CD34~+造血祖细胞及其亚群（CD34~+CD38~+和CD34~+CD38~-

分离纯化造血干祖细胞具有十分重要的理论和应用价值。本文利用吸附免疫微球的单克隆抗体分离系统，对不同来源造血组织的ＣＤ３４＋细胞进行纯化分离，经流式细胞仪检测，其纯度可达９５—９９％。在外源性生长因子的刺激下，ＣＤ３４＋细胞可形成大量各系造血集落，而ＣＤ３４－组分则几乎不含造血集落形成细胞。进一步的研究则是利用免疫荧光激活的流式细胞分选系统，将ＣＤ３４＋细胞群分为ＣＤ３４＋ＣＤ３８＋和ＣＤ３４＋ＣＤ３８－两个亚群，并比较正常人骨髓、脐带血、外周血来源的亚群细胞造血性能。结果表明，不同细胞亚群造血性能不均一，同一亚群不同来源的细胞也同样具有不均一性，从而为造血干细胞的基因治疗、建库、移植等提供了理论依据。     

Continuous cell introduction for the analysis of individual cells by capillary electrophoresis

Instrumentation for high-throughput analysis of single cells by capillary electrophoresis is described. A flow-based interface that uses electroosmotic flow (EOF) provides continuous injection of intact cells through an introduction capillary into a cell lysis junction and migration of the resulting cell lysate through a separation capillary for analysis. Specifically, two capillaries were coupled together with 5-mm-long Teflon tubing to create a approximately 5-microm gap, and the junction was immersed in a buffer reservoir. High voltage was applied across both capillaries so that cells were continuously pumped into the first capillary by EOF. Individual cells were lysed on-column at the junction without detergents, presumably owing to mechanical disruption caused by a dramatic change in flow properties at the gap. After each cell was lysed at the junction, the major proteins hemoglobin and carbonic anhydrase were separated by capillary electrophoresis and the resultant analyte zones were detected by laser-induced native fluorescence using 275-nm excitation. The detection limits of hemoglobin and carbonic anhydrase were 37 and 1.6 amol, respectively, which correlate well with the literature. The instrumentation was evaluated with intact red blood cells. The averaged time for complete analysis (i.e., continuous injection, lysis, separation, and detection) of one human erythrocyte was less than 4 min with this capillary-based setup. Moreover, this instrumentation simplifies the introduction of individual, intact cells without the use of a microscope.     

Aptamer-enabled efficient isolation of cancer cells from whole blood using a microfluidic device

Circulating tumor cells (CTC) in the peripheral blood could provide important information for diagnosis of cancer metastasis and monitoring treatment progress. However, CTC are extremely rare in the bloodstream, making their detection and characterization technically challenging. We report here the development of an aptamer-mediated, micropillar-based microfluidic device that is able to efficiently isolate tumor cells from unprocessed whole blood. High-affinity aptamers were used as an alternative to antibodies for cancer cell isolation. The microscope-slide-sized device consists of >59,000 micropillars, which enhanced the probability of the interactions between aptamers and target cancer cells. The device geometry and the flow rate were investigated and optimized by studying their effects on the isolation of target leukemia cells from a cell mixture. The device yielded a capture efficiency of ~95% with purity of ~81% at the optimum flow rate of 600 nL/s. Further, we exploited the device for isolating colorectal tumor cells from unprocessed whole blood; as few as 10 tumor cells were captured from 1 mL of whole blood. We also addressed the question of low throughput of a typical microfluidic device by processing 1 mL of blood within 28 min. In addition, we found that ~93% of the captured cells were viable, making them suitable for subsequent molecular and cellular studies.     

Biopolymer system for cell recovery from microfluidic cell capture devices

Microfluidic systems for affinity-based cell isolation have emerged as a promising approach for the isolation of specific cells from complex matrices (i.e., circulating tumor cells in whole blood). However, these technologies remain limited by the lack of reliable methods for the innocuous recovery of surface captured cells. Here, we present a biofunctional sacrificial hydrogel coating for microfluidic chips that enables the highly efficient release of isolated cells (99% ± 1%) following gel dissolution. This covalently cross-linked alginate biopolymer system is stable in a wide variety of physiologic solutions (including EDTA treated whole blood) and may be rapidly degraded via backbone cleavage with alginate lyase. The capture and release of EpCAM expressing cancer cells using this approach was found to have no significant effect on cell viability or proliferative potential, and recovered cells were demonstrated to be compatible with downstream immunostaining and FISH analysis.     

Automated in-tube solid-phase microextraction coupled with HPLC for the determination of N-nitrosamines in cell cultures

An automated in-tube solid-phase microextraction (SPME) HPLC analysis method for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and several metabolites has been developed. NNK is one of the tobacco-specific N-nitrosamines (TSNA), which has been linked to cancers associated with the use of or exposure to tobacco products. In-tube SPME is an on-line extraction technique in which analytes are extracted and concentrated from the sample directly into a coated capillary by repeated draw/eject steps. In this study, a tailor-made polypyrrole (PPY)-coated capillary and several commercially available capillaries (capillary GC columns) were used to evaluate their extraction efficiencies for NNK and several metabolites in cell cultures. Compared with commercial capillaries that were currently used for in-tube SPME, the PPY-coated capillary showed better extraction efficiency for all of the compounds studied. After optimization of the extraction conditions, NNK and five metabolite compounds were analyzed in spiked cell cultures, confirming the applicability of the developed method. Excellent linearity was observed for all compounds (av R2 = 0.9942) and detection limits that ranged from 20 to 250 ng/mL. The average within-day and between day variations (% RSD) were 2.9 and 3.6%, respectively. This automated extraction and analysis method simplified the determination of the TSNA, requiring a total sample analysis time of only approximately 30 min.     

Human endothelial progenitor cell attachment to polysaccharide-based hydrogels: A pre-requisite for vascular tissue engineering

A hydrogel was prepared from polysaccharides (pullulan/dextran/fucoidan) and evaluated as a novel biomaterial for Endothelial Progenitor Cell (EPC) culture. Using a cross-linking process with sodium trimetaphosphate in aqueous solution, homogeneous, transparent and easy to handle gels were obtained with a water content higher than 90%. Circular scaffolds (6 mm diameter and 2 mm thickness discs) were used for cell culture. Different types of EPCs were used: CD34+ derived ECs from cord blood and two sorts of CD133+ derived ECs from human bone marrow, old (30 days) and young (4 days) cells. EPCs were characterised as endothelial cells by immunofluorescent stainings for CD31 and Dil-Ac-LDL. CD133+ derived ECs from bone marrow were characterized by RT-PCR for CD31, VE-cadherin and KDR. HSVECs (Human Saphenous Vein Endothelial Cells) were used as control cells. We evaluated whether different kinds of EPCs could adhere on this novel hydrogel 4 h and 24 h after seeding, by a colorimetric quantitative test. EPCs adhered to hydrogels in serum- free conditions with values being over than 80% for young CD133+ cells at 4 h and 24 h. This pullulan-based hydrogel could constitute a suitable support for vascular cell adhesion as a pre-requisite for vascular tissue engineering.     

A Radial Flow Microfluidic Device for Ultra‐High‐Throughput Affinity‐Based Isolation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer‐related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time‐consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h^?1 in contrast to a flow rate of 1 mL h^?1 standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs.     

A Cellular Compatible Chitosan Nanoparticle Surface for Isolation and In Situ Culture of Rare Number CTCs

Circulating tumor cell (CTC) isolation has attracted a great deal of research interest in recent years. However, there are still some challenges, including purity as well as viability of the captured CTCs, resulting from nanoscale structures and inorganic nanomaterials. Here, a chitosan nanoparticle surface is first fabricated by electrospray to provide a cellular compatible interface. The “soft” substrate, further modified by polyethylene glycol (PEG) as an antifouling molecule and DNA aptamer as a specific capture molecule, has a hydrophilic nature and is capable of specific capture of viable rare CTCs from artificial white blood cell (WBC) samples. Furthermore, a subsequent in situ culture strategy based on the developed cellular compatible soft interface is introduced for further purification and proliferation of the captured rare number target cells. The WBCs are weeded out after 2 d, and after a 7 d proliferation nearly 200 MCF‐7 cells are obtained from 7 target cells with more than 90% purity. This work provides a promising strategy for viable isolation and purification of rare CTCs and it has great potential for achieving clinical validity.     

Development of a low-cost magnetic microfluidic chip for circulating tumour cell capture

The authors have developed a novel fabrication process for a selective micro-magnetic activated cell sorting (MACS) chip based on ferromagnetic material encapsulated micropillars (FMEMs), which is technically simple and low cost. The FMEM produces a high field gradient to magnetically attract, capture and hold cells on its interface. System test simulations were carried out to predict the efficacy of target capture and verify that the actual magnetic particles behaviour agreed well with model predictions. To determine the ability of the novel microMACS chip to capture circulating tumour cells (CTCs), SW620 human colon cancer cells were used in an in vitro flow model system and were able to be captured with the efficiency of 72.8%. The obvious accumulation of CTCs at a certain location on the chip suggested shear stress events at the pillar boundary may influence efficacy, and should be considered in further optimisation efforts.     

Validation of paper-based assay for rapid blood typing

We developed and validated a new paper-based assay for the detection of human blood type. Our method involves spotting a 3 μL blood sample on a paper surface where grouping antibodies have already been introduced. A thin film chromatograph tank was used to chromatographically elute the blood spot with 0.9% NaCl buffer for 10 min by capillary absorption. Agglutinated red blood cells (RBCs) were fixed on the paper substrate, resulting in a high optical density of the spot, with no visual trace in the buffer wicking path. Conversely, nonagglutinated RBCs could easily be eluted by the buffer and had low optical density of the spot and clearly visible trace of RBCs in the buffer wicking path. Different paper substrates had comparable ability to fix agglutinated blood, while a more porous substrate like Kleenex paper had enhanced ability to elute nonagglutinated blood. Using optimized conditions, a rapid assay for detection of blood groups was developed by spotting blood to antibodies absorbed to paper and eluted with 200 μL of 0.9% NaCl buffer directly by pipetting. RBCs fixation on paper accurately detected blood groups (ABO and RhD) using ascending buffer for 10 min or using a rapid elution step in 100/100 blood samples including 4 weak AB and 4 weak RhD samples. The assay has excellent reproducibility where the same blood group was obtained in 26 samples assessed in 2 different days. Agglutinated blood fixation on porous paper substrate provides a new, simple, and sensitive assay for rapid detection of blood group for point-of-care applications.     

Electrochromatographic retention studies on a flavin-binding RNA aptamer sorbent

Aptamers are oligonucleotides that are isolated and amplified on the basis of their recognition of a target molecule. In this study, an RNA aptamer isolated and amplified on the basis of its affinity for flavin mononucleotide (FMN) was covalently bound to the inner walls of fused-silica capillaries. This aptamer recognizes the flavin moiety of both FMN and flavin adenine dinucleotide (FAD). When an attempt was made to evaluate these capillaries according to existing theory, the theory proved to be insufficient. We describe a new method to evaluate capillaries for use in open-tubular capillary electrochromatography (OTCEC) of charged analytes, which combines OTCEC and flow-counterbalanced capillary electrophoresis. This method enabled us to extract k' and evaluate k(CEC) values for these capillaries, and the dependence of these values on Mg(2+) concentration was explored. The k' values for these capillaries ranged from 0.0951 to 0.2530 and from 0.0255 to 0.1118 for FMN and FAD, respectively.     

Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.     

Nanotentacle‐Structured Magnetic Particles for Efficient Capture of Circulating Tumor Cells

Circulating tumor cells (CTCs) have attracted considerable attention as promising markers for diagnosing and monitoring the cancer status. Despite many technological advances in isolating CTCs, the capture efficiency and purity still remain challenges that limit clinical practice. Here, the construction of “nanotentacle”‐structured magnetic particles using M13‐bacteriophage and their application for the efficient capturing of CTCs is demonstrated. The M13‐bacteriophage to magnetic particles followed by modification with PEG is conjugated, and further tethered monoclonal antibodies against the epidermal receptor 2 (HER2). The use of nanotentacle‐structured magnetic particles results in a high capture purity (>45%) and efficiency (>90%), even for a smaller number of cancer cells (≈25 cells) in whole blood. Furthermore, the cancer cells captured are shown to maintain a viability of greater than 84%. The approach can be effectively used for capturing CTCs with high efficiency and purity for the diagnosis and monitoring of cancer status.     

Microfluidic shear devices for quantitative analysis of cell adhesion

We describe the design, construction, and characterization of microfluidic devices for studying cell adhesion and cell mechanics. The method offers multiple advantages over previous approaches, including a wide range of distractive forces, high-throughput performance, simplicity in experimental setup and control, and potential for integration with other microanalytic modules. By manipulating the geometry and surface chemistry of the microdevices, we are able to vary the shear force and the biochemistry during an experiment. The dynamics of cell detachment under different conditions can be captured simultaneously using time-lapse videomicroscopy. We demonstrate assessment of cell adhesion to fibronectin-coated substrates as a function of the shear stress or fibronectin concentration in microchannels. Furthermore, a combined perfusion-shear device is designed to maintain cell viability for long-term culture as well as to introduce exogenous reagents for biochemical studies of cell adhesion regulation. In agreement with established literature, we show that fibroblasts cultured in the combined device reduced their adhesion strength to the substrate in response to epidermal growth factor stimulation.     

Influence of cell shape and aggregate formation on the optical properties of flowing whole blood

We studied the influence of shape and secondary, or intercellular, organization on the absorption and scattering properties of red blood cells to determine whether these properties are of any practical significance for optical evaluation of whole blood and its constituents. A series of measurements of transmittance and reflectance of light from bovine blood in a flow cuvette was conducted with a 650-900-nm integrating sphere at shear rates of 0-1600 s(-1), from which the influence of cell orientation, elongation, and aggregate formation on the absorption (mu(a)) and the reduced scattering (mu(s)') coefficients could be quantified. Aggregation was accompanied by a decrease of 4% in mu(s)' compared with the value in randomly oriented single cells. Increasing the degree of cell alignment and elongation as a result of increasing shear rate reduced mu(s)' by 6% and mu(a) by 3%, evaluated at a shear rate of 1600 s(-1). Comparison with T-matrix computations for oblate- and prolate-shaped cells with corresponding elongation and orientation indicates that the optical properties of whole blood are determined by those of its individual cells, though influenced by a collective scattering factor that depends on the cell-to-cell organization. We demonstrate that cell morphological changes must be taken into consideration when one is conducting whole blood spectroscopy.     

Sol-gel poly(ethylene glycol) stationary phase for high-resolution capillary gas chromatography

A sol-gel chemistry-based method was developed for the preparation of highly stable capillary gas chromatography (GC) columns with surface-bonded poly(ethylene glycol) (PEG) stationary phase. Through a single-step procedure, it concurrently provided column deactivation, stationary-phase coating, and chemical immobilization of the coated film. Sol-gel reactions were carried out within fused-silica capillaries that were filled with properly designed sol solutions containing two sol-gel precursors, two different triethoxysilyl-derivatized poly(ethylene glycol)s, two sol-gel catalysts, and a deactivation reagent. Hydrolytic polycondensation reactions led to the formation of a sol-gel coating chemically bonded to the inner walls of the capillary. A number of sol-gel coated fused-silica capillary columns were prepared using sol-gel-active PEG derivatives. These columns demonstrated many inherent advantages, the main being the strong anchoring of the coating to the capillary wall resulting from chemical bonding with the silanol groups on the fused-silica capillary inner surface. This chemical bonding yielded strongly immobilized PEG coatings with outstanding thermal stability (up to 320 degrees C). To our knowledge, such a high thermal stability has not been achieved so far on conventionally prepared PEG GC columns. Sol-gel PEG columns provided excellent chromatographic performances: high number of theoretical plates, excellent run-to-run and column-to-column reproducibility, and pronounced selectivity for a wide range of test solutes. Using n-octadecane as a test solute (k = 7.14), an efficiency value of 3200 theoretical plates/m was obtained on a 10 m x 0.25 mm i.d. fused-silica capillary column. Five sol-gel PEG columns provided RSD values of 1.09% for column efficiency (solute, n-octadecane), 1.37% for retention factor (solute, n-octadecane), and 0.9% for separation factor (for solute pair o- and p-xylene). In five replicate measurements using the same column, RSD values of less than 0.50% for the retention time and 1.36% for retention factor (k) were obtained.     

Tailoring elution of tetraalkylammonium ions. Ideal electrostatic selectivity elution order on a polymeric ion exchanger

Although ion exchange is often depicted as a process driven by electrostatic forces, ionic solvation or hydrophobic forces contribute greatly to ion exchange selectivity and is often the dominant factor. On a variety of commercial anion exchange columns, monovalent ClO4- elutes after doubly charged SO42- and even triply charged PO43-. For identically charged alkali metal ions, electrostatic charge densities based on crystal radii would suggest Li+ to be the most strongly retained on a cation exchanger. In practice, it is typically the least strongly held cation on most cation exchangers, because of its very high hydration energy and with most eluents its capacity factor approaches zero. Even when the ion is very poorly solvated, as with tetraalkylammonium (NR4+) cations, there has never been a report on a polymeric ion exchanger of an ideal electrostatic selectivity order where NR4+ cations elute in their increasing charge density order: R = n-butyl first, followed by n-propyl, ethyl, and last, methyl. We show that this selectivity order is easily achieved on recently described methracrylate-based monolithic capillary cation exchange columns (Ueki, Y.; Umemura, T.; Li, J. X.; Odake, T; Tsunoda, K. Anal. Chem. 2004, 76, 7007-7012) with minor amounts of hydroorganic modifiers. Indeed, under such conditions, Li+ (and other alkali cations) elutes after NMe4+.     

Analysis of carbonic anhydrase in human red Blood cells using capillary electrophoresis/ electrospray ionization-mass spectrometry

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of human red blood cells (RBCs) using the split-flow technique for interfacing CE to MS. By using a long (approximately125-cm) and narrow (approximately 15-microm-i.d.) capillary, the four major proteins of the RBC, which are hemoglobin (Hb, alpha- and beta-chains, 900 amol/chain), carbonic anhydrase I (CAI, approximately 7 amol/cell), and carbonic anhydrase II (CAII, approximately 0.8 amol/cell), were separated from each other and detected at low-attomole levels in one run and minimal sample preparation. Under these conditions, the detection limits for CAI and CAII in lysed RBCs were approximately 20 and approximately 44 amol, respectively. The approximately 20-amol detection limit of CAI was confirmed by the CE/ESI-MS analysis of three intact RBCs that had been drawn into the capillary under a microscope. A shorter capillary (approximately 55 cm long) provided faster analysis time but did not separate CAII from the beta-chain of hemoglobin, causing the CAII signal to be masked by the background chemical noise generated by the approximately 1,000 x molar excess of the beta-chain. Under this condition, the CAII detection limit increased to approximately 500 amol. From three methods of sample introduction (injection of lysed blood, injection of intact cells under microscope, and injection of intact cells suspended in saline solution), injection of lysed blood provided the optimum sensitivity. It was found that a background electrolyte (BGE) containing 0.1% acetic acid in water worked best for the analysis of intact cells, while a BGE containing 0.1% acetic acid in water + acetonitrile (50/50 by volume) worked best for the analysis of lysed blood.     

Cytocompatibility of magnesium and AZ31 alloy with three types of cell lines using a direct in vitro method

Magnesium alloys have been investigated by many researchers as a new absorbable biomaterial owing to their excellent degradability with non-maleficence or low-maleficence in living tissues. In the present work, the in vitro cytocompatibility of an Magnesium alloy was investigated by culturing cells directly on it. Investigations were carried out in terms of the cell viability along with the use of scanning electron microscopy to observe its morphology. The cell lines used were derived from fibroblast, endothelial, and smooth muscle cells. Pure magnesium and AZ31 alloy composed of magnesium (96?%), aluminum (3?%), and zinc (1?%) were adopted as models. The viability of cells on the metal samples and on the margin area of a multi-well plate was investigated. For direct culturing on metal, a depression in the viability and morphologically stressed cells were observed. In addition, the cell viability was also depressed for the margin area. To clarify the factors causing the negative effects, the amount of eluted metal ions and pH changes in the medium because of the erosion of the Magnesium samples were investigated, together with the cytotoxicity of sole metal ions corresponding to the composition of the metals. It was found that Mg²⁺, Zn²⁺, and Al³⁺ ions were less toxic at the investigated concentrations, and that these factors will not produce negative effects on cells. Consequently, these factors cannot fully explain the results.     

Generating nonlinear concentration gradients in microfluidic devices for cell studies

We describe a microfluidic device for generating nonlinear (exponential and sigmoidal) concentration gradients, coupled with a microwell array for cell storage and analysis. The device has two inputs for coflowing multiple aqueous solutions, a main coflow channel and an asymmetrical grid of fluidic channels that allows the two solutions to combine at intersection points without fully mixing. Due to this asymmetry and diffusion of the two species in the coflow channel, varying amounts of the two solutions enter each fluidic path. This induces exponential and sigmoidal concentration gradients at low and high flow rates, respectively, making the microfluidic device versatile. A key feature of this design is that it is space-saving, as it does not require multiplexing or a separate array of mixing channels. Furthermore, the gradient structure can be utilized in concert with cell experiments, to expose cells captured in microwells to various concentrations of soluble factors. We demonstrate the utility of this design to assess the viability of fibroblast cells in response to a range of hydrogen peroxide (H(2)O(2)) concentrations.