Continuous-Flow Fractionation of Animal Cells in Microfluidic Device Using Aqueous Two-Phase Extraction

Monitoring of live cells is important in the field of medical science, diagnostics, biology, and the pharmaceutical industry. In this study, live and dead CHO-K1 (Chinese Hamster Ovary) cells were fractionated by continuous-flow extraction in a microfluidic device using immiscible aqueous two-phase extraction technique. The polymer solutions offered stable two-phase flows in microchannel without diffusive mixing. The fundamentals of aqueous two-phase extraction can support stable and reproducible recovery and separation of biomolecules in microfluidic devices. Polyethylene glycol 8000 (PEG 8000, 4%) and dextran T500 (5%) were selected as model polymer solutions. The appropriate flow rates of polymer and cell solutions were suggested. The fractionation efficiency of live and dead CHO K-1 cells from the culture broth was compared in normal macroscale system and microfluidic device. The optimum pH for the fractionation was 6.6 in both the normal and micro-scale systems. The loss of target live cells by sedimentation was circumvented in microfluidic device because of the negligible effect of gravity on the sedimentation. Most live cells were distributed to PEG-rich phase, while dead cells were found at the interface of two polymer solutions in microchannel. In this case, the recovery and fractionation efficiency of live cells in the PDMS-based microfluidic device was 100% and 97.0%, respectively.     

Selective Surface Modification in Silicon Microfluidic Channels for Micromanipulation of Biological Macromolecules

Interactions between biological macromolecules and micrometer- and sub-micrometer-scale surface structures are directly influenced by the surface wettability, chemical reactivity and surface charge. Understanding these interactions is crucial for developing integrated microsystems for biological and biomedical processing and analysis. We report development of selective surface modification techniques based on microcontact printing and polyelectrolyte adsorption. These techniques were applied to lithographically patterned silicon microfluidic channels and flat silicon substrates to create surface microstructures with contrasting wetting properties and surface charges. These controls enabled us to devise various techniques for controlled loading and processing of biomaterials in the channels. Solutions containing long chain biological macromolecules DNA and microtubules were directly loaded into the microchannels by using a micromanipulator/microinjector system. Structural arrangements of these linear macromolecules, which were probed by using fluorescence and laser scanning confocal microscopy, were found to be quite different from bulk solutions. As expected, the filamentous molecules were observed to align linearly along the channels, with the degree of alignment dependent on channel width as well as the length of the molecule. This molecular alignment, which is induced by both the surface confinement effect and capillary flow during sample loading, may be used to enhance processing of biological materials in silicon biomedical microdevices. It also opens up the possibility of carrying out direct combinatorial structural characterization of proteins in the microchannels utilizing X-ray diffraction, which so far has not been possible.     

Time-of-Flight Optophoresis Analysis of Live Whole Cells in Microfluidic Channels

Optophoresis is a non-invasive cell analysis technique that is based on the interaction of live whole cells with optical gradient fields, typically generated by a near-infrared laser. The magnitude of the interaction depends upon the intrinsic physical properties of the cells, such as their refractive index, composition, size, and morphology. Time-of-flight (TOF) optophoresis is an implementation of this technique in a microfluidic environment. It measures cell travel times through a fixed distance with and without irradiation from a laser beam. The magnitude of the optical force from the laser, and therefore the change in transit time introduced by the presence of the infrared laser provides a signature for the cell. By accumulating such measurements for a population of cells (typically 200-300 cells per population), different cell types, drug treatments, or biological states can be compared quantitatively without the need for external labels or markers. An integrated TOF system has been constructed and characterized. The system typically uses square capillaries with 50-100 microm internal diameter and uses a syringe-pump-based flow system that generates initial bulk flow velocities between 200 and 600 microm/sec. Using this TOF technique, we have been able to consistently detect significant differences between normal skin and melanoma cell lines, CCD-1037 and A375, respectively. We have also been able to measure consistent differences in a cell differentiation model (HL60 cell line with DMSO treatment). These early results indicate the potential biological sensitivity of the TOF measurement technique for cellular analysis and cancer diagnostic applications.     

流式细胞术在母婴血型不合新生儿溶血病检测中的应用

目的建立流式细胞仪检测母婴血型不合新生儿溶血病（HDN）的方法。方法对临床送检的115例拟诊新生儿溶血病标本以流式细胞仪进行直接抗人-IgG试验、血清游离抗体检测及红细胞放散液抗体检测3项试验，选择FITC标记的单克隆二抗作为与红细胞特异性抗体结合的抗体。同时以试管抗球蛋白法进行3项试验作为对照，比较两方法的差异。结果建立了以流式细胞仪检测新生儿溶血病的实验方法：细胞采集比例为54．5％；阴性阈值2％；流式法对ABOHDN和RhHDN的检测阳性率分别为86％和100％。试管法检测阳性率分别为50％和100％。结论流式法具有敏感性高，特异性强，结果易判定，客观、标准等优点，为新生儿溶血病检测提供了一种可靠的诊断依据。     

Dispersion of Dynamic Biochemical Signals in Steady Flow in a Shallow Y-type Microfluidic Channel

This paper presents an analysis of dispersion of dynamic biochemical signals in steady flow in a shallow Y-type microfluidic channel. A method is presented to control the flow widths of two steady flows in the Y-type microchannel from two inlets. The transfer function for the Y-type microchannel is given by solving the governing e- quation for the Taylor-Aris dispersion in the microchannel. The amplitude-frequency and phase-frequency relations are provided which show that a shallow Y-type mi- crochannel acts as a low-pass filter. The transports of different dynamic biochemical sig- nals are investigated. In comparison with a fully mixing microfluidic channel, the magni- tudes of the dynamic signals at the outlets in a Y-type microchannel are much smaller than those in a fully mixing microchannel, which demonstrates that the amplitude atten- uation in a Y-type microchannel is larger than that of a fully mixing microchannel due to the transverse molecular diffusion. In order to control the desired signal in a microchannel, the solution of the inverse problem for the channel is also presented.     

再生障碍性贫血患者外周血T细胞亚群和T细胞受体表达水平及其意义

采用流式细胞仪及间接免疫荧光法检测30例再生障碍性贫血患者外周血中T细胞亚群及T细胞表面受体表达水平,并与健康对照组相比,结果表明:70％再障患者存在CD4/CD8比例倒置及CD8~+％异常增高;50％再障患者外周血γδ-T细胞亚群及其在T淋巴细胞总体中所占比例均显著增高;而αβT细胞亚群及TirA~+细胞百分率与正常对照组相比无显著性差异。提示:半数以上再障患者外周血中存在异常增多的γδT细胞及Ts细胞亚群。并可通过其直接或间接作用抑制造血,从而导致再障的发生。     

应用流式细胞术分析不同年龄大鼠脑组织细胞周期增殖特性

应用流式细胞术对不同年龄的大鼠的大脑、小脑和脑干组织的细胞周期进行分析。结果显示在相同年龄组不同部位的脑组织细胞增殖程度不均一,大脑细胞增殖明显高于小脑和脑干。同一部位的脑组织各年龄组之间细胞增殖情况亦存在差异。新生和幼年大鼠(出生后1天和21天)其脑细胞增殖较成年大鼠(6个月和1年)旺盛。实验还表明,特殊萤光素染色脑细胞DNA,然后用流式细胞仪测定DNA量,是一种简单、快速分析脑细胞增殖活性的可行技术。     

Use of antibody-coated polyacrylic plastic beads for semiquantitative determination of cell surface antigens

Polyacrylic plastic beads coated with specific antibodies, which had previously been shown to bind specifically to cells carrying the related antigen, are shown to react quantitatively with the antigen on individual cells. The number of beads per cell which can be counted by simple microscopy is a measure of the amount of antigen accessible on the cell surface. The method may also be used in combination with other methods for cell surface antigens, such as immunofluorescence, for multiple antigen determination.     

不同临床期多发性骨髓瘤患者细胞免疫功能流式细胞术分析

本文应用流式细胞仪（ＦＣＭ）分析了２３例不同临床期多发性骨髓瘤（ＭＭ）患者Ｔ细胞亚群、ＮＫ细胞及ＣＤ１９＋Ｂ淋巴细胞变化。结果显示：初诊未化疗ＭＭ患者ＣＤ４、ＣＤ２５、ＣＤ１９细胞百分率ＣＤ４／ＣＤ８比值均下降，ＣＤ８细胞百分率升高；稳定期ＭＭ患者除ＣＤ２５、ＣＤ１９细胞百分率下降外，其他指标均正常；进展期ＭＭ患者ＣＤ３、ＣＤ４细胞百分率和ＣＤ４／ＣＤ８比值下降，ＣＤ８、ＣＤ１９细胞百分率升高；不同临床期ＭＭ患者ＮＫ细胞与正常人比较无显著差异（Ｐ＞０．０５）。研究结果表明：不同临床期ＭＭ患者细胞免疫功能不同，细胞免疫调节异常在ＭＭ发病机制及稳定期脱逸过程中可能起一定作用。     

Flow cytometry methods for the study of cell‐cycle parameters of planarian stem cells

Due to their characteristic inaccessibility and low numbers, little is known about the cell‐cycle dynamics of most stem cells in vivo. A powerful, established methodology to study cell‐cycle dynamics is flow cytometry, which is used routinely to study the cell‐cycle dynamics of proliferating cells in vitro. Its use in heterogeneous mixtures of cells obtained from whole animals, however, is complicated by the relatively low abundance of cycling to non‐cycling cells. We report on flow cytometric methods that take advantage of the abundance of proliferating stem cells in the planarian Schmidtea mediterranea. The optimized protocols allow us to measure cell‐cycle dynamics and follow BrdU‐labeled cells specifically in complex mixtures of cells. These methods expand on the growing toolkit being developed to study stem cell biology in planarians, and open the door to detailed cytometric studies of a collectively totipotent population of adult stem cells in vivo. Developmental Dynamics 238:1111–1117, 2009. © 2009 Wiley‐Liss, Inc.     

高速螺旋流场中人体血液流动性能及红细胞力学行为分析

血泵的溶血程度主要受血液的运动流场影响,所以研究血液在血泵内腔的螺旋流动特性对于螺旋叶片血泵的设计和研究工作具有十分重要的意义。本文将血液流变理论和传统的力学分析方法相结合,对血液在低、高剪切变率两种条件下的环形空间螺旋流动性能进行了研究,给出了速度表达式,分析了各参数对流动性能的影响,同时还对高速螺旋流场中红细胞的力学行为进行了分析。结果表明,高速螺旋流场中的血液流动情况十分复杂,在进行高速螺旋血泵设计时,应综合考虑血液在不同剪切变率条件下的流动性能及红细胞的力学行为。     

CD44与CD62P在肾脏疾病中的表达及意义

目的 探讨肾小球变患者外周血Ｐ选择素（ＣＤ６２Ｐ）与细胞表面糖蛋白（ＣＤ４４）的表达特征及临床意义。方法 采用流式细胞术对９０例肾脏疾病患者外周血ＣＤ６２Ｐ和ＣＤ４４进行检测分析,以３０例下沉人作为对照。结果 不同病因肾病患者ＣＤ６２Ｐ和ＣＤ４４表达 较正常组显著增高,其中慢性肾衰组ＣＤ４４阳性细胞数增高极为显著,ＩｇＡ肾病组ＣＤ６２Ｐ和ＣＤ４４表达均较其它肾脏疾病组降低,,肾病患者ＣＤ６２Ｐ和Ｃ     

Flow cytometric analysis of cell proliferation dynamics in the B cell compartment of the mouse

Using a method which allows simultaneous flow cytometric detectionof cell surface markers and 5-bromo-2'-deoxyuridine (BrdU) incorporation,the distribution and proliferative behavior of B lineage subpopulationswas studied in intact adult mice. In the bone marrow we coulddefine two subsets of B cells on the basis of differential expressionof the pan-B cell marker B220 and of membrane-associated µand immunoglobulin heavy chains. B220^dullµ^+– Bcells were found to emerge from rapidly dividing cells and probablyrepresent B cells recently generated from B220^dullµ^–pie-B cells. In contrast, oniy few, If any, of the B220^brightµ⁺⁺B cells were labeled with BrdU after a period of 8 days, suggestingthat these cells represent long-lived B cells residing in thebone marrow. Analysis of BrdU-Incorporation into splenic B cellsshowed that only 20% of these cells had gone through cell divisionduring the preceding 8 days. Almost none of the B cells in theperitoneum, a large fraction of which belongs to the Ly1 B subset,were labeled with BrdU over a period of 7 days in 8-month-oldanimals.     

Use of an automatic cell harvester in a cellular radioimmunoassay

An automatic cell harvester was used in the final step of a cellular radioimmunoassay to collect cell bound anti-rat IgG ¹²⁵I-F(ab′)₂. Studies on the reliability of this collection method were performed with antibodies directed against cell surface antigens induced by the Gross murine leukemia virus and produced by immunization of W/Fu rats with the syngeneic (C58NT)D lymphoma. Glutaraldehyde-fixed as well as untreated Gross virus induced lymphoma cells could be used. Similar and specific antibody binding curves were obtained when the cells were incubated with the anti-(C58NT)D serum and anti-rat IgG ¹²⁵I-F(ab′)₂ in the presence of 0.1% NaN₃. Background levels of non-specific binding of anti-rat ¹²⁵I-F(ab′)₂ to mouse lymphoma cells or rat thymocytes were only a few cpm above the background of the gamma-counter. This allowed detection of surface immunoglobulin positive lymphocytes among as few as 30,000 rat splenocytes. In addition, this cellular radioimmunoassay was found to be suitable for the measurement of solubilized cell surface antigen by its capacity to inhibit binding of the specific antibodies to the target cells.     

CD24 and CD70 as differential markers of human hepatocellular carcinoma cells in culture

We developed a new method for evaluation of the purity of cell population in primary cultures of hepatocellular carcinoma. Expression of potential surface molecular markers on primary cultures of renal tumors was assayed by the method of flow cytofluorometry. CD24 and CD70 were identified as differential markers, which allowed us to distinguish cancer cells from tumor stromal cells in vitro. A negative marker CD90 was expressed on fibroblasts of the tumor stroma, but not on cancer cells. Combined use of these three markers allows evaluation of the severity of tumor culture contamination with stromal cells. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 12, pp. 668–671, December, 2007     

基于暗视场荧光图像分析的大鼠微血管流速测量系统的建立

将荧光标记的自身红细胞注入SD大鼠体内,在荧光显微镜下观测标记红细胞在大鼠微血管中的流动情况,并通过显微摄像系统将整个过程以视频信号的形式存贮。使用视频采集卡将流速变化过程回放采样,得到暗视场下的荧光图像。然后利用帧图像分离出奇偶场的图像分析方法测定血流速度。在该系统下测量流动小室中荧光小球的流速,得到的测量值与实际值之间的误差小于7%,两者没明显的差异(P>0.05),流速测量的上限为9.6mm/s。并在大鼠微循环障碍研究中,应用此系统得到了血流速度随时间变化的情况。     

A simple reliable method for producing electron dense markers of uniform size for use in immunoelectron microscopy

A method is presented for the preparation of electron dense markers of defined size for use in immunoelectron microscopy. The electron dense markers were formed by the covalent binding of rabbit IgG and horse spleen ferritin mediated by glutaraldehyde. We demonstrated that the conjugate size, i.e. the number of ferritin grains per unit conjugate, was directly related to the time of incubation af 4°C in a 1000 molar excess of glutaraldehyde. Conjugates were separated on the basis of molecular size by Sepharose 4B column chromatography. Three different sized conjugates were used to delineate pseudomonas toxin receptors on the surface of mouse LM fibroblasts; similar numbers of receptors were observed with all conjugates.     

大鼠骨髓基质细胞表面抗原CD71、CD44和CD45的检测

目的研究表面抗原CD71、CD44和CD45在体外培养的大鼠骨髓基质细胞的表达,了解骨髓基质细胞的生物学特性。方法采用全骨髓法分离培养大鼠骨髓基质细胞,待原代培养的细胞达70%以上汇合时,用含胰酶和EDTA的消化液消化细胞,进行传代培养。传至第3代(P3)的骨髓基质细胞采用流式细胞术检测表面抗原CD71、CD44和CD45的表达,专用配套软件计算细胞表面抗原阳性%率。结果骨髓基质细胞呈纺锤形、成纤维细胞样生长,漩涡状分布。传至第3代的细胞形态趋于一致。流式细胞仪检测结果显示,CD71、CD44和CD45阳性细胞百分比分别为99.07±7.6%、52.81±7.3%和10.85±5.0%。结论P3的骨髓基质细胞具有较强的增殖特性。     

Flow cytometric analysis of intraepithelial lymphocytes in the human nasal mucosa

Recently, much research has been carried out on phenotypes and the function of intraepithelial lymphocytes of the intestines. However, only few studies have been made of nasal intraepithelial lymphocytes (nIEL), and the results have been controversial. We examined the population of different subsets of the human nIEL by means of 2-color flow cytometry after culturing lymphocytes isolated from the inferior turbinates of hypertrophic rhinitis in incubation media with monoclonal antibody against CD3. As a result, CD8+ cells (suppressor/cytotoxic T cells) and double negative T cells (CD8- CD4- T cells) were the most predominant. The CD4+ cells (helper/inducer T cells) to CD8+ cells ratio was 0.6. Cytotoxic T cells predominated over suppressor T cells in CD8+ cells and helper T cells predominated over inducer T cells in CD4+ cells. Double positive T cells (CD8+CD4+ T cells) were nil and natural killer cells and CD8+ killer cells few. CD8+ cells and CD4+ cells were activated during cell culture. alpha beta receptor bearing T cells predominated over gamma delta receptor bearing T cells in CD8+ cells and CD4+ cells but gamma delta receptor bearing T cells predominated over alpha beta receptor bearing T cells in double negative T cells. To investigate the effect of cell culture on the population of each phenotype of nIEL we also examined peripheral blood lymphocytes by flow cytometry before and after culture, and found that activated T cells markedly increased and suppressor T cells, gamma delta bearing T cells and natural killer cells slightly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)