Methodological Study of Cell Separation with Domestic Immunomagnetic Beads

To establish the method of cell separation with domestic immuomagnetic beads, three methods were investigated. Direct method, SPA method and Biotin Avidin method were applied to separate cell strain Hut 78 and CD4 positive cells. Separation rate of strain Hut 78 was more than 90 % in direct method. Detachment rate with papain was over 95 %. Cell activity was well retained. SPA method and Biotin Avidin methods were also effective, but the direct method was superior to the other two techniques. Before separated by the direct method, CD4 positive cells constituted 46.4 %±6.4 % of mononuclear cells (MNC), but in eliminated suspension there was only 6.2 %±2.3 % CD4 positive cells left. In the separated part, 80.6 %±7.2 % of the cells combined with the beads. It is concluded that the direct method in separating cells had high sensitivity and specificity.

Microfluidic methods for cell separation and subsequent analysis

Cell is the most basic unit of the morphological structure and life activity of an organism. Learning the composition, structure and function of cells, exploring the life activities of cells and studying the interaction between cells are of great significance for human cognition and control of the life activities of organisms. Therefore, rapid, convenient, inexpensive, high-precision and reliable methods of cell separation and analysis are being developed to obtain accurate information for the study of cytology and pathology.Microfluidic chip is a new emerging technology in recent years. It has a micromanufacturing structure,which can not only realize the precise space-time control of fluid and cells, but also reproduces the threedimensional dynamic microenvironment of cell growth in the body. In addition, the microfluidic chip has unique microphysical properties and facilitates the integration of microdevices, which provides the possibility of real-time monitoring, continuous culture, separation and enrichment, and even in situ analysis of cells. In this review, we summarized recent advances in the development of different techniques for cell isolation and analysis on microfluidic platforms. Focus was put on biochemical and physical methods for cell separation on microfluidic chips. Subsequent cell analysis depending on fluorescence, Raman, cytomicroscopic imaging, mass spectrometry and electrochemical methods also was remarked. Through analyzing and learning the advantages and disadvantages of different technologies, we hope that microfluidic chips will continue to be improved and expanded for medical and clinical applications.

Computational Microuidic Channel for Separation of Escherichia coli from Blood-Cells

Microuidic channels play a vital role in separation of analytes of interest such as bacteria and platelet cells,etc.,in various biochemical diagnosis procedures including urinary tract infections(UTI)and bloodstream infections.This paper presents the multi physics computational model specifically designed to study the effects of design parameters of a microuidics channel for the separation of Escherichia coli(E.coli)from various blood constituents including red blood cells(RBC)and platelets.A standard two inlet and a two outlet microchannel of length 805µm with a channel width of 40µm is simulated.The effect of electrode potentials and the effect of electrode placement along the channel length and also the levitation of electrodes from the channel wall are studied to optimize the selective particle separation throughput.Simulated results show the efcient separation of E-coli with a mean diameter 0.68µm is achieved at low voltages(less than 20 V)when electrodes placed near to the micro channel and also noticed that the applied electric potential is inversely proportional to the number of electrodes placed along the microuidic channel.The computer aided multi physics simulations with multiple governing parameters could be advantage in design optimization of microuidics channels and support precise bioparticle separation for better diagnosis.

Effects of Three-Dimensional Platform Stiffness and Layer Dimensions on Separation of Carcinoma Cells

Cancer cell separation is highly desirable for cancer diagnosis and therapy.Besides biochemical methods,engineered platforms are effective alternatives for sorting carcinoma cells from normal cells based on their unique properties in responding to the physical changes of the surrounding microenvironment.In this work,three-dimensional(3D)biomimetic scaffold platforms were developed to separate nasopharyngeal carcinoma 43(NPC43)cells from immortalized nasopharyngeal epithelial 460(NP460)cells based on precisely controlled design parameters including stiffness,number of layers,and structural layout.The migration characteristics of NPC43 and NP460 cells on the scaffold platforms revealed that NPC43 cells could squeeze into 10 lm wide,15 lm deep trenches while NP460 cells could not.The different migration behavior was mainly due to cells having different interactions with the surrounding microenvironment.NPC43 cells had filopodia-like protrusions,while NP460 cells exhibited a sheet-like morphology.Using these 3D biomimetic platforms,89%separation efficiency of NPC43 cells from NP460 cells was achieved on stiffer two-layer scaffold platforms with a 40/10 lm ridge/trench(R/T)grating on the top layer and a 20/10 lm R/T grid on the bottom layer.Moreover,the separation efficiency was further increased to 93%by adding an active conditioned medium(ACM)that caused the cells to have higher motility and deformability.These results demonstrate the capability to apply biomimetic engineered platforms with appropriate designs to separate cancer cells from normal cells for potential cancer diagnosis and treatment.

Separation of CD34 Positive Cells and Determination of Surface Homing Antigen

To study the differences in homing potential between bone marrow cells and umbilical blood cells, CD34 positive cells were obtained from bone marrow (BM) and umbilical blood (UB) by the direct cell separation with domestic immunomagnetic beads. The expression of the two adhesion molecules CD11a/CD18 and CD44 were examined. After separation, CD34 positive cells accounted for 51 %-82 % of the harvested cells and dye-resistance rate was 82 %-88 %. The expression of CD11a/CD18 and CD44 on the surfaces of UB cells was 49. 6 % 1 10. 2 % and 37. 7 % ± 10. 3 % respectively. On BM cells they were 50. 2 % ± 6. 2 % and 34 % ± 13. 3 % respectively. There were no significant differences in the expression of these two molecules. It was concluded .that the cell separation method with domestic immunomagnetic beads was effective and the stem cells from UB could serve as an alternative source for transplantation.

Biocatalytic Buoyancy‑Driven Nanobots for Autonomous Cell Recognition and Enrichment

Autonomously self-propelled nanoswimmers represent the nextgeneration nano-devices for bio-and environmental technology.However,current nanoswimmers generate limited energy output and can only move in short distances and duration,thus are struggling to be applied in practical challenges,such as living cell transportation.Here,we describe the construction of biodegradable metal-organic framework based nanobots with chemically driven buoyancy to achieve highly efficient,long-distance,directional vertical motion to“find-and-fetch”target cells.Nanobots surface-functionalized with antibodies against the cell surface marker carcinoembryonic antigen are exploited to impart the nanobots with specific cell targeting capacity to recognize and separate cancer cells.We demonstrate that the self-propelled motility of the nanobots can sufficiently transport the recognized cells autonomously,and the separated cells can be easily collected with a customized glass column,and finally regain their full metabolic potential after the separation.The utilization of nanobots with easy synthetic pathway shows considerable promise in cell recognition,separation,and enrichment.

Preparation of Immuno-magnetic Beads and Their Separation & Detection to Ovary Cancer Cells

The organic monomer-molecule with nanometer magnetic powder by means of reforming the surface of nanometer magnetic powder have been synthesized. Magnetic beads in diameter of 2 mum or so am obtained by controlling conditions. Ovary cancer cells of ascites are separated and ovary cancer cells of blood arc detected by using immuno-magnetic beads linked with ovary cancer cell mono-antibodies. Results show that the specificity is 85% sensitivity is 87%, accuracy is 84%, cells acquiring purity is 90%, cells activity is 92% and detection sensitivity is 25 x 10(-7).

Cell biomechanics and its applications in human disease diagnosis

Certain diseases are known to cause changes in the physical and biomechanical properties of cells.These include cancer,malaria,and sickle cell anemia among others.Typically,such physical property changes can result in several fold increases or decreases in cell stiffness,which are significant and can result in severe pathology and eventual catastrophic breakdown of the bodily functions.While there are developed biochemical and biological assays to detect the onset or presence of diseases,there is always a need to develop more rapid,precise,and sensitive methods to detect and diagnose diseases.Biomechanical property changes can play a significant role in this regard.As such,research into disease biomechanics can not only give us an in-depth knowledge of the mechanisms underlying disease progression,but can also serve as a powerful tool for detection and diagnosis.This article provides some insights into opportunities for how significant changes in cellular mechanical properties during onset or progression of a disease can be utilized as useful means for detection and diagnosis.We will also showcase several technologies that have already been developed to perform such detection and diagnosis.

Prevention of central cell damage to isolated islets of Langerhans in hamsters by low temperature preconditioning

BACKGROUND: The efficacy of clinical islet transplanta- tion has been demonstrated with autografts, and although islet allografts have established insulin independence in a small number of IDDM patients, the treatment is con- founded by the necessity of central cell damage immuno- suppression, the lack of donor tissue, and recurring islet immunogenicity. These limitations underscore a need to develop therapies to serve the large population of diabetic patients. This study was designed to document central cell damage to isolated islets of Langerhans in hamsters and its prevention. METHODS: Islets were cultured at 37 °C for 7-14 days after isolation, and then at 26 °C for 2,4 and 7 days before addi- tional culture at 37 °C for an additional 7 days. Central cell damage in the isolated islets was monitored by video-mi- croscopy and analyzed quantitatively by a computer-assis- ted image analysis system. The analysis included daily measurement of the diameter and the area of the isolated is- lets and the area of the central cell damage that developed in those islets over time during culture. Histological exami- nation and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to characterize cell damage and to monitor islet function. RESULTS; Microscopic analysis showed that during the 7 to 14 days of culture at 37 °C, central cell damage appeared in the larger islets with diameters greater than 200 μm, which included both necrotic and apoptotic cell death. Low temperature (26 °C) culture prevented central cell damage of isolated islets. The 7-day culture procedure at 26 °C could inhibit most of the central cell ( excluding diameters greater than 300 μm) damage when the islets were re- warmed to 37 °C. CONCLUSIONS: Our results indicate that central cell da- mage to isolated islets of Langerhans correlates with the size of the islets. Low temperature (26 °C) culture can preventcentral cell damage to the isolated islets, and is capable to successfully precondition these islets for 37 °C culture. These novel findings may help to understand the patho- physiology of early loss of islet tissue after transplantation, and may provide a new strategy to improve graft function in the clinical setting of islet transplantation.

Comparison of conventional and inverted structures in fullerene-free organic solar cells

A n-type small molecule DC-IDT2E with 4,4,9,9-tetrakis（4-hexylphenyl）-indaceno[1,2-b：5,6-bt]dithiophene as a central building block, furan as rr-bridges, and 1,1 -dicyanomethylene-3-indanone as end acceptor groups, was synthesized and used as an electron acceptor in solution-processed organic solar cells （OSCs）. DC-IDT2F exhibited good thermal stability, broad and strong absorption in 500-850 rim, a narrow bandgap of 1.54 eV, LUMO of-3.88 eV, HOMO of-5.44 eV and an electron mobility of 6.5 × 10-4 cm2/（V.s）. DC-IDT2F-based OSCs with conventional and inverted structures exhibited power conversion efficiencies of 2.26 and 3.08% respec- tively. The effect of vertical phase separation and morphology of the active layer on the device performance in the two structures was studied.

Functionalization of high-moment magnetic nanodisks for cell manipulation and separation

Synthetic antiferromagnetic （SAF） nanoparticles are layer-structured particles with high single-particle magnetic moments. In order to covalently bind these nanopartides to cells, they were coated with a silica shell followed by conjugation with streptavidin. The silica coating generates both SAF@SiO2 core-shell nano- particles and silica core-free nanopartides. Using a simple magnetic separation, silica nanoparticles were removed and SAF@SiO2 nanoparticles were purified. After streptavidin conjugation, these particles were used to stain lung cancer cells, making them highly magnetically responsive. The stained cells can rotate in response to an external magnetic field and can be captured when a blood sample containing these cells flows through the sifter.

Cell Wall Microstructure Analysis Implicates Hemicellulose Polysaccharides in Cell Adhesion in Tomato Fruit Pericarp Parenchyma

Methods developed to isolate intact cells from both unripe and ripe tomato fruit pericarp parenchyma have allowed the cell biological analysis of polysaccharide epitopes at the surface of separated cells. The LM7 pectic homogalacturonan epitope is a marker of the junctions of adhesion planes and intercellular spaces in parenchyma systems. The LM7 epitope persistently marked the former edge of adhesion planes at the surface of cells separated from unripe and ripened tomato fruit and also from fruits with the Cnr mutation. The LM 11 xylan epitope was associated, in sections, with cell walls lining intercellular space but the epitope was not detected at the surface of isolated cells, being lost during cell isolation. The LM15 xyloglucan epitope was present at the surface of cells isolated from unripe fruit in a pattern reflecting the former edge of cell adhesion planes/intercellular space but with gaps and apparent breaks. An equivalent pattern of LM15 epitope occurrence was revealed at the surface of cells isolated by pectate lyase action but was not present in cells isolated from ripe fruit or from Cnr fruit. In contrast to wild-type cells, the LM5 galactan and LM21 mannan epitopes occurred predominantly in positions reflecting intercellular space in Cnr, suggesting a concerted alteration in cell wall microstructure in response to this mutation. Galactanase and mannanase, along with pectic homogalacturonan-degrading enzymes, were capable of releasing cells from unripe fruit parenchyma. These observations indicate that hemicellulose polymers are present in architectural contexts reflecting cell adhesion and that several cell wall polysaccharide classes are likely to contribute to cell adhesion/cell separation in tomato fruit pericarp parenchyma.

MACS-annexin V cell sorting of semen samples with high TUNEL values decreases the concentration of cells with abnormal chromosomal content:a pilot study

We question whether,in men with an abnormal rate of sperm DNA fragmentation,the magnetic-activated cell sorting(MACS)could select spermatozoa with lower rates of DNA fragmentation as well as spermatozoa with unbalanced chromosome content.Cryopreserved spermatozoa from six males were separated into nonapoptotic and apoptotic populations.We determined the percentages of spermatozoa with(i)externalization of phosphatidylserine(EPS)by annexin V-Fluorescein isothiocyanate(FITC)labeling,(ii)DNA fragmentation by TdT-mediated-dUTP nick-end labeling(TUNEL),and(iii)numerical abnormalities for chromosomes X,Y,13,18,and 21 by fluorescence in situ hybridization(FISH),on the whole ejaculate and selected spermatozoa in the same patient.Compared to the nonapoptotic fraction,the apoptotic fraction statistically showed a higher number of spermatozoa with EPS,with DNA fragmentation,and with numerical chromosomal abnormalities.Compared to the whole ejaculate,we found a significant decrease in the percentage of spermatozoa with EPS and decrease tendencies of the DNA fragmentation rate and the sum of disomy levels in the nonapoptotic fraction.Conversely,we observed statistically significant higher rates of these three parameters in the apoptotic fraction.MACS may help to select spermatozoa with lower rates of DNA fragmentation and unbalanced chromosome content in men with abnormal rates of sperm DNA fragmentation.

Single living cell manipulation and identification using microsystems technologies

The paper presents the principles and the results of the implementation of dielectrophoresis for separation and identification of rare cells such as circulation tumor cells(CTCs)from diluted blood specimens in media and further label-free identification of the origins of separated cells using radio-frequency(RF)imaging.The separation and the identification units use same fabrication methods which enable system integration on the same platform.The designs use the advantage of higher surface volume ratio which represents the particular feature for micro-and nanotechnologies.Diluted blood in solution of sucrose–dextrose 1–10 is used for cell separation that yields more than 95.3% efficiency.For enhanced sensitivity in identification,RF imaging is performed in 3.5–1 solution of glycerol and trypsin.Resonance cavity performance method is used to determine the constant permittivity of the cell lines.The results illustrated by the signature of specific cells subjected to RF imaging suggest a reliable label-free single cell detection method for identification of the type of CTC.

Letting Go is Never Easy:Abscission and Receptor- Like Protein Kinases

Abscission is the process by which plants discard organs in response to environmental cues/stressors, or as part of their normal development. Abscission has been studied throughout the history of the plant sciences and in numerous species. Although long studied at the anatomical and physiological levels, abscission has only been elucidated at the molecular and genetic levels within the last two decades, primarily with the use of the model plant Arabidopsis thaliana. This has led to the discovery of numerous genes involved at all steps of abscission, including key pathways involving receptor-like protein kinases （RLKs）. This review covers the current knowledge of abscission research, highlighting the role of RLKs.

Viscoelastic microfluidics: progress and challenges

The manipulation of cells and particles suspended in viscoelastic fluids in microchannels has drawn increasing attention,in part due to the ability for single-stream three-dimensional focusing in simple channel geometries.Improvement in the understanding of non-Newtonian effects on particle dynamics has led to expanding exploration of focusing and sorting particles and cells using viscoelastic microfluidics.Multiple factors,such as the driving forces arising from fluid elasticity and inertia,the effect of fluid rheology,the physical properties of particles and cells,and channel geometry,actively interact and compete together to govern the intricate migration behavior of particles and cells in microchannels.Here,we review the viscoelastic fluid physics and the hydrodynamic forces in such flows and identify three pairs of competing forces/effects that collectively govern viscoelastic migration.We discuss migration dynamics,focusing positions,numerical simulations,and recent progress in viscoelastic microfluidic applications as well as the remaining challenges.Finally,we hope that an improved understanding of viscoelastic flows in microfluidics can lead to increased sophistication of microfluidic platforms in clinical diagnostics and biomedical research.