微流控芯片上的肿瘤组织微阵列构建

研发了一种多层复合微流控芯片,包含64细胞培养微孔阵列,该微阵列集成了细胞进样、水凝胶三维支架形成和持续灌流培养的过程.以MCF-7乳腺癌细胞为模型,连续培养中监测细胞存活率、细胞密度、增殖率和细胞内pH值,并同时进行冰冻切片后免疫组化染色.实验结果显示,乳腺癌细胞在水凝胶微球中增殖形成了类组织结构.E-cadherin及Vinculin在细胞内、细胞间隙均出现较强表达,提示水凝胶微球中细胞建立了细胞-细胞、细胞-间质连接.芯片上连续培养15天内细胞存活率保持在85％以上,细胞增殖率随时间延长而递减.细胞内pH值检测显示芯片3D培养细胞内部呈现明显的酸化,其程度随着细胞密度增大而增加.这种芯片肿瘤组织微阵列构建方法简单高效,有望发展成为肿瘤研究的有力工具.     

微流控多层纸芯片研究乳腺癌组织的微环境酸化

将多层纸芯片技术用于肿瘤微环境酸化研究。将种植有乳腺癌细胞的8层硝酸纤维素薄膜叠放并封装于芯片中,用以模拟3D乳腺癌组织。灌流培养多层纸芯片乳腺癌组织数天后拆分多层纸芯片,以检测各层薄膜上细胞生存、增殖和胞内乳酸含量,解析不同深度下肿瘤细胞微环境酸化程度。实验表明,细胞酸化程度受灌流速度影响,高灌流速度可以增加纸层上细胞密度,酸性代谢产物排出增加。缺氧也是导致微环境酸化的重要因素。随着氧气扩散距离的增加,酸化程度加重,并且肿瘤细胞生存率和增殖率相应降低。     

阵列微流控浓度梯度网络用于细胞-化学刺激反应研究

设计和制作了具有5组平行浓度梯度形成网络和30个细胞培养池的微流控芯片,该芯片集成了细胞接种、培养、梯度浓度化学刺激、标记和检测等功能单元。芯片为玻璃-PDMS杂合结构,微流控通道刻蚀于玻璃层。芯片细胞培养池设计了系列围堰结构以利于细胞贴壁。细胞接种、灌流培养和试剂引入通过外接微量注射泵控制完成。该芯片可以生成连续、稳定的平行浓度梯度。观察发现,围堰结构有利于细胞接种和生长,乳腺癌MCF-7细胞在芯片灌流培养条件下生长良好。利用该芯片检测了在接受As2O3和乙酰丝氨酸(NAC)梯度浓度刺激后乳腺癌MCF-7细胞内谷胱甘肽(GSH)水平以及细胞阿霉素敏感性的变化,分析乳腺癌细胞阿霉素敏感性与细胞内GSH水平的关系。MCF-7细胞内GSH水平的变化与刺激药物浓度呈剂量-效应依赖关系,在接受As2O3刺激后GSH水平有所下降;而在接受NAC刺激后GSH水平有所升高。MCF-7细胞阿霉素敏感性与GSH水平相关。在降低GSH水平后药物敏感性升高;而升高细胞内GSH水平后敏感性降低。这种阵列微流控浓度梯度网络可以用于高通量细胞-化学刺激反应研究,有潜力成为细胞水平大规模药物筛选的技术平台。     

微流控芯片－质谱联用技术在细胞代谢与药物代谢中的研究进展

细胞代谢与药物代谢是新药筛选和研发的关键环节,在推动人类大健康发展进程中具有重要意义。通常情况下,细胞代谢和药物筛选以传统细胞培养测定研究为主,多为静态培养条件,无法很好地模拟体内细胞动态微环境。微流控芯片－质谱联用是近年发展起来的一种新型高通量分析技术。微流控芯片模块可高度模拟细胞体内动态微环境,与质谱联用可实时在线检测样品物质,具有高效、快速、简便、样品和试剂消耗低等特点,广泛应用于细胞代谢和药物代谢分析,有利于加速药物筛选研发进程。该文重点综述了微流控芯片－质谱联用技术及其在细胞代谢和药物代谢方面的应用概况,并对目前存在的局限性进行了讨论和展望,以期为微流控芯片－质谱联用技术在新药研发与细胞分析领域的发展提供参考。     

微流控芯片上细胞培养与分析方法研究进展

微流控芯片以其强大的微流体和微小物质控制能力成为研究单细胞、细胞群落乃至生物组织的重要手段。在本篇综述中,我们将以微流控芯片上细胞体外培养模型的建立为主,对近几年来重要的研究工作加以评述,全面地介绍微流控技术在细胞生命科学研究中应用的优势和未来发展方向,具体包括微流控芯片的细胞操控能力、细胞培养微环境的构建以及芯片联用检测手段,希望为从事这一领域研究工作的读者提供一些新的思路。     

微流控芯片技术在精细胞研究中的应用

具有多维网络微通道结构的微流控芯片可在微纳尺度上集成细胞进样、培养、分选、裂解和分离检测等多种功能单元,不仅在尺寸上与精细胞匹配,还可为精细胞提供相对封闭的接近生理状态的生长微环境。研究者已利用此系统的层流、微通道特殊几何结构等特点对精子进行了多方面研究。该文对微流控芯片技术在精细胞的培养、分选、胞内成分分析和人工授精中的应用进行了综述,介绍了用于精细胞研究的多种微流控芯片系统,并讨论了精细胞分选的各种方法。     

多层纸芯片乳腺癌组织微阵列用于抗肿瘤药物测试

开发了一种多层纸芯片细胞培养平台,将乳腺癌细胞分别接种于多层的图形化纸芯片的亲水区,折叠后构建了仿真实体肿瘤.多层纸芯片覆以微孔薄膜,用以仿真血管内皮层.培养不同时间后,拆解多层纸芯片检测乳腺癌组织内各层面的细胞形态、存活率、细胞周期分布以及细胞内乳酸含量.实验结果显示,各层纸芯片培养的乳腺癌细胞存活率均高于80％,并形成了类组织结构.芯片乳腺癌组织内部呈酸化倾向,且酸化程度随着培养时间的延长而升高.与二维(2D)培养细胞相比较,纸芯片乳腺癌组织内细胞增殖比例显著降低(15％ vs 60％).多层纸芯片乳腺癌组织显示了更接近体内情况的药物反应机制,细胞存活率随阿霉素浓度升高呈现缓慢下降趋势,IC50值显著高于2D培养细胞组(5.0 μmol/L vsl.144 μmol/L).这种多层纸芯片乳腺癌组织微阵列构建简便、仿真度高,有望成为抗肿瘤药物反应测试的有力工具.     

微流控芯片操纵传输及实时监测单细胞量子释放

微流控芯片技术用于细胞生化分析已引起了广泛关注.Harrison等首次在微流控芯片上对细胞群体进行操纵、传输及反应.yang等在微流控芯片上操纵细胞群体的排列,并用荧光检测细胞群体摄取钙的反应.至今还未见到微流控芯片对单个细胞进行操纵传输、定位及实时监测的报道.单细胞受激释放的监测对探索生物体神经传导具有重要意义.     

单细胞分析的研究

单细胞分析是分析化学、生物学和医学之间渗透发展形成的跨学科前沿领域。近年来,毛细管电泳及微流控芯片用于单细胞分析已取得显著进展,特别表现在微流控芯片用于细胞的培养、分选、操纵、定位、分离及检测细胞的组分,实时监测细胞释放,及高通量阵列检测等方面。芯片的单元操作可根据需要灵活组合,显示出其独特的优点。本文重点介绍作者研究组的工作,并对近三年来国内外在毛细管电泳及芯片毛细管电泳用于单细胞分析的新进展进行评论。最后从毛细管电泳与微流控芯片、微流控芯片与细胞界面以及量子点用于探测活细胞等方面,展望了单细胞分析的发展前景。     

基于微流控技术的单细胞生物物理特性表征

单细胞水平的生物物理特性表征,可有效阐明细胞的功能和状态,揭示细胞的单体差异性,对于细胞的分化和病理研究,以及疾病的早期临床诊断和治疗具有非常重要的意义。由于具有与细胞尺度相匹配的微米级腔道,微流控芯片比传统生化方法更适合单细胞样本的微环境精确控制、高通量定向操纵及多参数非特异性检测,已成为单细胞表征与分析的一项重要技术平台。本文总结了基于微流控技术的单细胞生物物理特性表征方法及其应用的最新进展,着重分析微流控芯片在常规方法难以达成的单细胞和高通量研究中的独特优势,最后探讨了微流控单细胞生物物理特性检测芯片在临床应用中面临的挑战和未来的发展动向,并提出一种新型的单细胞多参数同时表征的微流控分析器件。     

Dielectrophoresis-based cellular microarray chip for anticancer drug screening in perfusion microenvironments

We present a dielectrophoresis (DEP)-based cellular microarray chip for cell-based anticancer drug screening in perfusion microenvironments. Human breast cancer cells, MCF7, were seeded into the chip and patterned via DEP forces onto the planar interdigitated ring electrode (PIRE) arrays. Roughly, only one third of the cell amount was required for the chip compared to that for a 96-well plate control. Drug concentrations (cisplatin or docetaxel) were stably generated by functional integration of a concentration gradient generator (CGG) and an anti-crosstalk valve (ACV) to treat cells for 24 hours. Cell viability was quantified using a dual staining method. Results of cell patterning show substantial uniformity of patterned cells (92 ± 5 cells per PIRE). Furthermore, after 24 hour drug perfusion, no statistical significance in dose-responses between the chip and the 96-well plate controls was found. The IC(50) value from the chip also concurred with the values from the literature. Moreover, the perfusion culture exhibited reproducibility of drug responses of cells on different PIREs in the same chamber. The chip would enable applications where cells are of limited supply, and supplement microfluidic perfusion cultures for clinical practices.     

A microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow

Biological cells in vivo typically reside in a dynamic flowing microenvironment with extensive biomechanical and biochemical cues varying in time and space. These dynamic biomechanical and biochemical signals together act to regulate cellular behaviors and functions. Microfluidic technology is an important experimental platform for mimicking extracellular flowing microenvironment in vitro. However, most existing microfluidic chips for generating dynamic shear stress and biochemical signals require expensive, large peripheral pumps and external control systems, unsuitable for being placed inside cell incubators to conduct cell biology experiments. This study has developed a microfluidic generator of dynamic shear stress and biochemical signals based on autonomously oscillatory flow. Further, based on the lumped-parameter and distributed-parameter models of multiscale fluid dynamics, the oscillatory flow field and the concentration field of biochemical factors has been simulated at the cell culture region within the designed microfluidic chip. Using the constructed experimental system, the feasibility of the designed microfluidic chip has been validated by simulating biochemical factors with red dye. The simulation results demonstrate that dynamic shear stress and biochemical signals with adjustable period and amplitude can be generated at the cell culture chamber within the microfluidic chip. The amplitudes of dynamic shear stress and biochemical signals is proportional to the pressure difference and inversely proportional to the flow resistance, while their periods are correlated positively with the flow capacity and the flow resistance. The experimental results reveal the feasibility of the designed microfluidic chip. Conclusively, the proposed microfluidic generator based on autonomously oscillatory flow can generate dynamic shear stress and biochemical signals without peripheral pumps and external control systems. In addition to reducing the experimental cost, due to the tiny volume, it is beneficial to be integrated into cell incubators for cell biology experiments. Thus, the proposed microfluidic chip provides a novel experimental platform for cell biology investigations.     

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 s...     

一种直接测定微流控芯片电渗流速度的新方法

随着微芯片技术的成熟,越来越迫切地需要有一个准确而简洁的电渗流速度的检测方法。根据荧光物质罗丹明123（Rh123）在不同pH缓冲溶液中迁移时间的变化,推导出Rh123在pH 9和10条件下分别有中性分子存在,而中性分子的移动速度等于电渗流速度,因此建立了直接以Rh123中性分子为标记物测定电渗流速度的方法。通过直接检测Rh123中性分子的迁移时间,计算得出所用玻璃微流控芯片在pH 9.3和pH 10.1的电渗流速度为3.9×10-4 cm2/(s·V)和4.1×10-4 cm2/(s·V),与经典方法对照无明显差异。     

DNA fragment‐assisted microfluidic chip for capture and release of circulating tumor cells

Circulating tumor cells are specifically referred as cells that detached from the primary tumor and are present in the bloodstream. They could be isolated from blood and used as representative biomarker for predicting cancer prognoses. Here, we developed a microfluidic chip with multiple curved channels, in which DNA fragments and antibody‐based enrichment are exploited to capture circulating tumor cells in blood sample. By introducing DNA fragments as long tentacles, the active antibody could be extended into the microchannel stereoscopically, which could greatly increase the chances of adhesion in a multidirectional way and improve the capture efficacy. Several pivotal factors for cell capturing were optimized to the best state. Compared to conventional chips for planar capturing, the capture efficiency of MCF‐7 cells was greatly increased from 37.17 to 85.10%. For the detection of MCF‐7‐containing artificial blood sample detection, the capture efficiency of tumor cells was about 74.19 ± 2.13%, which was obviously better than the result of flow cytometry (29.67 ± 4.02%). Captured cells were easily released from the surface of microfluidic chip with high cell viability, which could be investigated for the molecular analysis in the field of tumor diagnosis.     

Biofunctionalization of electrowetting-on-dielectric digital microfluidic chips for miniaturized cell-based applications

In this paper we report on the controlled biofunctionalization of the hydrophobic layer of electrowetting-on-dielectric (EWOD) based microfluidic chips with the aim to execute (adherent) cell-based assays. The biofunctionalization technique involves a dry lift-off method with an easy to remove Parylene-C mask and allows the creation of spatially controlled micropatches of biomolecules in the Teflon-AF(?) layer of the chip. Compared to conventional methods, this method (i) is fully biocompatible; and (ii) leaves the hydrophobicity of the chip surface unaffected by the fabrication process, which is a crucial feature for digital microfluidic chips. In addition, full control of the geometry and the dimensions of the micropatches is achieved, allowing cells to be arrayed as cell clusters or as single cells on the digital microfluidic chip surface. The dry Parylene-C lift-off technique proves to have great potential for precise biofunctionalization of digital microfluidic chips, and can enhance their use for heterogeneous bio-assays that are of interest in various biomedical applications.     

Co-culture of tumor spheroids and monocytes in a collagen matrix-embedded microfluidic device to study the migration of breast cancer cells

A 3D co-culture microfluidic device was developed to study the effects of ECM stiffness and TAMs on tumor cells migration.     

聚二甲基硅氧烷-纸复合微流控芯片上的肝癌细胞三维培养

研发了一种聚二甲基硅氧烷-纸复合型微流控芯片用于肝癌细胞三维培养.芯片使用明胶处理硝酸纤维素薄膜作为细胞培养基底,以水凝胶网格作为三维培养支撑.结合微通道主动灌流与水凝胶中的被动扩散,模拟体内的流体运输形式实现细胞与外界物质交换.实验结果显示,芯片上的液滴生成以及细胞定位种植简便可靠.连续监测显示肝癌HcpG2细胞在水凝胶微球中增殖形成类似组织的三维结构.细胞增殖动力学分析以及生化检测结果显示了芯片三维培养与二维培养的差别.这种芯片三维细胞培养方法操作简便可靠,仿真度高,适合于肿瘤细胞研究.     

A serial dilution microfluidic device using a ladder network generating logarithmic or linear concentrations

In this paper, we propose a serial dilution microfluidic chip which is able to generate logarithmic or linear step-wise concentrations. These concentrations were generated via adjustments in the flow rate of two converging fluids at the channel junctions of the ladder network. The desired dilution ratios are almost independent of the flow rate or diffusion length of molecules, as the dilution device is influenced only by the ratio of volumetric flow rates. Given a set of necessary dilution ratios, whether linear or logarithmic, a serial dilution chip can be constructed via the modification of a microfluidic resistance network. The design principle was suggested and both the logarithmic and linear dilution chips were fabricated in order to verify their performance in accordance with the fluorescence intensity. The diluted concentrations of a fluorescein solution in the microfluidic device evidenced relatively high linearity, and the cytotoxicity test of MCF-7 breast cancer cells via the logarithmic dilution chip was generally consistent with the results generated with manual dilution.     

Automatic detecting and counting magnetic beads‐labeled target cells from a suspension in a microfluidic chip

A novel microfluidic method of continually detecting and counting beads‐labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre‐labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T‐lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell‐magnetic beads complex for beads‐labeled cell detection is simple, automatic, and particularly suitable for beads‐based immunoassay without using fluorescence labeling.