated exclusively yielding neutrophils of high purity (<10% RBC contamination). To our knowledge, this is the first ever demonstration of continuous label free sorting of neutrophils from whole blood. We believe this technology will be useful in developing point-of-care diagnostic devices and also for a host of cell sorting applications.

Separator for alkaline electric batteries and method of making

NASA Technical Reports Server (NTRS)

Pfluger, H. L. (Inventor); Hoyt, H. E.

1970-01-01

Battery separator membranes of high electrolytic conductivity comprising a cellulose ether and a compatible metallic salt of water soluble aliphatic acids and their hydroxy derivatives are described. It was found that methyl cellulose can be modified by another class of materials, nonpolymeric in nature, to form battery separator membranes of low electrolytic resistance but which have the flexibility of membranes made of unmodified methyl cellulose, and which in many cases enhance flexibility over membranes made with unmodified methyl cellulose. Separator membranes for electrochemical cells comprising a cellulose ether and a modified selected from the group consisting of metallic salts of water soluble alphatic acids and their hydroxy derivatives and to electrochemical cells utilizing said membranes are described.

Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues

PubMed Central

Scoville, Steven D.; Keller, Karen A.; Cheng, Stephanie; Zhang, Michael; Zhang, Xiaoli; Caligiuri, Michael A.; Freud, Aharon G.

2015-01-01

We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method. PMID:26223896

Silicon concentrator cell-assembly development

NASA Astrophysics Data System (ADS)

1982-08-01

The purpose was to develop an improved cell assembly design for photovoltaic concentrator receivers. Efforts were concentrated on a study of adhesive/separator systems that might be applied between cell and substrate, because this area holds the key to improved heat transfer, electrical isolation and adhesion. It is also the area in which simpler construction methods offer the greatest benefits for economy and reliability in the manufacturing process. Of the ten most promising designs subjected to rigorous environmental testing, eight designs featuring acrylic and silicon adhesives and fiberglass and polyester separators performed very well.

Simultaneous determination of superoxide and hydrogen peroxide in macrophage RAW 264.7 cell extracts by microchip electrophoresis with laser-induced fluorescence detection.

PubMed

Li, Hongmin; Li, Qingling; Wang, Xu; Xu, Kehua; Chen, Zhenzhen; Gong, Xiaocong; Liu, Xin; Tong, Lili; Tang, Bo

2009-03-15

A method for the first time to simultaneously determine superoxide and hydrogen peroxide in macrophage RAW 264.7 cell extracts by microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF) was developed. 2-Chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and bis(p-methylbenzenesulfonyl) dichlorofluorescein (FS), two probes that can be specifically derivatized by superoxide and hydrogen peroxide, respectively, were synthesized and used. Parameters influencing the derivatization and on-chip separation were optimized. With the use of a HEPES (20 mM, pH 7.4) running buffer, a 50 mm long separation channel, and a separation voltage of 1800 V, baseline separation was achieved within 48 s for the two derivatization products, DBZTC-oxide (DBO) and 2,7-dichlorofluorescein (DCF). The linearity ranges of the method were 0.08-5.0 and 0.02-5.0 microM with detection limits (signal-to-noise ratio = 3) of 10 nM (1.36 amol) and 5.6 nM (0.76 amol) for superoxide and hydrogen peroxide, respectively. The relative standard deviations (RSDs) of migration time and peak area were less than 2.0% and 5.0%, respectively. The recoveries of the cell extract samples spiked with 1.0 microM standard solutions were 96.1% and 93.0% for superoxide and hydrogen peroxide, respectively. With the use of this method, superoxide and hydrogen peroxide in phorbol myristate acetate (PMA)-stimulated macrophage RAW 264.7 cell extracts were found to be 0.78 and 1.14 microM, respectively. The method has paved a way for simultaneously determining two or more reactive oxygen species (ROS) in a biological system with high resolution.

Selective separation of arsenopyrite from pyrite by biomodulation in the presence of Acidithiobacillus ferrooxidans.

PubMed

Chandraprabha, M N; Natarajan, K A; Somasundaran, P

2004-08-15

Effective methods for selective separation using flotation or flocculation of arsenopyrite from pyrite by biomodulation using Acidithiobacillus ferrooxidans are presented here. Adhesion of the bacterium to the surface of arsenopyrite was very slow compared to that to pyrite, resulting in a difference in surface modification of the minerals subsequent to interaction with cells. The cells were able to effectively depress pyrite flotation in presence of collectors like potassium isopropyl xanthate and potassium amyl xanthate. On the other hand the flotability of arsenopyrite after conditioning with the cells was not significantly affected. The activation of pyrite by copper sulfate was reduced when the minerals were conditioned together, resulting in better selectivity. Selective separation could also be achieved by flocculation of biomodulated samples.

Centrifuge apparatus

DOEpatents

Sartory, Walter K.; Eveleigh, John W.

1976-01-01

A method and apparatus for operating a continuous flow blood separation centrifuge are provided. The hematocrit of the entrant whole blood is continuously maintained at an optimum constant value by the addition of plasma to the entrant blood. The hematocrit of the separated red cells is monitored to indicate the degree of separation taking place, thereby providing a basis for regulating the flow through the centrifuge.

Series interconnected photovoltaic cells and method for making same

DOEpatents

Albright, S.P.; Chamberlin, R.R.; Thompson, R.A.

1995-01-31

A novel photovoltaic module and method for constructing the same are disclosed. The module includes a plurality of photovoltaic cells formed on a substrate and laterally separated by interconnection regions. Each cell includes a bottom electrode, a photoactive layer and a top electrode layer. Adjacent cells are connected in electrical series by way of a conductive-buffer line. The buffer line is also useful in protecting the bottom electrode against severing during downstream layer cutting processes. 11 figs.

Numerical analysis of a dielectrophoresis field-flow fractionation device for the separation of multiple cell types.

PubMed

Shamloo, Amir; Kamali, Ali

2017-10-01

In this study, a dielectrophoresis field-flow fractionation device was analyzed using a numerical simulation method and the behaviors of a set of different cells were investigated. By reducing the alternating current frequency of the electrodes from the value used in the original setup configuration and increasing the number of exit channels, total discrimination in cell trajectories and subsequent separation of four cell types were achieved. Cells were differentiated based on their size and dielectric response that are represented in their real part of Clausius-Mossotti factor at different frequencies. A number of novel designs were also proposed based on the original setup configuration. It was seen that by reducing the length of the main channel and the number of electrodes at low frequencies and not changing the inlet flow velocities, cell separation was still achieved successfully, although with a slightly larger electrode voltage. The shorter main channel decreased the residence time for the cells on the chip and also reduced the overall size of the device-these were improvements over the original design. The obtained results can be used to analyze other cell types by knowing their size and dielectric properties to design geometries that can ensure separation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microfluidic antibody arrays for simultaneous cell separation and stimulus.

PubMed

Liu, Yan; Germain, Todd; Pappas, Dimitri

2014-12-01

A microfluidic chip containing stamped antibody arrays was developed for simultaneous cell separation and drug testing. Poly(dimethyl siloxane) (PDMS) stamping was used to deposit antibodies in a microfluidic channel, forming discrete cell-capture regions on the surface. Cell mixtures were then introduced, resulting in the separation of cells when specific antibodies were used. Anti-CD19 antibody regions resulted in 94 % capture purity for CD19+ Ramos cells. An antibody that captures multiple cell types, for example anti-CD71, can also be used to capture several cell types simultaneously. Cells could also be loaded onto the arrays with spatial control using laminar streams. Both Ramos B cells and HuT 78 T cells were isolated in the chip and exposed to staurosporine in the same channel. Both cell lines had similar responses to the drug, with 2-10 % of cells remaining viable after 20 h of drug treatment, depending on cell type. The chip can also be used to analyze the efficacy of antibody therapy against cancer cells. Anti-CD95 was deposited on the surface and used for simultaneous cell capture and apoptosis induction via the extrinsic pathway. Cells captured on anti-CD95 surfaces had significant viability loss (15 % viability after 24 h) when compared with a control anti-CD71 antibody (81 % viability after 24 h). This chip can be used for a variety of cell separation and/or drug testing studies, enabling researchers to isolate cells and test them against different anti-cancer compounds and to follow cell response using fluorescence or other readout methods.

Particle separation by phase modulated surface acoustic waves.

PubMed

Simon, Gergely; Andrade, Marco A B; Reboud, Julien; Marques-Hueso, Jose; Desmulliez, Marc P Y; Cooper, Jonathan M; Riehle, Mathis O; Bernassau, Anne L

2017-09-01

High efficiency isolation of cells or particles from a heterogeneous mixture is a critical processing step in lab-on-a-chip devices. Acoustic techniques offer contactless and label-free manipulation, preserve viability of biological cells, and provide versatility as the applied electrical signal can be adapted to various scenarios. Conventional acoustic separation methods use time-of-flight and achieve separation up to distances of quarter wavelength with limited separation power due to slow gradients in the force. The method proposed here allows separation by half of the wavelength and can be extended by repeating the modulation pattern and can ensure maximum force acting on the particles. In this work, we propose an optimised phase modulation scheme for particle separation in a surface acoustic wave microfluidic device. An expression for the acoustic radiation force arising from the interaction between acoustic waves in the fluid was derived. We demonstrated, for the first time, that the expression of the acoustic radiation force differs in surface acoustic wave and bulk devices, due to the presence of a geometric scaling factor. Two phase modulation schemes are investigated theoretically and experimentally. Theoretical findings were experimentally validated for different mixtures of polystyrene particles confirming that the method offers high selectivity. A Monte-Carlo simulation enabled us to assess performance in real situations, including the effects of particle size variation and non-uniform acoustic field on sorting efficiency and purity, validating the ability to separate particles with high purity and high resolution.

Selective digestion of Ba2+/Ca2+ alginate gel microdroplets for single-cell handling

NASA Astrophysics Data System (ADS)

Odaka, Masao; Hattori, Akihiro; Matsuura, Kenji; Yasuda, Kenji

2018-06-01

Cells encapsuled by polymer microdroplets are an effective platform for the identification and separation of individual cells for single-cell-based analysis. However, a key challenge is to maintain and release the captured cells in the microdroplets selectively, nondestructively, and noninvasively. We developed a simple method of encapsulating cells in alginate microdroplets having different digestion characteristics. Cells were diluted with an alginate polymer of sol state and encapsulated into microdroplets with Ba2+ and Ca2+ by a spray method. When a chelating buffer was applied, alginate gel microdroplets were digested according to the difference in chelating efficiency of linkage-divalent cations; hence, two types of alginate microdroplets were formed. Moreover, we examined the capability of the alginate gel to exchange linkage-divalent cations and found that both Ca2+ exchange in Ba-alginate microdroplets and Ba2+ exchange in Ca-alginate microdroplets occurred. These results indicate that the potential applications of a mixture of alginate microdroplets with different divalent cations control the selective digestion of microdroplets to improve the high-throughput, high-content microdroplet-based separation, analysis, or storage of single cells.

Apparatus and method for constant flow oxidizing of organic materials

DOEpatents

Surma, Jeffrey E.; Nelson, Norvell; Steward, G. Anthony; Bryan, Garry H.

1999-01-01

The invention is a method and apparatus using high cerium concentration in the anolyte of an electrochemical cell to oxidize organic materials. The method and apparatus further use an ultrasonic mixer to enhance the oxidation rate of the organic material in the electrochemical cell. A reaction vessel provides an advantage of independent reaction temperature control and electrochemical cell temperature control. A separate or independent reaction vessel may be used without an ultrasonic mixer to oxidize gaseous phase organic materials.

Simultaneous fermentation and separation in an immobilized cell trickle bed reactor: Acetone-butanol-ethane (ABE) and ethanol fermentation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, C.H.

1989-01-01

A novel process employing immobilized cells and in-situ product removal was studied for acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum and ethanol fermentation by Saccharomyces cerevisiae. Experimental studies of ABE fermentation in a trickle bed reactor without product separation showed that solvent production could be improved by one order of magnitude compared to conventional batch fermentation. Control of effluent pH near 4.3 and feed glucose concentrations higher than 10 g/L were the necessary conditions for cell growth and solvent production. A mathematical model using an equilibrium staged model predicted efficient separation of butanol from the fermentation broth. Activity coefficients of multicomponentmore » system were estimated by Wilson's equation or the ASOG method. Inhibition by butanol and organic acids was incorporated into the kinetic expression. Experimental performance of simultaneous fermentation and separation in an immobilized cell trickle bed reactor showed that glucose conversion was improved as predicted by mathematical modeling and analysis. The effect of pH and temperature on ethanol fermentation by Saccharomyces cerevisiae was studied in free and immobilized cell reactors. Conditions for the highest glucose conversion, cell viability and least glycerol yield were determined.« less

Simultaneous determination of reactive oxygen and nitrogen species in mitochondrial compartments of apoptotic HepG2 cells and PC12 cells based on microchip electrophoresis-laser-induced fluorescence.

PubMed

Chen, Zhenzhen; Li, Qingling; Sun, Qianqian; Chen, Hao; Wang, Xu; Li, Na; Yin, Miao; Xie, Yanxia; Li, Hongmin; Tang, Bo

2012-06-05

Determination of intracellular bioactive species will afford beneficial information related to cell metabolism, signal transduction, cell function, and disease treatment. In this study, the first application of a microchip electrophoresis-laser-induced fluorescence (MCE-LIF) method for concurrent determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS), i.e., superoxide (O(2)(-•)) and nitric oxide (NO) in mitochondria, was developed using fluorescent probes 2-chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and 3-amino,4-aminomethyl-2',7'-difluorescein (DAF-FM), respectively. Potential interference of intracellular dehydroascorbic acid (DHA) and ascorbic acid (AA) for NO detection with DAF-FM was eliminated through oxidation of AA with the addition of ascorbate oxidase, followed by subsequent MCE separation. Fluorescent products of O(2)(-•) and NO, DBZTC oxide (DBO), and DAF-FM triazole (DAF-FMT) showed excellent baseline separation within 1 min with a running buffer of 40 mM Tris solution (pH 7.4) and a separating electric field of 500 V/cm. The levels of DBO and DAF-FMT in mitochondria isolated from normal HepG2 cells and PC12 cells were evaluated using this method. Furthermore, the changes of DBO and DAF-FMT levels in mitochondria isolated from apoptotic HepG2 cells and PC12 cells could also be detected. The current approach was proved to be simple, fast, reproducible, and efficient. Measurement of the two species with the method will be beneficial to understand ROS/RNS distinctive functions. In addition, it will provide new insights into the role that both species play in biological systems.

System and method for networking electrochemical devices

DOEpatents

Williams, Mark C.; Wimer, John G.; Archer, David H.

1995-01-01

An improved electrochemically active system and method including a plurality of electrochemical devices, such as fuel cells and fluid separation devices, in which the anode and cathode process-fluid flow chambers are connected in fluid-flow arrangements so that the operating parameters of each of said plurality of electrochemical devices which are dependent upon process-fluid parameters may be individually controlled to provide improved operating efficiency. The improvements in operation include improved power efficiency and improved fuel utilization in fuel cell power generating systems and reduced power consumption in fluid separation devices and the like through interstage process fluid parameter control for series networked electrochemical devices. The improved networking method includes recycling of various process flows to enhance the overall control scheme.

Method for producing a secondary lithium cell comprising a heat-sensitive protective mechanism

DOEpatents

Ullrich, Matthias; Bechtold, Dieter; Rabenstein, Heinrich; Brohm, Thomas

2003-01-01

A method for producing a secondary lithium cell which has at least one lithium-cycling negative electrode, at least one lithium-intercalating positive electrode, at least one separator disposed between the positive and the negative electrode, and a nonaqueous lithium ion-conducting electrolyte. The method is carried out by the electrodes and/or the separator being coated, by means of electrostatic powder coating, with wax particles which are insoluble in the electrolyte and have a melting temperature of from about 50 to about 150 .degree. C. and a mean particle size of from about 6 to about 20 .mu.m, the amount of wax being between about 0.5 and about 2.5 mg/cm.sup.2 of electrode area.

Separation of periportal and perivenous rat hepatocytes by fluorescence-activated cell sorting: confirmation with colloidal gold as an exogenous marker.

PubMed

Braakman, I; Keij, J; Hardonk, M J; Meijer, D K; Groothuis, G M

1991-01-01

Periportal and perivenous hepatocytes are known to display various functional differences. In this study we present a new method to separate periportal and perivenous cells: after selectively loading zone 1 or zone 3 with the fluorescent label acridine orange in an antegrade or retrograde perfusion, respectively, we separated the isolated hepatocytes on a fluorescence-activated cell sorter. The common way to check on proper separation is to estimate activities of enzymes known to exhibit a heterogeneous acinar distribution. Using enzyme histochemistry, however, we found that already on short collagenase perfusion, some enzymes displayed a more shallow gradient than in vivo, making enzyme activities less suitable as zonal markers. We therefore used colloidal gold granules (17 nm) injected intravenously (2.5 mg) into the rat 2 to 3 hr before cell isolation. The gold is taken up predominantly by perivenous hepatocytes, probably because of the efficient removal of gold granules in zone 1 by competing Kupffer cells. We compared acridine orange fluorescence, presence of gold particles and activities of six marker enzymes, three biochemically and three histochemically determined. Acridine orange and gold both pointed to a high enrichment of the fractions, whereas most enzyme activities were more randomly distributed among the cells as a result of the isolation procedure. Our separation procedure yielded fractions highly enriched in either viable periportal or perivenous cells, both from one liver. The use of colloidal gold as a marker to monitor separation is a valuable alternative to the more risky estimation of enzyme activities.

Injection by hydrostatic pressure in conjunction with electrokinetic force on a microfluidic chip.

PubMed

Gai, Hongwei; Yu, Linfen; Dai, Zhongpeng; Ma, Yinfa; Lin, Bingcheng

2004-06-01

A simple method was developed for injecting a sample on a cross-form microfluidic chip by means of hydrostatic pressure combined with electrokinetic forces. The hydrostatic pressure was generated simply by adjusting the liquid level in different reservoirs without any additional driven equipment such as a pump. Two dispensing strategies using a floating injection and a gated injection, coupled with hydrostatic pressure loading, were tested. The fluorescence observation verified the feasibility of hydrostatic pressure loading in the separation of a mixture of fluorescein sodium salt and fluorescein isothiocyanate. This method was proved to be effective in leading cells to a separation channel for single cell analysis.

Extraction of the number of peroxisomes in yeast cells by automated image analysis.

PubMed

Niemistö, Antti; Selinummi, Jyrki; Saleem, Ramsey; Shmulevich, Ilya; Aitchison, John; Yli-Harja, Olli

2006-01-01

An automated image analysis method for extracting the number of peroxisomes in yeast cells is presented. Two images of the cell population are required for the method: a bright field microscope image from which the yeast cells are detected and the respective fluorescent image from which the number of peroxisomes in each cell is found. The segmentation of the cells is based on clustering the local mean-variance space. The watershed transformation is thereafter employed to separate cells that are clustered together. The peroxisomes are detected by thresholding the fluorescent image. The method is tested with several images of a budding yeast Saccharomyces cerevisiae population, and the results are compared with manually obtained results.

Normalization, bias correction, and peak calling for ChIP-seq

PubMed Central

Diaz, Aaron; Park, Kiyoub; Lim, Daniel A.; Song, Jun S.

2012-01-01

Next-generation sequencing is rapidly transforming our ability to profile the transcriptional, genetic, and epigenetic states of a cell. In particular, sequencing DNA from the immunoprecipitation of protein-DNA complexes (ChIP-seq) and methylated DNA (MeDIP-seq) can reveal the locations of protein binding sites and epigenetic modifications. These approaches contain numerous biases which may significantly influence the interpretation of the resulting data. Rigorous computational methods for detecting and removing such biases are still lacking. Also, multi-sample normalization still remains an important open problem. This theoretical paper systematically characterizes the biases and properties of ChIP-seq data by comparing 62 separate publicly available datasets, using rigorous statistical models and signal processing techniques. Statistical methods for separating ChIP-seq signal from background noise, as well as correcting enrichment test statistics for sequence-dependent and sonication biases, are presented. Our method effectively separates reads into signal and background components prior to normalization, improving the signal-to-noise ratio. Moreover, most peak callers currently use a generic null model which suffers from low specificity at the sensitivity level requisite for detecting subtle, but true, ChIP enrichment. The proposed method of determining a cell type-specific null model, which accounts for cell type-specific biases, is shown to be capable of achieving a lower false discovery rate at a given significance threshold than current methods. PMID:22499706

Use of bacteriophage cell wall-binding proteins for rapid diagnostics of Listeria.

PubMed

Schmelcher, Mathias; Loessner, Martin J

2014-01-01

Diagnostic protocols for food-borne bacterial pathogens such as Listeria need to be sensitive, specific, rapid, and inexpensive. Conventional culture methods are hampered by lengthy enrichment and incubation steps. Bacteriophage-derived high-affinity binding molecules (cell wall-binding domains, CBDs) specific for Listeria cells have recently been introduced as tools for detection and differentiation of this pathogen in foods. When coupled with magnetic separation, these proteins offer advantages in sensitivity and speed compared to the standard diagnostic methods. Furthermore, fusion of CBDs to differently colored fluorescent reporter proteins enables differentiation of Listeria strains in mixed cultures. This chapter provides protocols for detection of Listeria in food by CBD-based magnetic separation and subsequent multiplexed identification of strains of different serotypes with reporter-CBD fusion proteins.

Method of minimizing the effects of parasitic currents

DOEpatents

Chi, Michael C.; Carr, Peter

1983-02-01

A method of minimizing the effect of parasitic currents in secondary batteries having a plurality of cells connected electrically in series and a common electrolyte in communication with the cells is described. Specifically, the parasitic currents flowing through the battery cause a cell imbalance over the charge/discharge cycle. This cell imbalance is minimized by first separating the cells of the battery into two equal groups. Then the battery is charged with the two groups of cells connected electrically in series, and subsequently discharged with the two groups of cells reconnected electrically in series in an inverted sequence.

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system.

PubMed

Tagawa, M; Matoba, S; Narita, M; Saito, N; Nagai, T; Imai, K

2008-03-15

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.

Methods for using novel cathode and electrolyte materials for solid oxide fuel cells and ion transport membranes

DOEpatents

Jacobson, Allan J.; Wang, Shuangyan; Kim, Gun Tae

2016-01-12

Methods using novel cathode, electrolyte and oxygen separation materials operating at intermediate temperatures for use in solid oxide fuel cells and ion transport membranes include oxides with perovskite related structures and an ordered arrangement of A site cations. The materials have significantly faster oxygen kinetics than in corresponding disordered perovskites.

Separation of sperm and epithelial cells based on the hydrodynamic effect for forensic analysis

PubMed Central

Liu, Weiran; Chen, Weixing; Liu, Ran; Ou, Yuan; Liu, Haoran; Xie, Lan; Lu, Ying; Li, Caixia; Li, Bin; Cheng, Jing

2015-01-01

In sexual assault cases, forensic samples are a mixture of sperm from the perpetrator and epithelial cells from the victim. To obtain an independent short tandem repeat (STR) profile of the perpetrator, sperm cells must be separated from the mixture of cells. However, the current method used in crime laboratories, namely, differential extraction, is a time-consuming and labor-intensive process. To achieve a rapid and automated sample pretreatment process, we fabricated a microdevice for hydrodynamic and size-based separation of sperm and epithelial cells. When cells in suspension were introduced into the device's microfluidic channels, they were forced to flow along different streamlines and into different outlets due to their different diameters. With the proposed microdevice, sperm can be separated within a short period of time (0.5 h for a 50-μl mock sample). The STR profiles of the products in the sperm outlet reservoir demonstrated that a highly purified male DNA fraction could be obtained (94.0% male fraction). This microdevice is of low-cost and can be easily integrated with other subsequent analysis units, providing great potential in the process of analyzing sexual assault evidence as well as in other areas requiring cell sorting. PMID:26392829

21 CFR 640.16 - Processing.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.16 Processing. (a) Separation. Within the..., Red Blood Cells may be prepared either by centrifugation, done in a manner that will not tend to... for Red Blood Cells shall be the original blood containers unless the method of processing requires a...

21 CFR 640.16 - Processing.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.16 Processing. (a) Separation. Within the..., Red Blood Cells may be prepared either by centrifugation, done in a manner that will not tend to... for Red Blood Cells shall be the original blood containers unless the method of processing requires a...

21 CFR 640.16 - Processing.

Code of Federal Regulations, 2013 CFR

2013-04-01

... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.16 Processing. (a) Separation. Within the..., Red Blood Cells may be prepared either by centrifugation, done in a manner that will not tend to... for Red Blood Cells shall be the original blood containers unless the method of processing requires a...

21 CFR 640.16 - Processing.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.16 Processing. (a) Separation. Within the..., Red Blood Cells may be prepared either by centrifugation, done in a manner that will not tend to... for Red Blood Cells shall be the original blood containers unless the method of processing requires a...

Ultracapacitor electroyte

DOEpatents

Wei, Chang; LeBlanc, Jr., Oliver Harris; Jerabek, Elihu Calvin

2001-07-03

The invention relates to an ultracapacitor and to a method of making an ultracapacitor. The ultracapacitor of the invention includes two solid, nonporous current collectors, two porous electrodes separating the collectors, a porous separator between the electrodes and an electrolyte occupying the pores in the electrodes and separator. The electrolyte includes a cyclic carbonate solvent, a cyclic ester solvent and an electrolyte salt. The invention also relates to a stack of ultracapacitor cells.

Chemical-Reaction-Controlled Phase Separated Drops: Formation, Size Selection, and Coarsening

NASA Astrophysics Data System (ADS)

Wurtz, Jean David; Lee, Chiu Fan

2018-02-01

Phase separation under nonequilibrium conditions is exploited by biological cells to organize their cytoplasm but remains poorly understood as a physical phenomenon. Here, we study a ternary fluid model in which phase-separating molecules can be converted into soluble molecules, and vice versa, via chemical reactions. We elucidate using analytical and simulation methods how drop size, formation, and coarsening can be controlled by the chemical reaction rates, and categorize the qualitative behavior of the system into distinct regimes. Ostwald ripening arrest occurs above critical reaction rates, demonstrating that this transition belongs entirely to the nonequilibrium regime. Our model is a minimal representation of the cell cytoplasm.

The use of FDTD in establishing in vitro experimentation conditions representative of lifelike cell phone radiation on the spermatozoa.

PubMed

Mouradi, Rand; Desai, Nisarg; Erdemir, Ahmet; Agarwal, Ashok

2012-01-01

Recent studies have shown that exposing human semen samples to cell phone radiation leads to a significant decline in sperm parameters. In daily living, a cell phone is usually kept in proximity to the groin, such as in a trouser pocket, separated from the testes by multiple layers of tissue. The aim of this study was to calculate the distance between cell phone and semen sample to set up an in vitro experiment that can mimic real life conditions (cell phone in trouser pocket separated by multiple tissue layers). For this reason, a computational model of scrotal tissues was designed by considering these separating layers, the results of which were used in a series of simulations using the Finite Difference Time Domain (FDTD) method. To provide an equivalent effect of multiple tissue layers, these results showed that the distance between a cell phone and semen sample should be 0.8 cm to 1.8 cm greater than the anticipated distance between a cell phone and the testes.

Paper-based device for separation and cultivation of single microalga.

PubMed

Chen, Chih-Chung; Liu, Yi-Ju; Yao, Da-Jeng

2015-12-01

Single-cell separation is among the most useful techniques in biochemical research, diagnosis and various industrial applications. Microalgae species have great economic importance as industrial raw materials. Microalgae species collected from environment are typically a mixed and heterogeneous population of species that must be isolated and purified for examination and further application. Conventional methods, such as serial dilution and a streaking-plate method, are intensive of labor and inefficient. We developed a paper-based device for separation and cultivation of single microalga. The fabrication was simply conducted with a common laser printer and required only a few minutes without lithographic instruments and clean-room. The driving force of the paper device was simple capillarity without a complicated pump connection that is part of most devices for microfluidics. The open-structure design of the paper device makes it operable with a common laboratory micropipette for sample transfer and manipulation with a naked eye or adaptable to a robotic system with functionality of high-throughput retrieval and analysis. The efficiency of isolating a single cell from mixed microalgae species is seven times as great as with a conventional method involving serial dilution. The paper device can serve also as an incubator for microalgae growth on simply rinsing the paper with a growth medium. Many applications such as highly expressed cell selection and various single-cell analysis would be applicable. Copyright © 2015 Elsevier B.V. All rights reserved.

Sampling based State of Health estimation methodology for Li-ion batteries

NASA Astrophysics Data System (ADS)

Camci, Fatih; Ozkurt, Celil; Toker, Onur; Atamuradov, Vepa

2015-03-01

Storage and management of energy is becoming a more and more important problem every day, especially for electric and hybrid vehicle applications. Li-ion battery is one of the most important technological alternatives for high capacity energy storage and related industrial applications. State of Health (SoH) of Li-ion batteries plays a critical role in their deployment from economic, safety, and availability aspects. Most, if not all, of the studies related to SoH estimation focus on the measurement of a new parameter/physical phenomena related to SoH, or development of new statistical/computational methods using several parameters. This paper presents a new approach for SoH estimation for Li-ion battery systems with multiple battery cells: The main idea is a new circuit topology which enables separation of battery cells into two groups, main and test batteries, whenever a SoH related measurement is to be conducted. All battery cells will be connected to the main battery during the normal mode of operation. When a measurement is needed for SoH estimation, some of the cells will be separated from the main battery, and SoH estimation related measurements will be performed on these units. Compared to classical SoH measurement methods which deal with whole battery system, the proposed method estimates the SoH of the system by separating a small but representative set of cells. While SoH measurements are conducted on these isolated cells, remaining cells in the main battery continue to function in normal mode, albeit in slightly reduced performance levels. Preliminary experimental results are quite promising, and validate the feasibility of the proposed approach. Technical details of the proposed circuit architecture are also summarized in the paper.

Real-time PCR method combined with immunomagnetic separation for detecting healthy and heat-injured Salmonella Typhimurium on raw duck wings.

PubMed

Zheng, Qianwang; Mikš-Krajnik, Marta; Yang, Yishan; Xu, Wang; Yuk, Hyun-Gyun

2014-09-01

Conventional culture detection methods are time consuming and labor-intensive. For this reason, an alternative rapid method combining real-time PCR and immunomagnetic separation (IMS) was investigated in this study to detect both healthy and heat-injured Salmonella Typhimurium on raw duck wings. Firstly, the IMS method was optimized by determining the capture efficiency of Dynabeads(®) on Salmonella cells on raw duck wings with different bead incubation (10, 30 and 60 min) and magnetic separation (3, 10 and 30 min) times. Secondly, three Taqman primer sets, Sal, invA and ttr, were evaluated to optimize the real-time PCR protocol by comparing five parameters: inclusivity, exclusivity, PCR efficiency, detection probability and limit of detection (LOD). Thirdly, the optimized real-time PCR, in combination with IMS (PCR-IMS) assay, was compared with a standard ISO and a real-time PCR (PCR) method by analyzing artificially inoculated raw duck wings with healthy and heat-injured Salmonella cells at 10(1) and 10(0) CFU/25 g. Finally, the optimized PCR-IMS assay was validated for Salmonella detection in naturally contaminated raw duck wing samples. Under optimal IMS conditions (30 min bead incubation and 3 min magnetic separation times), approximately 85 and 64% of S. Typhimurium cells were captured by Dynabeads® from pure culture and inoculated raw duck wings, respectively. Although Sal and ttr primers exhibited 100% inclusivity and exclusivity for 16 Salmonella spp. and 36 non-Salmonella strains, the Sal primer showed lower LOD (10(3) CFU/ml) and higher PCR efficiency (94.1%) than the invA and ttr primers. Moreover, for Sal and invA primers, 100% detection probability on raw duck wings suspension was observed at 10(3) and 10(4) CFU/ml with and without IMS, respectively. Thus, the Sal primer was chosen for further experiments. The optimized PCR-IMS method was significantly (P=0.0011) better at detecting healthy Salmonella cells after 7-h enrichment than traditional PCR method. However there was no significant difference between the two methods with longer enrichment time (14 h). The diagnostic accuracy of PCR-IMS was shown to be 98.3% through the validation study. These results indicate that the optimized PCR-IMS method in this study could provide a sensitive, specific and rapid detection method for Salmonella on raw duck wings, enabling 10-h detection. However, a longer enrichment time could be needed for resuscitation and reliable detection of heat-injured cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Polyvinyl alcohol membranes as alkaline battery separators

NASA Technical Reports Server (NTRS)

Sheibley, D. W.; Gonzalez-Sanabria, O.; Manzo, M. A.

1982-01-01

Polyvinly alcohol (PVA) cross-linked with aldehyde reagents yields membranes that demonstrate properties that make them suitable for use as alkaline battery separators. Film properties can be controlled by the choice of cross-linker, cross-link density and the method of cross-linking. Three methods of cross-linking and their effects on film properties are discussed. Film properties can also be modified by using a copolymer of vinyl alcohol and acrylic acid as the base for the separator and cross-linking it similarly to the PVA. Fillers can be incorporated into the films to further modify film properties. Results of separator screening tests and cell tests for several variations of PBA films are discussed.

Separation of biogenic materials by electrophoresis under zero gravity (L-3)

NASA Technical Reports Server (NTRS)

Kuroda, Masao

1993-01-01

Electrophoresis separates electrically charged materials by imposing a voltage between electrodes. Though free-flow electrophoresis is used without carriers such as colloids to separate and purify biogenic materials including biogenic cells and proteins in blood, its resolving power and separation efficiency is very low on Earth due to sedimentation, flotation, and thermal convection caused by the specific gravity differences between separated materials and buffer solutions. The objective of this experiment is to make a comparative study of various electrophoresis conditions on the ground and in zero-gravity in order to ultimately develop a method for separating various important 'vial' components which are difficult to separate on the ground.

Charge separation at disordered semiconductor heterojunctions from random walk numerical simulations.

PubMed

Mandujano-Ramírez, Humberto J; González-Vázquez, José P; Oskam, Gerko; Dittrich, Thomas; Garcia-Belmonte, Germa; Mora-Seró, Iván; Bisquert, Juan; Anta, Juan A

2014-03-07

Many recent advances in novel solar cell technologies are based on charge separation in disordered semiconductor heterojunctions. In this work we use the Random Walk Numerical Simulation (RWNS) method to model the dynamics of electrons and holes in two disordered semiconductors in contact. Miller-Abrahams hopping rates and a tunnelling distance-dependent electron-hole annihilation mechanism are used to model transport and recombination, respectively. To test the validity of the model, three numerical "experiments" have been devised: (1) in the absence of constant illumination, charge separation has been quantified by computing surface photovoltage (SPV) transients. (2) By applying a continuous generation of electron-hole pairs, the model can be used to simulate a solar cell under steady-state conditions. This has been exploited to calculate open-circuit voltages and recombination currents for an archetypical bulk heterojunction solar cell (BHJ). (3) The calculations have been extended to nanostructured solar cells with inorganic sensitizers to study, specifically, non-ideality in the recombination rate. The RWNS model in combination with exponential disorder and an activated tunnelling mechanism for transport and recombination is shown to reproduce correctly charge separation parameters in these three "experiments". This provides a theoretical basis to study relevant features of novel solar cell technologies.

Biopolymer - A beginning towards back to nature

NASA Astrophysics Data System (ADS)

Gautam, S.; Gautam, A.

2018-05-01

Biopolymer is regarded as a polymer which can be biodegradable. Polyhydroxyalkanoates (PHAs) is one of the biopolymer which can be recovered from biomass. PHAs are naturally conserved in the cytoplasm of the bacterial cell during the growth. Bacteria/microbes store their energy from carbon sources in the form of hydrocarbons. Intracellular stored compounds are tightly linked with entire cell resulting difficulty of separation. The work aims to extract PHAs from biomass effectively. Chemical and mechanical separation of PHA can be done from biomass. A pretreatment of cells before chemical and mechanical separation is also effective for separation of PHA and has been carried out. Chemical extraction of PHA includes digestion of cell wall in acidic or alkaline medium and releasing PHA in broth, later sedimentation recovers PHA. In recent work different chemical methods were carried out to extract PHA of medium chain length. In one of these, sodium hypochlorite was used to denature the protein and chloroform was used for extraction of purified PHA. A recovery upto 96.6%, PHA by dried weight of cell, was obtained which is quite high comparing to reported literature. Other chemical disruption by sodium chloride, sodium hydroxide and hydrogen peroxide with and without pretreatment have also been carried out.

Isolation and genetic analysis of pure cells from forensic biological mixtures: The precision of a digital approach.

PubMed

Fontana, F; Rapone, C; Bregola, G; Aversa, R; de Meo, A; Signorini, G; Sergio, M; Ferrarini, A; Lanzellotto, R; Medoro, G; Giorgini, G; Manaresi, N; Berti, A

2017-07-01

Latest genotyping technologies allow to achieve a reliable genetic profile for the offender identification even from extremely minute biological evidence. The ultimate challenge occurs when genetic profiles need to be retrieved from a mixture, which is composed of biological material from two or more individuals. In this case, DNA profiling will often result in a complex genetic profile, which is then subject matter for statistical analysis. In principle, when more individuals contribute to a mixture with different biological fluids, their single genetic profiles can be obtained by separating the distinct cell types (e.g. epithelial cells, blood cells, sperm), prior to genotyping. Different approaches have been investigated for this purpose, such as fluorescent-activated cell sorting (FACS) or laser capture microdissection (LCM), but currently none of these methods can guarantee the complete separation of different type of cells present in a mixture. In other fields of application, such as oncology, DEPArray™ technology, an image-based, microfluidic digital sorter, has been widely proven to enable the separation of pure cells, with single-cell precision. This study investigates the applicability of DEPArray™ technology to forensic samples analysis, focusing on the resolution of the forensic mixture problem. For the first time, we report here the development of an application-specific DEPArray™ workflow enabling the detection and recovery of pure homogeneous cell pools from simulated blood/saliva and semen/saliva mixtures, providing full genetic match with genetic profiles of corresponding donors. In addition, we assess the performance of standard forensic methods for DNA quantitation and genotyping on low-count, DEPArray™-isolated cells, showing that pure, almost complete profiles can be obtained from as few as ten haploid cells. Finally, we explore the applicability in real casework samples, demonstrating that the described approach provides complete separation of cells with outstanding precision. In all examined cases, DEPArray™ technology proves to be a groundbreaking technology for the resolution of forensic biological mixtures, through the precise isolation of pure cells for an incontrovertible attribution of the obtained genetic profiles. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Method of constructing an improved electrochemical cell

DOEpatents

Grimes, Patrick G.; Einstein, Harry

1984-10-09

An electrochemical cell construction features a novel co-extruded plastic electrode in an interleaved construction with a novel integral separator-spacer. Also featured is a leak and impact resistant construction for preventing the spill of corrosive materials in the event of rupture.

Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation.

PubMed

Bhagat, Ali Asgar S; Hou, Han Wei; Li, Leon D; Lim, Chwee Teck; Han, Jongyoon

2011-06-07

Blood is a highly complex bio-fluid with cellular components making up >40% of the total volume, thus making its analysis challenging and time-consuming. In this work, we introduce a high-throughput size-based separation method for processing diluted blood using inertial microfluidics. The technique takes advantage of the preferential cell focusing in high aspect-ratio microchannels coupled with pinched flow dynamics for isolating low abundance cells from blood. As an application of the developed technique, we demonstrate the isolation of cancer cells (circulating tumor cells (CTCs)) spiked in blood by exploiting the difference in size between CTCs and hematologic cells. The microchannel dimensions and processing parameters were optimized to enable high throughput and high resolution separation, comparable to existing CTC isolation technologies. Results from experiments conducted with MCF-7 cells spiked into whole blood indicate >80% cell recovery with an impressive 3.25 × 10(5) fold enrichment over red blood cells (RBCs) and 1.2 × 10(4) fold enrichment over peripheral blood leukocytes (PBL). In spite of a 20× sample dilution, the fast operating flow rate allows the processing of ∼10(8) cells min(-1) through a single microfluidic device. The device design can be easily customized for isolating other rare cells from blood including peripheral blood leukocytes and fetal nucleated red blood cells by simply varying the 'pinching' width. The advantage of simple label-free separation, combined with the ability to retrieve viable cells post enrichment and minimal sample pre-processing presents numerous applications for use in clinical diagnosis and conducting fundamental studies.

Immunomagnetic cell separation, imaging, and analysis using Captivate ferrofluids

NASA Astrophysics Data System (ADS)

Jones, Laurie; Beechem, Joseph M.

2002-05-01

We have developed applications of CaptivateTM ferrofluids, paramagnetic particles (approximately 200 nm diameter), for isolating and analyzing cell populations in combination with fluorescence-based techniques. Using a microscope-mounted magnetic yoke and sample insertion chamber, fluorescent images of magnetically captured cells were obtained in culture media, buffer, or whole blood, while non-magnetically labeled cells sedimented to the bottom of the chamber. We combined this immunomagnetic cell separation and imaging technique with fluorescent staining, spectroscopy, and analysis to evaluate cell surface receptor-containing subpopulations, live/dead cell ratios, apoptotic/dead cell ratios, etc. The acquired images were analyzed using multi-color parameters, as produced by nucleic acid staining, esterase activity, or antibody labeling. In addition, the immunomagnetically separated cell fractions were assessed through microplate analysis using the CyQUANT Cell Proliferation Assay. These methods should provide an inexpensive alternative to some flow cytometric measurements. The binding capacities of the streptavidin- labled Captivate ferrofluid (SA-FF) particles were determined to be 8.8 nmol biotin/mg SA-FF, using biotin-4- fluorescein, and > 106 cells/mg SA-FF, using several cell types labeled with biotinylated probes. For goat anti- mouse IgG-labeled ferrofluids (GAM-FF), binding capacities were established to be approximately 0.2 - 7.5 nmol protein/mg GAM-FF using fluorescent conjugates of antibodies, protein G, and protein A.

Method of forming components for a high-temperature secondary electrochemical cell

DOEpatents

Mrazek, Franklin C.; Battles, James E.

1983-01-01

A method of forming a component for a high-temperature secondary electrochemical cell having a positive electrode including a sulfide selected from the group consisting of iron sulfides, nickel sulfides, copper sulfides and cobalt sulfides, a negative electrode including an alloy of aluminum and an electrically insulating porous separator between said electrodes. The improvement comprises forming a slurry of solid particles dispersed in a liquid electrolyte such as the lithium chloride-potassium chloride eutetic, casting the slurry into a form having the shape of one of the components and smoothing the exposed surface of the slurry, cooling the cast slurry to form the solid component, and removing same. Electrodes and separators can be thus formed.

Feasibility study of red blood cell debulking by magnetic field-flow fractionation with step-programmed flow

PubMed Central

Moore, Lee R.; Williams, P. Stephen; Nehl, Franziska; Abe, Koji; Chalmers, Jeffrey J.; Zborowski, Maciej

2013-01-01

Emerging applications of rare cell separation and analysis, such as separation of mature red blood cells from hematopoietic cell cultures require efficient methods of red blood cell (RBC) debulking. We have tested the feasibility of magnetic RBC separation as an alternative to centrifugal separation using an approach based on the mechanism of magnetic field-flow fractionation (MgFFF). A specially designed permanent magnet assembly generated a quadrupole field having a maximum field of 1.68 T at the magnet pole tips, zero field at the aperture axis, and a nearly constant radial field gradient of 1.75 T/mm (with a negligible angular component) inside a cylindrical aperture of 1.9 mm (diameter) and 76 mm (length). The cell samples included high-spin hemoglobin RBCs obtained by chemical conversion of hemoglobin to methemoglobin (met RBC) or by exposure to anoxic conditions (deoxy RBC), low-spin hemoglobin obtained by exposure of RBC suspension to ambient air (oxy RBC), and mixtures of deoxy RBC and cells from a KG-1a white blood cell (WBC) line. The observation that met RBCs did not elute from the channel at the lower flow rate of 0.05 mL/min applied for 15 min but quickly eluted at the subsequent higher flow rate of 2.0 mL/min was in agreement with FFF theory. The well-defined experimental conditions (precise field and flow characteristics) and a well-established FFF theory verified by studies with model cell systems provided us with a strong basis for making predictions about potential practical applications of the magnetic RBC separation. PMID:24141316

Feasibility study of red blood cell debulking by magnetic field-flow fractionation with step-programmed flow.

PubMed

Moore, Lee R; Williams, P Stephen; Nehl, Franziska; Abe, Koji; Chalmers, Jeffrey J; Zborowski, Maciej

2014-02-01

Emerging applications of rare cell separation and analysis, such as separation of mature red blood cells from hematopoietic cell cultures, require efficient methods of red blood cell (RBC) debulking. We have tested the feasibility of magnetic RBC separation as an alternative to centrifugal separation using an approach based on the mechanism of magnetic field-flow fractionation (MgFFF). A specially designed permanent magnet assembly generated a quadrupole field having a maximum field of 1.68 T at the magnet pole tips, zero field at the aperture axis, and a nearly constant radial field gradient of 1.75 T/mm (with a negligible angular component) inside a cylindrical aperture of 1.9 mm (diameter) and 76 mm (length). The cell samples included high-spin hemoglobin RBCs obtained by chemical conversion of hemoglobin to methemoglobin (met RBC) or by exposure to anoxic conditions (deoxy RBC), low-spin hemoglobin obtained by exposure of RBC suspension to ambient air (oxy RBC), and mixtures of deoxy RBC and cells from a KG-1a white blood cell (WBC) line. The observation that met RBCs did not elute from the channel at the lower flow rate of 0.05 mL/min applied for 15 min but quickly eluted at the subsequent higher flow rate of 2.0 mL/min was in agreement with FFF theory. The well-defined experimental conditions (precise field and flow characteristics) and a well-established FFF theory verified by studies with model cell systems provided us with a strong basis for making predictions about potential practical applications of the magnetic RBC separation.

Whole Blood Cell Staining Device

NASA Technical Reports Server (NTRS)

Sams, Clarence F.; Clift, Vaughan L.; McDonald, Kelly E.

2000-01-01

An apparatus and method for staining particular cell markers is disclosed. The apparatus includes a flexible tube that is reversibly pinched into compartments with one or more clamps. Each compartment of the tube contains a separate reagent and is in selective fluid communication with adjoining compartments.

Series interconnected photovoltaic cells and method for making same

DOEpatents

Albright, Scot P.; Chamberlin, Rhodes R.; Thompson, Roger A.

1995-01-01

A novel photovoltaic module (10) and method for constructing the same are disclosed. The module (10) includes a plurality of photovoltaic cells (12) formed on a substrate (14) and laterally separated by interconnection regions (15). Each cell (12) includes a bottom electrode (16), a photoactive layer (18) and a top electrode layer (20). Adjacent cells (12) are connected in electrical series by way of a conductive-buffer line (22). The buffer line (22) is also useful in protecting the bottom electrode (16) against severing during downstream layer cutting processes.

Development of Lectin-Linked Immunomagnetic Separation for the Detection of Hepatitis A Virus

PubMed Central

Ko, Sang-Mu; Kwon, Joseph; Vaidya, Bipin; Choi, Jong Soon; Lee, Hee-Min; Oh, Myung-Joo; Bae, Hyeun-Jong; Cho, Se-Young; Oh, Kyung-Seo; Kim, Duwoon

2014-01-01

The accuracy and sensitivity of PCR-based methods for detection of hepatitis A virus (HAV) are dependent on the methods used to separate and concentrate the HAV from the infected cells. The pH and ionic strength affect the binding affinity of the virus to cells. In this study, we initially investigated the effects of pH (4.0–10.0) and metal ions (Fe2+, Co2+, Cu2+, Mg2+, K+, and Ca2+) on the binding of HAV to oyster digestive cells. The lowest relative binding (RB) of HAV to the cells was found at pH 4.0 and in FeSO4 solution (64.6% and 68.1%, respectively). To develop an alternative to antibody-dependent immunomagnetic separation prior to detection of HAV using RT-PCR, the binding of HAV to five lectins, peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA), Ulex europaeus agglutinin (UEA-1) and soybean agglutinin (SBA), was evaluated using ELISAs. SBA showed significantly higher RB to HAV than the other lectins tested. In addition, HAV could be concentrated within 30 min using SBA-linked magnetic bead separation (SMS) prior to the RT-PCR assay. Our findings demonstrate the feasibility of using SMS combined with RT-PCR to detect HAV at dilutions ranging from 10−1–10−4 of a HAV stock (titer: 104 TCID50/mL). PMID:24599279

Stationary semi-solid battery module and method of manufacture

DOEpatents

Slocum, Alexander; Doherty, Tristan; Bazzarella, Ricardo; Cross, III, James C.; Limthongkul, Pimpa; Duduta, Mihai; Disko, Jeffry; Yang, Allen; Wilder, Throop; Carter, William Craig; Chiang, Yet-Ming

2015-12-01

A method of manufacturing an electrochemical cell includes transferring an anode semi-solid suspension to an anode compartment defined at least in part by an anode current collector and an separator spaced apart from the anode collector. The method also includes transferring a cathode semi-solid suspension to a cathode compartment defined at least in part by a cathode current collector and the separator spaced apart from the cathode collector. The transferring of the anode semi-solid suspension to the anode compartment and the cathode semi-solid to the cathode compartment is such that a difference between a minimum distance and a maximum distance between the anode current collector and the separator is maintained within a predetermined tolerance. The method includes sealing the anode compartment and the cathode compartment.

A Minimally Invasive Method for Retrieving Single Adherent Cells of Different Types from Cultures

PubMed Central

Zeng, Jia; Mohammadreza, Aida; Gao, Weimin; Merza, Saeed; Smith, Dean; Kelbauskas, Laimonas; Meldrum, Deirdre R.

2014-01-01

The field of single-cell analysis has gained a significant momentum over the last decade. Separation and isolation of individual cells is an indispensable step in almost all currently available single-cell analysis technologies. However, stress levels introduced by such manipulations remain largely unstudied. We present a method for minimally invasive retrieval of selected individual adherent cells of different types from cell cultures. The method is based on a combination of mechanical (shear flow) force and biochemical (trypsin digestion) treatment. We quantified alterations in the transcription levels of stress response genes in individual cells exposed to varying levels of shear flow and trypsinization. We report optimal temperature, RNA preservation reagents, shear force and trypsinization conditions necessary to minimize changes in the stress-related gene expression levels. The method and experimental findings are broadly applicable and can be used by a broad research community working in the field of single cell analysis. PMID:24957932

Single-molecule two-colour coincidence detection to probe biomolecular associations.

PubMed

Orte, Angel; Clarke, Richard; Klenerman, David

2010-08-01

Two-colour coincidence detection (TCCD) is a form of single-molecule fluorescence developed to sensitively detect and characterize associated biomolecules without any separation, in solution, on the cell membrane and in live cells. In the present short review, we first explain the principles of the method and then describe the application of TCCD to a range of biomedical problems and how this method may be developed further in the future to try to monitor biological processes in live cells.

Plasmid DNA production combining antibiotic-free selection, inducible high yield fermentation, and novel autolytic purification.

PubMed

Carnes, Aaron E; Hodgson, Clague P; Luke, Jeremy M; Vincent, Justin M; Williams, James A

2009-10-15

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage lambdaR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non-ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid-liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts.

A simple method to obtain low density marrow cells for human marrow transplantation.

PubMed

de Witte, T; Plas, A; Vet, J; Koekman, E; Preyers, F; Wessels, J

1987-01-01

Removal of more than 99% of the erythrocytes and 74% of the nucleated cells from marrow grafts was achieved by density floatation separation in Percoll gradients with a density of 1.070 g/ml in eight 250-ml tubes, containing up to 3 X 10(9) nucleated cells per gradient. More than 90% of the myeloid and erythroid progenitor cells were recovered in the low density fraction. It appeared mandatory to use a centrifuge with the possibility of a gradual acceleration and deceleration. Twenty-five patients received a marrow graft from a histocompatible sibling after additional lymphocyte depletion by counterflow centrifugation, and 5 patients with T lymphoblastic malignancies received an autograft after in vitro purging with immunotoxins. All evaluable patients engrafted within normal limits, except 1 patient with an autoimmune pancytopenia who responded to steroids and 1 patient with a CMV infection. Four patients died too early for complete evaluation. The described separation method is easy, cheap and requires only 2 h for the complete processing of a marrow graft.

Preparation of genomic DNA from a single species of uncultured magnetotactic bacterium by multiple-displacement amplification.

PubMed

Arakaki, Atsushi; Shibusawa, Mie; Hosokawa, Masahito; Matsunaga, Tadashi

2010-03-01

Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using phi29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, "Magnetospirillum magneticum AMB-1," whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.

Effective Dual Polysulfide Rejection by a Tannic Acid/FeIII Complex-Coated Separator in Lithium-Sulfur Batteries.

PubMed

Zhang, Hong; Lin, Chuner; Hu, Xuanhe; Zhu, Baoku; Yu, Dingshan

2018-04-18

The solubility behaviour of polysulfides in electrolyte solutions is a major bottleneck prior to the practical application of the lithium-sulfur battery. To address this issue, we fabricate a tannic acid/Fe III complex-coated polypropylene (PP) separator (TA/Fe III -PP separator) via a simple, fast, and green method. Benefiting from dual-confinement effects based on Lewis acid-base interactions between Fe III and polysulfides as well as the dipole-dipole interactions between rich phenol groups and polysulfides, the migration of polysulfides is effectively suppressed. Meanwhile, the porous structure of the PP separator is not destroyed by an additional coating layer. Thus, the TA/Fe III -PP separator can retain rapid lithium ion transport, eventually leading to a significant improvement in both the discharge capacity and rate performance of the corresponding lithium-sulfur cells. The cell with the TA/Fe III -PP separator presents a low capacity fade of 0.06% per cycle over 1000 cycles at 2.0 C, along with a high Coulombic efficiency of >97% over 300 cycles at 0.5 C. With respect to the one with the bare PP separator, the cell with the TA/Fe III -PP separator exhibits a 1.7-fold increase in the discharge capacity at 3.0 C. The proposed simple and economical approach shows great potential in constructing advanced separators to retard the shuttle effect of polysulfides for lithium-sulfur batteries.

Method for producing a biological reagent

NASA Technical Reports Server (NTRS)

Yen, Shiao-Ping S. (Inventor); Rembaum, Alan (Inventor); Molday, Robert S. (Inventor)

1980-01-01

Polymeric functional microspheres containing metal or metal compounds are formed by addition polymerization of a covalently bondable olefinic monomer such as hydroxyethylmethacrylate in the presence of finely divided metal or metal oxide particles, such as iron, gold, platinum or magnetite, which are embedded in the resulting microspheres. The microspheres can be covalently bonded to chemotherapeutic agents, antibodies, or other proteins providing a means for labeling or separating labeled cells. Labeled cells or microspheres can be concentrated at a specific body location such as in the vicinity of a malignant tumor by applying a magnetic field to the location and then introducing the magnetically attractable microspheres or cells into the circulatory system of the subject. Labeled cells can be separated from a cell mixture by applying a predetermined magnetic field to a tube in which the mixture is flowing. After collection of the labeled cells, the magnetic field is discontinued and the labeled sub-cell population recovered.

Cellular and Pectin Dynamics during Abscission Zone Development and Ripe Fruit Abscission of the Monocot Oil Palm

PubMed Central

Roongsattham, Peerapat; Morcillo, Fabienne; Fooyontphanich, Kim; Jantasuriyarat, Chatchawan; Tragoonrung, Somvong; Amblard, Philippe; Collin, Myriam; Mouille, Gregory; Verdeil, Jean-Luc; Tranbarger, Timothy J.

2016-01-01

The oil palm (Elaeis guineensis Jacq.) fruit primary abscission zone (AZ) is a multi-cell layered boundary region between the pedicel (P) and mesocarp (M) tissues. To examine the cellular processes that occur during the development and function of the AZ cell layers, we employed multiple histological and immunohistochemical methods combined with confocal, electron and Fourier-transform infrared (FT-IR) microspectroscopy approaches. During early fruit development and differentiation of the AZ, the orientation of cell divisions in the AZ was periclinal compared with anticlinal divisions in the P and M. AZ cell wall width increased earlier during development suggesting cell wall assembly occurred more rapidly in the AZ than the adjacent P and M tissues. The developing fruit AZ contain numerous intra-AZ cell layer plasmodesmata (PD), but very few inter-AZ cell layer PD. In the AZ of ripening fruit, PD were less frequent, wider, and mainly intra-AZ cell layer localized. Furthermore, DAPI staining revealed nuclei are located adjacent to PD and are remarkably aligned within AZ layer cells, and remain aligned and intact after cell separation. The polarized accumulation of ribosomes, rough endoplasmic reticulum, mitochondria, and vesicles suggested active secretion at the tip of AZ cells occurred during development which may contribute to the striated cell wall patterns in the AZ cell layers. AZ cells accumulated intracellular pectin during development, which appear to be released and/or degraded during cell separation. The signal for the JIM5 epitope, that recognizes low methylesterified and un-methylesterified homogalacturonan (HG), increased in the AZ layer cell walls prior to separation and dramatically increased on the separated AZ cell surfaces. Finally, FT-IR microspectroscopy analysis indicated a decrease in methylesterified HG occurred in AZ cell walls during separation, which may partially explain an increase in the JIM5 epitope signal. The results obtained through a multi-imaging approach allow an integrated view of the dynamic developmental processes that occur in a multi-layered boundary AZ and provide evidence for distinct regulatory mechanisms that underlie oil palm fruit AZ development and function. PMID:27200017

Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry1[W][OA

PubMed Central

Alonso, Ana P.; Piasecki, Rebecca J.; Wang, Yan; LaClair, Russell W.; Shachar-Hill, Yair

2010-01-01

The biosynthesis of cell wall polymers involves enormous fluxes through central metabolism that are not fully delineated and whose regulation is poorly understood. We have established and validated a liquid chromatography tandem mass spectrometry method using multiple reaction monitoring mode to separate and quantify the levels of plant cell wall precursors. Target analytes were identified by their parent/daughter ions and retention times. The method allows the quantification of precursors at low picomole quantities with linear responses up to the nanomole quantity range. When applying the technique to Arabidopsis (Arabidopsis thaliana) T87 cell cultures, 16 hexose-phosphates (hexose-Ps) and nucleotide-sugars (NDP-sugars) involved in cell wall biosynthesis were separately quantified. Using hexose-P and NDP-sugar standards, we have shown that hot water extraction allows good recovery of the target metabolites (over 86%). This method is applicable to quantifying the levels of hexose-Ps and NDP-sugars in different plant tissues, such as Arabidopsis T87 cells in culture and fenugreek (Trigonella foenum-graecum) endosperm tissue, showing higher levels of galacto-mannan precursors in fenugreek endosperm. In Arabidopsis cells incubated with [U-13CFru]sucrose, the method was used to track the labeling pattern in cell wall precursors. As the fragmentation of hexose-Ps and NDP-sugars results in high yields of [PO3]−/or [H2PO4]− ions, mass isotopomers can be quantified directly from the intensity of selected tandem mass spectrometry transitions. The ability to directly measure 13C labeling in cell wall precursors makes possible metabolic flux analysis of cell wall biosynthesis based on dynamic labeling experiments. PMID:20442274

Flow analysis of human chromosome sets by means of mixing-stirring device

NASA Astrophysics Data System (ADS)

Zenin, Valeri V.; Aksenov, Nicolay D.; Shatrova, Alla N.; Klopov, Nicolay V.; Cram, L. Scott; Poletaev, Andrey I.

1997-05-01

A new mixing and stirring device (MSD) was used to perform flow karyotype analysis of single human mitotic chromosomes analyzed so as to maintain the identity of chromosomes derived from the same cell. An improved method for cell preparation and intracellular staining of chromosomes was developed. The method includes enzyme treatment, incubation with saponin and separation of prestained cells from debris on a sucrose gradient. Mitotic cells are injected one by one in the MSD which is located inside the flow chamber where cells are ruptured, thereby releasing chromosomes. The set of chromosomes proceeds to flow in single file fashion to the point of analysis. The device works in a stepwise manner. The concentration of cells in the sample must be kept low to ensure that only one cell at a time enters the breaking chamber. Time-gated accumulation of data in listmode files makes it possible to separate chromosome sets comprising of single cells. The software that was developed classifies chromosome sets according to different criteria: total number of chromosomes, overall DNA content in the set, and the number of chromosomes of certain types. This approach combines the high performance of flow cytometry with the advantages of image analysis. Examples obtained with different human cell lines are presented.

Computed aided system for separation and classification of the abnormal erythrocytes in human blood

NASA Astrophysics Data System (ADS)

Wąsowicz, Michał; Grochowski, Michał; Kulka, Marek; Mikołajczyk, Agnieszka; Ficek, Mateusz; Karpieńko, Katarzyna; Cićkiewicz, Maciej

2017-12-01

The human peripheral blood consists of cells (red cells, white cells, and platelets) suspended in plasma. In the following research the team assessed an influence of nanodiamond particles on blood elements over various periods of time. The material used in the study consisted of samples taken from ten healthy humans of various age, different blood types and both sexes. The markings were leaded by adding to the blood unmodified diamonds and oxidation modified. The blood was put under an impact of two diamond concentrations: 20μl and 100μl. The amount of abnormal cells increased with time. The percentage of echinocytes as a result of interaction with nanodiamonds in various time intervals for individual specimens was scarce. The impact of the two diamond types had no clinical importance on red blood cells. It is supposed that as a result of longlasting exposure a dehydratation of red cells takes place, because of the function of the cells. The analysis of an influence of nanodiamond particles on blood elements was supported by computer system designed for automatic counting and classification of the Red Blood Cells (RBC). The system utilizes advanced image processing methods for RBCs separation and counting and Eigenfaces method coupled with the neural networks for RBCs classification into normal and abnormal cells purposes.

Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry.

PubMed

Keserue, Hans-Anton; Baumgartner, Andreas; Felleisen, Richard; Egli, Thomas

2012-11-01

We developed a rapid detection method for Legionella pneumophila (Lp) by filtration, immunomagnetic separation, double fluorescent staining, and flow cytometry (IMS-FCM method). The method requires 120 min and can discriminate 'viable' and 'membrane-damaged' cells. The recovery is over 85% of spiked Lp SG 1 cells in 1 l of tap water and detection limits are around 50 and 15 cells per litre for total and viable Lp, respectively. The method was compared using water samples from house installations in a blind study with three environmental laboratories performing the ISO 11731 plating method. In 53% of the water samples from different taps and showers significantly higher concentrations of Lp were detected by flow cytometry. No correlation to the plate culture method was found. Since also 'viable but not culturable' (VNBC) cells are detected by our method, this result was expected. The IMS-FCM method is limited by the specificity of the used antibodies; in the presented case they target Lp serogroups 1-12. This and the fact that no Lp-containing amoebae are detected may explain why in 21% of all samples higher counts were observed using the plate culture method. Though the IMS-FCM method is not yet fit to completely displace the established plating method (ISO 11731) for routine Lp monitoring, it has major advantages to plating and can quickly provide important insights into the ecology of this pathogen in water distribution systems. © 2012 The Authors. Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Single Cell Total RNA Sequencing through Isothermal Amplification in Picoliter-Droplet Emulsion.

PubMed

Fu, Yusi; Chen, He; Liu, Lu; Huang, Yanyi

2016-11-15

Prevalent single cell RNA amplification and sequencing chemistries mainly focus on polyadenylated RNAs in eukaryotic cells by using oligo(dT) primers for reverse transcription. We develop a new RNA amplification method, "easier-seq", to reverse transcribe and amplify the total RNAs, both with and without polyadenylate tails, from a single cell for transcriptome sequencing with high efficiency, reproducibility, and accuracy. By distributing the reverse transcribed cDNA molecules into 1.5 × 10 5 aqueous droplets in oil, the cDNAs are isothermally amplified using random primers in each of these 65-pL reactors separately. This new method greatly improves the ease of single-cell RNA sequencing by reducing the experimental steps. Meanwhile, with less chance to induce errors, this method can easily maintain the quality of single-cell sequencing. In addition, this polyadenylate-tail-independent method can be seamlessly applied to prokaryotic cell RNA sequencing.

Method of forming components for a high-temperature secondary electrochemical cell

DOEpatents

Mrazek, F.C.; Battles, J.E.

1981-05-22

A method of forming a component for a high-temperature secondary electrochemical cell having a positive electrode including a sulfide selected from the group consisting of iron sulfides, nickel sulfides, copper sulfides and cobalt sulfides, a negative electrode including an alloy of aluminum and an electrically insulating porous separator between said electrodes is described. The improvement comprises forming a slurry of solid particles dispersed in a liquid electrolyte such as the lithium chloride-potassium chloride eutectic, casting the slurry into a form having the shape of one of the components and smoothing the exposed surface of the slurry, cooling the cast slurry to form the solid component, and removing same. Electrodes and separators can be thus formed.

Evaluation of Wet Chemical ICP-AES Elemental Analysis Methods usingSimulated Hanford Waste Samples-Phase I Interim Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coleman, Charles J.; Edwards, Thomas B.

2005-04-30

The wet chemistry digestion method development for providing process control elemental analyses of the Hanford Tank Waste Treatment and Immobilization Plant (WTP) Melter Feed Preparation Vessel (MFPV) samples is divided into two phases: Phase I consists of: (1) optimizing digestion methods as a precursor to elemental analyses by ICP-AES techniques; (2) selecting methods with the desired analytical reliability and speed to support the nine-hour or less turnaround time requirement of the WTP; and (3) providing baseline comparison to the laser ablation (LA) sample introduction technique for ICP-AES elemental analyses that is being developed at the Savannah River National Laboratory (SRNL).more » Phase II consists of: (1) Time-and-Motion study of the selected methods from Phase I with actual Hanford waste or waste simulants in shielded cell facilities to ensure that the methods can be performed remotely and maintain the desired characteristics; and (2) digestion of glass samples prepared from actual Hanford Waste tank sludge for providing comparative results to the LA Phase II study. Based on the Phase I testing discussed in this report, a tandem digestion approach consisting of sodium peroxide fusion digestions carried out in nickel crucibles and warm mixed-acid digestions carried out in plastic bottles has been selected for Time-and-Motion study in Phase II. SRNL experience with performing this analytical approach in laboratory hoods indicates that well-trained cell operator teams will be able to perform the tandem digestions in five hours or less. The selected approach will produce two sets of solutions for analysis by ICP-AES techniques. Four hours would then be allocated for performing the ICP-AES analyses and reporting results to meet the nine-hour or less turnaround time requirement. The tandem digestion approach will need to be performed in two separate shielded analytical cells by two separate cell operator teams in order to achieve the nine-hour or less turnaround time. Because of the simplicity of the warm mixed-acid method, a well-trained cell operator team may in time be able to perform both sets of digestions. However, having separate shielded cells for each of the methods is prudent to avoid overcrowding problems that would impede a minimal turnaround time.« less

Characterization of human skeletal stem and bone cell populations using dielectrophoresis.

PubMed

Ismail, A; Hughes, M P; Mulhall, H J; Oreffo, R O C; Labeed, F H

2015-02-01

Dielectrophoresis (DEP) is a non-invasive cell analysis method that uses differences in electrical properties between particles and surrounding medium to determine a unique set of cellular properties that can be used as a basis for cell separation. Cell-based therapies using skeletal stem cells are currently one of the most promising areas for treating a variety of skeletal and muscular disorders. However, identifying and sorting these cells remains a challenge in the absence of unique skeletal stem cell markers. DEP provides an ideal method for identifying subsets of cells without the need for markers by using their dielectric properties. This study used a 3D dielectrophoretic well chip device to determine the dielectric characteristics of two osteosarcoma cell lines (MG-63 and SAOS-2) and an immunoselected enriched skeletal stem cell fraction (STRO-1 positive cell) of human bone marrow. Skeletal cells were exposed to a series of different frequencies to induce dielectrophoretic cell movement, and a model was developed to generate the membrane and cytoplasmic properties of the cell populations. Differences were observed in the dielectric properties of MG-63, SAOS-2 and STRO-1 enriched skeletal populations, which could potentially be used to sort cells in mixed populations. This study provide evidence of the ability to characterize different human skeletal stem and mature cell populations, and acts as a proof-of-concept that dielectrophoresis can be exploited to detect, isolate and separate skeletal cell populations from heterogeneous bone marrow cell populations. Copyright © 2012 John Wiley & Sons, Ltd.

Foam vessel for cryogenic fluid storage

DOEpatents

Spear, Jonathan D [San Francisco, CA

2011-07-05

Cryogenic storage and separator vessels made of polyolefin foams are disclosed, as are methods of storing and separating cryogenic fluids and fluid mixtures using these vessels. In one embodiment, the polyolefin foams may be cross-linked, closed-cell polyethylene foams with a density of from about 2 pounds per cubic foot to a density of about 4 pounds per cubic foot.

Short communication: Improved method for centrifugal recovery of bacteria from raw milk applied to sensitive real-time quantitative PCR detection of Salmonella spp

USDA-ARS?s Scientific Manuscript database

Centrifugation of milk is widely used as a separation/concentration step in assays for pathogenic microorganisms. Separation of the cream and liquid supernate from the pellet containing sedimented solids, somatic cells and microorganisms eliminates many interfering substances, and resuspension of th...

Cell bioprocessing in space - Applications of analytical cytology

NASA Technical Reports Server (NTRS)

Todd, P.; Hymer, W. C.; Goolsby, C. L.; Hatfield, J. M.; Morrison, D. R.

1988-01-01

Cell bioprocessing experiments in space are reviewed and the development of on-board cell analytical cytology techniques that can serve such experiments is discussed. Methods and results of experiments involving the cultivation and separation of eukaryotic cells in space are presented. It is suggested that an advanced cytometer should be developed for the quantitative analysis of large numbers of specimens of suspended eukaryotic cells and bioparticles in experiments on the Space Station.

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice.

PubMed

Maridas, David E; Rendina-Ruedy, Elizabeth; Le, Phuong T; Rosen, Clifford J

2018-01-06

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

Accounting for host cell protein behavior in anion-exchange chromatography.

PubMed

Swanson, Ryan K; Xu, Ruo; Nettleton, Daniel S; Glatz, Charles E

2016-11-01

Host cell proteins (HCP) are a problematic set of impurities in downstream processing (DSP) as they behave most similarly to the target protein during separation. Approaching DSP with the knowledge of HCP separation behavior would be beneficial for the production of high purity recombinant biologics. Therefore, this work was aimed at characterizing the separation behavior of complex mixtures of HCP during a commonly used method: anion-exchange chromatography (AEX). An additional goal was to evaluate the performance of a statistical methodology, based on the characterization data, as a tool for predicting protein separation behavior. Aqueous two-phase partitioning followed by two-dimensional electrophoresis provided data on the three physicochemical properties most commonly exploited during DSP for each HCP: pI (isoelectric point), molecular weight, and surface hydrophobicity. The protein separation behaviors of two alternative expression host extracts (corn germ and E. coli) were characterized. A multivariate random forest (MVRF) statistical methodology was then applied to the database of characterized proteins creating a tool for predicting the AEX behavior of a mixture of proteins. The accuracy of the MVRF method was determined by calculating a root mean squared error value for each database. This measure never exceeded a value of 0.045 (fraction of protein populating each of the multiple separation fractions) for AEX. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1453-1463, 2016. © 2016 American Institute of Chemical Engineers.

Structure and compressive strength of silicon open-cell foam obtained by a centrifugal separation method

NASA Astrophysics Data System (ADS)

Cho, Ju-Young; Kim, Ki-Young

2013-03-01

The present study describes a new way to make an open-cell silicon foam from an Al-Si alloy melt by centrifugation during its solidification. The effects of the silicon content and the chute diameter of the crucible on the morphology, the density and the compressive strength of the silicon foams were investigated. A vertical-type centrifugal separator was designed to push the unfrozen Al-Si melt outside, leaving only the silicon foam inside the crucible during rotation. Alloys in the Al-Si system with silicon contents of 40 and 50 wt% were prepared by an electrical resistance furnace, and the revolution of the centrifugal separator was controlled to fabricate the foam. Open-cell silicon foams could be obtained successfully. The apparent density and the compressive strength were in the ranges of 620-820 kg/m3 and 7.5-14.5 MPa, respectively.

Method of enhancing the wettability of boron nitride for use as an electrochemical cell separator

DOEpatents

McCoy, L.R.

1981-01-23

A felt or other fabric of boron nitride suitable for use as an interelectrode separator within an electrochemical cell is wetted with a solution containing a thermally decomposable organic salt of an alkaline earth metal. An aqueous solution of magnesium acetate is the preferred solution for this purpose. After wetting the boron nitride, the solution is dried by heating at a sufficiently low temperature to prevent rapid boiling and the creation of voids within the separator. The dried material is then calcined at an elevated temperature in excess of 400/sup 0/C to provide a coating of an oxide of magnesium on the surface of the boron nitride fibers. A fabric or felt of boron nitride treated in this manner is easily wetted by molten electrolytic salts, such as the alkali metal halides or alkaline earth metal halides, that are used in high temperature, secondary electrochemical cells.

Method of enhancing the wettability of boron nitride for use as an electrochemical cell separator

DOEpatents

McCoy, Lowell R.

1982-01-01

A felt or other fabric of boron nitride suitable for use as an interelecte separator within an electrochemical cell is wetted with a solution containing a thermally decomposable organic salt of an alkaline earth metal. An aqueous solution of magnesium acetate is the preferred solution for this purpose. After wetting the boron nitride, the solution is dried by heating at a sufficiently low temperature to prevent rapid boiling and the creation of voids within the separator. The dried material is then calcined at an elevated temperature in excess of 400.degree. C. to provide a coating of an oxide of magnesium on the surface of the boron nitride fibers. A fabric or felt of boron nitride treated in this manner is easily wetted by molten electrolytic salts, such as the alkali metal halides or alkaline earth metal halides, that are used in high temperature, secondary electrochemical cells.

Electrophoresis tests on STS-3 and ground control experiments - A basis for future biological sample selections

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Lewis, M. L.

1982-01-01

Static zone electrophoresis is an electrokinetic method of separating macromolecules and small particles. However, its application for the isolation of biological cells and concentrated protein solutions is limited by sedimentation and convection. Microgravity eliminates or reduces sedimentation, floatation, and density-driven convection arising from either Joule heating or concentration differences. The advantages of such an environment were first demonstrated in space during the Apollo 14 and 16 missions. In 1975 the Electrophoresis Technology Experiment (MA-011) was conducted during the Apollo-Soyuz Test Project flight. In 1979 a project was initiated to repeat the separations of human kidney cells. One of the major objectives of the Electrophoresis Equipment Verification Tests (EEVT) on STS-3 was to repeat and thereby validate the first successful electrophoretic separation of human kidney cells. Attention is given to the EEVT apparatus, the preflight electrophoresis, and inflight operational results.

Segmentation of clustered cells in negative phase contrast images with integrated light intensity and cell shape information.

PubMed

Wang, Y; Wang, C; Zhang, Z

2018-05-01

Automated cell segmentation plays a key role in characterisations of cell behaviours for both biology research and clinical practices. Currently, the segmentation of clustered cells still remains as a challenge and is the main reason for false segmentation. In this study, the emphasis was put on the segmentation of clustered cells in negative phase contrast images. A new method was proposed to combine both light intensity and cell shape information through the construction of grey-weighted distance transform (GWDT) within preliminarily segmented areas. With the constructed GWDT, the clustered cells can be detected and then separated with a modified region skeleton-based method. Moreover, a contour expansion operation was applied to get optimised detection of cell boundaries. In this paper, the working principle and detailed procedure of the proposed method are described, followed by the evaluation of the method on clustered cell segmentation. Results show that the proposed method achieves an improved performance in clustered cell segmentation compared with other methods, with 85.8% and 97.16% accuracy rate for clustered cells and all cells, respectively. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

On-chip Magnetic Separation and Cell Encapsulation in Droplets†

PubMed Central

Chen, Aaron; Byvank, Tom; Chang, Woo-Jin; Bharde, Atul; Vieira, Greg; Miller, Brandon; Chalmers, Jeffrey J.; Bashir, Rashid; Sooryakumar, Ratnasingham

2014-01-01

The demand for high-throughput single cell assays is gaining importance because of the heterogeneity of many cell suspensions, even after significant initial sorting. These suspensions may display cell-to-cell variability at the gene expression level that could impact single cell functional genomics, cancer, stem-cell research and drug screening. The on-chip monitoring of individual cells in an isolated environment would prevent cross-contamination, provide high recovery yield, and enable study of biological traits at a single cell level. These advantages of on-chip biological experiments is a significant improvement for myriad of cell analyses over conventional methods, which require bulk samples providing only averaged information on cell metabolism. We report on a device that integrates mobile magnetic trap array with microfluidic technology to provide, combined functionality of separation of immunomagnetically labeled cells or magnetic beads and their encapsulation with reagents into pico-liter droplets. This scheme of simultaneous reagent delivery and compartmentalization of the cells immediately after sorting, all performed seamlessly within the same chip, offers unique advantages such as the ability to capture cell traits as originated from its native environment, reduced chance of contamination, minimal use and freshness of the reagent solution that reacts only with separated objects, and tunable encapsulation characteristics independent of the input flow. In addition to the demonstrated preliminary cell viability assay, the device can potentially be integrated with other up- or downstream on-chip modules to become a powerful single-cell analysis tool. PMID:23370785

Energy Materials Center at Cornell: Final Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abruña, Héctor; Mutolo, Paul F

2015-01-02

The mission of the Energy Materials Center at Cornell (emc 2) was to achieve a detailed understanding, via a combination of synthesis of new materials, experimental and computational approaches, of how the nature, structure, and dynamics of nanostructured interfaces affect energy conversion and storage with emphasis on fuel cells, batteries and supercapacitors. Our research on these systems was organized around a full system strategy for; the development and improved performance of materials for both electrodes at which storage or conversion occurs; understanding their internal interfaces, such as SEI layers in batteries and electrocatalyst supports in fuel cells, and methods formore » structuring them to enable high mass transport as well as high ionic and electronic conductivity; development of ion-conducting electrolytes for batteries and fuel cells (separately) and other separator components, as needed; and development of methods for the characterization of these systems under operating conditions (operando methods) Generally, our work took industry and DOE report findings of current materials as a point of departure to focus on novel material sets for improved performance. In addition, some of our work focused on studying existing materials, for example observing battery solvent degradation, fuel cell catalyst coarsening or monitoring lithium dendrite growth, employing in operando methods developed within the center.« less

Hollow silica microspheres for buoyancy-assisted separation of infectious pathogens from stool.

PubMed

Weigum, Shannon E; Xiang, Lichen; Osta, Erica; Li, Linying; López, Gabriel P

2016-09-30

Separation of cells and microorganisms from complex biological mixtures is a critical first step in many analytical applications ranging from clinical diagnostics to environmental monitoring for food and waterborne contaminants. Yet, existing techniques for cell separation are plagued by high reagent and/or instrumentation costs that limit their use in many remote or resource-poor settings, such as field clinics or developing countries. We developed an innovative approach to isolate infectious pathogens from biological fluids using buoyant hollow silica microspheres that function as "molecular buoys" for affinity-based target capture and separation by floatation. In this process, antibody functionalized glass microspheres are mixed with a complex biological sample, such as stool. When mixing is stopped, the target-bound, low-density microspheres float to the air/liquid surface, which simultaneously isolates and concentrates the target analytes from the sample matrix. The microspheres are highly tunable in terms of size, density, and surface functionality for targeting diverse analytes with separation times of ≤2min in viscous solutions. We have applied the molecular buoy technique for isolation of a protozoan parasite that causes diarrheal illness, Cryptosporidium, directly from stool with separation efficiencies over 90% and low non-specific binding. This low-cost method for phenotypic cell/pathogen separation from complex mixtures is expected to have widespread use in clinical diagnostics as well as basic research. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhanced Microchip Electrophoresis Separations Combined with Electrochemical Detection Utilizing a Capillary Embedded in Polystyrene.

PubMed

Mehl, Benjamin T; Martin, R Scott

2018-01-07

The ability to use microchip-based electrophoresis for fast, high-throughput separations provides researchers with a tool for close-to real time analysis of biological systems. While PDMS-based electrophoresis devices are popular, the separation efficiency is often an issue due to the hydrophobic nature of PDMS. In this study, a hybrid microfluidic capillary device was fabricated to utilize the positive features of PDMS along with the electrophoretic performance of fused silica. A capillary loop was embedded in a polystyrene base that can be coupled with PDMS microchannels at minimal dead volume interconnects. A method for cleaning out the capillaries after a wet-polishing step was devised through the use of 3D printed syringe attachment. By comparing the separation efficiency of fluorescein and CBI-glycine with both a PDMS-based serpentine device and the embedded capillary loop device, it was shown that the embedded capillary loop device maintained higher theoretical plates for both analytes. A Pd decoupler with a carbon or Pt detection electrode were embedded along with the loop allowing integration of the electrophoretic separation with electrochemical detection. A series of catecholamines were separated to show the ability to resolve similar analytes and detect redox active species. The release of dopamine and norepinephrine from PC 12 cells was also analyzed showing the compatibility of these improved microchip separations with high ionic cell buffers associated with cell culture.

Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mortimer, Monika; Petersen, Elijah; Buchholz, Bruce

Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here in this paper, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstratemore » separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. Lastly, the optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.« less

Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

DOE PAGES

Mortimer, Monika; Petersen, Elijah; Buchholz, Bruce; ...

2016-10-12

Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here in this paper, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstratemore » separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. Lastly, the optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation.« less

Separation of Bacteria, Protozoa and Carbon Nanotubes by Density Gradient Centrifugation

PubMed Central

Mortimer, Monika; Petersen, Elijah J.; Buchholz, Bruce A.; Holden, Patricia A.

2016-01-01

Sustainable production and use of carbon nanotube (CNT)-enabled materials require efficient assessment of CNT environmental hazards, including the potential for CNT bioaccumulation and biomagnification in environmental receptors. Microbes, as abundant organisms responsible for nutrient cycling in soil and water, are important ecological receptors for studying the effects of CNTs. Quantification of CNT association with microbial cells requires efficient separation of CNT-associated cells from individually dispersed CNTs and CNT agglomerates. Here, we designed, optimized, and demonstrated procedures for separating bacteria (Pseudomonas aeruginosa) from unbound multiwall carbon nanotubes (MWCNTs) and MWCNT agglomerates using sucrose density gradient centrifugation. We demonstrate separation of protozoa (Tetrahymena thermophila) from MWCNTs, bacterial agglomerates, and protozoan fecal pellets by centrifugation in an iodixanol solution. The presence of MWCNTs in the density gradients after centrifugation was determined by quantification of 14C-labeled MWCNTs; the recovery of microbes from the density gradient media was confirmed by optical microscopy. Protozoan intracellular contents of MWCNTs and of bacteria were also unaffected by the designed separation process. The optimized methods contribute to improved efficiency and accuracy in quantifying MWCNT association with bacteria and MWCNT accumulation in protozoan cells, thus supporting improved assessment of CNT bioaccumulation. PMID:27917301

Immobilization of Pichia pastoris cells containing alcohol oxidase activity

PubMed Central

Maleknia, S; Ahmadi, H; Norouzian, D

2011-01-01

Background and Objectives The attempts were made to describe the development of a whole cell immobilization of P. pastoris by entrapping the cells in polyacrylamide gel beads. The alcohol oxidase activity of the whole cell Pichia pastoris was evaluated in comparison with yeast biomass production. Materials and Methods Methylotrophic yeast P. pastoris was obtained from Collection of Standard Microorganisms, Department of Bacterial Vaccines, Pasteur Institute of Iran (CSMPI). Stock culture was maintained on YPD agar plates. Alcohol oxidase was strongly induced by addition of 0.5% methanol as the carbon source. The cells were harvested by centrifugation then permeabilized. Finally the cells were immobilized in polyacrylamide gel beads. The activity of alcohol oxidase was determined by method of Tane et al. Results At the end of the logarithmic phase of cell culture, the alcohol oxidase activity of the whole cell P. Pastoris reached the highest level. In comparison, the alcohol oxidase activity was measured in an immobilized P. pastoris when entrapped in polyacrylamide gel beads. The alcohol oxidase activity of cells was induced by addition of 0.5% methanol as the carbon source. The cells were permeabilized by cetyltrimethylammonium bromide (CTAB) and immobilized. CTAB was also found to increase the gel permeability. Alcohol oxidase activity of immobilized cells was then quantitated by ABTS/POD spectrophotometric method at OD 420. There was a 14% increase in alcohol oxidase activity in immobilized cells as compared with free cells. By addition of 2-butanol as a substrate, the relative activity of alcohol oxidase was significantly higher as compared with other substrates added to the reaction media. Conclusion Immobilization of cells could eliminate lengthy and expensive procedures of enzyme separation and purification, protect and stabilize enzyme activity, and perform easy separation of the enzyme from the reaction media. PMID:22530090

Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

PubMed

Martínez Martínez, Joaquín; Poulton, Nicole J; Stepanauskas, Ramunas; Sieracki, Michael E; Wilson, William H

2011-01-01

Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

Proton Exchange Membrane (PEM) Fuel Cells for Space Applications

NASA Technical Reports Server (NTRS)

Bradley, Karla

2004-01-01

This presentation will provide a summary of the PEM fuel cell development at the National Aeronautics and Space Administration, Johnson Space Center (NASA, JSC) in support of future space applications. Fuel cells have been used for space power generation due to their high energy storage density for multi-day missions. The Shuttle currently utilizes the alkaline fuel cell technology, which has highly safe and reliable performance. However, the alkaline technology has a limited life due to the corrosion inherent to the alkaline technology. PEM fuel cells are under development by industry for transportation, residential and commercial stationary power applications. NASA is trying to incorporate some of this stack technology development in the PEM fuel cells for space. NASA has some unique design and performance parameters which make developing a PEM fuel cell system more challenging. Space fuel cell applications utilize oxygen, rather than air, which yields better performance but increases the hazard level. To reduce the quantity of reactants that need to be flown in space, NASA also utilizes water separation and reactant recirculation. Due to the hazards of utilizing active components for recirculation and water separation, NASA is trying to develop passive recirculation and water separation methods. However, the ability to develop recirculation components and water separators that are gravity-independent and successfully operate over the full range of power levels is one of the greatest challenges to developing a safe and reliable PEM fuel cell system. PEM stack, accessory component, and system tests that have been performed for space power applications will be discussed.

High Speed Size Sorting of Subcellular Organelles by Flow Field-Flow Fractionation.

PubMed

Yang, Joon Seon; Lee, Ju Yong; Moon, Myeong Hee

2015-06-16

Separation/isolation of subcellular species, such as mitochondria, lysosomes, peroxisomes, Golgi apparatus, and others, from cells is important for gaining an understanding of the cellular functions performed by specific organelles. This study introduces a high speed, semipreparative scale, biocompatible size sorting method for the isolation of subcellular organelle species from homogenate mixtures of HEK 293T cells using flow field-flow fractionation (FlFFF). Separation of organelles was achieved using asymmetrical FlFFF (AF4) channel system at the steric/hyperlayer mode in which nuclei, lysosomes, mitochondria, and peroxisomes were separated in a decreasing order of hydrodynamic diameter without complicated preprocessing steps. Fractions in which organelles were not clearly separated were reinjected to AF4 for a finer separation using the normal mode, in which smaller sized species can be well fractionated by an increasing order of diameter. The subcellular species contained in collected AF4 fractions were examined with scanning electron microscopy to evaluate their size and morphology, Western blot analysis using organelle specific markers was used for organelle confirmation, and proteomic analysis was performed with nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS). Since FlFFF operates with biocompatible buffer solutions, it offers great flexibility in handling subcellular components without relying on a high concentration sucrose solution for centrifugation or affinity- or fluorescence tag-based sorting methods. Consequently, the current study provides an alternative, competitive method for the isolation/purification of subcellular organelle species in their intact states.

Assessment of Telomere Length, Phenotype, and DNA Content

PubMed Central

Kelesidis, Theodoros; Schmid, Ingrid

2017-01-01

Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe. Cells with long telomeres are included as internal standards. Addition of a DNA dye permits exclusion of proliferating cells during data analysis. DNA ploidy measurements of cells of interest and internal standard are performed on separate aliquots in parallel to Flow-FISH. Telomere fluorescence of G0/1 cells of subpopulations and internal standards obtained from Flow-FISH are normalized for DNA ploidy, and telomere length in subsets of interest is expressed as a fraction of the internal standard telomere length. PMID:28055113

Assessment of Telomere Length, Phenotype, and DNA Content.

PubMed

Kelesidis, Theodoros; Schmid, Ingrid

2017-01-05

Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe. Cells with long telomeres are included as internal standards. Addition of a DNA dye permits exclusion of proliferating cells during data analysis. DNA ploidy measurements of cells of interest and internal standard are performed on separate aliquots in parallel to Flow-FISH. Telomere fluorescence of G 0/1 cells of subpopulations and internal standards obtained from Flow-FISH are normalized for DNA ploidy, and telomere length in subsets of interest is expressed as a fraction of the internal standard telomere length. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Separation and determination of peptide metabolite of Bacillus licheniformis in a microbial fuel cell by high-speed capillary micellar electrokinetic chromatography.

PubMed

Wang, Wei; Bai, Ruiguang; Cai, Xiaoyu; Lin, Ping; Ma, Lihong

2017-11-01

A method using high-speed capillary micellar electrokinetic chromatography and a microbial fuel cell was applied to determine the metabolite of the peptides released by Bacillus licheniformis. Two peptides, l-carnosine and l-alanyl-l-glutamine were used as the substrate to feed Bacillus licheniformis in a microbial fuel cell. The metabolism process of the bacterium was monitored by analyzing the voltage outputs of the microbial fuel cell. A home-made spontaneous injection device was applied to perform high-speed capillary micellar electrokinetic chromatography. Under the optimized conditions, tryptophan, glycine, valine, tyrosine and the two peptides could be rapidly separated within 2.5 min with micellar electrokinetic chromatography mode. Then the method was applied to analyze the solutions sampled from the microbial fuel cell. After 92 h running, valine, as the metabolite, was successfully detected with concentration 3.90 × 10 -5 M. The results demonstrated that Bacillus licheniformis could convert l-carnosine and l-alanyl-l-glutamine into valine. The method employed in this work was proved to have great potential in analysis of metabolites, such as amino acids, for microorganisms. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

PubMed

Sediq, A S; Klem, R; Nejadnik, M R; Meij, P; Jiskoot, Wim

2018-05-30

To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells.

Separation of abscission zone cells in detached Azolla roots depends on apoplastic pH.

PubMed

Fukuda, Kazuma; Yamada, Yoshiya; Miyamoto, Kensuke; Ueda, Junichi; Uheda, Eiji

2013-01-01

In studies on the mechanism of cell separation during abscission, little attention has been paid to the apoplastic environment. We found that the apoplastic pH surrounding abscission zone cells in detached roots of the water fern Azolla plays a major role in cell separation. Abscission zone cells of detached Azolla roots were separated rapidly in a buffer at neutral pH and slowly in a buffer at pH below 4.0. However, cell separation rarely occurred at pH 5.0-5.5. Light and electron microscopy revealed that cell separation was caused by a degradation of the middle lamella between abscission zone cells at both pH values, neutral and below 4.0. Low temperature and papain treatment inhibited cell separation. Enzyme(s) in the cell wall of the abscission zone cells might be involved in the degradation of the pectin of the middle lamella and the resultant, pH-dependent cell separation. By contrast, in Phaseolus leaf petioles, unlike Azolla roots, cell separation was slow and increased only at acidic pH. The rapid cell separation, as observed in Azolla roots at neutral pH, did not occur. Indirect immunofluorescence microscopy, using anti-pectin monoclonal antibodies, revealed that the cell wall pectins of the abscission zone cells of Azolla roots and Phaseolus leaf petioles looked similar and changed similarly during cell separation. Thus, the pH-related differences in cell separation mechanisms of Azolla and Phaseolus might not be due to differences in cell wall pectin, but to differences in cell wall-located enzymatic activities responsible for the degradation of pectic substances. A possible enzyme system is discussed. Copyright © 2012 Elsevier GmbH. All rights reserved.

Development of a Universal RNA Beacon for Exogenous Gene Detection

PubMed Central

Guo, Yuanjian; Lu, Zhongju; Cohen, Ira Stephen

2015-01-01

Stem cell therapy requires a nontoxic and high-throughput method to achieve a pure cell population to prevent teratomas that can occur if even one cell in the implant has not been transformed. A promising method to detect and separate cells expressing a particular gene is RNA beacon technology. However, developing a successful, specific beacon to a particular transfected gene can take months to develop and in some cases is impossible. Here, we report on an off-the-shelf universal beacon that decreases the time and cost of applying beacon technology to select any living cell population transfected with an exogenous gene. PMID:25769653

Development of a universal RNA beacon for exogenous gene detection.

PubMed

Guo, Yuanjian; Lu, Zhongju; Cohen, Ira Stephen; Scarlata, Suzanne

2015-05-01

Stem cell therapy requires a nontoxic and high-throughput method to achieve a pure cell population to prevent teratomas that can occur if even one cell in the implant has not been transformed. A promising method to detect and separate cells expressing a particular gene is RNA beacon technology. However, developing a successful, specific beacon to a particular transfected gene can take months to develop and in some cases is impossible. Here, we report on an off-the-shelf universal beacon that decreases the time and cost of applying beacon technology to select any living cell population transfected with an exogenous gene. ©AlphaMed Press.

Vibration-type particle separation device with piezoceramic vibrator

NASA Astrophysics Data System (ADS)

Ooe, Katsutoshi; Doi, Akihiro

2008-12-01

During hemanalysis, it is necessary to separate blood cells from whole blood. Many blood separation methods, for example, centrifugation and filtering, are in practical use. However, the use of these methods involves problems from the perspectives of processing speed and processing volume. We develop new types of blood separation devices that use piezo-ceramic vibrators. The first device uses a capillary. One end of the capillary is fixed to the device frame, and the other is fixed to a piezo-ceramic vibrator. The vibrator transmits bending waves to the capillary. This device can process only a small amount of solution; therefore, it is not suitable for hemanalysis. In order to solve this problem, we developed a second device; this device has a pair of thin glass plates with a small gap as a substitute for the capillary used in the first device. These devices are based on the fact that particles heavier than water move toward transverse velocity antinodes while those lighter than water move toward velocity nodes. In this report, we demonstrate the highspeed separation of silica microbeads and 50-vol% glycerol water by using these devices. The first device can separate the abovementioned solution within 3 min while the second can separate it within 1 min. Both devices are driven by a rectangular wave of 15 to 20 Vpp. Furthermore, it has been confirmed that red blood cells are separated from diluted whole blood using the first device within approximately 1 min. These devices have transparency, so they can compose as the analysis system with the chemical analyzer easily.

A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods.

PubMed

Greening, David W; Xu, Rong; Ji, Hong; Tauro, Bow J; Simpson, Richard J

2015-01-01

Exosomes are 40-150 nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of tumorigenic proteins, mRNA and miRNA. Exosomes are important regulators of the cellular niche, and their altered characteristics in many diseases, such as cancer, suggest their importance for diagnostic and therapeutic applications, and as drug delivery vehicles. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. In this chapter, we reveal the protocol and key insights into the isolation, purification and characterization of exosomes, distinct from shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, a comprehensive evaluation of exosome isolation methods including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM-coated magnetic beads (IAC-Exos) were examined. All exosome isolation methodologies contained 40-150 nm vesicles based on electron microscopy, and positive for exosome markers (Alix, TSG101, HSP70) based on immunoblotting. This protocol employed a proteomic profiling approach to characterize the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method in exosome isolation. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, IAC-Exos was shown to be the most effective method to isolate exosomes. However, the use of density-based separation (DG-Exos) provides significant advantages for exosome isolation when the use of immunoaffinity capture is limited (due to antibody availability and suitability of exosome markers).

Simultaneous separation of five major ribonucleic acids by capillary electrophoresis with laser-induced fluorescence in the presence of electroosmotic flow: application to the rapid screening of 5S rRNA from ovarian cancer cells.

PubMed

Shih, Ya-Chu; Liao, Ching-Ru; Chung, I-Che; Chang, Yu-Sun; Chang, Po-Ling

2014-10-17

RNA integrity is important in RNA studies because poor RNA quality may impact downstream methodologies. This study proposes a rapid and cost-effective method for the determination of RNA integrity based on CE-LIF in the presence of electroosmotic flow. The proposed method uses poly(ethylene) oxide (Mavg=4,000,000 Da) as a sieving matrix for total RNA separation. Ethidium bromide (μg mL(-1)) was dissolved in a polymer solution as an interchelating dye for on-column fluorescent labeling. The 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA and tRNA from the total human RNA extracted from the cells were fully separated using the proposed method. The lowest detectable concentration of total RNA achieved was 100 pg μL(-1) with a 6 min sample injection followed by on-column concentration. In addition, the temperature-induced degradation of total RNA was observed by CE-LIF. The electropherograms revealed more fragmentation of 28S and 18S rRNAs by temperature-induced hydrolysis compared with the 5.8S rRNA, 5S rRNA and tRNA. Therefore, the results indicated that RNA degradation should be considered for long-term, high-temperature incubations in RNA-related experiments involving RNA hybridization. The proposed method is furthermore, applied to the determination of 5S rRNA overexpressed in ovarian cancer cells as compared to the cervical cancer cells. Overall, CE-LIF is highly promising for rapid screening of ovarian cancers without tedious pre-amplification steps. Copyright © 2014 Elsevier B.V. All rights reserved.

Gliogenesis: historical perspectives, 1839-1985.

PubMed

Webster, Henry deF; Aström, Karl E

2009-01-01

This historical review of gliogenesis begins with Schwann's introduction of the cell doctrine in 1839. Subsequent microscopic studies revealed the cellular structure of many organs and tissues, but the CNS was thought to be different. In 1864, Virchow created the concept that nerve cells are held together by a "Nervenkitte" which he called"glia" (for glue). He and his contemporaries thought that "glia" was an unstructured, connective tissue-like ground substance that separated nerve cells from each other and from blood vessels. Dieters, a pupil of Virchow, discovered that this ground substance contained cells, which he described and illustrated. Improvements in microscopes and discovery of metallic impregnation methods finally showed convincingly that the "glia" was not a binding substance. Instead, it was composed of cells, each separate and distinct from neighboring cells and each with its own characteristic array of processes. Light microscopic studies of developing and mature nervous tissue led to the discovery of different types of glial cells-astroglia, oligodendroglia, microglia, and ependymal cells in the CNS, and Schwann cells in the peripheral nervous system (PNS). Subsequent studies characterized the origins and development of each type of glial cell. A new era began with the introduction of electron microscopy, immunostaining, and in vitro maintenance of both central and peripheral nervous tissue. Other methods and models greatly expanded our understanding of how glia multiply, migrate, and differentiate. In 1985, almost a century and a half of study had produced substantial progress in our understanding of glial cells, including their origins and development. Major advances were associated with the discovery of new methods. These are summarized first. Then the origins and development of astroglia, oligodendroglia, microglia, ependymal cells, and Schwann cells are described and discussed. In general, morphology is emphasized. Findings related to cytodifferentiation, cellular interactions, functions, and regulation of developing glia have also been included.

Long life, rechargeable nickel-zinc battery

NASA Technical Reports Server (NTRS)

Luksha, E.

1974-01-01

A production version of the inorganic separator was evaluated for improving the life of the nickel-zinc system. Nickel-zinc cells (7-10 Ah capacities) of different electrode separator configurations were constructed and tested. The nickel-zinc cells using the inorganic separator encasing the zinc electrode, the nickel electrode, or both electrodes had shorter lives than cells using Visking and cellophane separation. Cells with the inorganic separation all fell below 70% of their theoretical capacity within 30 cycles, but the cells constructed with organic separation required 80 cycles. Failure of the cells using the ceramic separator was irreversible capacity degradation due to zinc loss through cracks developed in the inorganic separator. Zinc loss through the separator was minimized with the use of combinations of the inorganic separator with Visking and cellophane. Cells using the combined separation operated 130 duty cycles before degrading to 70% of their theoretical capacity.

Selection of Shared and Neoantigen-Reactive T Cells for Adoptive Cell Therapy Based on CD137 Separation.

PubMed

Seliktar-Ofir, Sivan; Merhavi-Shoham, Efrat; Itzhaki, Orit; Yunger, Sharon; Markel, Gal; Schachter, Jacob; Besser, Michal J

2017-01-01

Adoptive cell therapy (ACT) of autologous tumor infiltrating lymphocytes (TIL) is an effective immunotherapy for patients with solid tumors, yielding objective response rates of around 40% in refractory patients with metastatic melanoma. Most clinical centers utilize bulk, randomly isolated TIL from the tumor tissue for ex vivo expansion and infusion. Only a minor fraction of the administered T cells recognizes tumor antigens, such as shared and mutation-derived neoantigens, and consequently eliminates the tumor. Thus, there are many ongoing effects to identify and select tumor-specific TIL for therapy; however, those approaches are very costly and require months, which is unreasonable for most metastatic patients. CD137 (4-1BB) has been identified as a co-stimulatory marker, which is induced upon the specific interaction of T cells with their target cell. Therefore, CD137 can be a useful biomarker and an important tool for the selection of tumor-reactive T cells. Here, we developed and validated a simple and time efficient method for the selection of CD137-expressing T cells for therapy based on magnetic bead separation. CD137 selection was performed with clinical grade compliant reagents, and TIL were expanded in a large-scale manner to meet cell numbers required for the patient setting in a GMP facility. For the first time, the methodology was designed to comply with both clinical needs and limitations, and its feasibility was assessed. CD137-selected TIL demonstrated significantly increased antitumor reactivity and were enriched for T cells recognizing neoantigens as well as shared tumor antigens. CD137-based selection enabled the enrichment of tumor-reactive T cells without the necessity of knowing the epitope specificity or the antigen type. The direct implementation of the CD137 separation method to the cell production of TIL may provide a simple way to improve the clinical efficiency of TIL ACT.

Selection of Shared and Neoantigen-Reactive T Cells for Adoptive Cell Therapy Based on CD137 Separation

PubMed Central

Seliktar-Ofir, Sivan; Merhavi-Shoham, Efrat; Itzhaki, Orit; Yunger, Sharon; Markel, Gal; Schachter, Jacob; Besser, Michal J.

2017-01-01

Adoptive cell therapy (ACT) of autologous tumor infiltrating lymphocytes (TIL) is an effective immunotherapy for patients with solid tumors, yielding objective response rates of around 40% in refractory patients with metastatic melanoma. Most clinical centers utilize bulk, randomly isolated TIL from the tumor tissue for ex vivo expansion and infusion. Only a minor fraction of the administered T cells recognizes tumor antigens, such as shared and mutation-derived neoantigens, and consequently eliminates the tumor. Thus, there are many ongoing effects to identify and select tumor-specific TIL for therapy; however, those approaches are very costly and require months, which is unreasonable for most metastatic patients. CD137 (4-1BB) has been identified as a co-stimulatory marker, which is induced upon the specific interaction of T cells with their target cell. Therefore, CD137 can be a useful biomarker and an important tool for the selection of tumor-reactive T cells. Here, we developed and validated a simple and time efficient method for the selection of CD137-expressing T cells for therapy based on magnetic bead separation. CD137 selection was performed with clinical grade compliant reagents, and TIL were expanded in a large-scale manner to meet cell numbers required for the patient setting in a GMP facility. For the first time, the methodology was designed to comply with both clinical needs and limitations, and its feasibility was assessed. CD137-selected TIL demonstrated significantly increased antitumor reactivity and were enriched for T cells recognizing neoantigens as well as shared tumor antigens. CD137-based selection enabled the enrichment of tumor-reactive T cells without the necessity of knowing the epitope specificity or the antigen type. The direct implementation of the CD137 separation method to the cell production of TIL may provide a simple way to improve the clinical efficiency of TIL ACT. PMID:29067023

Magnetophoretic separation ICP-MS immunoassay using Cs-doped multicore magnetic nanoparticles for the determination of salmonella typhimurium.

PubMed

Jeong, Arong; Lim, H B

2018-02-01

In this work, a magnetophoretic separation ICP-MS immunoassay using newly synthesized multicore magnetic nanoparticles (MMNPs) was developed for the determination of salmonella typhimurium (typhi). The uniqueness of this method was the use of MMNPs doped with Cs for both separation and detection, which enable us to achieve fast analysis, high sensitivity, and good reliability. For demonstration, heat-killed typhi in a phosphate buffer solution was determined by ICP-MS after the MMNP-typhi reaction product was separated from unreacted MMNPs in a micropipette tip filled with 25% polyethylene glycol through magnetophoretic separation. The calibration curve obtained by plotting 133 Cs intensity vs. the number of synthetic standard, showed a coefficient of determination (R 2 ) of 0.94 with a limit of detection (LOD) of 102 cells/mL without cell culturing. Excellent recoveries, between 98-100%, were obtained from four replicates and compared with a sandwich-type ICP-MS immunoassay for further confirmation. Copyright © 2017 Elsevier B.V. All rights reserved.

High performance a-Si solar cells and new fabrication methods for a-Si solar cells

NASA Astrophysics Data System (ADS)

Nakano, S.; Kuwano, Y.; Ohnishi, M.

1986-12-01

The super chamber, a separated UHV reaction-chamber system has been developed. A conversion efficiency of 11.7% was obtained for an a-Si solar cell using a high-quality i-layer deposited by the super chamber, and a p-layer fabricated by a photo-CVD method. As a new material, amorphous superlattice-structure films were fabricated by the photo-CVD method for the first time. Superlattice structure p-layer a-Si solar cells were fabricated, and a conversion efficiency of 10.5% was obtained. For the fabrication of integrated type a-Si solar cell modules, a laser pattering method was investigated. A thermal analysis of the multilayer structure was done. It was confirmed that selective scribing for a-Si, TCO and metal film is possible by controlling the laser power density. Recently developed a-Si solar power generation systems and a-Si solar cell roofing tiles are also described.

Rapid and continuous magnetic separation in droplet microfluidic devices.

PubMed

Brouzes, Eric; Kruse, Travis; Kimmerling, Robert; Strey, Helmut H

2015-02-07

We present a droplet microfluidic method to extract molecules of interest from a droplet in a rapid and continuous fashion. We accomplish this by first marginalizing functionalized super-paramagnetic beads within the droplet using a magnetic field, and then splitting the droplet into one droplet containing the majority of magnetic beads and one droplet containing the minority fraction. We quantitatively analysed the factors which affect the efficiency of marginalization and droplet splitting to optimize the enrichment of magnetic beads. We first characterized the interplay between the droplet velocity and the strength of the magnetic field and its effect on marginalization. We found that marginalization is optimal at the midline of the magnet and that marginalization is a good predictor of bead enrichment through splitting at low to moderate droplet velocities. Finally, we focused our efforts on manipulating the splitting profile to improve the enrichment provided by asymmetric splitting. We designed asymmetric splitting forks that employ capillary effects to preferentially extract the bead-rich regions of the droplets. Our strategy represents a framework to optimize magnetic bead enrichment methods tailored to the requirements of specific droplet-based applications. We anticipate that our separation technology is well suited for applications in single-cell genomics and proteomics. In particular, our method could be used to separate mRNA bound to poly-dT functionalized magnetic microparticles from single cell lysates to prepare single-cell cDNA libraries.

Rapid and continuous magnetic separation in droplet microfluidic devices

PubMed Central

Brouzes, Eric; Kruse, Travis; Kimmerling, Robert; Strey, Helmut H.

2015-01-01

We present a droplet microfluidic method to extract molecules of interest from a droplet in a rapid and continuous fashion. We accomplish this by first marginalizing functionalized super-paramagnetic beads within the droplet using a magnetic field, and then splitting the droplet into one droplet containing the majority of magnetic beads and one droplet containing the minority fraction. We quantitatively analysed the factors which affect the efficiency of marginalization and droplet splitting to optimize the enrichment of magnetic beads. We first characterized the interplay between the droplet velocity and the strength of the magnetic field and its effect on marginalization. We found that marginalization is optimal at the midline of the magnet and that marginalization is a good predictor of bead enrichment through splitting at low to moderate droplet velocities. Finally, we focused our efforts on manipulating the splitting profile to improve the enrichment provided by asymmetric splitting. We designed asymmetric splitting forks that employ capillary effects to preferentially extract the bead-rich regions of the droplets. Our strategy represents a framework to optimize magnetic bead enrichment methods tailored to the requirements of specific droplet-based applications. We anticipate that our separation technology is well suited for applications in single-cell genomics and proteomics. In particular, our method could be used to separate mRNA bound to poly-dT functionalized magnetic microparticles from single cell lysates to prepare single-cell cDNA libraries. PMID:25501881

Cloned Hemoglobin Genes Enhance Growth Of Cells

NASA Technical Reports Server (NTRS)

Khosla, Chaitan; Bailey, James E.

1991-01-01

Experiments show that portable deoxyribonucleic acid (DNA) sequences incorporated into host cells make them produce hemoglobins - oxygen-binding proteins essential to function of red blood cells. Method useful in several biotechnological applications. One, enhancement of growth of cells at higher densities. Another, production of hemoglobin to enhance supplies of oxygen in cells, for use in chemical reactions requiring oxygen, as additive to serum to increase transport of oxygen, and for binding and separating oxygen from mixtures of gases.

Cell wall layers delimit cell groups derived from cell division in the foliose trebouxiophycean alga Prasiola japonica.

PubMed

Mine, Ichiro; Kinoshita, Urara; Kawashima, Shigetaka; Sekida, Satoko

2018-01-22

The cells in the foliose thallus of trebouxiophycean alga Prasiola japonica apparently develop into 2 × 2 cell groups composed of two two-celled groups, each of which is a pair of derivative cells of the latest cell division. In the present study, the structural features of cell walls of the alga P. japonica concerning the formation of the cell groups were investigated using histochemical methods. Thin cell layers stained by Calcofluor White appeared to envelope the two-celled and four-celled groups separately and, hence, separated them from neighboring cell groups, and the Calcofluor White-negative gaps between neighboring four-celled groups were specifically stained by lectins, such as soybean agglutinin, jacalin, and Vicia villosa lectin conjugated with fluorescein. These results indicated that the Calcofluor White-positive cell wall layer of parent cell that existed during two successive cell divisions structurally distinguished two-celled and four-celled groups from others in this alga. Moreover, the results suggested that the cell wall components of the Calcofluor White-negative gaps would possibly contribute to the formation of the planar thallus through lateral union of the cell groups.

Kidney cell electrophoresis in space flight: Rationale, methods, results and flow cytometry applications

NASA Technical Reports Server (NTRS)

Todd, P.; Morrison, Dennis R.; Barlow, Grant H.; Lewis, Marian L.; Lanham, J. W.; Cleveland, C.; Williams, K.; Kunze, M. E.; Goolsby, C. L.

1988-01-01

Cultures of human embryonic kidney cells consistently contain an electrophoretically separable subpopulation of cells that produce high levels of urokinase and have an electrophoretic mobility about 85 percent as high as that of the most mobile human embryonic kidney cells. This subpopulation is rich in large epithelioid cells that have relatively little internal structure. When resolution and throughput are adequate, free fluid electrophoresis can be used to isolate a broad band of low mobility cells which also produces high levels of plasminogen activators (PAs). In the course of performing this, it was discovered that all electrophoretic subpopulations of cultured human embryonic kidney cells produce some PAs and that separate subpopulations produce high quantities of different types of PA's. This information and the development of sensitive assays for this project have provided new insights into cell secretion mechanisms related to fibrinolysis. These advances would probably not have been made without the NASA program to explore fundamental questions of free fluid electrophoresis in space.

In search of the skeletal stem cell: isolation and separation strategies at the macro/micro scale for skeletal regeneration.

PubMed

Gothard, David; Tare, Rahul S; Mitchell, Peter D; Dawson, Jonathan I; Oreffo, Richard O C

2011-04-07

Skeletal stem cells (SSCs) show great capacity for bone and cartilage repair however, current in vitro cultures are heterogeneous displaying a hierarchy of differentiation potential. SSCs represent the diminutive true multipotent stem cell fraction of bone marrow mononuclear cell (BMMNC) populations. Endeavours to isolate SSCs have generated a multitude of separation methodologies. SSCs were first identified and isolated by their ability to adhere to culture plastic. Once isolated, further separation is achieved via culture in selective or conditioned media (CM). Indeed, preferential SSC growth has been demonstrated through selective in vitro culture conditions. Other approaches have utilised cell morphology (size and shape) as selection criteria. Studies have also targeted SSCs based on their preferential adhesion to specified compounds, individually or in combination, on both macro and microscale platforms. Nevertheless, most of these methods which represent macroscale function with relatively high throughput, yield insufficient purity. Consequently, research has sought to downsize isolation methodologies to the microscale for single cell analysis. The central approach is identification of the requisite cell populations of SSC-specific surface markers that can be targeted for isolation by either positive or negative selection. SELEX and phage display technology provide apt means to sift through substantial numbers of candidate markers. In contrast, single cell analysis is the paramount advantage of microfluidics, a relatively new field for cell biology. Here cells can be separated under continuous or discontinuous flow according to intrinsic phenotypic and physicochemical properties. The combination of macroscale quantity with microscale specificity to generate robust high-throughput (HT) technology for pure SSC sorting, isolation and enrichment offers significant implications therein for skeletal regenerative strategies as a consequence of lab on chip derived methodology.

Magnetic bead-based separation of sperm from buccal epithelial cells using a monoclonal antibody against MOSPD3.

PubMed

Li, Xue-Bo; Wang, Qing-Shan; Feng, Yu; Ning, Shu-Hua; Miao, Yuan-Ying; Wang, Ye-Quan; Li, Hong-Wei

2014-11-01

Forensic DNA analysis of sexual assault evidence requires unambiguous differentiation of DNA profiles in mixed samples. To investigate the feasibility of magnetic bead-based separation of sperm from cell mixtures using a monoclonal antibody against MOSPD3 (motile sperm domain-containing protein 3), 30 cell samples were prepared by mixing 10(4) female buccal epithelial cells with sperm cells of varying densities (10(3), 10(4), or 10(5) cells/mL). Western blot and immunofluorescence assays showed that MOSPD3 was detectable on the membrane of sperm cells, but not in buccal epithelial cells. After biotinylated MOSPD3 antibody was incubated successively with the prepared cell mixtures and avidin-coated magnetic beads, microscopic observation revealed that each sperm cell was bound by two or more magnetic beads, in the head, neck, mid-piece, or flagellum. A full single-source short tandem repeat profile could be obtained in 80% of mixed samples containing 10(3) sperm cells/mL and in all samples containing ≥10(4) sperm cells/mL. For dried vaginal swab specimens, the rate of successful detection was 100% in both flocked and cotton swabs preserved for 1 day, 87.5% in flocked swabs and 40% in cotton swabs preserved for 3 days, and 40% in flocked swabs and 16.67% in cotton swabs preserved for 10 days. Our findings suggest that immunomagnetic bead-based separation is potentially a promising alternative to conventional methods for isolating sperm cells from mixed forensic samples.

Preparative liquid column electrophoresis of T and B lymphocytes at gravity = 1

NASA Technical Reports Server (NTRS)

Van Oss, C. J.; Bigazzi, P. E.; Gillman, C. F.; Allen, R. E.

1974-01-01

Vertical liquid columns containing low-molecular-weight dextran density gradients can be used for preparative lymphocyte electrophoresis on earth, in simulation of zero gravity conditions. Another method that has been tested at 1 g, is the electrophoresis of lymphocytes in an upward direction in vertical columns. By both methods up to 100 million lymphocytes can be separated at one time in a 30-cm glass column of 8-mm inside diameter, at 12 V/cm, in two hours. Due to convection and sedimentation problems, the separation at 1 g is less than ideal, but it is expected that at zero gravity electrophoresis will probe to be a uniquely powerful cell separation tool.

Bacteriophage-based nanoprobes for rapid bacteria separation

NASA Astrophysics Data System (ADS)

Chen, Juhong; Duncan, Bradley; Wang, Ziyuan; Wang, Li-Sheng; Rotello, Vincent M.; Nugen, Sam R.

2015-10-01

The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying concentrations were determined. The results indicated a similar bacteria capture efficiency between the two nanoprobes.The lack of practical methods for bacterial separation remains a hindrance for the low-cost and successful development of rapid detection methods from complex samples. Antibody-tagged magnetic particles are commonly used to pull analytes from a liquid sample. While this method is well-established, improvements in capture efficiencies would result in an increase of the overall detection assay performance. Bacteriophages represent a low-cost and more consistent biorecognition element as compared to antibodies. We have developed nanoscale bacteriophage-tagged magnetic probes, where T7 bacteriophages were bound to magnetic nanoparticles. The nanoprobe allowed the specific recognition and attachment to E. coli cells. The phage magnetic nanprobes were directly compared to antibody-conjugated magnetic nanoprobes. The capture efficiencies of bacteriophages and antibodies on nanoparticles for the separation of E. coli K12 at varying concentrations were determined. The results indicated a similar bacteria capture efficiency between the two nanoprobes. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr03779d

Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

PubMed Central

Tanase, Maya; Zolla, Valerio; Clement, Cristina C; Borghi, Francesco; Urbanska, Aleksandra M; Rodriguez-Navarro, Jose Antonio; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Cuervo, Ana Maria; Santambrogio, Laura

2016-01-01

Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions. PMID:25521790

Apparatus and method for electrochemical modification of liquids

DOEpatents

James, Patrick I

2015-04-21

An apparatus for electrochemical modification of liquid streams employing an electrolytic cell which includes an anode compartment defined by an anode structure where oxidation is effected, containing a liquid electrolyte anolyte, and a cathode compartment defined by a cathode structure where reduction is effected containing a liquid electrolyte catholyte. In addition, the electrolytic cell includes at least one additional compartment arranged at least partially between the anode compartment and the cathode compartment and separated from the anode compartment and the cathode compartment by a separator structure arranged to supports ionic conduction of current between the anode structure and the cathode structure.

Removal of algal blooms from freshwater by the coagulation-magnetic separation method.

PubMed

Liu, Dan; Wang, Peng; Wei, Guanran; Dong, Wenbo; Hui, Franck

2013-01-01

This research investigated the feasibility of changing waste into useful materials for water treatment and proposed a coagulation-magnetic separation technique. This technique was rapid and highly effective for clearing up harmful algal blooms in freshwater and mitigating lake eutrophication. A magnetic coagulant was synthesized by compounding acid-modified fly ash with magnetite (Fe(3)O(4)). Its removal effects on algal cells and dissolved organics in water were studied. After mixing, coagulation, and magnetic separation, the flocs obtained from the magnet surface were examined by SEM. Treated samples were withdrawn for the content determination of chlorophyll-a, turbidity, chemical oxygen demand (COD), total nitrogen, and total phosphorus. More than 99 % of algal cells were removed within 5 min after the addition of magnetic coagulant at optimal loadings (200 mg L(-1)). The removal efficiencies of COD, total nitrogen, and phosphorus were 93, 91, and 94 %, respectively. The mechanism of algal removal explored preliminarily showed that the magnetic coagulant played multiple roles in mesoporous adsorption, netting and bridging, as well as high magnetic responsiveness to a magnetic field. The magnetic-coagulation separation method can rapidly and effectively remove algae from water bodies and greatly mitigate eutrophication of freshwater using a new magnetic coagulant. The method has good performance, is low cost, can turn waste into something valuable, and provides reference and directions for future pilot and production scale-ups.

Heterogeneity in the growth hormone pituitary gland system of rats and humans: Implications to microgravity based research

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Grindeland, R.; Hayes, C.; Lanham, J. W.; Cleveland, C.; Todd, P.; Morrison, Dennis R.

1988-01-01

The cell separation techniques of velocity sedimentation, flow cytometry and continuous flow electrophoresis were used to obtain enriched populations of growth hormone (GH) cells. The goal was to isolate a GH cell subpopulation which releases GH molecules which are very high in biological activity, it was important to use a method which was effective in processing large numbers of cells over a short time span. The techniques based on sedimentation are limited by cell density overlaps and streaming. While flow cytometry is useful in the analytical mode for objectively establishing cell purity, the numbers of cells which can be processed in the sort mode are so small as to make this approach ineffective in terms of the long term goals. It was shown that continuous flow electrophoresis systems (CFES) can separate GH cells from other cell types on the basis of differences in surface charge. The bioreactive producers appear to be more electrophoretically mobile than the low producers. Current ground based CFES efforts are hampered by cell clumping in low ionic strength buffers and poor cell recoveries from the CFES device.

Determination of 90Sr / 238U ratio by double isotope dilution inductively coupled plasma mass spectrometer with multiple collection in spent nuclear fuel samples with in situ 90Sr / 90Zr separation in a collision-reaction cell

NASA Astrophysics Data System (ADS)

Isnard, H.; Aubert, M.; Blanchet, P.; Brennetot, R.; Chartier, F.; Geertsen, V.; Manuguerra, F.

2006-02-01

Strontium-90 is one of the most important fission products generated in nuclear industry. In the research field concerning nuclear waste disposal in deep geological environment, it is necessary to quantify accurately and precisely its concentration (or the 90Sr / 238U atomic ratio) in irradiated fuels. To obtain accurate analysis of radioactive 90Sr, mass spectrometry associated with isotope dilution is the most appropriated method. But, in nuclear fuel samples the interference with 90Zr must be previously eliminated. An inductively coupled plasma mass spectrometer with multiple collection, equipped with an hexapole collision cell, has been used to eliminate the 90Sr / 90Zr interference by addition of oxygen in the collision cell as a reactant gas. Zr + ions are converted into ZrO +, whereas Sr + ions are not reactive. A mixed solution, prepared from a solution of enriched 84Sr and a solution of enriched 235U was then used to quantify the 90Sr / 238U ratio in spent fuel sample solutions using the double isotope dilution method. This paper shows the results, the reproducibility and the uncertainties that can be obtained with this method to quantify the 90Sr / 238U atomic ratio in an UOX (uranium oxide) and a MOX (mixed oxide) spent fuel samples using the collision cell of an inductively coupled plasma mass spectrometer with multiple collection to perform the 90Sr / 90Zr separation. A comparison with the results obtained by inductively coupled plasma mass spectrometer with multiple collection after a chemical separation of strontium from zirconium using a Sr spec resin (Eichrom) has been performed. Finally, to validate the analytical procedure developed, measurements of the same samples have been performed by thermal ionization mass spectrometry, used as an independent technique, after chemical separation of Sr.

Antibody-immobilized column for quick cell separation based on cell rolling.

PubMed

Mahara, Atsushi; Yamaoka, Tetsuji

2010-01-01

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.

Detection of sepsis in patient blood samples using CD64 expression in a microfluidic cell separation device.

PubMed

Zhang, Ye; Li, Wenjie; Zhou, Yun; Johnson, Amanda; Venable, Amanda; Hassan, Ahmed; Griswold, John; Pappas, Dimitri

2017-12-18

A microfluidic affinity separation device was developed for the detection of sepsis in critical care patients. An affinity capture method was developed to capture cells based on changes in CD64 expression in a single, simple microfluidic chip for sepsis detection. Both sepsis patient samples and a laboratory CD64+ expression model were used to validate the microfluidic assay. Flow cytometry analysis showed that the chip cell capture had a linear relationship with CD64 expression in laboratory models. The Sepsis Chip detected an increase in upregulated neutrophil-like cells when the upregulated cell population is as low as 10% of total cells spiked into commercially available aseptic blood samples. In a proof of concept study, blood samples obtained from sepsis patients within 24 hours of diagnosis were tested on the chip to further validate its performance. On-chip CD64+ cell capture from 10 patient samples (619 ± 340 cells per chip) was significantly different from control samples (32 ± 11 cells per chip) and healthy volunteer samples (228 ± 95 cells per chip). In addition, the on-chip cell capture has a linear relationship with CD64 expression indicating our approach can be used to measure CD64 expression based on total cell capture on Sepsis Chip. Our method has proven to be sensitive, accurate, rapid, and cost-effective. Therefore, this device is a promising detection platform for neutrophil activation and sepsis diagnosis.

Flow-enhanced solution printing of all-polymer solar cells

DOE PAGES

Diao, Ying; Zhou, Yan; Kurosawa, Tadanori; ...

2015-08-12

Morphology control of solution coated solar cell materials presents a key challenge limiting their device performance and commercial viability. Here we present a new concept for controlling phase separation during solution printing using an all-polymer bulk heterojunction solar cell as a model system. The key aspect of our method lies in the design of fluid flow using a microstructured printing blade, on the basis of the hypothesis of flow-induced polymer crystallization. Our flow design resulted in a similar to 90% increase in the donor thin film crystallinity and reduced microphase separated donor and acceptor domain sizes. The improved morphology enhancedmore » all metrics of solar cell device performance across various printing conditions, specifically leading to higher short-circuit current, fill factor, open circuit voltage and significantly reduced device-to-device variation. However, we expect our design concept to have broad applications beyond all-polymer solar cells because of its simplicity and versatility.« less

Flow-enhanced solution printing of all-polymer solar cells

PubMed Central

Diao, Ying; Zhou, Yan; Kurosawa, Tadanori; Shaw, Leo; Wang, Cheng; Park, Steve; Guo, Yikun; Reinspach, Julia A.; Gu, Kevin; Gu, Xiaodan; Tee, Benjamin C. K.; Pang, Changhyun; Yan, Hongping; Zhao, Dahui; Toney, Michael F.; Mannsfeld, Stefan C. B.; Bao, Zhenan

2015-01-01

Morphology control of solution coated solar cell materials presents a key challenge limiting their device performance and commercial viability. Here we present a new concept for controlling phase separation during solution printing using an all-polymer bulk heterojunction solar cell as a model system. The key aspect of our method lies in the design of fluid flow using a microstructured printing blade, on the basis of the hypothesis of flow-induced polymer crystallization. Our flow design resulted in a ∼90% increase in the donor thin film crystallinity and reduced microphase separated donor and acceptor domain sizes. The improved morphology enhanced all metrics of solar cell device performance across various printing conditions, specifically leading to higher short-circuit current, fill factor, open circuit voltage and significantly reduced device-to-device variation. We expect our design concept to have broad applications beyond all-polymer solar cells because of its simplicity and versatility. PMID:26264528

21 CFR 864.9245 - Automated blood cell separator.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated blood cell separator. 864.9245 Section... Blood and Blood Products § 864.9245 Automated blood cell separator. (a) Identification. An automated blood cell separator is a device that uses a centrifugal or filtration separation principle to...

21 CFR 864.9245 - Automated blood cell separator.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated blood cell separator. 864.9245 Section... Blood and Blood Products § 864.9245 Automated blood cell separator. (a) Identification. An automated blood cell separator is a device that uses a centrifugal or filtration separation principle to...

21 CFR 864.9245 - Automated blood cell separator.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated blood cell separator. 864.9245 Section... Blood and Blood Products § 864.9245 Automated blood cell separator. (a) Identification. An automated blood cell separator is a device that uses a centrifugal or filtration separation principle to...

21 CFR 864.9245 - Automated blood cell separator.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Automated blood cell separator. 864.9245 Section... Blood and Blood Products § 864.9245 Automated blood cell separator. (a) Identification. An automated blood cell separator is a device that uses a centrifugal or filtration separation principle to...

21 CFR 864.9245 - Automated blood cell separator.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Automated blood cell separator. 864.9245 Section... Blood and Blood Products § 864.9245 Automated blood cell separator. (a) Identification. An automated blood cell separator is a device that uses a centrifugal or filtration separation principle to...

Exosome separation using microfluidic systems: size-based, immunoaffinity-based and dynamic methodologies.

PubMed

Yang, Fang; Liao, Xiangzhi; Tian, Yuan; Li, Guiying

2017-04-01

Exosomes, nanovesicles secreted by most types of cells, exist in virtually all bodily fluids. Their rich nucleic acid and protein content make them potentially valuable biomarkers for noninvasive molecular diagnostics. They also show promise, after further development, to serve as a drug delivery system. Unfortunately, existing exosome separation technologies, such as ultracentrifugation and methods incorporating magnetic beads, are time-consuming, laborious and separate only exosomes of low purity. Thus, a more effective separation method is highly desirable. Microfluidic platforms are ideal tools for exosome separation, since they enable fast, cost-efficient, portable and precise processing of nanoparticles and small volumes of liquid samples. Recently, several microfluidic-based exosome separation technologies have been studied. In this article, the advantages of the most recent technologies, as well as their limitations, challenges and potential uses in novel microfluidic exosome separation and collection applications is reviewed. This review outlines the uses of new powerful microfluidic exosome detection tools for biologists and clinicians, as well as exosome separation tools for microfluidic engineers. Current challenges of exosome separation methodologies are also described, in order to highlight areas for future research and development. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of 6-methylsulfinylhexyl isothiocyanate as an apoptosis-inducing component in wasabi.

PubMed

Watanabe, Makoto; Ohata, Masahiko; Hayakawa, Sumio; Isemura, Mamoru; Kumazawa, Shigenori; Nakayama, Tsutomu; Furugori, Michiyo; Kinae, Naohide

2003-03-01

The ethanol extract from Japanese horseradish wasabi was found to inhibit cell proliferation in human monoblastic leukemia U937 cells by inducing apoptotic cell death. Separation by methods including silica gel chromatography and preparative HPLC gave an active compound, which was identified as 6-methylsulfinylhexyl isothiocyanate (6-HITC). Several lines of evidence indicated that 6-HITC induced apoptosis in U937 cells and human stomach cancer MKN45 cells. Thus, 6-HITC is potentially useful as a natural anti-cancer agent.

Efficient collection of peripheral blood stem cells using the Fresenius AS104 in chronic myelocytic leukemia patients with very high numbers of platelets.

PubMed

Komatsu, F; Ishida, Y

1997-04-01

For chronic myelocytic leukemia patients with very high numbers of platelets, we describe an efficient method for the collection of peripheral blood stem cells (PBSC) using the Fresenius AS104 cell separator. In these patients, it is difficult to collect a sufficient number of PBSC, due to the platelet band interfering with the machine's red cell interface sensor. We, therefore, tried a manual adjustment of the device. The collection phase was set automatically. When the whole blood began to separate into the red cell layer and plasma (plus mononuclear cell) layer, the red cell interface setting of "7:1" was changed to "OFF," and the plasma pump flow rate was controlled manually in order to locate the interface position 1 cm from the outside wall of the centrifuge chamber. After the collection phase, the procedure was returned to the automatic setting. By repeating this procedure, we were able to collect large numbers of PBSC.

Design of microfluidic channels for magnetic separation of malaria-infected red blood cells

PubMed Central

Wu, Wei-Tao; Martin, Andrea Blue; Gandini, Alberto; Aubry, Nadine; Massoudi, Mehrdad; Antaki, James F.

2016-01-01

This study is motivated by the development of a blood cell filtration device for removal of malaria-infected, parasitized red blood cells (pRBCs). The blood was modeled as a multi-component fluid using the computational fluid dynamics discrete element method (CFD-DEM), wherein plasma was treated as a Newtonian fluid and the red blood cells (RBCs) were modeled as soft-sphere solid particles which move under the influence of drag, collisions with other RBCs, and a magnetic force. The CFD-DEM model was first validated by a comparison with experimental data from Han et al. 2006 (Han and Frazier 2006) involving a microfluidic magnetophoretic separator for paramagnetic deoxygenated blood cells. The computational model was then applied to a parametric study of a parallel-plate separator having hematocrit of 40% with a 10% of the RBCs as pRBCs. Specifically, we investigated the hypothesis of introducing an upstream constriction to the channel to divert the magnetic cells within the near-wall layer where the magnetic force is greatest. Simulations compared the efficacy of various geometries upon the stratification efficiency of the pRBCs. For a channel with nominal height of 100 µm, the addition of an upstream constriction of 80% improved the proportion of pRBCs retained adjacent to the magnetic wall (separation efficiency) by almost 2 fold, from 26% to 49%. Further addition of a downstream diffuser reduced remixing, hence improved separation efficiency to 72%. The constriction introduced a greater pressure drop (from 17 to 495 Pa), which should be considered when scaling-up this design for a clinical-sized system. Overall, the advantages of this design include its ability to accommodate physiological hematocrit and high throughput – which is critical for clinical implementation as a blood-filtration system. PMID:27761107

Nanofiber/ZrO2-based mixed matrix separator for high safety/high-rate lithium-ion batteries

NASA Astrophysics Data System (ADS)

Xiao, Wei; Liu, Jianguo; Yan, Chuanwei

2017-10-01

A novel asymmetric separator based on a thin bacterial cellulose nanofiber (BCF)/nano-ZrO2 composite layer and a non-woven support was prepared by paper-making method. Owing to the relatively polar constituents and well-developed, gradient porous structure, the separator exhibited the advantages of higher thermal resistance, electrolyte wettability, and ionic conductivity in comparison to polyethylene separator. Based on these advantages, the Li/LiFePO4 cells assembled from this composite separator showed excellent performance characteristics, including outstanding C-rate capability, high capacity and cycling performance. Production of the composite separator is simple, environmentally benign and economically viable. Therefore, it's a good candidate for creating improved lithium-ion batteries.

Fast label-free detection of Legionella spp. in biofilms by applying immunomagnetic beads and Raman spectroscopy.

PubMed

Kusić, Dragana; Rösch, Petra; Popp, Jürgen

2016-03-01

Legionellae colonize biofilms, can form a biofilm by itself and multiply intracellularly within the protozoa commonly found in water distribution systems. Approximately half of the known species are pathogenic and have been connected to severe multisystem Legionnaires' disease. The detection methods for Legionella spp. in water samples are still based on cultivation, which is time consuming due to the slow growth of this bacterium. Here, we developed a cultivation-independent, label-free and fast detection method for legionellae in a biofilm matrix based on the Raman spectroscopic analysis of isolated single cells via immunomagnetic separation (IMS). A database comprising the Raman spectra of single bacterial cells captured and separated from the biofilms formed by each species was used to build the identification method based on a support vector machine (SVM) discriminative classifier. The complete method allows the detection of Legionella spp. in 100 min. Cross-reactivity of Legionella spp. specific immunomagnetic beads to the other studied genera was tested, where only small cell amounts of Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli compared to the initial number of cells were isolated by the immunobeads. Nevertheless, the Raman spectra collected from isolated non-targeted bacteria were well-discriminated from the Raman spectra collected from isolated Legionella cells, whereby the Raman spectra of the independent dataset of Legionella strains were assigned with an accuracy of 98.6%. In addition, Raman spectroscopy was also used to differentiate between isolated Legionella species. Copyright © 2016 Elsevier GmbH. All rights reserved.

Systems and methods for separation and purification of products

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weiss, Michael Joseph; Gilliam, Ryan J.; Self, Kyle

There are provided methods and systems for an electrochemical cell including an anode and a cathode where the anode is contacted with a metal ion that converts the metal ion from a lower oxidation state to a higher oxidation state. The metal ion in the higher oxidation state is reacted with an unsaturated hydrocarbon and/or a saturated hydrocarbon to form products. Separation and/or purification of the products as well as of the metal ions in the lower oxidation state and the higher oxidation state, is provided herein.

The Effect of Created Local Hyperosmotic Microenvironment in Microcapsule for the Growth and Metabolism of Osmotolerant Yeast Candida krusei

PubMed Central

Chen, Guo; Yao, Shanjing

2013-01-01

Candida krusei is osmotolerant yeast used for the production of glycerol. Addition of osmolyte such as NaCl into culture medium can increase the production of glycerol from glucose, but osmolytes may burden the glycerol separation. A coencapsulation method was suggested to create local extracellular hyperosmotic stress for glycerol accumulation. Firstly, the influence of osmotic stress induced by the addition of PEG4000 on growth and metabolism of free cell was studied in detail. Glycerol accumulation could be improved by employing PEG4000 as osmoregulator. Secondly, cells and PEG4000 were coentrapped in NaCS/PDMDAAC capsules to create local hyperosmotic stress. The effects of local hyperosmotic microenvironment on the cell growth and metabolism were studied. The coentrapment method increased the glycerol concentration by 25%, and the glycerol concentration attained 50 gL−1 with productivity of 18.8 gL−1Day−1 in shake flask. More importantly, the glycerol could be directly separated from the encapsulated cells. The entrapped cells containing PEG4000 were also cultivated for 15 days in an airlift reactor. The yield and productivity were ca. 35% and 21 gL−1Day−1, respectively. PMID:24294610

The effect of created local hyperosmotic microenvironment in microcapsule for the growth and metabolism of osmotolerant yeast Candida krusei.

PubMed

Chen, Guo; Yao, Shanjing

2013-01-01

Candida krusei is osmotolerant yeast used for the production of glycerol. Addition of osmolyte such as NaCl into culture medium can increase the production of glycerol from glucose, but osmolytes may burden the glycerol separation. A coencapsulation method was suggested to create local extracellular hyperosmotic stress for glycerol accumulation. Firstly, the influence of osmotic stress induced by the addition of PEG4000 on growth and metabolism of free cell was studied in detail. Glycerol accumulation could be improved by employing PEG4000 as osmoregulator. Secondly, cells and PEG4000 were coentrapped in NaCS/PDMDAAC capsules to create local hyperosmotic stress. The effects of local hyperosmotic microenvironment on the cell growth and metabolism were studied. The coentrapment method increased the glycerol concentration by 25%, and the glycerol concentration attained 50 gL⁻¹ with productivity of 18.8 gL⁻¹Day⁻¹ in shake flask. More importantly, the glycerol could be directly separated from the encapsulated cells. The entrapped cells containing PEG4000 were also cultivated for 15 days in an airlift reactor. The yield and productivity were ca. 35% and 21 gL⁻¹Day⁻¹, respectively.

Extracting archaeal populations from iron oxidizing systems

NASA Astrophysics Data System (ADS)

Whitmore, L. M.; Hutchison, J.; Chrisler, W.; Jay, Z.; Moran, J.; Inskeep, W.; Kreuzer, H.

2013-12-01

Unique environments in Yellowstone National Park offer exceptional conditions for studying microorganisms in extreme and constrained systems. However, samples from some extreme systems often contain inorganic components that pose complications during microbial and molecular analysis. Several archaeal species are found in acidic, geothermal ferric-oxyhydroxide mats; these species have been shown to adhere to mineral surfaces in flocculated colonies. For optimal microbial analysis, (microscopy, flow cytometry, genomic extractions, proteomic analysis, stable isotope analysis, and others), improved techniques are needed to better facilitate cell detachment and separation from mineral surfaces. As a requirement, these techniques must preserve cell structure while simultaneously minimizing organic carryover to downstream analysis. Several methods have been developed for removing sediments from mixed prokaryotic populations, including ultra-centrifugation, nycodenz gradient, sucrose cushions, and cell straining. In this study we conduct a comparative analysis of mechanisms used to detach archaeal cell populations from the mineral interface. Specifically, we evaluated mechanical and chemical approaches for cell separation and homogenization. Methods were compared using confocal microscopy, flow cytometry analyses, and real-time PCR detection. The methodology and approaches identified will be used to optimize biomass collection from environmental specimens or isolates grown with solid phases.

Separation of In-Vitro-Derived Megakaryocytes and Platelets Using Spinning-Membrane Filtration

PubMed Central

Schlinker, Alaina C.; Radwanski, Katherine; Wegener, Christopher; Min, Kyungyoon; Miller, William M.

2015-01-01

In-vitro-derived platelets (PLTs) could potentially overcome problems associated with donated PLTs, including contamination and alloimmunization. Although several groups have produced functional PLTs from stem cells in vitro, the challenge of developing this technology to yield transfusable PLT units has yet to be addressed. The asynchronous nature of in vitro PLT generation makes a single harvest point infeasible for collecting PLTs as soon as they are formed. The current standard of performing manual centrifugations to separate PLTs from nucleated cells at multiple points during culture is labor-intensive, imprecise, and difficult to standardize in accordance with current Good Manufacturing Practices (cGMP). In an effort to develop a more effective method, we adapted a commercially-available, spinning-membrane filtration device to separate in-vitro-derived PLTs from nucleated cells and recover immature megakaryocytes (MKs), the precursor cells to PLTs, for continued culture. Processing a mixture of in-vitro-derived MKs and PLTs on the adapted device yielded a pure PLT population and did not induce PLT pre-activation. MKs recovered from the separation process were unaffected with respect to viability and ploidy, and were able to generate PLTs after reseeding in culture. Being able to efficiently harvest in-vitro-derived PLTs brings this technology one step closer to clinical relevance. PMID:25312394

Characterization of adhesive properties of red blood cells using surface acoustic wave induced flows for rapid diagnostics

NASA Astrophysics Data System (ADS)

Sivanantha, Ninnuja; Ma, Charles; Collins, David J.; Sesen, Muhsincan; Brenker, Jason; Coppel, Ross L.; Neild, Adrian; Alan, Tuncay

2014-09-01

This letter presents a method which employs surface acoustic wave induced acoustic streaming to differentially peel treated red blood cells (RBCs) off a substrate based on their adhesive properties and separate populations of pathological cells from normal ones. We demonstrate the principle of operation by comparing the applied power and time required to overcome the adhesion displayed by healthy, glutaraldehyde-treated or malaria-infected human RBCs. Our experiments indicate that the method can be used to differentiate between various cell populations contained in a 9 μl droplet within 30 s, suggesting potential for rapid diagnostics.

Ceramic distribution members for solid state electrolyte cells and method of producing

NASA Technical Reports Server (NTRS)

Clark, Douglas J. (Inventor); Galica, Leo M. (Inventor); Losey, Robert W. (Inventor); Suitor, Jerry W. (Inventor)

1993-01-01

A solid state electrolyte cells apparatus and method of producing is disclosed. The apparatus can be used for separating oxygen from an oxygen-containing feedstock or as a fuel cell for reacting fluids. Cells can be stacked so that fluids can be introduced and removed from the apparatus through ceramic distribution members having ports designed for distributing the fluids in parallel flow to and from each cell. The distribution members can also serve as electrodes to membranes or as membrane members between electrodes. The distribution member design does not contain any horizontal internal ports which allows the member to be thin. A method of tape casting in combination with an embossing method allows intricate radial ribs and bosses to be formed on each distribution member. The bosses serve as seals for the ports and allow the distribution members to be made without any horizontal internal ports.

Method of producing ceramic distribution members for solid state electrolyte cells

NASA Technical Reports Server (NTRS)

Clark, Douglas J. (Inventor); Galica, Leo M. (Inventor); Losey, Robert W. (Inventor); Suitor, Jerry W. (Inventor)

1995-01-01

A solid state electrolyte cells apparatus and method of producing is disclosed. The apparatus can be used for separating oxygen from an oxygen-containing feedstock or as a fuel cell for reacting fluids. Cells can be stacked so that fluids can be introduced and removed from the apparatus through ceramic distribution members having ports designed for distributing the fluids in parallel flow to and from each cell. The distribution members can also serve as electrodes to membranes or as membrane members between electrodes, The distribution member design does not contain any horizontal internal ports which allows the member to be thin. A method of tape casting in combination with an embossing method allows intricate radial ribs and bosses to be formed on each distribution member. The bosses serve as seals for the ports and allow the distribution members to be made without any horizontal internal ports.

Hydrophilic Strong Anion Exchange (hSAX) Chromatography for Highly Orthogonal Peptide Separation of Complex Proteomes

PubMed Central

2013-01-01

Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis. PMID:23294059

A facile method to enhance the uniformity and adhesion properties of water-based ceramic coating layers on hydrophobic polyethylene separators

NASA Astrophysics Data System (ADS)

Lee, Hoogil; Jeon, Hyunkyu; Gong, Seokhyeon; Ryou, Myung-Hyun; Lee, Yong Min

2018-01-01

To enhance the uniformity and adhesion properties of water-based ceramic coating layers on hydrophobic polyethylene (PE) separators, their surfaces were treated with thin and hydrophilic polydopamine layers. As a result, an aqueous ceramic coating slurry consisting of Al2O3 particles, carboxyl methyl cellulose (CMC) binders, and water solvent was easily spread on the separator surface, and a uniform ceramic layer was formed after solvent drying. Moreover, the ceramic coating layer showed greatly improved adhesion properties to the PE separator surface. Whereas the adhesion strength within the bulk coating layer (Fmid) ranged from 43 to 86 N m-1 depending on the binder content of 1.5-3.0 wt%, the adhesion strength at the interface between the ceramic coating layer and PE separator (Fsepa-Al2O3) was 245-360 N m-1, a value equivalent to an increase of four or five times. Furthermore, an additional ceramic coating layer of approximately 7 μm did not degrade the ionic conductivity and electrochemical properties of the bare PE separators. Thus, all the LiMn2O4/graphite cells with ceramic-coated separators delivered an improved cycle life and rate capability compared with those of the control cells with bare PE separators.

One-step fabrication of 3D silver paste electrodes into microfluidic devices for enhanced droplet-based cell sorting

NASA Astrophysics Data System (ADS)

Rao, Lang; Cai, Bo; Yu, Xiao-Lei; Guo, Shi-Shang; Liu, Wei; Zhao, Xing-Zhong

2015-05-01

3D microelectrodes are one-step fabricated into a microfluidic droplet separator by filling conductive silver paste into PDMS microchambers. The advantages of 3D silver paste electrodes in promoting droplet sorting accuracy are systematically demonstrated by theoretical calculation, numerical simulation and experimental validation. The employment of 3D electrodes also helps to decrease the droplet sorting voltage, guaranteeing that cells encapsulated in droplets undergo chip-based sorting processes are at better metabolic status for further potential cellular assays. At last, target droplet containing single cell are selectively sorted out from others by an appropriate electric pulse. This method provides a simple and inexpensive alternative to fabricate 3D electrodes, and it is expected our 3D electrode-integrated microfluidic droplet separator platform can be widely used in single cell operation and analysis.

Nonnegative matrix factorization: a blind sources separation method to extract content of fluorophores mixture media

NASA Astrophysics Data System (ADS)

Zhou, Kenneth J.; Chen, Jun

2014-03-01

The fluorophores of malignant human breast cells change their compositions that may be exposed in the fluorescence spectroscopy and blind source separation method. The content of the fluorophores mixture media such as tryptophan, collagen, elastin, NADH, and flavin were varied according to the cancer development. The native fluorescence spectra of these key fluorophores mixture media excited by the selective excitation wavelengths of 300 nm and 340 nm were analyzed using a blind source separation method: Nonnegative Matrix Factorization (NMF). The results show that the contribution from tryptophan, NADH and flavin to the fluorescence spectra of the mixture media is proportional to the content of each fluorophore. These data present a possibility that native fluorescence spectra decomposed by NMF can be used as potential native biomarkers for cancer detection evaluation of the cancer.

Multi-scale Gaussian representation and outline-learning based cell image segmentation

PubMed Central

2013-01-01

Background High-throughput genome-wide screening to study gene-specific functions, e.g. for drug discovery, demands fast automated image analysis methods to assist in unraveling the full potential of such studies. Image segmentation is typically at the forefront of such analysis as the performance of the subsequent steps, for example, cell classification, cell tracking etc., often relies on the results of segmentation. Methods We present a cell cytoplasm segmentation framework which first separates cell cytoplasm from image background using novel approach of image enhancement and coefficient of variation of multi-scale Gaussian scale-space representation. A novel outline-learning based classification method is developed using regularized logistic regression with embedded feature selection which classifies image pixels as outline/non-outline to give cytoplasm outlines. Refinement of the detected outlines to separate cells from each other is performed in a post-processing step where the nuclei segmentation is used as contextual information. Results and conclusions We evaluate the proposed segmentation methodology using two challenging test cases, presenting images with completely different characteristics, with cells of varying size, shape, texture and degrees of overlap. The feature selection and classification framework for outline detection produces very simple sparse models which use only a small subset of the large, generic feature set, that is, only 7 and 5 features for the two cases. Quantitative comparison of the results for the two test cases against state-of-the-art methods show that our methodology outperforms them with an increase of 4-9% in segmentation accuracy with maximum accuracy of 93%. Finally, the results obtained for diverse datasets demonstrate that our framework not only produces accurate segmentation but also generalizes well to different segmentation tasks. PMID:24267488

Automated morphological analysis of bone marrow cells in microscopic images for diagnosis of leukemia: nucleus-plasma separation and cell classification using a hierarchical tree model of hematopoesis

NASA Astrophysics Data System (ADS)

Krappe, Sebastian; Wittenberg, Thomas; Haferlach, Torsten; Münzenmayer, Christian

2016-03-01

The morphological differentiation of bone marrow is fundamental for the diagnosis of leukemia. Currently, the counting and classification of the different types of bone marrow cells is done manually under the use of bright field microscopy. This is a time-consuming, subjective, tedious and error-prone process. Furthermore, repeated examinations of a slide may yield intra- and inter-observer variances. For that reason a computer assisted diagnosis system for bone marrow differentiation is pursued. In this work we focus (a) on a new method for the separation of nucleus and plasma parts and (b) on a knowledge-based hierarchical tree classifier for the differentiation of bone marrow cells in 16 different classes. Classification trees are easily interpretable and understandable and provide a classification together with an explanation. Using classification trees, expert knowledge (i.e. knowledge about similar classes and cell lines in the tree model of hematopoiesis) is integrated in the structure of the tree. The proposed segmentation method is evaluated with more than 10,000 manually segmented cells. For the evaluation of the proposed hierarchical classifier more than 140,000 automatically segmented bone marrow cells are used. Future automated solutions for the morphological analysis of bone marrow smears could potentially apply such an approach for the pre-classification of bone marrow cells and thereby shortening the examination time.

Modern Approach to Medical Diagnostics - the Use of Separation Techniques in Microorganisms Detection.

PubMed

Chylewska, Agnieszka; Ogryzek, M; Makowski, Mariusz

2017-10-23

New analytical and molecular methods for microorganisms are being developed on various features of identification i.e. selectivity, specificity, sensitivity, rapidity and discrimination of the viable cell. The presented review was established following the current trends in improved pathogens separation and detection methods and their subsequent use in medical diagnosis. This contribution also focuses on the development of analytical and biological methods in the analysis of microorganisms, with special attention paid to bio-samples containing microbes (blood, urine, lymph, wastewater). First, the paper discusses microbes characterization, their structure, surface, properties, size and then it describes pivotal points in the bacteria, viruses and fungi separation procedure obtained by researchers in the last 30 years. According to the above, detection techniques can be classified into three categories, which were, in our opinion, examined and modified most intensively during this period: electrophoretic, nucleic-acid-based, and immunological methods. The review covers also the progress, limitations and challenges of these approaches and emphasizes the advantages of new separative techniques in selective fractionating of microorganisms. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Comparison of immunomagnetic separation/adenosine triphosphate rapid method to traditional culture-based method for E. coli and enterococci enumeration in wastewater

USGS Publications Warehouse

Bushon, R.N.; Likirdopulos, C.A.; Brady, A.M.G.

2009-01-01

Untreated wastewater samples from California, North Carolina, and Ohio were analyzed by the immunomagnetic separation/adenosine triphosphate (IMS/ATP) method and the traditional culture-based method for E. coli and enterococci concentrations. The IMS/ATP method concentrates target bacteria by immunomagnetic separation and then quantifies captured bacteria by measuring bioluminescence induced by release of ATP from the bacterial cells. Results from this method are available within 1 h from the start of sample processing. Significant linear correlations were found between the IMS/ATP results and results from traditional culture-based methods for E. coli and enterococci enumeration for one location in California, two locations in North Carolina, and one location in Ohio (r??values ranged from 0.87 to 0.97). No significant linear relation was found for a second location in California that treats a complex mixture of residential and industrial wastewater. With the exception of one location, IMS/ATP showed promise as a rapid method for the quantification of faecal-indicator organisms in wastewater.

The effects of stress on the enzymes of peripheral leukocytes

NASA Technical Reports Server (NTRS)

Leise, E. M.; Gray, I.

1973-01-01

Previous work showed an early response of rabbit and human leukocyte enzymes to the stress of bacterial infection. Since these represented a mixed population of leukocytes and since polymorphonuclear leukocytes (PMN) increased in these preparations, it was necessary to establish whether the observed increase in lactate dehydrenase (LDH) and protein was the result of an increase in any one particular cell type or in all cells. The need for the development of a simple reproducible method for the differential separation of peripheral leukocytes for the furtherance of our own studies was apparent. It was also becoming increasingly apparent that morphologically similar cells, such as small lymphocytes (L) and macrophages, were capable of different biological functions. A dextran gradient centrifugation method was developed which has provided an easily reproducible technique for separating L from PMN. During the course of this work, in which over 250 rabbits were examined, the pattern of daily leukocyte protein and enzyme variation became increasingly more apparent. This information could have some impact on future work with leukocyte enzymes, by our group and by other workers. The differences in normal protein and enzyme levels maintained by some individuals, and some inbred strains, were evaluated and reported separately. It has been shown that one type of leukocyte may react more to a given stress than other leukocytes.

Photoelectrochemical molecular comb

DOEpatents

Thundat, Thomas G [Knoxville, TN; Ferrell, Thomas L [Knoxville, TN; Brown,; Gilbert, M [Knoxville, TN

2007-05-01

A method, system, and apparatus are provided for separating molecules, such as biomolecules. The method, system, and apparatus utilize an electrochemical cell having at least to electrodes, one electrode comprising a photo-sensitive material capable of generating a photopotential. Molecules are moved through an electrolyte medium between the at least two electrodes based upon localized photopotentials.

Photoelectrochemical molecular comb

DOEpatents

Thundat, Thomas G [Knoxville, TN; Ferrell, Thomas L [Knoxville, TN; Brown, Gilbert M [Knoxville, TN

2012-02-07

A method, system, and apparatus are provided for separating molecules, such as biomolecules. The method, system, and apparatus utilize an electrochemical cell having at least two electrodes, one electrode comprising a photo-sensitive material capable of generating a photopotential. Molecules are moved through an electrolyte medium between the at least two electrodes based upon localized photopotentials.

Detection and cultivation of circulating tumor cells in malignant pleural mesothelioma.

PubMed

Bobek, Vladimir; Kacprzak, Grzegorz; Rzechonek, Adam; Kolostova, Katarina

2014-05-01

Malignant pleural mesothelioma (MPM) is an aggressive disease with very poor prognosis which tends to affect older patients. Progress in the management of this group of patients has been limited by the rarity of the disease and hence, difficulty in conducting randomized trials. The vast majority of cancer deaths occur due to metastasis of the primary tumor to distant sites via circulating tumor cells (CTCs) in the circulation. CTCs are extremely rare and limits in technology used to capture these cells hamper our complete understanding over the metastatic process. In the present study we present a new method for detection and cultivation of CTCs isolated from peripheral blood of MPM patients. Patients with diagnosed MPM were enrolled into this study. A size-based separation method for viable CTC enrichment from unclothed peripheral blood has been introduced; MetaCell. The size-based enrichment process was based on filtration of peripheral blood (PB) through porous polycarbonate membrane. The separated CTCs are cultured on the membrane in vitro under standard cancer cell culture conditions and observed by an inverted microscope. The reported methodology allows for quick and easy enrichment of CTCs and their cultivation. The cultivated cells can be used for next specification of gene expression and histological/biological specificity of concrete mesothelioma.

Comparing cooling systems for the COBE 2991 cell separator used in the purification of human pancreatic islets of Langerhans.

PubMed

Adewola, A; Mage, R; Hansen, M; Barbaro, B; Mendoza-Elias, J; Harvat, T; Morel, P H; Oberholzer, J; Wang, Y

2010-01-01

Two different approaches of controlled cooling of the COBE 2991 cell-separator for islet purification were evaluated. The first method is the new Geneva COBE cooling system (GCCS), which consists of an electronically controlled liquid nitrogen injection system. The second is the University of Illinois at Chicago cooling system (UICCS), which consists of a specially designed "Cold Room" maintained at 1-8 C. For the GCCS, the mean temperatures of the gradient solutions were measured at the beginning and end of centrifugation were found to be 7 +/-0.7 C and 6.8 +/-0.6 C respectively. For the UICCS, the mean temperature of the gradients at the beginning and end of centrifugation were 4.7 +/-0.53 C and 7.03 C+/-0.91 C respectively. The presented COBE cooling systems can easily be adapted to a COBE 2991 cell-separator and are efficient in maintaining gradient solutions at a defined low temperature during centrifugation.

New procedure for epidermal cell isolation using kiwi fruit actinidin, and improved culture of melanocytes in the presence of leukaemia inhibitory factor and forskolin.

PubMed

Yarani, Reza; Mansouri, Kamran; Mohammadi-Motlagh, Hamid Reza; Bakhtiari, Mitra; Mostafaie, Ali

2013-06-01

Conventional isolation of epidermis from the dermis and disruption of epidermal sheets to liberate the cells, are performed using proteolytic enzymes such as thermolysin or collagenase. Selective population expansion of melanocytes is achieved by suppressing proliferation of keratinocytes and fibroblasts in epidermal cell suspensions, using phorbol esters and cholera toxin. Here, we introduce a new procedure for isolation of epidermal cells, using proteolytic activity of kiwi fruit actinidin, and also an improved growth medium for melanocytes in the presence of leukaemia inhibitory factor (LIF) and forskolin. Dermo-epidermal separation and epidermal sheet cell dispersion were performed using actinidin compared to conventional proteases including collagenase, thermolysin or trypsin. Thereafter, melanocyte culture was performed in two common media and one modified medium to discover optimization for these cells. We found that dermo-epidermal separation and epidermal sheet cell dispersion using kiwi fruit actinidin were considerably better than previously used methods, both from the aspect of less fibroblast and keratinocyte contamination, and of more viable native cells. Also, melanocytes proliferated better in phorbol ester- and cholera toxin-free proliferation medium supplemented with LIF and forskolin. Less contamination and higher numbers of viable cells were actinidin preferential for separation of epidermis and isolation of epidermal cells. Supplementation of LIF and forskolin to new medium increased proliferation potential of melanocytes in comparison to exogenous mitogens. © 2013 Blackwell Publishing Ltd.

From Molecules to Cells to Organisms: Understanding Health and Disease with Multidimensional Single-Cell Methods

NASA Astrophysics Data System (ADS)

Candia, Julián

2013-03-01

The multidimensional nature of many single-cell measurements (e.g. multiple markers measured simultaneously using Fluorescence-Activated Cell Sorting (FACS) technologies) offers unprecedented opportunities to unravel emergent phenomena that are governed by the cooperative action of multiple elements across different scales, from molecules and proteins to cells and organisms. We will discuss an integrated analysis framework to investigate multicolor FACS data from different perspectives: Singular Value Decomposition to achieve an effective dimensional reduction in the data representation, machine learning techniques to separate different patient classes and improve diagnosis, as well as a novel cell-similarity network analysis method to identify cell subpopulations in an unbiased manner. Besides FACS data, this framework is versatile: in this vein, we will demonstrate an application to the multidimensional single-cell shape analysis of healthy and prematurely aged cells.

Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP).

PubMed

Moon, Hui-Sung; Kwon, Kiho; Kim, Seung-Il; Han, Hyunju; Sohn, Joohyuk; Lee, Soohyeon; Jung, Hyo-Il

2011-03-21

Circulating tumor cells (CTCs) are highly correlated with the invasive behavior of cancer, so their isolations and quantifications are important for biomedical applications such as cancer prognosis and measuring the responses to drug treatments. In this paper, we present the development of a microfluidic device for the separation of CTCs from blood cells based on the physical properties of cells. For use as a CTC model, we successfully separated human breast cancer cells (MCF-7) from a spiked blood cell sample by combining multi-orifice flow fractionation (MOFF) and dielectrophoretic (DEP) cell separation technique. Hydrodynamic separation takes advantage of the massive and high-throughput filtration of blood cells as it can accommodate a very high flow rate. DEP separation plays a role in precise post-processing to enhance the efficiency of the separation. The serial combination of these two different sorting techniques enabled high-speed continuous flow-through separation without labeling. We observed up to a 162-fold increase in MCF-7 cells at a 126 µL min(-1) flow rate. Red and white blood cells were efficiently removed with separation efficiencies of 99.24% and 94.23% respectively. Therefore, we suggest that our system could be used for separation and detection of CTCs from blood cells for biomedical applications. This journal is © The Royal Society of Chemistry 2011

A cell-vertex multigrid method for the Navier-Stokes equations

NASA Technical Reports Server (NTRS)

Radespiel, R.

1989-01-01

A cell-vertex scheme for the Navier-Stokes equations, which is based on central difference approximations and Runge-Kutta time stepping, is described. Using local time stepping, implicit residual smoothing, a multigrid method, and carefully controlled artificial dissipative terms, very good convergence rates are obtained for a wide range of two- and three-dimensional flows over airfoils and wings. The accuracy of the code is examined by grid refinement studies and comparison with experimental data. For an accurate prediction of turbulent flows with strong separations, a modified version of the nonequilibrium turbulence model of Johnson and King is introduced, which is well suited for an implementation into three-dimensional Navier-Stokes codes. It is shown that the solutions for three-dimensional flows with strong separations can be dramatically improved, when a nonequilibrium model of turbulence is used.

Rapid and continuous magnetic separation in droplet microfluidic devices

DOE PAGES

Brouzes, Eric; Kruse, Travis; Kimmerling, Robert; ...

2014-12-03

Here, we present a droplet microfluidic method to extract molecules of interest from a droplet in a rapid and continuous fashion. We accomplish this by first marginalizing functionalized super-paramagnetic beads within the droplet using a magnetic field, and then splitting the droplet into one droplet containing the majority of magnetic beads and one droplet containing the minority fraction. We quantitatively analysed the factors which affect the efficiency of marginalization and droplet splitting to optimize the enrichment of magnetic beads. We first characterized the interplay between the droplet velocity and the strength of the magnetic field and its effect on marginalization.more » We found that marginalization is optimal at the midline of the magnet and that marginalization is a good predictor of bead enrichment through splitting at low to moderate droplet velocities. Finally, we focused our efforts on manipulating the splitting profile to improve the enrichment provided by asymmetric splitting. We designed asymmetric splitting forks that employ capillary effects to preferentially extract the bead-rich regions of the droplets. Our strategy represents a framework to optimize magnetic bead enrichment methods tailored to the requirements of specific droplet-based applications. We anticipate that our separation technology is well suited for applications in single-cell genomics and proteomics. In particular, our method could be used to separate mRNA bound to poly-dT functionalized magnetic microparticles from single cell lysates to prepare single-cell cDNA libraries.« less

Device and method for automated separation of a sample of whole blood into aliquots

DOEpatents

Burtis, Carl A.; Johnson, Wayne F.

1989-01-01

A device and a method for automated processing and separation of an unmeasured sample of whole blood into multiple aliquots of plasma. Capillaries are radially oriented on a rotor, with the rotor defining a sample chamber, transfer channels, overflow chamber, overflow channel, vent channel, cell chambers, and processing chambers. A sample of whole blood is placed in the sample chamber, and when the rotor is rotated, the blood moves outward through the transfer channels to the processing chambers where the blood is centrifugally separated into a solid cellular component and a liquid plasma component. When the rotor speed is decreased, the plasma component backfills the capillaries resulting in uniform aliquots of plasma which may be used for subsequent analytical procedures.

Novel platform for minimizing cell loss on separation process: Droplet-based magnetically activated cell separator

NASA Astrophysics Data System (ADS)

Kim, Youngho; Hong, Su; Lee, Sang Ho; Lee, Kangsun; Yun, Seok; Kang, Yuri; Paek, Kyeong-Kap; Ju, Byeong-Kwon; Kim, Byungkyu

2007-07-01

To reduce the problem of cell loss due to adhesion, one of the basic phenomena in microchannel, we proposed the droplet-based magnetically activated cell separator (DMACS). Based on the platform of the DMACS—which consists of permanent magnets, a coverslip with a circle-shaped boundary, and an injection tube—we could collect magnetically (CD45)-labeled (positive) cells with high purity and minimize cell loss due to adhesion. To compare separation efficiency between the MACS and the DMACS, the total number of cells before and after separation with both the separators was counted by flow cytometry. We could find that the number (3241/59940) of cells lost in the DMACS is much less than that (22360/59940) in the MACS while the efficiency of cell separation in the DMACS (96.07%) is almost the same as that in the MACS (96.72%). Practically, with fluorescent images, it was visually confirmed that the statistical data are reliable. From the viability test by using Hoechst 33 342, it was also demonstrated that there was no cell damage on a gas-liquid interface. Conclusively, DMACS will be a powerful tool to separate rare cells and applicable as a separator, key component of lab-on-a-chip.

Capillary zone electrophoresis method for a highly glycosylated and sialylated recombinant protein: development, characterization and application for process development.

PubMed

Zhang, Le; Lawson, Ken; Yeung, Bernice; Wypych, Jette

2015-01-06

A purity method based on capillary zone electrophoresis (CZE) has been developed for the separation of isoforms of a highly glycosylated protein. The separation was found to be driven by the number of sialic acids attached to each isoform. The method has been characterized using orthogonal assays and shown to have excellent specificity, precision and accuracy. We have demonstrated the CZE method is a useful in-process assay to support cell culture and purification development of this glycoprotein. Compared to isoelectric focusing (IEF), the CZE method provides more quantitative results and higher sample throughput with excellent accuracy, qualities that are required for process development. In addition, the CZE method has been applied in the stability testing of purified glycoprotein samples.

Method for identifying mutagenic agents which induce large, multilocus deletions in DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradley, W.E.C.; Belouchi, A.; Dewyse, P.

1993-07-13

A method of identifying a mutagenic agent is described which includes a large, multilocus deletions in DNA in mammalian cells comprising: (i) exposing a class III heterozygous CHO cell line to a potential mutagenic agent under investigation, and allowing any mutation of the cell line to proceed, said cell line being characterized in that a restriction fragment length variation exists in on mutation it becomes resistant to 2,6-diaminopurine and in that the DNA sequence adjacent to the two alleles of the APRT gene such that the DNA sequence adjacent to one of the two alleles can be digested with themore » enzyme BclI but the DNA sequence variation adjacent to the other of the two alleles cannot be digested with BclI, (ii) isolating induced mutations of the cell line deficient in APRT function, (iii) isolating DNA from the induced mutants, (iv) digesting the isolated DNA with BclI enzyme to produce digested fragments including a 19 kb fragment and any 2 kb fragment, which fragments hybridize with the labeled probe derived from DNA fragment PDI, (v) separating any digested fragments, (vi) transferring the separated fragments of (v) to a solid support, (vii) hybridizing the supported separated fragments with a labeled probe derived from the clone DNA fragment PD 1, (viii) determining fragments having undergone loss of the 2 kb band identified by the probe, as an identification of parent mutants in which the loss occurred, and (ix) evaluating the mutating ability of the potential mutagenic agent.« less

Separation of cells from the rat anterior pituitary gland

NASA Technical Reports Server (NTRS)

Hymer, Wesley C.; Hatfield, J. Michael

1983-01-01

Various techniques for separating the hormone-producing cell types from the rat anterior pituitary gland are examined. The purity, viability, and responsiveness of the separated cells depend on the physiological state of the donor, the tissue dissociation procedures, the staining technique used for identification of cell type, and the cell separation technique. The chamber-gradient setup and operation, the characteristics of the gradient materials, and the separated cell analysis of velocity sedimentation techniques (in particular Staput and Celsep) are described. Consideration is given to the various types of materials used in density gradient centrifugation and the operation of a gradient generating device. The use of electrophoresis to separate rat pituitary cells is discussed.

Two-dimensional single-cell patterning with one cell per well driven by surface acoustic waves

PubMed Central

Collins, David J.; Morahan, Belinda; Garcia-Bustos, Jose; Doerig, Christian; Plebanski, Magdalena; Neild, Adrian

2015-01-01

In single-cell analysis, cellular activity and parameters are assayed on an individual, rather than population-average basis. Essential to observing the activity of these cells over time is the ability to trap, pattern and retain them, for which previous single-cell-patterning work has principally made use of mechanical methods. While successful as a long-term cell-patterning strategy, these devices remain essentially single use. Here we introduce a new method for the patterning of multiple spatially separated single particles and cells using high-frequency acoustic fields with one cell per acoustic well. We characterize and demonstrate patterning for both a range of particle sizes and the capture and patterning of cells, including human lymphocytes and red blood cells infected by the malarial parasite Plasmodium falciparum. This ability is made possible by a hitherto unexplored regime where the acoustic wavelength is on the same order as the cell dimensions. PMID:26522429

Microfluidic DNA sample preparation method and device

DOEpatents

Krulevitch, Peter A.; Miles, Robin R.; Wang, Xiao-Bo; Mariella, Raymond P.; Gascoyne, Peter R. C.; Balch, Joseph W.

2002-01-01

Manipulation of DNA molecules in solution has become an essential aspect of genetic analyses used for biomedical assays, the identification of hazardous bacterial agents, and in decoding the human genome. Currently, most of the steps involved in preparing a DNA sample for analysis are performed manually and are time, labor, and equipment intensive. These steps include extraction of the DNA from spores or cells, separation of the DNA from other particles and molecules in the solution (e.g. dust, smoke, cell/spore debris, and proteins), and separation of the DNA itself into strands of specific lengths. Dielectrophoresis (DEP), a phenomenon whereby polarizable particles move in response to a gradient in electric field, can be used to manipulate and separate DNA in an automated fashion, considerably reducing the time and expense involved in DNA analyses, as well as allowing for the miniaturization of DNA analysis instruments. These applications include direct transport of DNA, trapping of DNA to allow for its separation from other particles or molecules in the solution, and the separation of DNA into strands of varying lengths.

Novel microfluidic device for the continuous separation of cancer cells using dielectrophoresis.

PubMed

Alazzam, Anas; Mathew, Bobby; Alhammadi, Falah

2017-03-01

We describe the design, microfabrication, and testing of a microfluidic device for the separation of cancer cells based on dielectrophoresis. Cancer cells, specifically green fluorescent protein-labeled MDA-MB-231, are successfully separated from a heterogeneous mixture of the same and normal blood cells. MDA-MB-231 cancer cells are separated with an accuracy that enables precise detection and counting of circulating tumor cells present among normal blood cells. The separation is performed using a set of planar interdigitated transducer electrodes that are deposited on the surface of a glass wafer and slightly protrude into the separation microchannel at one side. The device includes two parts, namely, a glass wafer and polydimethylsiloxane element. The device is fabricated using standard microfabrication techniques. All experiments are conducted with low conductivity sucrose-dextrose isotonic medium. The variation in response between MDA-MB-231 cancer cells and normal cells to a certain band of alternating-current frequencies is used for continuous separation of cells. The fabrication of the microfluidic device, preparation of cells and medium, and flow conditions are detailed. The proposed microdevice can be used to detect and separate malignant cells from heterogeneous mixture of cells for the purpose of early screening for cancer. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Selective Label-free Electrokinetic Cell Tracker (SELECT): a novel liquid platform for cell characterization

NASA Astrophysics Data System (ADS)

Taruvai Kalyana Kumar, Rajeshwari; de Mello Gindri, Izabelle; Kinnamon, David; Kanchustambham, Pradyotha; Rodrigues, Danieli; Prasad, Shalini; BiomaterialsOsseointegration; Novel Engineering Lab Collaboration

2015-03-01

Characterization and analysis of rare cells provide critical cues for early diagnosis of diseases. Electrokinetic cell separation has been previously established to have greater efficiency when compared to traditional flow cytometry methods. It has been shown by many researchers that buffer solutions in which cells are suspended in, have enormous effects on producing required dielectrophoretic (DEP) forces to characterize cells. Most commonly used suspension buffers used are deionized water and cell media. However, these solutions exhibit high level of intrinsic noise, which greatly masks the electrokinetic signals from cells under study. Ionic liquids (ILs) show promise towards the creation of conductive fluids with required electrical properties. The goal of this project is to design and test ILs for enhancing DEP forces on cells while creating an environment for preserving their integrity. We analyzed two methylimidazolium based ILs as suspension medium for cell separation. These dicationic ILs possess slight electrical and structural differences with high thermal stability. The two ILs were tested for cytotoxicity using HeLa and bone cells. The effects of electrical neutrality, free charge screening due to ILs towards enhanced electrokinetic signals from cells were studied with improved system resolution and no harmful effects.

Rapid separation on copper powder of total mercury in blood and determination of mercury by flameless atomic absorption spectrometry.

PubMed

Dogan, S; Haerdi, W

1979-01-01

The determination of mercury in blood by flameless atomic absorption spectrometry (FAAS) has been described. Prior to its analysis, the sample was decomposed by combustion and separated on a copper powder micro-column. A special type of cell has been used which gives a better sensitivity compared with the types of cells described in the literature and the method of FAAS analysis has been improved. The sensitivity of 0.1 ng for 1% absorbance was observed and the standard deviation for six determinations at this level was found to be +/- 0.05 ng, for 95% probability.

Methods of cell purification: a critical juncture for laboratory research and translational science.

PubMed

Amos, Peter J; Cagavi Bozkulak, Esra; Qyang, Yibing

2012-01-01

Research in cell biology and the development of translational technologies are driven by competition, public expectations, and regulatory oversight, putting these fields at a critical juncture. Success in these fields is quickly becoming dependent on the ability of researchers to identify and isolate specific cell populations from heterogeneous mixtures accurately and efficiently. Many methods for cell purification have been developed, and each has advantages and disadvantages that must be considered in light of the intended application. Current cell separation strategies make use of surface proteins, genetic expression, and physics to isolate specific cells by phenotypic traits. Cell purification is also dependent on the cellular reagents available for use and the intended application, as these factors may preclude certain mechanisms used in the processes of labeling and sorting cells. Copyright © 2011 S. Karger AG, Basel.

Recycling of CdTe photovoltaic waste

DOEpatents

Goozner, Robert E.; Long, Mark O.; Drinkard, Jr., William F.

1999-01-01

A method for extracting and reclaiming metals from scrap CdTe photovoltaic cells and manufacturing waste by leaching the waste with a leaching solution comprising nitric acid and water, skimming any plastic material from the top of the leaching solution, separating the glass substrate from the liquid leachate and electrolyzing the leachate to separate Cd from Te, wherein the Te is deposits onto a cathode while the Cd remains in solution.

ELECTROCHEMICAL DECONTAMINATION AND RECOVERY OF URANIUM VALUES

DOEpatents

McLaren, J.A.; Goode, J.H.

1958-05-13

An electrochemical process is described for separating uranium from fission products. The method comprises subjecting the mass of uranium to anodic dissolution in an electrolytic cell containing aqueous alkali bicarbonate solution as its electrolyte, thereby promoting a settling from the solution of a solid sludge from about the electrodes and separating the resulting electrolyte solution containing the anodically dissolved uranium from the sludge which contains the rare earth fission products.

Method and apparatus for rebalancing a redox flow cell system

NASA Technical Reports Server (NTRS)

Gahn, Randall F. (Inventor)

1986-01-01

A rebalance cell is provided for a REDOX electrochemical system of the type having anode and cathode fluids which are aqueous HCl solutions with two metal species in each. The rebalance cell has a cathode compartment and a chlorine compartment separated by an ion permeable membrane. By applying an electrical potential to the rebalance cell while circulating cathode fluid through the cathode compartment and while circulating an identical fluid through the chlorine compartment, any significant imbalance of the REDOX system is prevented.

Method and apparatus for rebalancing a REDOX flow cell system

NASA Technical Reports Server (NTRS)

Gahn, R. F. (Inventor)

1985-01-01

A rebalance cell is provided for a REDOX electrochemical system of the type with anode and cathode fluids which are aqueous HC1 solutions with two metal species in each. The rebalance cell has a cathode compartment and a chlorine compartment separated by an ion permeable membrane. By applying an electrical potential to the rebalance cell while circulating cathode fluid through the cathode compartment and while circulating an identical fluid through the chlorine compartment, any significant imbalance of the REDOX system is prevented.

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. Furthermore, this platform has great potential in both medical diagnostics and researchmore » applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.« less

A Centrifugal Microfluidic Platform That Separates Whole Blood Samples into Multiple Removable Fractions Due to Several Discrete but Continuous Density Gradient Sections

DOE PAGES

Moen, Scott T.; Hatcher, Christopher L.; Singh, Anup K.

2016-04-07

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. Furthermore, this platform has great potential in both medical diagnostics and researchmore » applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.« less

Cs-Ba separation using N 2O as a reactant gas in a Multiple Collector-Inductively Coupled Plasma Mass Spectrometer collision-reaction cell: Application to the measurements of Cs isotopes in spent nuclear fuel samples

NASA Astrophysics Data System (ADS)

Granet, M.; Nonell, A.; Favre, G.; Chartier, F.; Isnard, H.; Moureau, J.; Caussignac, C.; Tran, B.

2008-11-01

In the general frameworks of the nuclear fuel cycle and environmental research field, the Cs isotopic composition must be known with high precision and accuracy. The direct determination of Cs isotopes by mass spectrometry techniques is generally hampered by the presence of Ba isobaric interferences however. Here we present a new method which takes advantage of the collision-reaction cell based Multiple Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICPMS) and allows to analyse Cs isotopes in the presence of Ba without prior separation step. The addition of N 2O gas in the cell leads to an antagonistic behavior of Cs + and Ba + as the latter reacts with the gas to form BaO + and BaOH + products whereas Cs + remains unreactive. The efficiency of the method was demonstrated for an UOx sample by comparing the results obtained (1) from the measurements of pure Cs fractions and (2) from Fission Products fractions containing more than 30 ionisable elements in addition to Cs, Ba, and where U and Pu were previously removed by using ion exchange resin. An excellent agreement is achieved between each set of experiments with an external reproducibility always better than 0.5% (RSD, k = 2). This study confirms the strong potential of collision-reaction cell to measure Cs isotopes in presence of interfering Ba, precluding therefore former systematic chemical separations.

Multi-layer coatings for bipolar rechargeable batteries with enhanced terminal voltage

DOEpatents

Farmer, Joseph C.; Kaschmitter, James; Pierce, Steve

2017-06-06

A method for producing a multi-layer bipolar coated cell according to one embodiment includes applying a first active cathode material above a substrate to form a first cathode; applying a first solid-phase ionically-conductive electrolyte material above the first cathode to form a first electrode separation layer; applying a first active anode material above the first electrode separation layer to form a first anode; applying an electrically conductive barrier layer above the first anode; applying a second active cathode material above the anode material to form a second cathode; applying a second solid-phase ionically-conductive electrolyte material above the second cathode to form a second electrode separation layer; applying a second active anode material above the second electrode separation layer to form a second anode; and applying a metal material above the second anode to form a metal coating section. In another embodiment, the anode is formed prior to the cathode. Cells are also disclosed.

Droplet microfluidics with magnetic beads: a new tool to investigate drug-protein interactions.

PubMed

Lombardi, Dario; Dittrich, Petra S

2011-01-01

In this study, we give the proof of concept for a method to determine binding constants of compounds in solution. By implementing a technique based on magnetic beads with a microfluidic device for segmented flow generation, we demonstrate, for individual droplets, fast, robust and complete separation of the magnetic beads. The beads are used as a carrier for one binding partner and hence, any bound molecule is separated likewise, while the segmentation into small microdroplets ensures fast mixing, and opens future prospects for droplet-wise analysis of drug candidate libraries. We employ the method for characterization of drug-protein binding, here warfarin to human serum albumin. The approach lays the basis for a microfluidic droplet-based screening device aimed at investigating the interactions of drugs with specific targets including enzymes and cells. Furthermore, the continuous method could be employed for various applications, such as binding assays, kinetic studies, and single cell analysis, in which rapid removal of a reactive component is required.

Cytologic separation of branchial cleft cyst from metastatic cystic squamous cell carcinoma: A multivariate analysis of nineteen cytomorphologic features.

PubMed

Layfield, Lester J; Esebua, Magda; Schmidt, Robert L

2016-07-01

The separation of branchial cleft cysts from metastatic cystic squamous cell carcinomas in adults can be clinically and cytologically challenging. Diagnostic accuracy for separation is reported to be as low as 75% prompting some authors to recommend frozen section evaluation of suspected branchial cleft cysts before resection. We evaluated 19 cytologic features to determine which were useful in this distinction. Thirty-three cases (21 squamous carcinoma and 12 branchial cysts) of histologically confirmed cystic lesions of the lateral neck were graded for the presence or absence of 19 cytologic features by two cytopathologists. The cytologic features were analyzed for agreement between observers and underwent multivariate analysis for correlation with the diagnosis of carcinoma. Interobserver agreement was greatest for increased nuclear/cytoplasmic (N/C) ratio, pyknotic nuclei, and irregular nuclear membranes. Recursive partitioning analysis showed increased N/C ratio, small clusters of cells, and irregular nuclear membranes were the best discriminators. The distinction of branchial cleft cysts from cystic squamous cell carcinoma is cytologically difficult. Both digital image analysis and p16 testing have been suggested as aids in this separation, but analysis of cytologic features remains the main method for diagnosis. In an analysis of 19 cytologic features, we found that high nuclear cytoplasmic ratio, irregular nuclear membranes, and small cell clusters were most helpful in their distinction. Diagn. Cytopathol. 2016;44:561-567. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

SU-E-T-398: Evaluation of Radiobiological Parameters Using Serial Tumor Imaging During Radiotherapy as An Inverse Ill-Posed Problem

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chvetsov, A; Sandison, G; Schwartz, J

Purpose: Combination of serial tumor imaging with radiobiological modeling can provide more accurate information on the nature of treatment response and what underlies resistance. The purpose of this article is to improve the algorithms related to imaging-based radiobilogical modeling of tumor response. Methods: Serial imaging of tumor response to radiation therapy represents a sum of tumor cell sensitivity, tumor growth rates, and the rate of cell loss which are not separated explicitly. Accurate treatment response assessment would require separation of these radiobiological determinants of treatment response because they define tumor control probability. We show that the problem of reconstruction ofmore » radiobiological parameters from serial imaging data can be considered as inverse ill-posed problem described by the Fredholm integral equation of the first kind because it is governed by a sum of several exponential processes. Therefore, the parameter reconstruction can be solved using regularization methods. Results: To study the reconstruction problem, we used a set of serial CT imaging data for the head and neck cancer and a two-level cell population model of tumor response which separates the entire tumor cell population in two subpopulations of viable and lethally damage cells. The reconstruction was done using a least squared objective function and a simulated annealing algorithm. Using in vitro data for radiobiological parameters as reference data, we shown that the reconstructed values of cell surviving fractions and potential doubling time exhibit non-physical fluctuations if no stabilization algorithms are applied. The variational regularization allowed us to obtain statistical distribution for cell surviving fractions and cell number doubling times comparable to in vitro data. Conclusion: Our results indicate that using variational regularization can increase the number of free parameters in the model and open the way to development of more advanced algorithms which take into account tumor heterogeneity, for example, related to hypoxia.« less

Thermal design of a Mars oxygen production plant

NASA Technical Reports Server (NTRS)

Sridhar, K. R.; Iyer, Venkatesh A.

1991-01-01

The optimal design of the thermal components of a system that uses carbon dioxide from the Martian atmosphere to produce oxygen for spacecraft propulsion and/or life support is discussed. The gases are pressurized, heated and passed through an electrochemical cell. Carbon dioxide is reduced to carbon monoxide and oxygen due to thermal dissociation and electrocatalysis. The oxygen thus formed is separated from the gas mixture by the electrochemical cell. The objective of the design is to optimize both the overall mass and the power consumption of the system. The analysis shows that at electrochemical cell efficiencies of about 50 percent and lower, the optimal system would require unspent carbon dioxide in the exhaust gases to be separated and recycled. Various methods of efficiently compressing the intake gases to system pressures of 0.1 MPa are investigated. The total power requirement for oxygen production rates of 1, 5, and 10 kg/day at various cell efficiencies are presented.

Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii.

PubMed

Everson, William V; Ware, Michael W; Dubey, J P; Lindquist, H D Alan

2002-01-01

Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.

Estimating error rates for firearm evidence identifications in forensic science

PubMed Central

Song, John; Vorburger, Theodore V.; Chu, Wei; Yen, James; Soons, Johannes A.; Ott, Daniel B.; Zhang, Nien Fan

2018-01-01

Estimating error rates for firearm evidence identification is a fundamental challenge in forensic science. This paper describes the recently developed congruent matching cells (CMC) method for image comparisons, its application to firearm evidence identification, and its usage and initial tests for error rate estimation. The CMC method divides compared topography images into correlation cells. Four identification parameters are defined for quantifying both the topography similarity of the correlated cell pairs and the pattern congruency of the registered cell locations. A declared match requires a significant number of CMCs, i.e., cell pairs that meet all similarity and congruency requirements. Initial testing on breech face impressions of a set of 40 cartridge cases fired with consecutively manufactured pistol slides showed wide separation between the distributions of CMC numbers observed for known matching and known non-matching image pairs. Another test on 95 cartridge cases from a different set of slides manufactured by the same process also yielded widely separated distributions. The test results were used to develop two statistical models for the probability mass function of CMC correlation scores. The models were applied to develop a framework for estimating cumulative false positive and false negative error rates and individual error rates of declared matches and non-matches for this population of breech face impressions. The prospect for applying the models to large populations and realistic case work is also discussed. The CMC method can provide a statistical foundation for estimating error rates in firearm evidence identifications, thus emulating methods used for forensic identification of DNA evidence. PMID:29331680

Estimating error rates for firearm evidence identifications in forensic science.

PubMed

Song, John; Vorburger, Theodore V; Chu, Wei; Yen, James; Soons, Johannes A; Ott, Daniel B; Zhang, Nien Fan

2018-03-01

Estimating error rates for firearm evidence identification is a fundamental challenge in forensic science. This paper describes the recently developed congruent matching cells (CMC) method for image comparisons, its application to firearm evidence identification, and its usage and initial tests for error rate estimation. The CMC method divides compared topography images into correlation cells. Four identification parameters are defined for quantifying both the topography similarity of the correlated cell pairs and the pattern congruency of the registered cell locations. A declared match requires a significant number of CMCs, i.e., cell pairs that meet all similarity and congruency requirements. Initial testing on breech face impressions of a set of 40 cartridge cases fired with consecutively manufactured pistol slides showed wide separation between the distributions of CMC numbers observed for known matching and known non-matching image pairs. Another test on 95 cartridge cases from a different set of slides manufactured by the same process also yielded widely separated distributions. The test results were used to develop two statistical models for the probability mass function of CMC correlation scores. The models were applied to develop a framework for estimating cumulative false positive and false negative error rates and individual error rates of declared matches and non-matches for this population of breech face impressions. The prospect for applying the models to large populations and realistic case work is also discussed. The CMC method can provide a statistical foundation for estimating error rates in firearm evidence identifications, thus emulating methods used for forensic identification of DNA evidence. Published by Elsevier B.V.

Evaluating the efficacy of DNA differential extraction methods for sexual assault evidence.

PubMed

Klein, Sonja B; Buoncristiani, Martin R

2017-07-01

Analysis of sexual assault evidence, often a mixture of spermatozoa and victim epithelial cells, represents a significant portion of a forensic DNA laboratory's case load. Successful genotyping of sperm DNA from these mixed cell samples, particularly with low amounts of sperm, depends on maximizing sperm DNA recovery and minimizing non-sperm DNA carryover. For evaluating the efficacy of the differential extraction, we present a method which uses a Separation Potential Ratio (SPRED) to consider both sperm DNA recovery and non-sperm DNA removal as variables for determining separation efficiency. In addition, we describe how the ratio of male-to-female DNA in the sperm fraction may be estimated by using the SPRED of the differential extraction method in conjunction with the estimated ratio of male-to-female DNA initially present on the mixed swab. This approach may be useful for evaluating or modifying differential extraction methods, as we demonstrate by comparing experimental results obtained from the traditional differential extraction and the Erase Sperm Isolation Kit (PTC © ) procedures. Copyright © 2017 Elsevier B.V. All rights reserved.

Production of alpha 1,3-galactosyltransferase gene-deficient pigs by somatic cell nuclear transfer: a novel selection method for gal alpha 1,3-Gal antigen-deficient cells.

PubMed

Fujimura, Tatsuya; Takahagi, Yoichi; Shigehisa, Tamotsu; Nagashima, Hiroshi; Miyagawa, Shuji; Shirakura, Ryota; Murakami, Hiroshi

2008-09-01

The objective of the present study was to isolate alpha 1,3-galactosyltransferase (GalGT)-gene double knockout (DKO) cells using a novel simple method of cell selection method. To obtain GalGT-DKO cells, GalGT-gene single knockout (SKO) fetal fibroblast cells were cultured for three to nine passages and GalGT-null cells were separated using a biotin-labeled IB4 lectin attached to streptavidin-coated magnetic beads. After 15-17 days of additional cultivation, seven GalGT-DKO cell colonies were obtained from a total of 2.5 x 10(7) GalGT-SKO cells. A total of 926 somatic nuclear transferred embryos reconstructed with the DKO cells were transferred into eight recipient pigs, producing four farrowed, three liveborns, and six stillborns. Absence of GalGT gene in the cloned pigs was confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that alphaGal antigens were not present in the cells of the cloned DKO pigs.

Label-free cancer cell separation from human whole blood using inertial microfluidics at low shear stress.

PubMed

Lee, Myung Gwon; Shin, Joong Ho; Bae, Chae Yun; Choi, Sungyoung; Park, Je-Kyun

2013-07-02

We report a contraction-expansion array (CEA) microchannel device that performs label-free high-throughput separation of cancer cells from whole blood at low Reynolds number (Re). The CEA microfluidic device utilizes hydrodynamic field effect for cancer cell separation, two kinds of inertial effects: (1) inertial lift force and (2) Dean flow, which results in label-free size-based separation with high throughput. To avoid cell damages potentially caused by high shear stress in conventional inertial separation techniques, the CEA microfluidic device isolates the cells with low operational Re, maintaining high-throughput separation, using nondiluted whole blood samples (hematocrit ~45%). We characterized inertial particle migration and investigated the migration of blood cells and various cancer cells (MCF-7, SK-BR-3, and HCC70) in the CEA microchannel. The separation of cancer cells from whole blood was demonstrated with a cancer cell recovery rate of 99.1%, a blood cell rejection ratio of 88.9%, and a throughput of 1.1 × 10(8) cells/min. In addition, the blood cell rejection ratio was further improved to 97.3% by a two-step filtration process with two devices connected in series.

Characterization of fission gas bubbles in irradiated U-10Mo fuel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Casella, Andrew M.; Burkes, Douglas E.; MacFarlan, Paul J.

2017-09-01

Irradiated U-10Mo fuel samples were prepared with traditional mechanical potting and polishing methods with in a hot cell. They were then removed and imaged with an SEM located outside of a hot cell. The images were then processed with basic imaging techniques from 3 separate software packages. The results were compared and a baseline method for characterization of fission gas bubbles in the samples is proposed. It is hoped that through adoption of or comparison to this baseline method that sample characterization can be somewhat standardized across the field of post irradiated examination of metal fuels.

Method for recovering catalytic elements from fuel cell membrane electrode assemblies

DOEpatents

Shore, Lawrence [Edison, NJ; Matlin, Ramail [Berkeley Heights, NJ; Heinz, Robert [Ludwigshafen, DE

2012-06-26

A method for recovering catalytic elements from a fuel cell membrane electrode assembly is provided. The method includes converting the membrane electrode assembly into a particulate material, wetting the particulate material, forming a slurry comprising the wetted particulate material and an acid leachate adapted to dissolve at least one of the catalytic elements into a soluble catalytic element salt, separating the slurry into a depleted particulate material and a supernatant containing the catalytic element salt, and washing the depleted particulate material to remove any catalytic element salt retained within pores in the depleted particulate material.

Further analyses of human kidney cell populations separated on the Space Shuttle

NASA Technical Reports Server (NTRS)

Stewart, Robin M.; Todd, Paul; Cole, Kenneth D.; Morrison, Dennis R.

1992-01-01

Cultured human embryonic kidney cells were separated into electrophoretic subpopulations in laboratory experiments and in two separation experiments on the STS-8 (Challenger) Space Shuttle flight using the mid-deck Continuous Flow Electrophoretic Separator (CFES). Populations of cells from each fraction were cultured for the lifetime of the cells, and supernatant medium was withdrawn and replaced at 4-day intervals. Withdrawn medium was frozen at -120 C for subsequent analysis. Enzyme assays, antibodies and gel electrophoresis were used as analytical tools for the detection and quantization of plasminogen activators in these samples. These assays of frozen-culture supernatant fluids confirmed the electrophoretic separation of plasminogen-activator-producing cells from nonproducing cells, the isolation of cells capable of sustained production, and the separation of cells that produce different plasminogen activators from one other.

Quality testing of an innovative cascade separation system for multiple cell separation

NASA Astrophysics Data System (ADS)

Pierzchalski, Arkadiusz; Moszczynska, Aleksandra; Albrecht, Bernd; Heinrich, Jan-Michael; Tarnok, Attila

2012-03-01

Isolation of different cell types from mixed samples in one separation step by FACS is feasible but expensive and slow. It is cheaper and faster but still challenging by magnetic separation. An innovative bead-based cascade-system (pluriSelect GmbH, Leipzig, Germany) relies on simultaneous physical separation of different cell types. It is based on antibody-mediated binding of cells to beads of different size and isolation with sieves of different mesh-size. We validated pluriSelect system for single parameter (CD3) and simultaneous separation of CD3 and CD15 cells from EDTA blood-samples. Results were compared with those obtained by MACS (Miltenyi-Biotech) magnetic separation (CD3 separation). pluriSelect separation was done in whole blood, MACS on Ficoll gradient isolated leukocytes, according to the manufacturer's protocols. Isolated and residual cells were immunophenotyped (7-color 8-antibody panel (CD3; CD16/56; CD4; CD8; CD14; CD19; CD45; HLADR) on a CyFlowML flow cytometer (Partec GmbH). Cell count (Coulter), purity, yield and viability (7-AAD exclusion) were determined. There were no significant differences between both systems regarding purity (92-98%), yield (50-60%) and viability (92-98%) of isolated cells. PluriSelect separation was slightly faster than MACS (1.15 h versus 1.5h). Moreover, no preenrichment steps were necessary. In conclusion, pluriSelect is a fast, simple and gentle system for efficient simultaneous separation of two cell subpopulation directly from whole blood and can provide a simple alternative to FACS. The isolated cells can be used for further research applications.

Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow

NASA Astrophysics Data System (ADS)

O'Brien, Christine M.; Rood, Kyle D.; Bhattacharyya, Kiran; DeSouza, Thiago; Sengupta, Shramik; Gupta, Sagar K.; Mosley, Jeffrey D.; Goldschmidt, Benjamin S.; Sharma, Nikhilesh; Viator, John A.

2012-06-01

Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are unable to detect early onset of metastatic disease. Patients must wait until macroscopic secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and travel through the blood or lymph system can provide data for diagnosing and monitoring metastatic disease. By irradiating enriched blood samples spiked with cultured melanoma cells with nanosecond duration laser light, we induced photoacoustic responses in the pigmented cells. Thus, we can detect and enumerate melanoma cells in blood samples to demonstrate a paradigm for a photoacoustic flow cytometer. Furthermore, we capture the melanoma cells using microfluidic two phase flow, a technique that separates a continuous flow into alternating microslugs of air and blood cell suspension. Each slug of blood cells is tested for the presence of melanoma. Slugs that are positive for melanoma, indicated by photoacoustic waves, are separated from the cytometer for further purification and isolation of the melanoma cell. In this paper, we evaluate the two phase photoacoustic flow cytometer for its ability to detect and capture metastastic melanoma cells in blood.

A multivariate approach using attenuated total reflectance mid-infrared spectroscopy to measure the surface mannoproteins and β-glucans of yeast cell walls during wine fermentations.

PubMed

Moore, John P; Zhang, Song-Lei; Nieuwoudt, Hélène; Divol, Benoit; Trygg, Johan; Bauer, Florian F

2015-11-18

Yeast cells possess a cell wall comprising primarily glycoproteins, mannans, and glucan polymers. Several yeast phenotypes relevant for fermentation, wine processing, and wine quality are correlated with cell wall properties. To investigate the effect of wine fermentation on cell wall composition, a study was performed using mid-infrared (MIR) spectroscopy coupled with multivariate methods (i.e., PCA and OPLS-DA). A total of 40 yeast strains were evaluated, including Saccharomyces strains (laboratory and industrial) and non-Saccharomyces species. Cells were fermented in both synthetic MS300 and Chardonnay grape must to stationery phase, processed, and scanned in the MIR spectrum. PCA of the fingerprint spectral region showed distinct separation of Saccharomyces strains from non-Saccharomyces species; furthermore, industrial wine yeast strains separated from laboratory strains. PCA loading plots and the use of OPLS-DA to the data sets suggested that industrial strains were enriched with cell wall proteins (e.g., mannoproteins), whereas laboratory strains were composed mainly of mannan and glucan polymers.

Elastomeric negative acoustic contrast particles for affinity capture assays.

PubMed

Cushing, Kevin W; Piyasena, Menake E; Carroll, Nick J; Maestas, Gian C; López, Beth Ann; Edwards, Bruce S; Graves, Steven W; López, Gabriel P

2013-02-19

This report describes the development of elastomeric capture microparticles (ECμPs) and their use with acoustophoretic separation to perform microparticle assays via flow cytometry.We have developed simple methods to form ECμPs by cross-linking droplets of common commercially available silicone precursors in suspension followed by surface functionalization with biomolecular recognition reagents. The ECμPs are compressible particles that exhibit negative acoustic contrast in ultrasound when suspended in aqueous media, blood serum, or diluted blood. In this study, these particles have been functionalized with antibodies to bind prostate specific antigen and immunoglobulin (IgG). Specific separation of the ECμPs from blood cells is achieved by flowing them through a microfluidic acoustophoretic device that uses an ultrasonic standing wave to align the blood cells, which exhibit positive acoustic contrast, at a node in the acoustic pressure distribution while aligning the negative acoustic contrast ECμPs at the antinodes. Laminar flow of the separated particles to downstream collection ports allows for collection of the separated negative contrast (ECμPs) and positive contrast particles (cells). Separated ECμPs were analyzed via flow cytometry to demonstrate nanomolar detection for prostate specific antigen in aqueous buffer and picomolar detection for IgG in plasma and diluted blood samples. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types.

Elastomeric Negative Acoustic Contrast Particles for Affinity Capture Assays

PubMed Central

Cushing, Kevin W.; Piyasena, Menake E.; Carroll, Nick J.; Maestas, Gian C.; López, Beth Ann; Edwards, Bruce S.; Graves, Steven W.; López, Gabriel P.

2013-01-01

This report describes the development of elastomeric capture microparticles (ECμPs) and their use with acoustophoretic separation to perform microparticle assays via flow cytometry. We have developed simple methods to form ECμPsby crosslinking droplets of common commercially available silicone precursors in suspension followed by surface functionalization with biomolecular recognition reagents. The ECμPs are compressible particles that exhibit negative acoustic contrast in ultrasound when suspended in aqueous media, blood serum or diluted blood. In this study, these particles have been functionalized with antibodies to bind prostate specific antigen and immunoglobulin (IgG). Specific separation of the ECμPs from blood cells is achieved by flowing them through a microfluidic acoustophoretic device that uses an ultrasonic standing wave to align the blood cells, which exhibit positive acoustic contrast, at a node in the acoustic pressure distribution while aligning the negative acoustic contrast ECμPs at the antinodes. Laminar flow of the separated particles to downstream collection ports allows for collection of the separated negative contrast (ECμPs) and positive contrast particles (cells). Separated ECμPs were analyzed via flow cytometry to demonstrate nanomolar detection for prostate specific antigen in aqueous buffer and picomolar detection for IgG in plasma and diluted blood samples. This approach has potential applications in the development of rapid assays that detect the presence of low concentrations of biomarkers in a number of biological sample types. PMID:23331264

Recycling of used perfluorosulfonic acid membranes

DOEpatents

Grot, Stephen [Middletown, DE; Grot, Walther [Chadds Ford, PA

2007-08-14

A method for recovering and recycling catalyst coated fuel cell membranes includes dissolving the used membranes in water and solvent, heating the dissolved membranes under pressure and separating the components. Active membranes are produced from the recycled materials.

Mechanical Behavior of Free-Standing Fuel Cell Electrodes on Water Surface.

PubMed

Kim, Sanwi; Kim, Jae-Han; Oh, Jong-Gil; Jang, Kyung-Lim; Jeong, Byeong-Heon; Hong, Bo Ki; Kim, Taek-Soo

2016-06-22

Fundamental understanding of the mechanical behavior of polymer electrolyte fuel cell electrodes as free-standing materials is essential to develop mechanically robust fuel cells. However, this has been a significant challenge due to critical difficulties, such as separating the pristine electrode from the substrate without damage and precisely measuring the mechanical properties of the very fragile and thin electrodes. We report the mechanical behavior of free-standing fuel cell electrodes on the water surface through adopting an innovative ice-assisted separation method to separate the electrode from decal transfer film. It is found that doubling the ionomer content in electrodes increases not only the tensile stress at the break and the Young's modulus (E) of the electrodes by approximately 2.1-3.5 and 1.7-2.4 times, respectively, but also the elongation at the break by approximately 1.5-1.7 times, which indicates that stronger, stiffer, and tougher electrodes are attained with increasing ionomer content, which have been of significant interest in materials research fields. The scaling law relationship between Young's modulus and density (ρ) has been unveiled as E ∼ ρ(1.6), and it is compared with other materials. These findings can be used to develop mechanically robust electrodes for fuel cell applications.

Stability of DNA Origami Nanoarrays in Cell Lysate

PubMed Central

Mei, Qian; Wei, Xixi; Su, Fengyu; Liu, Yan; Youngbull, Cody; Johnson, Roger; Lindsay, Stuart; Yan, Hao; Meldrum, Deirdre

2012-01-01

Scaffolded DNA origami, a method to create self-assembled nanostructures with spatially addressable features, has recently been used to develop water-soluble molecular chips for label-free RNA detection, platforms for deterministic protein positioning, and single molecule reaction observatories. These applications highlight the possibility of exploiting the unique properties and biocompatibility of DNA nanostructures in live, cellular systems. Herein, we assembled several DNA origami nanostructures of differing shape, size and probes, and investigated their interaction with lysate obtained from various normal and cancerous cell lines. We separated and analyzed the origami–lysate mixtures using agarose gel electrophoresis and recovered the DNA structures for functional assay and subsequent microscopic examination. Our results demonstrate that DNA origami nanostructures are stable in cell lysate and can be easily separated from lysate mixtures, in contrast to natural, single- and double-stranded DNA. Atomic force microscope (AFM) and transmission electron microscope (TEM) images show that the DNA origami structures are fully intact after separation from cell lysates and hybridize to their targets, verifying the superior structural integrity and functionality of self-assembled DNA origami nanostructures relative to conventional oligonucleotides. The stability and functionality of DNA origami structures in cell lysate validate their use for biological applications, for example, as programmable molecular rafts or disease detection platforms. PMID:21366226

Size and deformability based separation of circulating tumor cells from castrate resistant prostate cancer patients using resettable cell traps.

PubMed

Qin, Xi; Park, Sunyoung; Duffy, Simon P; Matthews, Kerryn; Ang, Richard R; Todenhöfer, Tilman; Abdi, Hamid; Azad, Arun; Bazov, Jenny; Chi, Kim N; Black, Peter C; Ma, Hongshen

2015-05-21

The enumeration and capture of circulating tumor cells (CTCs) are potentially of great clinical value as they offer a non-invasive means to access tumor materials to diagnose disease and monitor treatment efficacy. Conventional immunoenrichment of CTCs may fail to capture cells with low surface antigen expression. Micropore filtration presents a compelling label-free alternative that enriches CTCs using their biophysical rather than biochemical characteristics. However, this strategy is prone to clogging of the filter microstructure, which dramatically reduces the selectivity after processing large numbers of cells. Here, we use the resettable cell trap (RCT) mechanism to separate cells based on their size and deformability using an adjustable aperture that can be periodically cleared to prevent clogging. After separation, the output sample is stained and analyzed using multi-spectral analysis, which provides a more sensitive and unambiguous method to identify CTC biomarkers than traditional immunofluorescence. We tested the RCT device using blood samples obtained from 22 patients with metastatic castrate-resistant prostate cancer while comparing the results with the established CellSearch® system. The RCT mechanism was able to capture ≥5 CTCs in 18/22 (82%) patients with a mean count of 257 in 7.5 ml of whole blood, while the CellSearch system found ≥5 CTCs in 9/22 (41%) patients with a mean count of 25. The ~10× improvement in the CTC capture rate provides significantly more materials for subsequent analysis of these cells such as immunofluorescence, propagation by tissue culture, and genetic profiling.

Preparative electrophoresis of living lymphocytes

NASA Technical Reports Server (NTRS)

Vanoss, C. J.; Bigazzi, P. E.; Gillman, C. F.; Allen, R. E.

1974-01-01

Vertical liquid columns containing low molecular weight dextran density gradients can be used for preparative lymphocyte electrophoresis on earth, in simulation of 0 gravity conditions. Another method that has been tested at 1 G, is the electrophoresis of lymphocytes in a upward direction in vertical columns. By both methods up to 10 to the 7th power lymphocytes can be separated at one time in a 30 cm glass column of 8 mm inside diameter, at 12 v/cm, in 2 hours. Due to convection and sedimentation problems, the separation at 1 G is less than ideal, but it is expected that at 0 gravity electrophoresis will prove to be a uniquely powerful cell separation tool. The technical feasibility of electrophoresing inert particles at 0 G has been proven earlier, during the flight of Apollo 16.

High speed flow cytometric separation of viable cells

DOEpatents

Sasaki, D.T.; Van den Engh, G.J.; Buckie, A.M.

1995-11-14

Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

High speed flow cytometric separation of viable cells

DOEpatents

Sasaki, Dennis T.; Van den Engh, Gerrit J.; Buckie, Anne-Marie

1995-01-01

Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

Deterministic Migration-Based Separation of White Blood Cells.

PubMed

Kim, Byeongyeon; Choi, Young Joon; Seo, Hyekyung; Shin, Eui-Cheol; Choi, Sungyoung

2016-10-01

Functional and phenotypic analyses of peripheral white blood cells provide useful clinical information. However, separation of white blood cells from peripheral blood requires a time-consuming, inconvenient process and thus analyses of separated white blood cells are limited in clinical settings. To overcome this limitation, a microfluidic separation platform is developed to enable deterministic migration of white blood cells, directing the cells into designated positions according to a ridge pattern. The platform uses slant ridge structures on the channel top to induce the deterministic migration, which allows efficient and high-throughput separation of white blood cells from unprocessed whole blood. The extent of the deterministic migration under various rheological conditions is explored, enabling highly efficient migration of white blood cells in whole blood and achieving high-throughput separation of the cells (processing 1 mL of whole blood less than 7 min). In the separated cell population, the composition of lymphocyte subpopulations is well preserved, and T cells secrete cytokines without any functional impairment. On the basis of the results, this microfluidic platform is a promising tool for the rapid enrichment of white blood cells, and it is useful for functional and phenotypic analyses of peripheral white blood cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A miniaturized multipurpose platform for rapid, label-free, and simultaneous separation, patterning, and in vitro culture of primary and rare cells.

PubMed

Didar, Tohid Fatanat; Bowey, Kristen; Almazan, Guillermina; Tabrizian, Maryam

2014-02-01

Given that current cell isolation techniques are expensive, time consuming, yield low isolation purities, and/or alter target cell properties, a versatile, cost effective, and easy-to-operate microchip with the capability to simultaneously separate, capture, pattern, and culture rare and primary cells in vitro is developed. The platform is based on target cell adhesion onto the micro-fabricated interfaces produced by microcontact printing of cell-specific antibodies. Results show over 95% separation efficiency in less than 10 min for the separation of oligodendrocyte progenitor cells (OPCs) and cardiomyocytes from rat brain and heart mixtures, respectively. Target cell attachment and single cell spreading can be precisely controlled on the basis of the designed patterns. Both cell types can maintain their biofunctionality. Indeed, isolated OPCs can proliferate and differentiate into mature oligodendrocytes, while isolated cardiomyocytes retain their contractile properties on the separation platform. Successful separation of two dissimilar cell types present in varying concentrations in their respective cell mixtures and the demonstration of their integrity after separation open new avenues for time and cost-effective sorting of various cell types using the developed miniaturized platform. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molten carbonate fuel cell separator

DOEpatents

Nickols, Richard C.

1986-09-02

In a stacked array of molten carbonate fuel cells, a fuel cell separator is positioned between adjacent fuel cells to provide isolation as well as a conductive path therebetween. The center portion of the fuel cell separator includes a generally rectangular, flat, electrical conductor. Around the periphery of the flat portion of the separator are positioned a plurality of elongated resilient flanges which form a gas-tight seal around the edges of the fuel cell. With one elongated flange resiliently engaging a respective edge of the center portion of the separator, the sealing flanges, which are preferably comprised of a noncorrosive material such as an alloy of yttrium, iron, aluminum or chromium, form a tight-fitting wet seal for confining the corrosive elements of the fuel cell therein. This arrangement permits a good conductive material which may be highly subject to corrosion and dissolution to be used in combination with a corrosion-resistant material in the fuel cell separator of a molten carbonate fuel cell for improved fuel cell conductivity and a gas-tight wet seal.

Molten carbonate fuel cell separator

DOEpatents

Nickols, R.C.

1984-10-17

In a stacked array of molten carbonate fuel cells, a fuel cell separator is positioned between adjacent fuel cells to provide isolation as well as a conductive path therebetween. The center portion of the fuel cell separator includes a generally rectangular, flat, electrical conductor. Around the periphery of the flat portion of the separator are positioned a plurality of elongated resilient flanges which form a gas-tight seal around the edges of the fuel cell. With one elongated flange resiliently engaging a respective edge of the center portion of the separator, the sealing flanges, which are preferably comprised of a noncorrosive material such as an alloy of yttrium, iron, aluminum or chromium, form a tight-fitting wet seal for confining the corrosive elements of the fuel cell therein. This arrangement permits a good conductive material which may be highly subject to corrosion and dissolution to be used in combination with a corrosion-resistant material in the fuel cell separator of a molten carbonate fuel cell for improved fuel cell conductivity and a gas-tight wet seal.

Separation of CHO cells using hydrocyclones.

PubMed

Pinto, Rodrigo C V; Medronho, Ricardo A; Castilho, Leda R

2008-01-01

Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.

Cell separator for use in bipolar-stack energy storage devices

DOEpatents

Mayer, Steven T.; Feikert, John H.; Kachmitter, James L.; Pekala, Richard W.

1995-01-01

An improved multi-cell electrochemical energy storage device, such as a battery, fuel cell, or double layer capacitor using a cell separator which allows cells to be stacked and interconnected with low electrical resistance and high reliability while maximizing packaging efficiency. By adding repeating cells, higher voltages can be obtained. The cell separator is formed by applying an organic adhesive on opposing surfaces of adjacent carbon electrodes or surfaces of aerogel electrodes of a pair of adjacent cells prior to or after pyrolysis thereof to form carbon aerogel electrodes. The cell separator is electronically conductive, but ionically isolating, preventing an electrolytic conduction path between adjacent cells in the stack.

PCR-based detection of a rare linear DNA in cell culture.

PubMed

Saveliev, Sergei V.

2002-11-11

The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 10(7) or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.

PCR-based detection of a rare linear DNA in cell culture

PubMed Central

2002-01-01

The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials. PMID:12734566

A Fast, Automatic Segmentation Algorithm for Locating and Delineating Touching Cell Boundaries in Imaged Histopathology

PubMed Central

Qi, Xin; Xing, Fuyong; Foran, David J.; Yang, Lin

2013-01-01

Summary Background Automated analysis of imaged histopathology specimens could potentially provide support for improved reliability in detection and classification in a range of investigative and clinical cancer applications. Automated segmentation of cells in the digitized tissue microarray (TMA) is often the prerequisite for quantitative analysis. However overlapping cells usually bring significant challenges for traditional segmentation algorithms. Objectives In this paper, we propose a novel, automatic algorithm to separate overlapping cells in stained histology specimens acquired using bright-field RGB imaging. Methods It starts by systematically identifying salient regions of interest throughout the image based upon their underlying visual content. The segmentation algorithm subsequently performs a quick, voting based seed detection. Finally, the contour of each cell is obtained using a repulsive level set deformable model using the seeds generated in the previous step. We compared the experimental results with the most current literature, and the pixel wise accuracy between human experts' annotation and those generated using the automatic segmentation algorithm. Results The method is tested with 100 image patches which contain more than 1000 overlapping cells. The overall precision and recall of the developed algorithm is 90% and 78%, respectively. We also implement the algorithm on GPU. The parallel implementation is 22 times faster than its C/C++ sequential implementation. Conclusion The proposed overlapping cell segmentation algorithm can accurately detect the center of each overlapping cell and effectively separate each of the overlapping cells. GPU is proven to be an efficient parallel platform for overlapping cell segmentation. PMID:22526139

The role of RhD agglutination for the detection of weak D red cells by anti-D flow cytometry.

PubMed

Grey, D E; Davies, J I; Connolly, M; Fong, E A; Erber, W N

2005-04-01

Anti-D flow cytometry is an accurate method for quantifying feto-maternal haemorrhage (FMH). However, weak D red cells with <1000 RhD sites are not detectable using this methodology but are immunogenic. As quantitation of RhD sites is not practical, an alternative approach is required to identify those weak D fetal red cells where anti-D flow cytometry is inappropriate. We describe a simple algorithm based on RhD agglutination and flow cytometry peak separation. All weak D (n = 34) gave weak agglutination with RUM-1 on immediate spin (grading

Anisotropic properties of phase separation in two-component dipolar Bose-Einstein condensates

NASA Astrophysics Data System (ADS)

Wang, Wei; Li, Jinbin

2018-03-01

Using Crank-Nicolson method, we calculate ground state wave functions of two-component dipolar Bose-Einstein condensates (BECs) and show that, due to dipole-dipole interaction (DDI), the condensate mixture displays anisotropic phase separation. The effects of DDI, inter-component s-wave scattering, strength of trap potential and particle numbers on the density profiles are investigated. Three types of two-component profiles are present, first cigar, along z-axis and concentric torus, second pancake (or blood cell), in xy-plane, and two non-uniform ellipsoid, separated by the pancake and third two dumbbell shapes.

Crystal separation from mother solution and conservation under microgravity conditions using inert liquid

NASA Astrophysics Data System (ADS)

Regel, L. L.; Vedernikov, A. A.; Queeckers, P.; Legros, J.-C.

1991-12-01

The problem of the separation of crystals from their feeding solutions and their conservation at the end of the crystallization under microgravity is investigated. The goal to be reached is to propose an efficient and simple system. This method has to be applicable for an automatic separation on board a spacecraft, without using a centrifuge. The injection of an immiscible and inert liquid into the cell is proposed to solve the problem. The results of numerical modeling, earth simulation tests and experiments under short durations of weightlessness (using aircraft parabolic flights) are described.

Nano-optical conveyor belt, part I: Theory.

PubMed

Hansen, Paul; Zheng, Yuxin; Ryan, Jason; Hesselink, Lambertus

2014-06-11

We propose a method for peristaltic transport of nanoparticles using the optical force field over a nanostructured surface. Nanostructures may be designed to produce strong near-field hot spots when illuminated. The hot spots function as optical traps, separately addressable by their resonant wavelengths and polarizations. By activating closely packed traps sequentially, nanoparticles may be handed off between adjacent traps in a peristaltic fashion. A linear repeating structure of three separately addressable traps forms a "nano-optical conveyor belt"; a unit cell with four separately addressable traps permits controlled peristaltic transport in the plane. Using specifically designed activation sequences allows particle sorting.

Automated cell disruption is a reliable and effective method of isolating RNA from fresh snap-frozen normal and malignant oral mucosa samples.

PubMed

Van der Vorst, Sébastien; Dekairelle, Anne-France; Irenge, Léonid; Hamoir, Marc; Robert, Annie; Gala, Jean-Luc

2009-01-01

This study compared automated vs. manual tissue grinding in terms of RNA yield obtained from oral mucosa biopsies. A total of 20 patients undergoing uvulectomy for sleep-related disorders and 10 patients undergoing biopsy for head and neck squamous cell carcinoma were enrolled in the study. Samples were collected, snap-frozen in liquid nitrogen, and divided into two parts of similar weight. Sample grinding was performed on one sample from each pair, either manually or using an automated cell disruptor. The performance and efficacy of each homogenization approach was compared in terms of total RNA yield (spectrophotometry, fluorometry), mRNA quantity [densitometry of specific TP53 amplicons and TP53 quantitative reverse-transcribed real-time PCR (qRT-PCR)], and mRNA quality (functional analysis of separated alleles in yeast). Although spectrophotometry and fluorometry results were comparable for both homogenization methods, TP53 expression values obtained by amplicon densitometry and qRT-PCR were significantly and consistently better after automated homogenization (p<0.005) for both uvula and tumor samples. Functional analysis of separated alleles in yeast results was better with the automated technique for tumor samples. Automated tissue homogenization appears to be a versatile, quick, and reliable method of cell disruption and is especially useful in the case of small malignant samples, which show unreliable results when processed by manual homogenization.

Enhanced Methodologies for Detecting Phenotypic Resistance in Mycobacteria.

PubMed

Hammond, Robert J H; Baron, Vincent O; Lipworth, Sam; Gillespie, Stephen H

2018-01-01

Lipid droplets found in algae and other microscopic organisms have become of interest to many researchers partially because they carry the capacity to produce bio-oil for the mass market. They are of importance in biology and clinical practice because their presence can be a phenotypic marker of an altered metabolism, including reversible resistance to antibiotics, prompting intense research.A useful stain for detecting lipid bodies in the lab is Nile red. It is a dye that exhibits solvatochromism; its absorption band varies in spectral position, shape and intensity with the nature of its solvent environment, it will fluoresce intensely red in polar environment and blue shift with the changing polarity of its solvent. This makes it ideal for the detection of lipid bodies within Mycobacterium spp. This is because mycobacterial lipid bodies' primary constituents are nonpolar lipids such as triacylglycerols but bacterial cell membranes are primarily polar lipid species. In this chapter we describe an optimal method for using Nile red to distinguish lipid containing (Lipid rich or LR cells) from those without lipid bodies (Lipid Poor or LP). As part of the process we have optimized a method for separating LP and LR cells that does not require the use of an ultracentrifuge or complex separation media. We believe that these methods will facilitate further research in these enigmatic, transient and important cell states.

Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.

PubMed

Koning, Roman I; Celler, Katherine; Willemse, Joost; Bos, Erik; van Wezel, Gilles P; Koster, Abraham J

2014-01-01

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. © 2014 Elsevier Inc. All rights reserved.

Characterization of winemaking yeast by cell number-size distribution analysis through flow field-flow fractionation with multi-wavelength turbidimetric detection.

PubMed

Zattoni, Andrea; Melucci, Dora; Reschiglian, Pierluigi; Sanz, Ramsés; Puignou, Lluís; Galceran, Maria Teresa

2004-10-29

Yeasts are widely used in several areas of food industry, e.g. baking, beer brewing, and wine production. Interest in new analytical methods for quality control and characterization of yeast cells is thus increasing. The biophysical properties of yeast cells, among which cell size, are related to yeast cell capabilities to produce primary and secondary metabolites during the fermentation process. Biophysical properties of winemaking yeast strains can be screened by field-flow fractionation (FFF). In this work we present the use of flow FFF (FlFFF) with turbidimetric multi-wavelength detection for the number-size distribution analysis of different commercial winemaking yeast varieties. The use of a diode-array detector allows to apply to dispersed samples like yeast cells the recently developed method for number-size (or mass-size) analysis in flow-assisted separation techniques. Results for six commercial winemaking yeast strains are compared with data obtained by a standard method for cell sizing (Coulter counter). The method here proposed gives, at short analysis time, accurate information on the number of cells of a given size, and information on the total number of cells.

Cell partition in two phase polymer systems

NASA Technical Reports Server (NTRS)

Brooks, D. E.

1979-01-01

Aqueous phase-separated polymer solutions can be used as support media for the partition of biological macromolecules, organelles and cells. Cell separations using the technique have proven to be extremely sensitive to cell surface properties but application of the systems are limited to cells or aggregates which do not significantly while the phases are settling. Partition in zero g in principle removes this limitation but an external driving force must be applied to induce the phases to separate since their density difference disappears. We have recently shown that an applied electric field can supply the necessary driving force. We are proposing to utilize the NASA FES to study field-driven phase separation and cell partition on the ground and in zero g to help define the separation/partition process, with the ultimate goal being to develop partition as a zero g cell separation technique.

Magnetic Separation and Antibiotics Selection Enable Enrichment of Cells with ZFN/TALEN-Induced Mutations

PubMed Central

Lee, Choong-il; Kim, Hyongbum; Kim, Jin-Soo

2013-01-01

The ability to enrich cells with targeted mutations greatly facilitates the process of using engineered nucleases, including zinc-finger nucleases and transcription activator-like effector nucleases, to construct such cells. We previously used surrogate reporters to enrich cells containing nuclease-induced mutations via flow cytometry. This method is, however, limited by the availability of flow cytometers. Furthermore, sorted cells occasionally fail to form colonies after exposure to a strong laser and hydrostatic pressure. Here we describe two different types of novel reporters that enable mutant cell enrichment without the use of flow cytometers. We designed reporters that express H-2Kk, a surface antigen, and the hygromycin resistance protein (HygroR), respectively, when insertions or deletions are generated at the target sequences by the activity of engineered nucleases. After cotransfection of these reporters and the engineered nuclease-encoding plasmids, H-2Kk- and HygroR-expressing cells were isolated using magnetic separation and hygromycin treatment, respectively. We found that mutant cells were drastically enriched in the isolated cells, suggesting that these two reporters enable efficient enrichment of mutants. We propose that these two reporters will greatly facilitate the use of engineered nucleases in a wider range of biomedical research. PMID:23441197

Evaluation of results of cell electrophoresis experiments on space shuttle STS-3 including pre-flight and post-flight laboratory experiments

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

The objectives of the red blood cell experiments were to provide a visual check on the electrophoretic process and especially electroosmotic flow in space as well as to provide test separations of non-degradable standard particles for comparison with the separations of the three viable cell types studied on the Apollo-Soyuz Test Project. Determination of the maximum concentrations of cells that can be separated in column electrophore was a significant goal. Two of the eight columns were available for red cell experiments, so two concentrations of human and rabbit RBC mixtures were used. The objectives of another experiment were to evaluate the reproducibility of microgravity electrophoretic separation of living kidney cells, to separate cells with highly viability despite two freeze-thaw cycles, and to optimize the physical conditions of cell separation. Owing to the uncertain heterogeneity of the starting material, the experimental design does not assess resolution in microgravity, but improved separability was sought in comparison to density-gradient electrophoresis or continuous-flow electrophoresis. Efforts were made to increase cell yield and cell viability and to assess reproducibility directly.

Phase separated membrane bioreactor: Results from model system studies

NASA Astrophysics Data System (ADS)

Petersen, G. R.; Seshan, P. K.; Dunlop, E. H.

The operation and evaluation of a bioreactor designed for high intensity oxygen transfer in a microgravity environment is described. The reactor itself consists of a zero headspace liquid phase separated from the air supply by a long length of silicone rubber tubing through which the oxygen diffuses in and the carbon dioxide diffuses out. Mass transfer studies show that the oxygen is film diffusion controlled both externally and internally to the tubing and not by diffusion across the tube walls. Methods of upgrading the design to eliminate these resistances are proposed. Cell growth was obtained in the fermenter using Saccharomyces cerevisiae showing that this concept is capable of sustaining cell growth in the terrestial simulation.

Chromatographic Separation, and Characteristics of Nucleic Acids from HeLa Cells

PubMed Central

Philipson, Lennart

1961-01-01

The application of the phenol-duponol method to extraction of nucleic acids from HeLa cells is described. Chromatography of the phenol extract on an esterified bovine serum albumin column with a salt gradient of sodium chloride gives separation of soluble RNA, DNA, and two different high molecular RNA fractions. Ultracentrifugation of the DNA eluted from the column gives a sedimentation coefficient (s 20 o,w) of 38, which agrees with ultracentrifugation data on the phenol extract. The eluted RNA appears polydisperse at low ionic strength, but at high ionic strength and after alcohol precipitation two fractions with the sedimentation coefficients of 16 and 25 to 29, respectively, were obtained. PMID:13735276

Phase separated membrane bioreactor - Results from model system studies

NASA Technical Reports Server (NTRS)

Petersen, G. R.; Seshan, P. K.; Dunlop, E. H.

1989-01-01

The operation and evaluation of a bioreactor designed for high intensity oxygen transfer in a microgravity environment is described. The reactor itself consists of a zero headspace liquid phase separated from the air supply by a long length of silicone rubber tubing through which the oxygen diffuses in and the carbon dioxide diffuses out. Mass transfer studies show that the oxygen is film diffusion controlled both externally and internally to the tubing and not by diffusion across the tube walls. Methods of upgrading the design to eliminate these resistances are proposed. Cell growth was obtained in the fermenter using Saccharomyces cerevisiae showing that this concept is capable of sustaining cell growth in the terrestrial simulation.

Model system studies with a phase separated membrane bioreactor

NASA Technical Reports Server (NTRS)

Petersen, G. R.; Seshan, P. K.; Dunlop, Eric H.

1989-01-01

The operation and evaluation of a bioreactor designed for high intensity oxygen transfer in a microgravity environment is described. The reactor itself consists of a zero headspace liquid phase separated from the air supply by a long length of silicone rubber tubing through which the oxygen diffuses in and the carbon dioxide diffuses out. Mass transfer studies show that the oxygen is film diffusion controlled both externally and internally to the tubing and not by diffusion across the tube walls. Methods of upgrading the design to eliminate these resistances are proposed. Cell growth was obtained in the fermenter using Saccharomyces cerevisiae showing that this concept is capable of sustaining cell growth in the terrestial simulation.

Noncontact Measurement of the Local Mechanical Properties of Living Cells Using Pressure Applied via a Pipette

PubMed Central

Sánchez, Daniel; Johnson, Nick; Li, Chao; Novak, Pavel; Rheinlaender, Johannes; Zhang, Yanjun; Anand, Uma; Anand, Praveen; Gorelik, Julia; Frolenkov, Gregory I.; Benham, Christopher; Lab, Max; Ostanin, Victor P.; Schäffer, Tilman E.; Klenerman, David; Korchev, Yuri E.

2008-01-01

Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1–150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells. PMID:18515369

Multi-scale Gaussian representation and outline-learning based cell image segmentation.

PubMed

Farhan, Muhammad; Ruusuvuori, Pekka; Emmenlauer, Mario; Rämö, Pauli; Dehio, Christoph; Yli-Harja, Olli

2013-01-01

High-throughput genome-wide screening to study gene-specific functions, e.g. for drug discovery, demands fast automated image analysis methods to assist in unraveling the full potential of such studies. Image segmentation is typically at the forefront of such analysis as the performance of the subsequent steps, for example, cell classification, cell tracking etc., often relies on the results of segmentation. We present a cell cytoplasm segmentation framework which first separates cell cytoplasm from image background using novel approach of image enhancement and coefficient of variation of multi-scale Gaussian scale-space representation. A novel outline-learning based classification method is developed using regularized logistic regression with embedded feature selection which classifies image pixels as outline/non-outline to give cytoplasm outlines. Refinement of the detected outlines to separate cells from each other is performed in a post-processing step where the nuclei segmentation is used as contextual information. We evaluate the proposed segmentation methodology using two challenging test cases, presenting images with completely different characteristics, with cells of varying size, shape, texture and degrees of overlap. The feature selection and classification framework for outline detection produces very simple sparse models which use only a small subset of the large, generic feature set, that is, only 7 and 5 features for the two cases. Quantitative comparison of the results for the two test cases against state-of-the-art methods show that our methodology outperforms them with an increase of 4-9% in segmentation accuracy with maximum accuracy of 93%. Finally, the results obtained for diverse datasets demonstrate that our framework not only produces accurate segmentation but also generalizes well to different segmentation tasks.

Optical tweezers for measuring the interaction of the two single red blood cells in flow condition

NASA Astrophysics Data System (ADS)

Lee, Kisung; Muravyov, Alexei; Semenov, Alexei; Wagner, Christian; Priezzhev, Alexander

2017-03-01

Aggregation of red blood cells (RBCs) is an intrinsic property of blood, which has direct effect on the blood viscosity and therefore affects overall the blood circulation throughout the body. It is attracting interest for the research in both fundamental science and clinical application. Despite of the intensive research, the aggregation mechanism is remaining not fully clear. Recent advances in methods allowed measuring the interaction between single RBCs in a well-defined configuration leading the better understanding of the mechanism of the process. However the most of the studies were made on the static cells. Thus, the measurements in flow mimicking conditions are missing. In this work, we aim to study the interaction of two RBCs in the flow conditions. We demonstrate the characterization of the cells interaction strength (or flow tolerance) by measuring the flow velocity to be applied to separate two aggregated cells trapped by double channel optical tweezers in a desired configuration. The age-separated cells were used for this study. The obtained values for the minimum flow velocities needed to separate the two cells were found to be 78.9 +/- 6.1 μm/s and 110 +/- 13 μm/s for old and young cells respectively. The data obtained is in agreement with the observations reported by other authors. The significance of our results is in ability for obtaining a comprehensible and absolute physical value characterizing the cells interaction in flow conditions (not like the Aggregation Index measured in whole blood suspensions by other techniques, which is some abstract parameter)

Nanoparticles: synthesis and applications in life science and environmental technology

NASA Astrophysics Data System (ADS)

Luong Nguyen, Hoang; Nguyen, Hoang Nam; Hai Nguyen, Hoang; Quynh Luu, Manh; Hieu Nguyen, Minh

2015-03-01

This work focuses on the synthesis, functionalization, and application of gold and silver nanoparticles, magnetic nanoparticles Fe3O4, combination of 4-ATP-coated silver nanoparticles and Fe3O4 nanoparticles. The synthesis methods such as chemical reduction, seeding, coprecipitation,and inverse microemulsion will be outlined. Silica- and amino-coated nanoparticles are suitable for several applications in biomedicine and the environment. The applications of the prepared nanoparticles for early detection of breast cancer cells, basal cell carcinoma, antibacterial test, arsenic removal from water, Herpes DNA separation, CD4+ cell separation and isolation of DNA of Hepatitis virus type B (HBV) and Epstein-Barr virus (EBV) are discussed. Finally, some promising perspectives will be pointed out. Invited talk at the 7th International Workshop on Advanced Materials Science and Nanotechnology IWAMSN2014, 2-6 November, 2014, Ha Long, Vietnam.

Determination of a mutational spectrum

DOEpatents

Thilly, William G.; Keohavong, Phouthone

1991-01-01

A method of resolving (physically separating) mutant DNA from nonmutant DNA and a method of defining or establishing a mutational spectrum or profile of alterations present in nucleic acid sequences from a sample to be analyzed, such as a tissue or body fluid. The present method is based on the fact that it is possible, through the use of DGGE, to separate nucleic acid sequences which differ by only a single base change and on the ability to detect the separate mutant molecules. The present invention, in another aspect, relates to a method for determining a mutational spectrum in a DNA sequence of interest present in a population of cells. The method of the present invention is useful as a diagnostic or analytical tool in forensic science in assessing environmental and/or occupational exposures to potentially genetically toxic materials (also referred to as potential mutagens); in biotechnology, particularly in the study of the relationship between the amino acid sequence of enzymes and other biologically-active proteins or protein-containing substances and their respective functions; and in determining the effects of drugs, cosmetics and other chemicals for which toxicity data must be obtained.

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, M.; Watkins, B.E.; Stanker, L.H.

1991-10-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described. 14 figures.

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, Martin; Watkins, Bruce E.; Stanker, Larry H.

1991-01-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described.

Separator development and testing of nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Gonzalez-Sanabria, O. D.; Manzo, M. A.

1984-01-01

The components, design, and operating characteristics of Ni-H2 cells batteries were improved. A separator development program was designed to develop a separator that is resistant to penetration by oxygen and loose active material from then nickel electrode, while retraining the required chemical and thermal stability, reservoir capability, and high ionic conductivity. The performance of the separators in terms of cell operating voltage was to at least match that of state-of-the-art separators while eliminating the separator problems. The separators were submitted to initial screening tests and those which successfully completed the tests were built into Ni-H2 cells for short term testing. The separators with the best performance are tested for long term performance and life.

The Tumor Suppressor Protein p53 and its Physiological Splicing Variant p53as in a Mouse Mammary Cancer Model

DTIC Science & Technology

1997-10-01

and Biomedical Laboratories. PI - Signature Date TABLE OF CONTENTS Report Documentation Page ii Foreword m Introduction 1-2 Experimental Methods...life studies of P53 or p53as in G1, S, and G2/ M was unsuccessful as reported last year. Long cell cycle times may be responsible for lack of separation...of S-phase 5 cells from G2/ M -phase cells by centrifugal elutriation. Attempts to synchronize cells by density arrest and/or serum starvation resulted

Cell separator for use in bipolar-stack energy storage devices

DOEpatents

Mayer, S.T.; Feikert, J.H.; Kachmitter, J.L.; Pekala, R.W.

1995-02-28

An improved multi-cell electrochemical energy storage device is described, such as a battery, fuel cell, or double layer capacitor using a cell separator which allows cells to be stacked and interconnected with low electrical resistance and high reliability while maximizing packaging efficiency. By adding repeating cells, higher voltages can be obtained. The cell separator is formed by applying an organic adhesive on opposing surfaces of adjacent carbon electrodes or surfaces of aerogel electrodes of a pair of adjacent cells prior to or after pyrolysis thereof to form carbon aerogel electrodes. The cell separator is electronically conductive, but ionically isolating, preventing an electrolytic conduction path between adjacent cells in the stack. 2 figs.

Separation of rare oligodendrocyte progenitor cells from brain using a high-throughput multilayer thermoplastic-based microfluidic device.

PubMed

Didar, Tohid Fatanat; Li, Kebin; Veres, Teodor; Tabrizian, Maryam

2013-07-01

Despite the advances made in the field of regenerative medicine, the progress in cutting-edge technologies for separating target therapeutic cells are still at early stage of development. These cells are often rare, such as stem cells or progenitor cells that their overall properties should be maintained during the separation process for their subsequent application in regenerative medicine. This work, presents separation of oligodendrocyte progenitor cells (OPCs) from rat brain primary cultures using an integrated thermoplastic elastomeric (TPE)- based multilayer microfluidic device fabricated using hot-embossing technology. OPCs are frequently used in recovery, repair and regeneration of central nervous system after injuries. Indeed, their ability to differentiate in vitro into myelinating oligodendrocytes, are extremely important for myelin repair. OPCs form 5-10% of the glial cells population. The traditional macroscale techniques for OPCs separation require pre-processing of cells and/or multiple time consuming steps with low efficiency leading very often to alteration of their properties. The proposed methodology implies to separate OPCs based on their smaller size compared to other cells from the brain tissue mixture. Using aforementioned microfluidic chip embedded with a 5 μm membrane pore size and micropumping system, a separation efficiency more than 99% was achieved. This microchip was able to operate at flow rates up to 100 μl/min, capable of separating OPCs from a confluent 75 cm(2) cell culture flask in less than 10 min, which provides us with a high-throughput and highly efficient separation expected from any cell sorting techniques. Copyright © 2013 Elsevier Ltd. All rights reserved.

Rapid cell separation with minimal manipulation for autologous cell therapies

NASA Astrophysics Data System (ADS)

Smith, Alban J.; O'Rorke, Richard D.; Kale, Akshay; Rimsa, Roberts; Tomlinson, Matthew J.; Kirkham, Jennifer; Davies, A. Giles; Wälti, Christoph; Wood, Christopher D.

2017-02-01

The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of >98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pintauro, Peter N.; Ballengee, Jason; Brodt, Matthew

In one aspect of the present invention, a method of fabricating a fuel cell membrane-electrode-assembly (MEA) having an anode electrode, a cathode electrode, and a membrane disposed between the anode electrode and the cathode electrode, includes fabricating each of the anode electrode, the cathode electrode, and the membrane separately by electrospinning; and placing the membrane between the anode electrode and the cathode electrode, and pressing then together to form the fuel cell MEA.

Magnetic separation of encapsulated islet cells labeled with superparamagnetic iron oxide nano particles.

PubMed

Mettler, Esther; Trenkler, Anja; Feilen, Peter J; Wiegand, Frederik; Fottner, Christian; Ehrhart, Friederike; Zimmermann, Heiko; Hwang, Yong Hwa; Lee, Dong Yun; Fischer, Stefan; Schreiber, Laura M; Weber, Matthias M

2013-01-01

Islet cell transplantation is a promising option for the restoration of normal glucose homeostasis in patients with type 1 diabetes. Because graft volume is a crucial issue in islet transplantations for patients with diabetes, we evaluated a new method for increasing functional tissue yield in xenogeneic grafts of encapsulated islets. Islets were labeled with three different superparamagnetic iron oxide nano particles (SPIONs; dextran-coated SPION, siloxane-coated SPION, and heparin-coated SPION). Magnetic separation was performed to separate encapsulated islets from the empty capsules, and cell viability and function were tested. Islets labeled with 1000 μg Fe/ml dextran-coated SPIONs experienced a 69.9% reduction in graft volume, with a 33.2% loss of islet-containing capsules. Islets labeled with 100 μg Fe/ml heparin-coated SPIONs showed a 46.4% reduction in graft volume, with a 4.5% loss of capsules containing islets. No purification could be achieved using siloxane-coated SPIONs due to its toxicity to the primary islets. SPION labeling of islets is useful for transplant purification during islet separation as well as in vivo imaging after transplantation. Furthermore, purification of encapsulated islets can also reduce the volume of the encapsulated islets without impairing their function by removing empty capsules. © 2013 John Wiley & Sons A/S.

Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue

NASA Technical Reports Server (NTRS)

Slocum, R. D.; Flores, H. E.; Galston, A. W.; Weinstein, L. H.

1989-01-01

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

Review of free software tools for image analysis of fluorescence cell micrographs.

PubMed

Wiesmann, V; Franz, D; Held, C; Münzenmayer, C; Palmisano, R; Wittenberg, T

2015-01-01

An increasing number of free software tools have been made available for the evaluation of fluorescence cell micrographs. The main users are biologists and related life scientists with no or little knowledge of image processing. In this review, we give an overview of available tools and guidelines about which tools the users should use to segment fluorescence micrographs. We selected 15 free tools and divided them into stand-alone, Matlab-based, ImageJ-based, free demo versions of commercial tools and data sharing tools. The review consists of two parts: First, we developed a criteria catalogue and rated the tools regarding structural requirements, functionality (flexibility, segmentation and image processing filters) and usability (documentation, data management, usability and visualization). Second, we performed an image processing case study with four representative fluorescence micrograph segmentation tasks with figure-ground and cell separation. The tools display a wide range of functionality and usability. In the image processing case study, we were able to perform figure-ground separation in all micrographs using mainly thresholding. Cell separation was not possible with most of the tools, because cell separation methods are provided only by a subset of the tools and are difficult to parametrize and to use. Most important is that the usability matches the functionality of a tool. To be usable, specialized tools with less functionality need to fulfill less usability criteria, whereas multipurpose tools need a well-structured menu and intuitive graphical user interface. © 2014 Fraunhofer-Institute for Integrated Circuits IIS Journal of Microscopy © 2014 Royal Microscopical Society.

The preservation of living cells with biocompatible microparticles

NASA Astrophysics Data System (ADS)

Yang, Jing; Zhu, Yingnan; Xu, Tong; Pan, Chao; Cai, Nana; Huang, He; Zhang, Lei

2016-07-01

Biomedical applications of living cells have rapidly expanded in many fields such as toxic detection, drug screening, and regenerative medicine, etc. Efficient methods to support cell survival and maintain activity in vitro have become increasingly important. However, traditional cryopreservation for living cell-based applications is limited by several problems. Here, we report that magnetic hydrogel microparticles can physically assemble into a 3D environment for efficient cell preservation in physiological conditions, avoiding any chemical reactions that would damage the cells. Two representative cell lines (loosely and firmly adherent) were tested to evaluate the versatility of this method. The results showed that cell longevity was significantly extended to at least 15 days, while the control cell samples without microparticles quickly died within 3 days. Moreover, after preservation, cells can be easily retrieved by applying a magnet to separate the magnetic particles. This strategy can also inhibit cell over-proliferation while avoiding the use of temperature extremes or toxic cryoprotectants that are essential in cryopreservation.

Single-cell MALDI-MS as an analytical tool for studying intrapopulation metabolic heterogeneity of unicellular organisms.

PubMed

Amantonico, Andrea; Urban, Pawel L; Fagerer, Stephan R; Balabin, Roman M; Zenobi, Renato

2010-09-01

Heterogeneity is a characteristic feature of all populations of living organisms. Here we make an attempt to validate a single-cell mass spectrometric method for detection of changes in metabolite levels occurring in populations of unicellular organisms. Selected metabolites involved in central metabolism (ADP, ATP, GTP, and UDP-Glucose) could readily be detected in single cells of Closterium acerosum by means of negative-mode matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The analytical capabilities of this approach were characterized using standard compounds. The method was then used to study populations of individual cells with different levels of the chosen metabolites. With principal component analysis and support vector machine algorithms, it was possible to achieve a clear separation of individual C. acerosum cells in different metabolic states. This study demonstrates the suitability of mass spectrometric analysis of metabolites in single cells to measure cell-population heterogeneity.

Method and apparatus for capacitive deionization and electrochemical purification and regeneration of electrodes

DOEpatents

Tran, Tri D.; Farmer, Joseph C.; Murguia, Laura

2001-01-01

An electrically regeneratable electrochemical cell (30) for capacitive deionization and electrochemical purification and regeneration of electrodes includes two end plates (31, 32), one at each end of the cell (30). A new regeneration method is applied to the cell (30) which includes slowing or stopping the purification cycle, electrically desorbing contaminants and removing the desorbed contaminants. The cell (30) further includes a plurality of generally identical double-sided intermediate electrodes (37-43) that are equidistally separated from each other, between the two end electrodes (35, 36). As the electrolyte enters the cell, it flows through a continuous open serpentine channel (65-71) defined by the electrodes, substantially parallel to the surfaces of the electrodes. By polarizing the cell (30), ions are removed from the electrolyte and are held in the electric double layers formed at the carbon aerogel surfaces of the electrodes. The cell (30) is regenerated electrically to desorb such previously removed ions.

Lineage mapper: A versatile cell and particle tracker

NASA Astrophysics Data System (ADS)

Chalfoun, Joe; Majurski, Michael; Dima, Alden; Halter, Michael; Bhadriraju, Kiran; Brady, Mary

2016-11-01

The ability to accurately track cells and particles from images is critical to many biomedical problems. To address this, we developed Lineage Mapper, an open-source tracker for time-lapse images of biological cells, colonies, and particles. Lineage Mapper tracks objects independently of the segmentation method, detects mitosis in confluence, separates cell clumps mistakenly segmented as a single cell, provides accuracy and scalability even on terabyte-sized datasets, and creates division and/or fusion lineages. Lineage Mapper has been tested and validated on multiple biological and simulated problems. The software is available in ImageJ and Matlab at isg.nist.gov.

Solution-assisted ultrafast transfer of graphene-based thin films for solar cells and humidity sensors.

PubMed

Sun, Jiawei; Xie, Xiao; Bi, Hengchang; Jia, Haiyang; Zhu, Chongyang; Wan, Neng; Huang, Jianqiu; Nie, Meng; Li, Dan; Sun, Litao

2017-03-01

Vacuum filtration enables the fabrication of large-area graphene-based membranes (GBMs), possessing a smoother surface than that by spray, spin coating or drop casting. However, due to the strong interaction with substrates, the separation of thin GBMs from the filter is problematic. Conventional stamping separation/transfer of graphene oxide (GO) thin films requires another substrate and pressing for >10 h, which may damage the delicate structure of the transfer substrates. Other methods require GO to be reduced on filters before separation, thus limiting the reduction methods. Inspired by a coagulation bath that enables rapid formation of ultrastrong GO fibers, we present an ultrafast (<1 min) and solution-assisted strategy to fabricate smooth and freestanding GO films. The diverse interfacial energy of hydrogen bonds also demonstrates another reason for the successful separation. The film thickness ranges from 45 nm to several micrometers. When used as a composite of counter electrodes in dye sensitized solar cells, it showed higher (8.58%) power conversion efficiency than its spin-(7.71%) and spray-coated (8.07%) counterparts. It also showed promising performance in capacitive humidity sensors. The capacitance varied by three orders of magnitude in the range of the relative humidity of 15%-95%. Therefore the strategy realizes an ultrafast and high-quality film production which is suitable for various applications.

Solution-assisted ultrafast transfer of graphene-based thin films for solar cells and humidity sensors

NASA Astrophysics Data System (ADS)

Sun, Jiawei; Xie, Xiao; Bi, Hengchang; Jia, Haiyang; Zhu, Chongyang; Wan, Neng; Huang, Jianqiu; Nie, Meng; Li, Dan; Sun, Litao

2017-03-01

Vacuum filtration enables the fabrication of large-area graphene-based membranes (GBMs), possessing a smoother surface than that by spray, spin coating or drop casting. However, due to the strong interaction with substrates, the separation of thin GBMs from the filter is problematic. Conventional stamping separation/transfer of graphene oxide (GO) thin films requires another substrate and pressing for >10 h, which may damage the delicate structure of the transfer substrates. Other methods require GO to be reduced on filters before separation, thus limiting the reduction methods. Inspired by a coagulation bath that enables rapid formation of ultrastrong GO fibers, we present an ultrafast (<1 min) and solution-assisted strategy to fabricate smooth and freestanding GO films. The diverse interfacial energy of hydrogen bonds also demonstrates another reason for the successful separation. The film thickness ranges from 45 nm to several micrometers. When used as a composite of counter electrodes in dye sensitized solar cells, it showed higher (8.58%) power conversion efficiency than its spin-(7.71%) and spray-coated (8.07%) counterparts. It also showed promising performance in capacitive humidity sensors. The capacitance varied by three orders of magnitude in the range of the relative humidity of 15%-95%. Therefore the strategy realizes an ultrafast and high-quality film production which is suitable for various applications.

Harnessing electrical forces for separation. Capillary zone electrophoresis, isoelectric focusing, field-flow fractionation, split-flow thin-cell continuous-separation and other techniques.

PubMed

Giddings, J C

1989-10-20

A simple analysis, first presented twenty years ago, showed that the effectiveness of a field-driven separation like electrophoresis, as expressed by the maximum number of theoretical plates (N), is given by the dimensionless ratio of two energies N = -delta mu ext/2RT in which -delta mu ext is the electrical potential energy drop of a charged species and RT is the thermal energy (R is the gas constant and T is the absolute temperature). Quantity -delta mu ext is the product of the force F acting on the species and the path length X of separation. The exceptional power of electrophoresis, for which often N approximately 10(6), can be traced directly to the enormous magnitude of the electrical force F. This paper explores the fundamentals underlying several different means for utilizing these powerful electrical forces for separation, including capillary zone electrophoresis, gel electrophoresis, isoelectric focusing, electrical field-flow fractionation and split-flow thin continuous separation cells. Remarkably, the above equation and its relatives are found to describe the approximate performance of all these diverse electrically driven systems. Factors affecting both the resolving power and separation speed of the systems are addressed; from these considerations some broad optimization criteria emerge. The capabilities of the different methods are compared using numerical examples.

Automated Microfluidic Instrument for Label-Free and High-Throughput Cell Separation.

PubMed

Zhang, Xinjie; Zhu, Zhixian; Xiang, Nan; Long, Feifei; Ni, Zhonghua

2018-03-20

Microfluidic technologies for cell separation were reported frequently in recent years. However, a compact microfluidic instrument enabling thoroughly automated cell separation is still rarely reported until today due to the difficult hybrid between the macrosized fluidic control system and the microsized microfluidic device. In this work, we propose a novel and automated microfluidic instrument to realize size-based separation of cancer cells in a label-free and high-throughput manner. Briefly, the instrument is equipped with a fully integrated microfluidic device and a set of robust fluid-driven and control units, and the instrument functions of precise fluid infusion and high-throughput cell separation are guaranteed by a flow regulatory chip and two cell separation chips which are the key components of the microfluidic device. With optimized control programs, the instrument is successfully applied to automatically sort human breast adenocarcinoma cell line MCF-7 from 5 mL of diluted human blood with a high recovery ratio of ∼85% within a rapid processing time of ∼23 min. We envision that our microfluidic instrument will be potentially useful in many biomedical applications, especially cell separation, enrichment, and concentration for the purpose of cell culture and analysis.

A Rapid Method for Engineering Recombinant Polioviruses or Other Enteroviruses.

PubMed

Bessaud, Maël; Pelletier, Isabelle; Blondel, Bruno; Delpeyroux, Francis

2016-01-01

The cloning of large enterovirus RNA sequences is labor-intensive because of the frequent instability in bacteria of plasmidic vectors containing the corresponding cDNAs. In order to circumvent this issue we have developed a PCR-based method that allows the generation of highly modified or chimeric full-length enterovirus genomes. This method relies on fusion PCR which enables the concatenation of several overlapping cDNA amplicons produced separately. A T7 promoter sequence added upstream the fusion PCR products allows its transcription into infectious genomic RNAs directly in transfected cells constitutively expressing the phage T7 RNA polymerase. This method permits the rapid recovery of modified viruses that can be subsequently amplified on adequate cell-lines.

Automated processing of human bone marrow can result in a population of mononuclear cells capable of achieving engraftment following transplantation.

PubMed

Areman, E M; Cullis, H; Spitzer, T; Sacher, R A

1991-10-01

A concentrate of mononuclear bone marrow cells is often desired for ex vivo treatment with pharmacologic agents, monoclonal antibodies, cytokines, and other agents prior to transplantation. A method has been developed for automated separation of mononuclear cells from large volumes of harvested bone marrow. A programmable instrument originally designed for clinical ex vivo cell separation and the plasma-pheresis of patients and blood donors was adapted to permit rapid preparation, in a closed sterile system, of a bone marrow product enriched with mononuclear cells. A mean (+/- SEM) of 53 +/- 30 percent of the original mononuclear cells was recovered in a volume of 125 +/- 42 mL containing 82 +/- 12 percent mononuclear cells. This technique removed 95 +/- 9 percent of the red cells in the original marrow. No density gradient materials or sedimenting agents were employed in this process. Of 36 marrows processed by this technique, 19 autologous (6 of which were purged with 4-hydroperoxycyclophosphamide) and 7 allogeneic marrows have been transplanted, with all evaluable patients achieving a neutrophil count of 0.5 x 10(9) per L in a mean (+/- SEM) of 21 +/- 6 days.

Measuring cell cycle progression kinetics with metabolic labeling and flow cytometry.

PubMed

Fleisig, Helen; Wong, Judy

2012-05-22

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments. Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

A High-Efficiency Superhydrophobic Plasma Separator

PubMed Central

Liu, Changchun; Liao, Shih-Chuan; Song, Jinzhao; Mauk, Michael G.; Li, Xuanwen; Wu, Gaoxiang; Ge, Dengteng; Greenberg, Robert M.; Yang, Shu; Bau, Haim H.

2016-01-01

To meet stringent limit-of-detection specifications for low abundance target molecules, a relatively large volume of plasma is needed for many blood-based clinical diagnostics. Conventional centrifugation methods for plasma separation are not suitable for on-site testing or bedside diagnostics. Here, we report a simple, yet high-efficiency, clamshell-style, superhydrophobic plasma separator that is capable of separating a relatively large volume of plasma from several hundred microliters of whole blood (finger-prick blood volume). The plasma separator consists of a superhydrophobic top cover with a separation membrane and a superhydrophobic bottom substrate. Unlike previously reported membrane-based plasma separators, the separation membrane in our device is positioned at the top of the sandwiched whole blood film to increase the membrane separation capacity and plasma yield. In addition, the device’s superhydrophobic characteristics (i) facilitates the formation of well-defined, contracted, thin blood film with a high contact angle; (ii) minimizes biomolecular adhesion to surfaces; (iii) increases blood clotting time; and (iv) reduces blood cell hemolysis. The device demonstrated a “blood in-plasma out” capability, consistently extracting 65±21.5 μL of plasma from 200 μL of whole blood in less than 10 min without electrical power. The device was used to separate plasma from Schistosoma mansoni genomic DNA-spiked whole blood with a recovery efficiency of > 84.5 ± 25.8 %. The S. mansoni genomic DNA in the separated plasma was successfully tested on our custom-made microfluidic chip by using loop mediated isothermal amplification (LAMP) method. PMID:26732765

Large-scale separation of single-walled carbon nanotubes by electronic type using click chemistry

NASA Astrophysics Data System (ADS)

Um, Jo-Eun; Song, Sun Gu; Yoo, Pil J.; Song, Changsik; Kim, Woo-Jae

2018-01-01

Single-walled carbon nanotubes (SWCNTs) can be either metallic or semiconducting, making their separation critical for applications in nanoelectronics, biomedical materials, and solar cells. Herein, we investigate a novel solution-phase separation method based on click chemistry (azide-alkyne Huisgen cycloaddition) and determine its efficiency and scalability. In this method, metallic SWCNTs in metallic/semiconducting SWCNT mixtures are selectively functionalized with alkyne groups by being reacted with 4-propargyloxybenezenediazonium tetrafluoroborate. Subsequently, silica nanoparticles are functionalized with azide groups and reacted with alkyne-bearing metallic SWCNTs in the SWCNT mixture in the presence of a Cu catalyst. As a result, metallic SWCNTs are anchored on silica powder, whereas non-functionalized semiconducting SWCNTs remain in solution. Low-speed centrifugation effectively removes the silica powder with attached metallic SWCNTs, furnishing a solution of highly pure semiconducting SWCNTs, as confirmed by Raman and UV-vis/near-infrared absorption measurements. This novel separation scheme exhibits the advantage of simultaneously separating both metallic and semiconducting SWCNTs from their mixtures, being cost-effective and therefore applicable at an industrial scale.

Thinner, More-Efficient Oxygen-Separation Cells

NASA Technical Reports Server (NTRS)

Clark, Douglas J.; Galica, Leo M.; Losey, Robert W.

1992-01-01

Better gas-distribution plates fabricated more easily. Oxygen-separation cell redesigned to make it more efficient, smaller, lighter, and easier to manufacture. Potential applications include use as gas separators, filters, and fuel cells.

Continuous field-flow separation of particle populations in a dielectrophoretic chip with three dimensional electrodes

NASA Astrophysics Data System (ADS)

Iliescu, Ciprian; Tresset, Guillaume; Xu, Guolin

2007-06-01

This letter presents a dielectrophoretic (DEP) separation method of particles under continuous flow. The method consists of flowing two particle populations through a microfluidic channel, in which the vertical walls are the electrodes of the DEP device. The irregular shape of the electrodes generates both electric field and fluid velocity gradients. As a result, the particles that exhibit negative DEP can be trapped in the fluidic dead zones, while the particles that experience positive DEP are concentrated in the regions with high velocity and collected at the outlet. The device was tested with dead and living yeast cells.

Novel duplex vapor-electrochemical method for silicon solar cells

NASA Technical Reports Server (NTRS)

Nanis, L.; Sanjurjo, A.; Sancier, K. M.; Kapur, V. K.; Bartlett, R. W.; Westphal, S.

1980-01-01

A process was developed for the economic production of high purity Si from inexpensive reactants, based on the Na reduction of SiF4 gas. The products of reaction (NaF, Si) are separated by either aqueous leaching or by direct melting of the NaF-Si product mixture. Impurities known to degrade solar cell performance are all present at sufficiently low concentrations so that melt solidification (e.g., Czochralski) will provide a silicon material suitable for solar cells.

Electrokinetic gated injection-based microfluidic system for quantitative analysis of hydrogen peroxide in individual HepG2 cells.

PubMed

Zhang, Xinyuan; Li, Qingling; Chen, Zhenzhen; Li, Hongmin; Xu, Kehua; Zhang, Lisheng; Tang, Bo

2011-03-21

A microfluidic system to determine hydrogen peroxide (H(2)O(2)) in individual HepG2 cells based on the electrokinetic gated injection was developed for the first time. A home-synthesized fluorescent probe, bis(p-methylbenzenesulfonate)dichlorofluorescein (FS), was employed to label intracellular H(2)O(2) in the intact cells. On a simple cross microchip, multiple single-cell operations, including single cell injection, cytolysis, electrophoresis separation and detection of H(2)O(2), were automatically carried out within 60 s using the electrokinetic gated injection and laser-induced fluorescence detection (LIFD). The performance of the method was evaluated under the optimal conditions. The linear calibration curve was over a range of 4.39-610 amol (R(2)=0.9994). The detection limit was 0.55 amol or 9.0×10(-10) M (S/N=3). The relative standard deviations (RSDs, n=6) of migration time and peak area were 1.4% and 4.8%, respectively. With the use of this method, the average content of H(2)O(2) in single HepG2 cells was found to be 16.09±9.84 amol (n=15). Separation efficiencies in excess of 17,000 theoretical plates for the cells were achieved. These results demonstrated that the efficient integration and automation of these single-cell operations enabled the sensitive, reproducible, and quantitative examination of intracellular H(2)O(2) at single-cell level. Owing to the advantages of simple microchip structure, controllable single-cell manipulation and ease in building, this platform provides a universal way to automatically determine other intracellular constituents within single cells. This journal is © The Royal Society of Chemistry 2011

A 3D cell culture system: separation distance between INS-1 cell and endothelial cell monolayers co-cultured in fibrin influences INS-1 cells insulin secretion.

PubMed

Sabra, Georges; Vermette, Patrick

2013-02-01

The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72 h, INS-1 cells co-cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered. Copyright © 2012 Wiley Periodicals, Inc.

Biology Notes.

ERIC Educational Resources Information Center

School Science Review, 1984

1984-01-01

Describes mushroom growing as a school project, a method for illustrating need for carbon dioxide in photosynthesis, construction of a simple phytoplankton sampler, cell division activity using playing cards, blood separation activity, measurement of adaptation and selection pressures, computations in field ecology, and an activity demonstrating…

The evaluation of layered separators for nickel-hydrogen cells

NASA Technical Reports Server (NTRS)

Gahn, Randall F.

1991-01-01

The concept of using layered separators to achieve the required electrolyte retention and bubble pressure fo nickel-hydrogen cells was evaluated in a boilerplate cell test. Zircar cloth, polyethylene paper and polypropylene felt were combined with a layer of radiation-grafted polyethylene film to achieve the required properties. Three cells of each layered separator were built and tested by characterization cycling and by low earth orbit cycling for 5000 cycles at 80 percent DOD. Three cells containing asbestos separators were used as the reference.

Cell separation: Terminology and practical considerations

PubMed Central

Tomlinson, Sophie; Yang, Xuebin B; Kirkham, Jennifer

2013-01-01

Cell separation is a powerful tool in biological research. Increasing usage, particularly within the tissue engineering and regenerative medicine communities, means that researchers from a diverse range of backgrounds are utilising cell separation technologies. This review aims to offer potential solutions to cell sorting problems and to clarify common ambiguities in terminology and experimental design. The frequently used cell separation terms of ‘purity’, ‘recovery’ and ‘viability’ are discussed, and attempts are made to reach a consensus view of their sometimes ambiguous meanings. The importance of appropriate experimental design is considered, with aspects such as marker expression, tissue isolation and original cell population analysis discussed. Finally, specific technical issues such as cell clustering, dead cell removal and non-specific antibody binding are considered and potential solutions offered. The solutions offered may provide a starting point to improve the quality of cell separations achieved by both the novice and experienced researcher alike. PMID:23440031

Towards a Viscous Wall Model for Immersed Boundary Methods

NASA Technical Reports Server (NTRS)

Brehm, Christoph; Barad, Michael F.; Kiris, Cetin C.

2016-01-01

Immersed boundary methods are frequently employed for simulating flows at low Reynolds numbers or for applications where viscous boundary layer effects can be neglected. The primary shortcoming of Cartesian mesh immersed boundary methods is the inability of efficiently resolving thin turbulent boundary layers in high-Reynolds number flow application. The inefficiency of resolving the thin boundary is associated with the use of constant aspect ratio Cartesian grid cells. Conventional CFD approaches can efficiently resolve the large wall normal gradients by utilizing large aspect ratio cells near the wall. This paper presents different approaches for immersed boundary methods to account for the viscous boundary layer interaction with the flow-field away from the walls. Different wall modeling approaches proposed in previous research studies are addressed and compared to a new integral boundary layer based approach. In contrast to common wall-modeling approaches that usually only utilize local flow information, the integral boundary layer based approach keeps the streamwise history of the boundary layer. This allows the method to remain effective at much larger y+ values than local wall modeling approaches. After a theoretical discussion of the different approaches, the method is applied to increasingly more challenging flow fields including fully attached, separated, and shock-induced separated (laminar and turbulent) flows.

Microspheres

NASA Technical Reports Server (NTRS)

1990-01-01

Vital information on a person's physical condition can be obtained by identifying and counting the population of T-cells and B-cells, lymphocytes of the same shape and size that help the immune system protect the body from the invasion of disease. The late Dr. Alan Rembaum developed a method for identifying the cells. The method involved tagging the T-cells and B-cells with microspheres of different fluorescent color. Microspheres, which have fluorescent dye embedded in them, are chemically treated so that they can link with antibodies. With the help of a complex antibody/antigen reaction, the microspheres bind themselves to specific 'targets,' in this case the T-cells or B-cells. Each group of cells can then be analyzed by a photoelectronic instrument at different wavelengths emitted by the fluorescent dyes. Same concept was applied to the separation of cancer cells from normal cells. Microspheres were also used to conduct many other research projects. Under a patent license Magsphere, Inc. is producing a wide spectrum of microspheres on a large scale and selling them worldwide for various applications.

Rapid prototyping and parametric optimization of plastic acoustofluidic devices for blood-bacteria separation.

PubMed

Silva, R; Dow, P; Dubay, R; Lissandrello, C; Holder, J; Densmore, D; Fiering, J

2017-09-01

Acoustic manipulation has emerged as a versatile method for microfluidic separation and concentration of particles and cells. Most recent demonstrations of the technology use piezoelectric actuators to excite resonant modes in silicon or glass microchannels. Here, we focus on acoustic manipulation in disposable, plastic microchannels in order to enable a low-cost processing tool for point-of-care diagnostics. Unfortunately, the performance of resonant acoustofluidic devices in plastic is hampered by a lack of a predictive model. In this paper, we build and test a plastic blood-bacteria separation device informed by a design of experiments approach, parametric rapid prototyping, and screening by image-processing. We demonstrate that the new device geometry can separate bacteria from blood while operating at 275% greater flow rate as well as reduce the power requirement by 82%, while maintaining equivalent separation performance and resolution when compared to the previously published plastic acoustofluidic separation device.

A microchip electrophoresis-mass spectrometric platform with double cell lysis nano-electrodes for automated single cell analysis.

PubMed

Li, Xiangtang; Zhao, Shulin; Hu, Hankun; Liu, Yi-Ming

2016-06-17

Capillary electrophoresis-based single cell analysis has become an essential approach in researches at the cellular level. However, automation of single cell analysis has been a challenge due to the difficulty to control the number of cells injected and the irreproducibility associated with cell aggregation. Herein we report the development of a new microfluidic platform deploying the double nano-electrode cell lysis technique for automated analysis of single cells with mass spectrometric detection. The proposed microfluidic chip features integration of a cell-sized high voltage zone for quick single cell lysis, a microfluidic channel for electrophoretic separation, and a nanoelectrospray emitter for ionization in MS detection. Built upon this platform, a microchip electrophoresis-mass spectrometric method (MCE-MS) has been developed for automated single cell analysis. In the method, cell introduction, cell lysis, and MCE-MS separation are computer controlled and integrated as a cycle into consecutive assays. Analysis of large numbers of individual PC-12 neuronal cells (both intact and exposed to 25mM KCl) was carried out to determine intracellular levels of dopamine (DA) and glutamic acid (Glu). It was found that DA content in PC-12 cells was higher than Glu content, and both varied from cell to cell. The ratio of intracellular DA to Glu was 4.20±0.8 (n=150). Interestingly, the ratio drastically decreased to 0.38±0.20 (n=150) after the cells are exposed to 25mM KCl for 8min, suggesting the cells released DA promptly and heavily while they released Glu at a much slower pace in response to KCl-induced depolarization. These results indicate that the proposed MCE-MS analytical platform may have a great potential in researches at the cellular level. Copyright © 2016 Elsevier B.V. All rights reserved.

Integrated fuel cell stack shunt current prevention arrangement

DOEpatents

Roche, Robert P.; Nowak, Michael P.

1992-01-01

A fuel cell stack includes a plurality of fuel cells juxtaposed with one another in the stack and each including a pair of plate-shaped anode and cathode electrodes that face one another, and a quantity of liquid electrolyte present at least between the electrodes. A separator plate is interposed between each two successive electrodes of adjacent ones of the fuel cells and is unified therewith into an integral separator plate. Each integral separator plate is provided with a circumferentially complete barrier that prevents flow of shunt currents onto and on an outer peripheral surface of the separator plate. This barrier consists of electrolyte-nonwettable barrier members that are accommodated, prior to the formation of the integral separator plate, in corresponding edge recesses situated at the interfaces between the electrodes and the separator plate proper. Each barrier member extends over the entire length of the associated marginal portion and is flush with the outer periphery of the integral separator plate. This barrier also prevents cell-to-cell migration of any electrolyte that may be present at the outer periphery of the integral separator plate while the latter is incorporated in the fuel cell stack.

Quantitative Magnetic Separation of Particles and Cells using Gradient Magnetic Ratcheting

PubMed Central

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-01-01

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting (MACS), are robust but perform coarse, qualitative separations based on surface antigen expression. We report a quantitative magnetic separation technology using high-force magnetic ratcheting over arrays of magnetically soft micro-pillars with gradient spacing, and use the system to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micro-pillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic-field. Particles with higher IOC separate and equilibrate along the miro-pillar array at larger pitches. We develop a semi-analytical model that predicts behavior for particles and cells. Using the system, LNCaP cells were separated based on the bound quantity of 1μm anti-EpCAM particles as a metric for expression. The ratcheting cytometry system was able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof of concept, EpCAM-labeled cells from patient blood were isolated with 74% purity, demonstrating potential towards a quantitative magnetic separation instrument. PMID:26890496

Classification of Acute Myelogenous Leukemia (AML M2 and AML M3) using Momentum Back Propagation from Watershed Distance Transform Segmented Images

NASA Astrophysics Data System (ADS)

Suryani, Esti; Wiharto; Palgunadi, Sarngadi; Nurcahya Pradana, TP

2017-01-01

This study uses image processing to analyze white blood cell with leukemia indicated that includes the identification, analysis of shapes and sizes, as well as white blood cell count indicated the symptoms of leukemia. A case study in this research was blood cells, from the type of leukemia Acute Myelogenous Leukemia (AML), M2 and M3 in particular. Image processing operations used for segmentation by utilizing the color conversion from RGB (Red, Green dab Blue) to obtain white blood cell candidates. Furthermore, the white blood cells candidates are separated by other cells with active contour without edge. WBC (White Blood Cell) results still have intersected or overlap condition. Watershed distance transform method can separate overlap of WBC. Furthermore, the separation of the nucleus from the cytoplasm using the HSI (Hue Saturation Intensity). The further characteristic extraction process is done by calculating the area WBC, WBC edge, roundness, the ratio of the nucleus, the mean and standard deviation of pixel intensities. The feature extraction results are used for training and testing in determining the classification of AML: M2 and M3 by using the momentum backpropagation algorithm. The classification process is done by testing the numeric data input from the feature extraction results that have been entered in the database. K-Fold validation is used to divide the amount of training data and to test the classification of AML M2 and M3. The experiment results of eight images trials, the result, was 94.285% per cell accuracy and 75% per image accuracy

Tailored liquid chromatography-mass spectrometry analysis improves the coverage of the intracellular metabolome of HepaRG cells.

PubMed

Cuykx, Matthias; Negreira, Noelia; Beirnaert, Charlie; Van den Eede, Nele; Rodrigues, Robim; Vanhaecke, Tamara; Laukens, Kris; Covaci, Adrian

2017-03-03

Metabolomics protocols are often combined with Liquid Chromatography-Mass Spectrometry (LC-MS) using mostly reversed phase chromatography coupled to accurate mass spectrometry, e.g. quadrupole time-of-flight (QTOF) mass spectrometers to measure as many metabolites as possible. In this study, we optimised the LC-MS separation of cell extracts after fractionation in polar and non-polar fractions. Both phases were analysed separately in a tailored approach in four different runs (two for the non-polar and two for the polar-fraction), each of them specifically adapted to improve the separation of the metabolites present in the extract. This approach improves the coverage of a broad range of the metabolome of the HepaRG cells and the separation of intra-class metabolites. The non-polar fraction was analysed using a C18-column with end-capping, mobile phase compositions were specifically adapted for each ionisation mode using different co-solvents and buffers. The polar extracts were analysed with a mixed mode Hydrophilic Interaction Liquid Chromatography (HILIC) system. Acidic metabolites from glycolysis and the Krebs cycle, together with phosphorylated compounds, were best detected with a method using ion pairing (IP) with tributylamine and separation on a phenyl-hexyl column. Accurate mass detection was performed with the QTOF in MS-mode only using an extended dynamic range to improve the quality of the dataset. Parameters with the greatest impact on the detection were the balance between mass accuracy and linear range, the fragmentor voltage, the capillary voltage, the nozzle voltage, and the nebuliser pressure. By using a tailored approach for the intracellular HepaRG metabolome, consisting of three different LC techniques, over 2200 metabolites can be measured with a high precision and acceptable linear range. The developed method is suited for qualitative untargeted LC-MS metabolomics studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Large silver-cadmium technology program

NASA Technical Reports Server (NTRS)

Charlip, S.; Lerner, S.

1971-01-01

The effects of varying cell design on operation factors on the electrochemical performance of sealed, silver-cadmium cells were determined. A factorial experiment was conducted for all test cells constructed with organic separators. Three operating factors were evaluated: temperature, depth of discharge, and charge rate. The six construction factors considered were separator, absorber, electrolyte quantity, cadmium electrode type, cadmium-to-silver ratio, and auxiliary electrode. Test cells of 4 ampere-hour capacity were fabricated and cycled. The best performing cells, on a 94 minute orbit, at 40% depth of discharge, were those containing silver-treated fibrous sausage casings as the separator, and Teflon-ated, pressed cadmium electrodes. Cycling data of cells with inorganic separators (Astroset) are given. Best performance was shown by cells with nonwoven nylon absorbers. Rigid inorganic separators provided the best barrier to silver migration.

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

PubMed

Chong, B E; Hamler, R L; Lubman, D M; Ethier, S P; Rosenspire, A J; Miller, F R

2001-03-15

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

Random breakup of microdroplets for single-cell encapsulation

NASA Astrophysics Data System (ADS)

Um, Eujin; Lee, Seung-Goo; Park, Je-Kyun

2010-10-01

Microfluidic droplet-based technology enables encapsulation of cells in the isolated aqueous chambers surrounded by immiscible fluid but single-cell encapsulation efficiency is usually less than 30%. In this letter, we introduce a simple microgroove structure to break droplets into random sizes which further allows collecting of single-cell [Escherichia coli (E. coli)] containing droplets by their size differences. Pinched-flow separation method is integrated to sort out droplets of certain sizes which have high probability of containing one cell. Consequently, we were able to obtain more than 50% of droplets having single E. coli inside, keeping the proportion of multiple-cell containing droplets less than 16%.

Improved Separators For Rechargeable Lithium Cells

NASA Technical Reports Server (NTRS)

Shen, David; Surampudi, Subbarao; Huang, Chen-Kuo; Halpert, Gerald

1994-01-01

Improved pairs of separators proposed for use in rechargeable lithium cells operating at ambient temperature. Block growth of lithium dendrites and help prevent short circuits. Each cell contains one separator made of microporous polypropylene placed next to anode, and one separator made of microporous polytetrafluoroethylene (PTFE) next to cathode. Separators increase cycle lives of secondary lithium cells. Cells to which concept applicable those of Li/TiS(2), Li/NbSe(3), Li/CoO(2), Li/MoS(2), Li/VO(x), and Li/MnO(2) chemical systems. Advantageous in spacecraft, military, communications, automotive, and other applications in which high energy density and rechargeability needed.

Fuel-Cell Water Separator

NASA Technical Reports Server (NTRS)

Burke, Kenneth Alan; Fisher, Caleb; Newman, Paul

2010-01-01

The main product of a typical fuel cell is water, and many fuel-cell configurations use the flow of excess gases (i.e., gases not consumed by the reaction) to drive the resultant water out of the cell. This two-phase mixture then exits through an exhaust port where the two fluids must again be separated to prevent the fuel cell from flooding and to facilitate the reutilization of both fluids. The Glenn Research Center (GRC) has designed, built, and tested an innovative fuel-cell water separator that not only removes liquid water from a fuel cell s exhaust ports, but does so with no moving parts or other power-consuming components. Instead it employs the potential and kinetic energies already present in the moving exhaust flow. In addition, the geometry of the separator is explicitly intended to be integrated into a fuel-cell stack, providing a direct mate with the fuel cell s existing flow ports. The separator is also fully scalable, allowing it to accommodate a wide range of water removal requirements. Multiple separators can simply be "stacked" in series or parallel to adapt to the water production/removal rate. GRC s separator accomplishes the task of water removal by coupling a high aspect- ratio flow chamber with a highly hydrophilic, polyethersulfone membrane. The hydrophilic membrane readily absorbs and transports the liquid water away from the mixture while simultaneously resisting gas penetration. The expansive flow path maximizes the interaction of the water particles with the membrane while minimizing the overall gas flow restriction. In essence, each fluid takes its corresponding path of least resistance, and the two fluids are effectively separated. The GRC fuel-cell water separator has a broad range of applications, including commercial hydrogen-air fuel cells currently being considered for power generation in automobiles.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

PubMed

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-11-14

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

PubMed Central

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-01-01

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.

PubMed

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-04-13

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of ozone and peroxone on algal separation via dispersed air flotation.

PubMed

Nguyen, Truc Linh; Lee, D J; Chang, J S; Liu, J C

2013-05-01

Effects of pre-oxidation on algal separation by dispersed air flotation were examined. Ozone (O3) and peroxone (O3 and H2O2) could induce cell lysis, release of intracellular organic matter (IOM), and mineralization of organic substances. Separation efficiency of algal cells improved when pre-oxidized. Total of 76.4% algal cells was separated at 40 mg/L of N-cetyl-N-N-N-trimethylammonium bromide (CTAB), while 95% were separated after 30-min ozonation. Pre-oxidation by ozone and peroxone also enhanced flotation separation efficiency of dissolved organic carbon (DOC), polysaccharide, and protein, in which peroxone process exerted more significantly than O3. Two main mechanisms were involved in flotation separation of unoxidized algal suspension, namely hydrophobic cell surface and cell flocculation resulting from CTAB adsorption. However, flocculation by CTAB was hindered for pre-oxidized algal suspensions. It implied that the compositional changes in extracellular organic matter (EOM) by pre-oxidation were more determined for flotation separation of pre-oxidized cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Magnetic-Activated Cell Sorting for the Fast and Efficient Separation of Human and Rodent Schwann Cells from Mixed Cell Populations.

PubMed

Ravelo, Kristine M; Andersen, Natalia D; Monje, Paula V

2018-01-01

To date, magnetic-activated cell sorting (MACS) remains a powerful method to isolate distinct cell populations based on differential cell surface labeling. Optimized direct and indirect MACS protocols for cell immunolabeling are presented here as methods to divest Schwann cell (SC) cultures of contaminating cells (specifically, fibroblast cells) and isolate SC populations at different stages of differentiation. This chapter describes (1) the preparation of single-cell suspensions from established human and rat SC cultures, (2) the design and application of cell selection strategies using SC-specific (p75 NGFR , O4, and O1) and fibroblast-specific (Thy-1) markers, and (3) the characterization of both the pre- and post-sorting cell populations. A simple protocol for the growth of hybridoma cell cultures as a source of monoclonal antibodies for cell surface immunolabeling of SCs and fibroblasts is provided as a cost-effective alternative for commercially available products. These steps allow for the timely and efficient recovery of purified SC populations without compromising the viability and biological activity of the cells.

Fuel cell system with separating structure bonded to electrolyte

DOEpatents

Bourgeois, Richard Scott; Gudlavalleti, Sauri; Quek, Shu Ching; Hasz, Wayne Charles; Powers, James Daniel

2010-09-28

A fuel cell assembly comprises a separating structure configured for separating a first reactant and a second reactant wherein the separating structure has an opening therein. The fuel cell assembly further comprises a fuel cell comprising a first electrode, a second electrode, and an electrolyte interposed between the first and second electrodes, and a passage configured to introduce the second reactant to the second electrode. The electrolyte is bonded to the separating structure with the first electrode being situated within the opening, and the second electrode being situated within the passage.

Stripe-patterned thermo-responsive cell culture dish for cell separation without cell labeling.

PubMed

Kumashiro, Yoshikazu; Ishihara, Jun; Umemoto, Terumasa; Itoga, Kazuyoshi; Kobayashi, Jun; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2015-02-11

A stripe-patterned thermo-responsive surface is prepared to enable cell separation without labeling. The thermo-responsive surface containing a 3 μm striped pattern exhibits various cell adhesion and detachment properties. A mixture of three cell types is separated on the patterned surface based on their distinct cell-adhesion properties, and the composition of the cells is analyzed by flow cytometry. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of relative amounts of ribosome and subunits in Escherichia coli using asymmetrical flow field-flow fractionation.

PubMed

Nilsson, M; Birnbaum, S; Wahlund, K G

1996-10-15

The 30S and 50S subunits and the 70S ribosome of Escherichia coli were separated in 6 minutes by using asymmetrical flow field-flow fractionation (FFF). The total analysis time for determination of the relative amounts of ribosomes and free subunits in a preparation from a cell suspension was 8 min. The method can detect a change in the mass fraction of ribosomes if it exceeds approx, 10%. The separation is based on differences in diffusion coefficients, i.e., hydrodynamic diameters, and these can be determined from observed retention times. The hydrodynamic diameters were in good agreement with literature values obtained from electron microscopy. The mass fraction of ribosomes changed as a function of the magnesium ion concentration which confirms previous knowledge and shows the accuracy of the method. The method appears as an alternative to ultracentrifugation analysis and avoids some of its drawbacks and artefacts. An obvious application can be the optimisation of cell design in metabolic engineering in order to maximise translation and protein production.

On the behavior of reduced graphene oxide based electrodes coated with dispersed platinum by alternate current methods in the electrochemical degradation of reactive dyes.

PubMed

Del Río, A I; García, C; Molina, J; Fernández, J; Bonastre, J; Cases, F

2017-09-01

The electrochemical behavior of different carbon-based electrodes with and without nanoparticles of platinum electrochemically dispersed on their surface has been studied. Among others, reduced graphene oxide based electrodes was used to determine the best conditions for the decolorization/degradation of the reactive dye C.I. Reactive Orange 4 in sulfuric medium. Firstly, the electrochemical behavior was evaluated by cyclic voltammetry. Secondly, different electrolyses were performed using two cell configurations: cell with anodic and cathodic compartments separated (divided configuration) and without any separation (undivided configuration). The best results were obtained when reduced graphene oxide based anodes were used. The degree of decolorization was monitored by spectroscopic methods and high performance liquid chromatography. It was found that all of them followed pseudo-first order kinetics. When reduced graphene oxide-based electrodes coated with dispersed platinum by alternate current methods electrodes were used, the lowest energy consumption and the higher decolorization kinetics rate were obtained. Scanning Electronic Microscopy was used to observe the morphological surface differences. Copyright © 2017 Elsevier Ltd. All rights reserved.

Label-free microfluidic enrichment of ring-stage Plasmodium falciparum-infected red blood cells using non-inertial hydrodynamic lift.

PubMed

Geislinger, Thomas M; Chan, Sherwin; Moll, Kirsten; Wixforth, Achim; Wahlgren, Mats; Franke, Thomas

2014-09-20

Understanding of malaria pathogenesis caused by Plasmodium falciparum has been greatly deepened since the introduction of in vitro culture system, but the lack of a method to enrich ring-stage parasites remains a technical challenge. Here, a novel way to enrich red blood cells containing parasites in the early ring stage is described and demonstrated. A simple, straight polydimethylsiloxane microchannel connected to two syringe pumps for sample injection and two height reservoirs for sample collection is used to enrich red blood cells containing parasites in the early ring stage (8-10 h p.i.). The separation is based on the non-inertial hydrodynamic lift effect, a repulsive cell-wall interaction that enables continuous and label-free separation with deformability as intrinsic marker. The possibility to enrich red blood cells containing P. falciparum parasites at ring stage with a throughput of ~12,000 cells per hour and an average enrichment factor of 4.3 ± 0.5 is demonstrated. The method allows for the enrichment of red blood cells early after the invasion by P. falciparum parasites continuously and without any need to label the cells. The approach promises new possibilities to increase the sensitivity of downstream analyses like genomic- or diagnostic tests. The device can be produced as a cheap, disposable chip with mass production technologies and works without expensive peripheral equipment. This makes the approach interesting for the development of new devices for field use in resource poor settings and environments, e.g. with the aim to increase the sensitivity of microscope malaria diagnosis.

High-throughput separation of cells by dielectrophoresis enhanced with 3D gradient AC electric field.

PubMed

Tada, Shigeru; Hayashi, Masako; Eguchi, Masanori; Tsukamoto, Akira

2017-11-01

We propose a novel, high-performance dielectrophoretic (DEP) cell-separation flow chamber with a parallel-plate channel geometry. The flow chamber, consisting of a planar electrode on the top and an interdigitated-pair electrode array at the bottom, was developed to facilitate the separation of cells by creating a nonuniform AC electric field throughout the volume of the flow chamber. The operation and performance of the device were evaluated using live and dead human epithermal breast (MCF10A) cells. The separation dynamics of the cell suspension in the flow chamber was also investigated by numerically simulating the trajectories of individual cells. A theoretical model to describe the dynamic cell behavior under the action of DEP, including dipole-dipole interparticle, viscous, and gravitational forces, was developed. The results demonstrated that the live cells traveling through the flow chamber congregated into sites where the electric field gradient was minimal, in the middle of the flow stream slightly above the centerlines of the grounded electrodes at the bottom. Meanwhile, the dead cells were trapped on the edges of the high-voltage electrodes at the bottom. Cells were thus successfully separated with a remarkably high separation ratio (∼98%) at the appropriately tuned field frequency and applied voltage. The numerically predicted behavior and spatial distribution of the cells during separation also showed good agreement with those observed experimentally.

High-Density Droplet Microarray of Individually Addressable Electrochemical Cells.

PubMed

Zhang, Huijie; Oellers, Tobias; Feng, Wenqian; Abdulazim, Tarik; Saw, En Ning; Ludwig, Alfred; Levkin, Pavel A; Plumeré, Nicolas

2017-06-06

Microarray technology has shown great potential for various types of high-throughput screening applications. The main read-out methods of most microarray platforms, however, are based on optical techniques, limiting the scope of potential applications of such powerful screening technology. Electrochemical methods possess numerous complementary advantages over optical detection methods, including its label-free nature, capability of quantitative monitoring of various reporter molecules, and the ability to not only detect but also address compositions of individual compartments. However, application of electrochemical methods for the purpose of high-throughput screening remains very limited. In this work, we develop a high-density individually addressable electrochemical droplet microarray (eDMA). The eDMA allows for the detection of redox-active reporter molecules irrespective of their electrochemical reversibility in individual nanoliter-sized droplets. Orthogonal band microelectrodes are arranged to form at their intersections an array of three-electrode systems for precise control of the applied potential, which enables direct read-out of the current related to analyte detection. The band microelectrode array is covered with a layer of permeable porous polymethacrylate functionalized with a highly hydrophobic-hydrophilic pattern, forming spatially separated nanoliter-sized droplets on top of each electrochemical cell. Electrochemical characterization of single droplets demonstrates that the underlying electrode system is accessible to redox-active molecules through the hydrophilic polymeric pattern and that the nonwettable hydrophobic boundaries can spatially separate neighboring cells effectively. The eDMA technology opens the possibility to combine the high-throughput biochemical or living cell screenings using the droplet microarray platform with the sequential electrochemical read-out of individual droplets.

Matrix-assisted laser desorption/ionization mass spectrometry for the evaluation of the C-terminal lysine distribution of a recombinant monoclonal antibody.

PubMed

Lazar, Alexandru C; Kloczewiak, Marek A; Mazsaroff, Istvan

2004-01-01

Recombinant monoclonal antibodies produced using mammalian cell lines contain multiple chemical modifications. One specific modification resides on the C-terminus of the heavy chain. Enzymes inside the cell can cleave the C-terminal lysine from the heavy-chain molecules, and variants with and without C-terminal lysine can be produced. In order to fully characterize the protein, there is a need for analytical methods that are able to account for the different product variants. Conventional analytical methods used for the measurement of the distribution of the two different variants are based on chemical or enzymatic degradation of the protein followed by chromatographic separation of the degradation products. Chromatographic separations with gradient elution have long run times, and analyses of multiple samples are time-consuming. This paper reports development of a novel method for the determination of the relative amounts of the two C-terminal heavy-chain variants based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) measurements of the cyanogen bromide degraded recombinant monoclonal antibody products. The distribution of the variants is determined from the MALDI-TOF mass spectra by measuring the peak areas of the two C-terminal peptides. The assay was used for the assessment of the C-terminal lysine distribution in different development lots. The method was able to differentiate between the products obtained using the same cell line as well as between products obtained from different cell lines. Copyright 2004 John Wiley & Sons, Ltd.

Prenatal diagnosis of hemoglobinopathies: evaluation of techniques for analysing globin-chain synthesis in blood samples obtained by fetoscopy.

PubMed Central

Congote, L. F.; Hamilton, E. F.; Chow, J. C.; Perry, T. B.

1982-01-01

Three techniques for analysing hemoglobin synthesis in blood samples obtained by fetoscopy were evaluated. Of the fetuses studied, 12 were not at risk of genetic disorders, 10 were at risk of beta-thalassemia, 2 were at risk of sickle cell anemia and 1 was at risk of both diseases. The conventional method of prenatal diagnosis of hemoglobinopathies, involving the separation of globin chains labelled with a radioactive isotope on carboxymethyl cellulose (CMC) columns, was compared with a method involving globin-chain separation by high-pressure liquid chromatography (HPLC) and with direct analysis of labelled hemoglobin tetramers obtained from cell lysates by chromatography on ion-exchange columns. The last method is technically the simplest and can be used for diagnosing beta-thalassemia and sickle cell anemia. However, it gives spuriously high levels of adult hemoglobin in samples containing nonlabelled adult hemoglobin. HPLC is the fastest method for prenatal diagnosis of beta-thalassemia and may prove as reliable as the CMC method. Of the 13 fetuses at risk for hemoglobinopathies, 1 was predicted to be affected, and the diagnosis was confirmed in the abortus. Of 12 predicted to be unaffected, 1 was aborted spontaneously and was unavailable for confirmatory studies, as were 3 of the infants; however, the diagnosis was confirmed in seven cases and is awaiting confirmation when the infant in 6 months old in one case. Couples at risk of bearing a child with a hemoglobinopathy should be referred for genetic counselling before pregnancy or, at the latest, by the 12th week of gestation so that prenatal diagnosis can be attempted by amniocentesis, safer procedure, with restriction endonuclease analysis of the amniotic fluid cells. PMID:7139502

Methods for natural gas and heavy hydrocarbon co-conversion

DOEpatents

Kong, Peter C [Idaho Falls, ID; Nelson, Lee O [Idaho Falls, ID; Detering, Brent A [Idaho Falls, ID

2009-02-24

A reactor for reactive co-conversion of heavy hydrocarbons and hydrocarbon gases and includes a dielectric barrier discharge plasma cell having a pair of electrodes separated by a dielectric material and passageway therebetween. An inlet is provided for feeding heavy hydrocarbons and other reactive materials to the passageway of the discharge plasma cell, and an outlet is provided for discharging reaction products from the reactor. A packed bed catalyst may optionally be used in the reactor to increase efficiency of conversion. The reactor can be modified to allow use of a variety of light sources for providing ultraviolet light within the discharge plasma cell. Methods for upgrading heavy hydrocarbons are also disclosed.

Nonthermal plasma systems and methods for natural gas and heavy hydrocarbon co-conversion

DOEpatents

Kong, Peter C.; Nelson, Lee O.; Detering, Brent A.

2005-05-24

A reactor for reactive co-conversion of heavy hydrocarbons and hydrocarbon gases and includes a dielectric barrier discharge plasma cell having a pair of electrodes separated by a dielectric material and passageway therebetween. An inlet is provided for feeding heavy hydrocarbons and other reactive materials to the passageway of the discharge plasma cell, and an outlet is provided for discharging reaction products from the reactor. A packed bed catalyst may optionally be used in the reactor to increase efficiency of conversion. The reactor can be modified to allow use of a variety of light sources for providing ultraviolet light within the discharge plasma cell. Methods for upgrading heavy hydrocarbons are also disclosed.

Quantitative phase microscopy for cellular dynamics based on transport of intensity equation.

PubMed

Li, Ying; Di, Jianglei; Ma, Chaojie; Zhang, Jiwei; Zhong, Jinzhan; Wang, Kaiqiang; Xi, Teli; Zhao, Jianlin

2018-01-08

We demonstrate a simple method for quantitative phase imaging of tiny transparent objects such as living cells based on the transport of intensity equation. The experiments are performed using an inverted bright field microscope upgraded with a flipping imaging module, which enables to simultaneously create two laterally separated images with unequal defocus distances. This add-on module does not include any lenses or gratings and is cost-effective and easy-to-alignment. The validity of this method is confirmed by the measurement of microlens array and human osteoblastic cells in culture, indicating its potential in the applications of dynamically measuring living cells and other transparent specimens in a quantitative, non-invasive and label-free manner.

Two-stage microfluidic chip for selective isolation of circulating tumor cells (CTCs).

PubMed

Hyun, Kyung-A; Lee, Tae Yoon; Lee, Su Hyun; Jung, Hyo-Il

2015-05-15

Over the past few decades, circulating tumor cells (CTCs) have been studied as a means of overcoming cancer. However, the rarity and heterogeneity of CTCs have been the most significant hurdles in CTC research. Many techniques for CTC isolation have been developed and can be classified into positive enrichment (i.e., specifically isolating target cells using cell size, surface protein expression, and so on) and negative enrichment (i.e., specifically eluting non-target cells). Positive enrichment methods lead to high purity, but could be biased by their selection criteria, while the negative enrichment methods have relatively low purity, but can isolate heterogeneous CTCs. To compensate for the known disadvantages of the positive and negative enrichments, in this study we introduced a two-stage microfluidic chip. The first stage involves a microfluidic magnetic activated cell sorting (μ-MACS) chip to elute white blood cells (WBCs). The second stage involves a geometrically activated surface interaction (GASI) chip for the selective isolation of CTCs. We observed up to 763-fold enrichment in cancer cells spiked into 5 mL of blood sample using the μ-MACS chip at 400 μL/min flow rate. Cancer cells were successfully separated with separation efficiencies ranging from 10.19% to 22.91% based on their EpCAM or HER2 surface protein expression using the GASI chip at a 100 μL/min flow rate. Our two-stage microfluidic chips not only isolated CTCs from blood cells, but also classified heterogeneous CTCs based on their characteristics. Therefore, our chips can contribute to research on CTC heterogeneity of CTCs, and, by extension, personalized cancer treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

Determination of residual cell culture media components by MEKC.

PubMed

Zhang, Junge; Chakraborty, Utpal; Foley, Joe P

2009-11-01

Folic acid, hypoxanthine, mycophenolic acid, nicotinic acid, riboflavin, and xanthine are widely used as cell culture media components in monoclonal antibody manufacturing. These components are subsequently removed during the downstream purification processes. This article describes a single MEKC method that can simultaneously determine all the listed compounds with acceptable LOD and LOQ. All the analytes were successfully separated by MEKC using running buffer containing 40 mM SDS, 20 mM sodium phosphate, and 20 mM sodium borate at pH 9.0. The MEKC method was compared to the corresponding CZE method using the same running buffer containing no SDS. The effect of SDS concentration on separation, the pH of the running buffer, and the detection wavelength were studied and optimal MEKC conditions were established. Good linearity was obtained with correlation coefficients of more than 0.99 for all analytes. Specificity, accuracy, and precision were also evaluated. The recovery was in the range of 89-112%. The precision results were in the range of 1.7-4.8%. The experimentally determined data demonstrated that the MEKC method is applicable to the determination of the six analytes in in-process samples from monoclonal antibody manufacturing processes.

Flow cytometry for receptor analysis from ex-vivo brain tissue in adult rat.

PubMed

Benoit, A; Guillamin, M; Aitken, P; Smith, P F; Philoxene, B; Sola, B; Poulain, L; Coquerel, A; Besnard, S

2018-07-01

Flow cytometry allows single-cell analysis of peripheral biological samples and is useful in many fields of research and clinical applications, mainly in hematology, immunology, and oncology. In the neurosciences, the flow cytometry separation method was first applied to stem cell extraction from healthy or cerebral tumour tissue and was more recently tested in order to phenotype brain cells, hippocampal neurogenesis, and to detect prion proteins. However, it remains sparsely applied in quantifying membrane receptors in relation to synaptic plasticity. We aimed to optimize a flow cytometric procedure for receptor quantification in neurons and non-neurons. A neural dissociation process, myelin separation, fixation, and membrane permeability procedures were optimized to maximize cell survival and analysis in hippocampal tissue obtained from adult rodents. We then aimed to quantify membrane muscarinic acetylcholine receptors (mAChRs) in rats with and without bilateral vestibular loss (BVL). mAChR's were quantified for neuronal and non-neuronal cells in the hippocampus and striatum following BVL. At day 30 but not at day 7 following BVL, there was a significant increase (P ≤ 0.05) in the percentage of neurons expressing M 2/4 mAChRs in both the hippocampus and the striatum. Here, we showed that flow cytometry appears to be a reliable method of membrane receptor quantification in ex-vivo brain tissue. Copyright © 2018 Elsevier B.V. All rights reserved.

Observation of Bright Ring Phenomenon for Red Blood Cells by Lattice Boltzmann Method

NASA Astrophysics Data System (ADS)

Kim, Young Woo; Moon, Ji Young; Lee, Joon Sang

2017-11-01

RBC (Red Blood Cell) aggregation is one of interests for various biomechanical fields such as cell chip or visualization. The unique phenomenon called ``bright ring'' is due to RBC aggregation in pulsatile flow of blood. Shear rate and flow acceleration on RBC causes them to repeat aggregating and scattering from center of the channel. The reason that this phenomenon is called bright ring is because that when observed by ultrasound imaging, the bright ring occurs periodically. Many studies tried to observe this bright ring phenomenon experimentally. However, there are yet not many studies trying to make use of this phenomenon for practical purposes. Bright ring phenomenon has high potential when used for cell separation or other microchip devices. In this paper, the Lattice Boltzmann method is used to control this bright ring phenomenon. The purpose of this paper is to find conditions when bright ring phenomenon occurs, and to control the aggregating-scattering frequency and degree. Deformability of RBC is calculated following the work of Moon JY et al. (2016). The result of this paper could be further extended to the optimization of cell-separating microchips. This work was also supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (No. 2015R1A5A1037668) and Brain Korea 21 Plus.

Tailored Core Shell Cathode Powders for Solid Oxide Fuel Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swartz, Scott

2015-03-23

In this Phase I SBIR project, a “core-shell” composite cathode approach was evaluated for improving SOFC performance and reducing degradation of lanthanum strontium cobalt ferrite (LSCF) cathode materials, following previous successful demonstrations of infiltration approaches for achieving the same goals. The intent was to establish core-shell cathode powders that enabled high performance to be obtained with “drop-in” process capability for SOFC manufacturing (i.e., rather than adding an infiltration step to the SOFC manufacturing process). Milling, precipitation and hetero-coagulation methods were evaluated for making core-shell composite cathode powders comprised of coarse LSCF “core” particles and nanoscale “shell” particles of lanthanum strontiummore » manganite (LSM) or praseodymium strontium manganite (PSM). Precipitation and hetero-coagulation methods were successful for obtaining the targeted core-shell morphology, although perfect coverage of the LSCF core particles by the LSM and PSM particles was not obtained. Electrochemical characterization of core-shell cathode powders and conventional (baseline) cathode powders was performed via electrochemical impedance spectroscopy (EIS) half-cell measurements and single-cell SOFC testing. Reliable EIS testing methods were established, which enabled comparative area-specific resistance measurements to be obtained. A single-cell SOFC testing approach also was established that enabled cathode resistance to be separated from overall cell resistance, and for cathode degradation to be separated from overall cell degradation. The results of these EIS and SOFC tests conclusively determined that the core-shell cathode powders resulted in significant lowering of performance, compared to the baseline cathodes. Based on the results of this project, it was concluded that the core-shell cathode approach did not warrant further investigation.« less

Dual-wavelength common-path digital holographic microscopy for quantitative phase imaging of biological cells

NASA Astrophysics Data System (ADS)

Di, Jianglei; Song, Yu; Xi, Teli; Zhang, Jiwei; Li, Ying; Ma, Chaojie; Wang, Kaiqiang; Zhao, Jianlin

2017-11-01

Biological cells are usually transparent with a small refractive index gradient. Digital holographic interferometry can be used in the measurement of biological cells. We propose a dual-wavelength common-path digital holographic microscopy for the quantitative phase imaging of biological cells. In the proposed configuration, a parallel glass plate is inserted in the light path to create the lateral shearing, and two lasers with different wavelengths are used as the light source to form the dual-wavelength composite digital hologram. The information of biological cells for different wavelengths is separated and extracted in the Fourier domain of the hologram, and then combined to a shorter wavelength in the measurement process. This method could improve the system's temporal stability and reduce speckle noises simultaneously. Mouse osteoblastic cells and peony pollens are measured to show the feasibility of this method.

Optimization of yield in magnetic cell separations using nickel nanowires of different lengths.

PubMed

Hultgren, Anne; Tanase, Monica; Felton, Edward J; Bhadriraju, Kiran; Salem, Aliasger K; Chen, Christopher S; Reich, Daniel H

2005-01-01

Ferromagnetic nanowires are shown to perform both high yield and high purity single-step cell separations on cultures of NIH-3T3 mouse fibroblast cells. The nanowires are made by electrochemical deposition in nanoporous templates, permitting detailed control of their chemical and physical properties. When added to fibroblast cell cultures, the nanowires are internalized by the cells via the integrin-mediated adhesion pathway. The effectiveness of magnetic cell separations using Ni nanowires 350 nm in diameter and 5-35 micrometers long in field gradients of 40 T/m was compared to commercially available superparamagnetic beads. The percent yield of the separated populations is found to be optimized when the length of the nanowire is matched to the diameter of the cells in the culture. Magnetic cell separations performed under these conditions achieve 80% purity and 85% yield, a 4-fold increase over the beads. This effect is shown to be robust when the diameter of the cell is changed within the same cell line using mitomycin-C.

Innovative heating of large-size automotive Li-ion cells

NASA Astrophysics Data System (ADS)

Yang, Xiao-Guang; Liu, Teng; Wang, Chao-Yang

2017-02-01

Automotive Li-ion cells are becoming much larger and thicker in order to reduce the cell count and increase battery reliability, posing a new challenge to battery heating from the cold ambient due to poor through-plane heat transfer across a cell's multiple layers of electrodes and separators. In this work, widely used heating methods, including internal heating using the cell's resistance and external heating by resistive heaters, are compared with the recently developed self-heating Li-ion battery (SHLB) with special attention to the heating speed and maximum local temperature critical to battery safety. Both conventional methods are found to be slow due to low heating power required to maintain battery safety. The heating power in the external heating method is limited by the risk of local over-heating, in particular for thick cells. As a result, the external heating method is restricted to ∼20 min slow heating for a 30 °C temperature rise. In contrast, the SHLB is demonstrated to reach a heating speed of 1-2 °C/sec, ∼40 times faster for large-size thick cells, with nearly 100% heating efficiency and spatially uniform heating free from safety concerns.

Free flow electrophoresis in space shuttle program (biotex)

NASA Astrophysics Data System (ADS)

Hannig, Kurt; Bauer, Johann

In the space shuttle program free flow electrophoresis will be applied for separation of proteins, biopolymers and cells. Proteins are to be separated according to the ``Feldsprung-Gradienten'' procedure by Prof. H. Wagner, University of Saarbruecken, biopolymers are to be separated by the isotachophoresis technique by Prof. Schmitz, University of Muenster and we intend to separate cells in order to increase the efficiency of recovery of hybrid cells after electrofusion performed under microgravity in collaboration with Prof. U. Zimmermann, University of Wuerzburg. There are supposed two ways for reaching this goal: Enrichment of cells before electrofusion may enhance the probability that the cells of interest are immortalized. Separation of cells after electrofusion may help to clone the hybrid cells of interest. Under microgravity, the combination of improved electrophoresis with higher electrofusion rates may provide new possibilities for immortalization of cells. This may be a new way to obtain cellular products, which are physiologically glycosylated.

Semi-solid electrode cell having a porous current collector and methods of manufacture

DOEpatents

Chiang, Yet-Ming; Carter, William Craig; Cross, III, James C.; Bazzarella, Ricardo; Ota, Naoki

2017-11-21

An electrochemical cell includes an anode, a semi-solid cathode, and a separator disposed therebetween. The semi-solid cathode includes a porous current collector and a suspension of an active material and a conductive material disposed in a non-aqueous liquid electrolyte. The porous current collector is at least partially disposed within the suspension such that the suspension substantially encapsulates the porous current collector.

[Screening of anti-lung cancer bioactive compounds from Curcuma longa by target cell extraction and UHPLC/LTQ Orbitrap MS].

PubMed

Zhou, Jian-Liang; Wu, Ye-Qing; Tan, Chun-Mei; Zhu, Ming; Ma, Lin-Ke

2016-10-01

A target cell extraction-chemical profiling method based on human alveolar adenocarcinoma cell line (A549 cells) and UHPLC/LTQ Orbitrap MS for screening the anti-lung cancer bioactive compounds from Curcuma longa has been developed in this paper. According to the hypothesis that when cells are incubated together with the extract of Curcuma longa, the potential bioactive compounds in the extract should selectively combine with the cells, then the cell-binding compounds could be separated and analyzed by LC-MS. The bioactive compounds in C. longa are lipophilic components. They intend to be absorbed on the inner wall of cell culture flask when they were incubated with A549 cells, which will produce interference in the blank solution. In this paper, by using cells digestion and multi-step centrifugation and transfer strategy, the interference problem has been solved. Finally, using the developed method, three cell-binding compounds were screened out and were identified as bisdemethoxycurcumin, demethoxycurcumin, and curcumin. These compounds are the main bioactive compounds with anti-lung cancer bioactivity in C. longa. The improved method developed in this paper could avoid the false positive results due to the absorption of lipophilic compounds on the inner wall of cell culture flask, which will to be an effective complementary method for current target cell extraction-chemical profiling technology. Copyright© by the Chinese Pharmaceutical Association.

A method for isolation of rat lymphocyte-rich mononuclear cells from lung tissue useful for determination of nucleoside triphosphate diphosphohydrolase activity.

PubMed

Jaques, Jeandre Augusto Dos S; Peres Rezer, João Felipe; Ruchel, Jader Betsch; Gutierres, Jessié; Bairros, André Valle; Gomes Farias, Iria Luiza; Almeida da Luz, Sonia Cristina; Mello Bertoncheli, Claudia de; Chitolina Schetinger, Maria Rosa; Morsch, Vera Maria; Leal, Daniela Bitencourt Rosa

2011-03-01

Methods for the isolation of peripheral blood mononuclear cells (PBMCs) and human lung mononuclear cells (LMCs) have been proposed previously. This study describes a method that allows the separation of lymphocyte-rich LMCs from rats. Trypan blue was applied to determine cell viability. White blood cell and differential cell counts were also performed. Relationships between nucleoside triphosphate diphosphohydrolase (NTPDase, EC 3.6.1.5) activities expressed in milligrams of protein, millions of cells, and millions of viable cells were examined as linear correlations. The lung tissue yielded 82.46% lymphocytes, 8.6% macrophages, 2.20% monocytes, and 1.27% polymorphonuclear cells (PMNs). In LMCs, a very strong correlation was observed as follows: between NTPDase activity, as determined using ATP or ADP as a substrate, expressed in milligrams of protein and that expressed in millions of cells (r ≥ 0.91), between that expressed in milligrams of protein and that expressed in millions of viable cells (r ≥ 0.91), and between that expressed in millions of cells and that expressed in millions of viable cells (r ≥ 0.98). Based on our results, we affirm that NTPDase activity could be expressed in millions of viable cells, millions of cells, or milligrams of protein. 2010 Elsevier Inc. All rights reserved.

Red blood cell transport mechanisms in polyester thread-based blood typing devices.

PubMed

Nilghaz, Azadeh; Ballerini, David R; Guan, Liyun; Li, Lizi; Shen, Wei

2016-02-01

A recently developed blood typing diagnostic based on a polyester thread substrate has shown great promise for use in medical emergencies and in impoverished regions. The device is easy to use and transport, while also being inexpensive, accurate, and rapid. This study used a fluorescent confocal microscope to delve deeper into how red blood cells were behaving within the polyester thread-based diagnostic at the cellular level, and how plasma separation could be made to visibly occur on the thread, making it possible to identify blood type in a single step. Red blood cells were stained and the plasma phase dyed with fluorescent compounds to enable them to be visualised under the confocal microscope at high magnification. The mechanisms uncovered were in surprising contrast with those found for a similar, paper-based method. Red blood cell aggregates did not flow over each other within the thread substrate as expected, but suffered from a restriction to their flow which resulted in the chromatographic separation of the RBCs from the liquid phase of the blood. It is hoped that these results will lead to the optimisation of the method to enable more accurate and sensitive detection, increasing the range of blood systems that can be detected.

Improved native UV laser induced fluorescence detection for single cell analysis in poly(dimethylsiloxane) microfluidic devices.

PubMed

Hellmich, Wibke; Greif, Dominik; Pelargus, Christoph; Anselmetti, Dario; Ros, Alexandra

2006-10-20

Single cell analytics is a key method in the framework of proteom research allowing analyses, which are not subjected to ensemble-averaging, cell-cycle or heterogeneous cell-population effects. Our previous studies on single cell analysis in poly(dimethylsiloxane) microfluidic devices with native label-free laser induced fluorescence detection [W. Hellmich, C. Pelargus, K. Leffhalm, A. Ros, D. Anselmetti, Electrophoresis 26 (2005) 3689] were extended in order to improve separation efficiency and detection sensitivity. Here, we particularly focus on the influence of poly(oxyethylene) based coatings on the separation performance. In addition, the influence on background fluorescence is studied by the variation of the incident laser power as well as the adaptation of the confocal volume to the microfluidic channel dimensions. Last but not least, the use of carbon black particles further enhanced the detection limit to 25 nM, thereby reaching the relevant concentration ranges necessary for the label-free detection of low abundant proteins in single cells. On the basis of these results, we demonstrate the first electropherogram from an individual Spodoptera frugiperda (Sf9) cell with native label-free UV-LIF detection in a microfluidic chip.

Streamline-based microfluidic device

NASA Technical Reports Server (NTRS)

Tai, Yu-Chong (Inventor); Zheng, Siyang (Inventor); Kasdan, Harvey (Inventor)

2013-01-01

The present invention provides a streamline-based device and a method for using the device for continuous separation of particles including cells in biological fluids. The device includes a main microchannel and an array of side microchannels disposed on a substrate. The main microchannel has a plurality of stagnation points with a predetermined geometric design, for example, each of the stagnation points has a predetermined distance from the upstream edge of each of the side microchannels. The particles are separated and collected in the side microchannels.

The separation of leukotrienes and hydroxyeicosatetraenoic acid metabolites of arachidonic acid by high performance liquid chromatography (HPLC).

PubMed

Osborne, D J; Peters, B J; Meade, C J

1983-11-01

The following high performance liquid chromatography system was found suitable for separating most lipoxygenase metabolites of arachidonic acid: Techsphere 5-C18 column, eluting solvent methanol:water:acetic acid (65:35:0.06 v/v), pH 5.3. Comparisons with other packing materials and solvent systems are described. The method could be used to identify lipoxygenase products released from mouse macrophage cells stimulated with gamma-hexachlorocyclohexane. Detection limits between 1 and 10 ng were obtained.

Recycling of CdTe photovoltaic waste

DOEpatents

Goozner, Robert E.; Long, Mark O.; Drinkard, Jr., William F.

1999-04-27

A method for extracting and reclaiming metals from scrap CdTe photovoltaic cells and manufacturing waste by leaching the metals in dilute nitric acid, leaching the waste with a leaching solution comprising nitric acid and water, skimming any plastic material from the top of the leaching solution, separating the glass substrate from the liquid leachate, adding a calcium containing base to the leachate to precipitate Cd and Te, separating the precipitated Cd and Te from the leachate, and recovering the calcium-containing base.

Recycling of CdTe photovoltaic waste

DOEpatents

Goozner, R.E.; Long, M.O.; Drinkard, W.F. Jr.

1999-04-27

A method for extracting and reclaiming metals from scrap CdTe photovoltaic cells and manufacturing waste by leaching the metals in dilute nitric acid, leaching the waste with a leaching solution comprising nitric acid and water, skimming any plastic material from the top of the leaching solution, separating the glass substrate from the liquid leachate, adding a calcium containing base to the leachate to precipitate Cd and Te, separating the precipitated Cd and Te from the leachate, and recovering the calcium-containing base. 3 figs.

Microtubules as platforms for probing liquid-liquid phase separation in cells: application to RNA-binding proteins.

PubMed

Maucuer, Alexandre; Desforges, Bénédicte; Joshi, Vandana; Boca, Mirela; Kretov, Dmitry; Hamon, Loic; Bouhss, Ahmed; Curmi, Patrick A; Pastré, David

2018-05-04

Liquid-liquid phase separation enables compartmentalization of biomolecules in cells, notably RNA and associated proteins in the nucleus. Besides critical functions in RNA processing, there is a major interest in deciphering the molecular mechanisms of compartmentalization orchestrated by RNA-binding proteins such as TDP-43 and FUS due to their link to neuron diseases. However, tools for probing compartmentalization in cells are lacking. Here we developed a method to analyze the mixing:demixing of two different phases in a cellular context. The principle is the following: mRNA-binding proteins are confined on microtubules and quantitative parameters defining their spatial segregation are measured along the microtubule network. Through this approach, we found that four mRNA binding proteins, HuR, G3BP1, TDP-43 and FUS form mRNA-rich liquid-like compartments on microtubules. TDP-43 is partly miscible with FUS but immiscible with either HuR or G3BP1. We also demonstrate that mRNA is essential to capture the mixing:demixing behavior of RNA-binding proteins in cells. Altogether we show that microtubules can be used as platforms to understand the mechanisms underlying liquid-liquid phase separation and their deregulation in human diseases. © 2018. Published by The Company of Biologists Ltd.

Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: electrophoretic mobility studies.

PubMed

Wangsa-Wirawan, N D; O'Neill, B K; Middelberg, A P

2001-01-01

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.

DNA-protein crosslinking by trans-platinum(II)diamminedichloride in mammalian cells, a new method of analysis.

PubMed

Kohn, K W; Ewig, R A

1979-03-28

DNA-protien crosslinks produced in mouse leukemia L1210 cells by trans-Pt(II)diamminedichloride were quantitated using the technique of DNA alkaline elution. DNA single-strand segments that were or were not linked to protein were separable into distinct components by alkaline elution after exposure of the cells to 2--15 kR of X-ray. Protein-linked DNA strands were separated on the basis of their retention of filters at pH 12 while free DNA strands of the size generated by 2--15 kR of X-ray passed rapidly through the filters. The retention of protein-linked DNA strands was attributable to adsorption of protein to the filter under the conditions of alkaline elution. The results obeyed a simple quantitative model according to which the frequency of DNA-protein crosslinks could be calculated.

Advances in microfluidic devices made from thermoplastics used in cell biology and analyses.

PubMed

Gencturk, Elif; Mutlu, Senol; Ulgen, Kutlu O

2017-09-01

Silicon and glass were the main fabrication materials of microfluidic devices, however, plastics are on the rise in the past few years. Thermoplastic materials have recently been used to fabricate microfluidic platforms to perform experiments on cellular studies or environmental monitoring, with low cost disposable devices. This review describes the present state of the development and applications of microfluidic systems used in cell biology and analyses since the year 2000. Cultivation, separation/isolation, detection and analysis, and reaction studies are extensively discussed, considering only microorganisms (bacteria, yeast, fungi, zebra fish, etc.) and mammalian cell related studies in the microfluidic platforms. The advantages/disadvantages, fabrication methods, dimensions, and the purpose of creating the desired system are explained in detail. An important conclusion of this review is that these microfluidic platforms are still open for research and development, and solutions need to be found for each case separately.

[Isolation and purification of nonspecific nuclease of cyanobacterium Plectonema boryanum CALU 465].

PubMed

Tsymbal, N V; Samoĭlenko, V A; Syrchin, S A; Mendzhul, M I

2004-01-01

Nonspecific nuclease has been isolated from the cells of cyanobacterium Plectonema boryanum and purified to homogenic state. It has been established that the method of centrifugation of cell-free culture extract in the sucrose density gradient is efficient for the separation of pigment proteins and enzyme concentration. Under the successive use of two ion-exchangers the nuclease activity was determined in the concentration range of NaCl 0.065-0.085 M after separation of the cell-free cyanobacterium extract on the column with phosphocellulose in the range of 0.2-0.25 M, on the column with DEAE--Toyopearl respectively. The molecular mass of nuclease which is 40 kDa, has been determined by electrophoresis in polyacrylamide gel under denaturating conditions and gel-filtration on Sephadex G-100. It has been also established that the given enzyme is monosubunitary as to its structure.

Multi-stage microbial system for continuous hydrogen production

DOEpatents

Kosourov, Sergey; Ghirardi, Maria L.; Seibert, Michael

2010-06-08

A method of using sequential chemostat culture vessels to provide continuous H.sub.2 production, in which photosynthetic O.sub.2 evolution and H.sub.2 photoproduction are separated physically into two separate bioreactors, comprising: a) growing a microorganism culture able to continuously generate H.sub.2 by photosynthetically producing cells at about the early-to-late log state in a first photobioreactor operating as a sulfur chemostat under aerobic and/or conditions; b) continuously feeding cells from the first photobioreactor to a second photobioreactor operating under anaerobic conditions and sulfur deprivation conditions resulting from constant uptake of sulfate in the first bioreactor and a low rate of culture flow between the first and second bioreactors, for induction of hydrogenase and H.sub.2 photoproduction to allow for continuous cultivation of the microorganism's cells in the first photobioreactor and constant H.sub.2 production in the second photobioreactor, and c) H.sub.2 gas from the second photobioreactor.

Role of pectolytic enzymes in the programmed separation of cells from the root cap of higher plants. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hawes, M.C.

1995-03-01

The objective of this research was to develop a model system to study border cell separation in transgenic pea roots. In addition, the hypothesis that genes encoding pectolytic enzymes in the root cap play a role in the programmed separation of root border cells from the root tip was tested. The following objectives have been accomplished: (1) the use of transgenic hairy roots to study border cell separation has been optimized for Pisum sativum; (2) a cDNA encoding a root cap pectinmethylesterase (PME) has been cloned; (3) PME and polygalacturonase activities in cell walls of the root cap have beenmore » characterized and shown to be correlated with border cell separation. A fusion gene encoding pectate lyase has also been transformed into pea hairy root cells.« less

Electrolytic method to make alkali alcoholates using ion conducting alkali electrolyte/separator

DOEpatents

Joshi, Ashok V [Salt Lake City, UT; Balagopal, Shekar [Sandy, UT; Pendelton, Justin [Salt Lake City, UT

2011-12-13

Alkali alcoholates, also called alkali alkoxides, are produced from alkali metal salt solutions and alcohol using a three-compartment electrolytic cell. The electrolytic cell includes an anolyte compartment configured with an anode, a buffer compartment, and a catholyte compartment configured with a cathode. An alkali ion conducting solid electrolyte configured to selectively transport alkali ions is positioned between the anolyte compartment and the buffer compartment. An alkali ion permeable separator is positioned between the buffer compartment and the catholyte compartment. The catholyte solution may include an alkali alcoholate and alcohol. The anolyte solution may include at least one alkali salt. The buffer compartment solution may include a soluble alkali salt and an alkali alcoholate in alcohol.

Differentiation of Bovine Spermatogonial Stem Cells into Osteoblasts

PubMed Central

Qasemi-Panahi, Babak; Tajik, Parviz; Movahedin, Mansoureh; Moghaddam, Gholamali; Barzgar, Younes; Heidari-Vala, Hamed

2011-01-01

Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 days. Sertoli cells and SSCs were identified by Vimentin and Oct-4 immunocytochemical staining method, respectively. In order to differentiate SSCs into osteoblasts, we used consecutive inducer media without separation of the colonies. We characterized osteoblasts using Alizarin red staining. PMID:23408761

Differentiation of bovine spermatogonial stem cells into osteoblasts.

PubMed

Qasemi-Panahi, Babak; Tajik, Parviz; Movahedin, Mansoureh; Moghaddam, Gholamali; Barzgar, Younes; Heidari-Vala, Hamed

2011-07-01

Spermatogonial Stem Cell (SSC) technologies provide multiple opportunities for research in the field of biotechnology and regenerative medicine. The therapeutic use of Embryonic Stem Cells (ESCs) is restricted due to severe ethical and immunological concerns. Therefore, we need a new pluripotent cell type. Despite well-known role of germ cells in the gametogenesis, some facts apparently show their multipotentiality. In the present study, bovine SSCs were co-cultured with Sertoli cell for 7 days. Sertoli cells and SSCs were identified by Vimentin and Oct-4 immunocytochemical staining method, respectively. In order to differentiate SSCs into osteoblasts, we used consecutive inducer media without separation of the colonies. We characterized osteoblasts using Alizarin red staining.

Analysis of glycosaminoglycan-derived disaccharides by capillary electrophoresis using laser-induced fluorescence detection

PubMed Central

Chang, Yuqing; Yang, Bo; Zhao, Xue; Linhardt, Robert J.

2012-01-01

A quantitative and highly sensitive method for the analysis of glycosaminoglycan (GAG)-derived disaccharides is presented that relies on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. This method enables complete separation of seventeen GAG-derived disaccharides in a single run. Unsaturated disaccharides were derivatized with 2-aminoacridone (AMAC) to improve sensitivity. The limit of detection was at the attomole level and about 100-fold more sensitive than traditional CE-ultraviolet detection. A CE separation timetable was developed to achieve complete resolution and shorten analysis time. The RSD of migration time and peak areas at both low and high concentrations of unsaturated disaccharides are all less than 2.7% and 3.2%, respectively, demonstrating that this is a reproducible method. This analysis was successfully applied to cultured Chinese hamster ovary cell samples for determination of GAG disaccharides. The current method simplifies GAG extraction steps, and reduces inaccuracy in calculating ratios of heparin/heparan sulfate to chondroitin sulfate/dermatan sulfate, resulting from the separate analyses of a single sample. PMID:22609076

Development of a novel cell sorting method that samples population diversity in flow cytometry.

PubMed

Osborne, Geoffrey W; Andersen, Stacey B; Battye, Francis L

2015-11-01

Flow cytometry based electrostatic cell sorting is an important tool in the separation of cell populations. Existing instruments can sort single cells into multi-well collection plates, and keep track of cell of origin and sorted well location. However currently single sorted cell results reflect the population distribution and fail to capture the population diversity. Software was designed that implements a novel sorting approach, "Slice and Dice Sorting," that links a graphical representation of a multi-well plate to logic that ensures that single cells are sampled and sorted from all areas defined by the sort region/s. Therefore the diversity of the total population is captured, and the more frequently occurring or rarer cell types are all sampled. The sorting approach was tested computationally, and using functional cell based assays. Computationally we demonstrate that conventional single cell sorting can sample as little as 50% of the population diversity dependant on the population distribution, and that Slice and Dice sorting samples much more of the variety present within a cell population. We then show by sorting single cells into wells using the Slice and Dice sorting method that there are cells sorted using this method that would be either rarely sorted, or not sorted at all using conventional single cell sorting approaches. The present study demonstrates a novel single cell sorting method that samples much more of the population diversity than current methods. It has implications in clonal selection, stem cell sorting, single cell sequencing and any areas where population heterogeneity is of importance. © 2015 International Society for Advancement of Cytometry.

Gravity separation of fat, somatic cells, and bacteria in raw and pasteurized milks.

PubMed

Caplan, Z; Melilli, C; Barbano, D M

2013-04-01

The objective of experiment 1 was to determine if the extent of gravity separation of milk fat, bacteria, and somatic cells is influenced by the time and temperature of gravity separation or the level of contaminating bacteria present in the raw milk. The objective of experiment 2 was to determine if different temperatures of milk heat treatment affected the gravity separation of milk fat, bacteria, and somatic cells. In raw milk, fat, bacteria, and somatic cells rose to the top of columns during gravity separation. About 50 to 80% of the fat and bacteria were present in the top 8% of the milk after gravity separation of raw milk. Gravity separation for 7h at 12°C or for 22h at 4°C produced equivalent separation of fat, bacteria, and somatic cells. The completeness of gravity separation of fat was influenced by the level of bacteria in the milk before separation. Milk with a high bacterial count had less (about 50 to 55%) gravity separation of fat than milk with low bacteria count (about 80%) in 22h at 4°C. Gravity separation caused fat, bacteria, and somatic cells to rise to the top of columns for raw whole milk and high temperature, short-time pasteurized (72.6°C, 25s) whole milk. Pasteurization at ≥76.9°C for 25s prevented all 3 components from rising, possibly due to denaturation of native bovine immunoglobulins that normally associate with fat, bacteria, and somatic cells during gravity separation. Gravity separation can be used to produce reduced-fat milk with decreased bacterial and somatic cell counts, and may be a critical factor in the history of safe and unique traditional Italian hard cheeses produced from gravity-separated raw milk. A better understanding of the mechanism of this natural process could lead to the development of new nonthermal thermal technology (that does not involve heating the milk to high temperatures) to remove bacteria and spores from milk or other liquids. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Electrochemical cell and method of assembly

DOEpatents

Shimotake, Hiroshi; Voss, Ernst C. H.; Bartholme, Louis G.

1979-01-01

A method of preparing an electrochemical cell is disclosed which permits the assembly to be accomplished in air. The cell includes a metal sulfide as the positive electrode reactant, lithium alloy as the negative electrode reactant and an alkali metal, molten salt electrolyte. Positive electrode reactant is introduced as Li.sub.2 FeS.sub.2, a single-phase compound produced by the reaction of Li.sub.2 S and FeS. The use of this compound permits introduction of lithium in an oxidized form. Additional lithium can be introduced in the negative electrode structure enclosed within an aluminum foil envelope between layers of porous aluminum. Molten salt electrolyte is added after assembly and evacuation of the cell by including an interelectrode separator that has been prewet with an organic solution of KCl.