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-质谱联用细胞分析方法研究进展

细胞分析和代谢物分析在生物系统中起着重要的作用。微流控技术已成为细胞生物学研究的一个重要工具。该文总结了最近微流控芯片在细胞和代谢物的分析,尤其是微流控芯片与质谱联用技术的应用。同时对微流控芯片上细胞的生物学研究提出了见解和看法,希望能对感兴趣者提供一些启发。     

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。     

基于双向浓度梯度的微流控芯片系统对 肿瘤细胞侵袭能力的多重分析

利用微流控芯片易于模拟体内生理环境、 流体控制精确及易于集成等优势, 将基于扩散原理的浓度梯度形成结构与经典的圣诞树形浓度梯度发生器相集成, 建立了在垂直和水平方向上形成连续、 双向浓度梯度的微流控芯片系统, 采用该系统对不同类型细胞(HEK-293, MCF-7, SGC-7901)的侵袭力进行了定量分析; 通过在垂直方向上施加血清浓度梯度, 在水平方向上施加抗肿瘤药物十字孢碱浓度梯度, 分析了在连续药物浓度作用下的人胃癌SGC-7901细胞侵袭能力被抑制的情况, 同时观察并定量评价了伴随细胞侵袭力变化过程中细胞增殖能力受抑制的情况. 研究结果表明, 该系统可形成稳定的双向物质浓度梯度; 在血清浓度梯度存在情况下, 伴随十字孢碱浓度梯度的升高, 肿瘤细胞侵袭(P<0.0001)和增殖能力(P<0.001)均呈现浓度依赖性的连续降低. 建立的双向浓度梯度微流控芯片系统可用于评价复杂环境对细胞的多重影响, 也为研究细胞间相互作用、 多种药物联用及药物筛选等提供了良好的研究平台.     

惯性效应在微流控芯片中的应用

作为一种操控粒子或流体的新技术,基于流体惯性的操控技术已被应用于微流控芯片中粒子的输运、分选、聚焦及试样的混合和反应等操作,而在微尺度惯性效应基础上的惯性微流控芯片由于具有高通量、无需外场介入、低成本、易集成及微型化等众多优点,可用于解决医疗诊断、生化分析、合成化学及环境监测等领域的检测分析和微量操控问题,因此对该技术的机理及应用研究已成为目前微流控技术领域一个重要的研究热点。本文在介绍惯性微流控芯片机理及其研究进展的同时,从惯性聚焦、惯性分选及基于Dean流的微混合器和微流控光学器件等几个方面对惯性微流控芯片的最新应用研究进展进行了较为详细的介绍和分析比较。在此基础上,分析了惯性微流控芯片的局限和未来需要解决的问题。     

微流控芯片测定单细胞内化学组分的进展

细胞是生命的基本单元。由于细胞的个体差异,传统分析群体细胞的方法难以得到单细胞的重要信息。准确可靠地测定单细胞内化学组分的含量能大大提高从正常细胞中辨别不正常细胞的能力,为进一步研究和发展生物化学、医学和临床检验等领域奠定基础。近年来,用微流控芯片进行单细胞分析已引起广泛的兴趣。微流控芯片可以集成单细胞进样、溶膜、电泳分离胞内化学组分和高灵敏度测定等一系列操作步骤,为分析单细胞内的化学组分提供了新的技术平台。本文主要综述了近年来微流控芯片测定单细胞内化学组分的进展。重点在于利用电渗流、压力结合电渗流和激光镊子等技术操控单细胞在微流控芯片上完成单细胞进样、溶膜、细胞内化学组分的电泳分离和高灵敏度测定等一系列操作步骤。对在微流控芯片上的衍生技术也做了较为详细的阐述。     

微流控芯片上的免疫分析

近20年来,随着微流控芯片加工技术的不断发展,微流控分析已从一个概念发展为当前世界上最前沿的科技领域之一,微流控芯片上免疫分析的方法研究也取得重要进展。这些芯片包含传输流体的微通道和免疫分析程序中部分或全部的必要组件。微流控技术用于免疫分析在减少试剂用量、缩短分析时间、自动化等方面提高了分析性能。本文综述了微流控芯片上免疫分析的发展、分类,并评述了各类微流控免疫分析芯片的性能及优缺点。     

微流控芯片细胞实验室

以作者所在课题组近年开展的研究工作为基础,阐述了微流控芯片细胞实验室的平台特征,并从细胞个体、群体和多细胞生命体研究等三个方面概述微流控芯片细胞实验室的应用对象特征,显示其在生物医学领域的应用前景。     

一种微流控芯片中大体积进样方法及专用芯片

本发明涉及微流控芯片系统的进样方法。在使用固定相的微流控芯片系统中,于芯片分离通道与流动相人口之间设置两个或两个以上的分流通道。通过控制其中流体的切换和截止,实现对微流控芯片系统的大体积进样。该方法适合于微流控芯片中电色谱、加压电色谱和液相色谱模式下的大体积进样。该方法的优点为：可实现微流控芯片中大体积样品的上样;克服了系统中的梯度延迟效应,     

近期微流控芯片疾病诊断技术的研究进展

微流控芯片具有液流可控、样品消耗量小、反应速度快、易于集成化等特点,在临床诊断和疾病筛查领域具有广阔的发展前景。本文针对近年来微流控芯片技术在疾病诊断方面的最新研究进展,从疾病标志物检测、细胞筛选和药物代谢研究及疾病诊断微流控芯片装置的发展现状等方面概述其在疾病诊断方面的应用和发展。     

Macroporous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) matrices for cartilage tissue engineering

Polymer scaffold systems consisting of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) were investigated for possible application as a matrix for the three-dimensional growth of chondrocyte culture. The PHBV scaffolds were fabricated by a compression moulding, thermal processing and salt particulate leaching method without using organic solvent. The porous structure of the scaffolds was investigated with an optical microscope (OM) and scanning electron microscope (SEM) and the porosity was calculated. Then, the chondrocytes were cultured on the PHBV scaffolds for lone time to investigate whether it can be applied to construct the cartilage tissue in vitro. The results showed that the chondrocytes maintained their activity, fully expressed their phenotype and produced the extracellular matrix after incubation in vitro on the scaffolds for 7 days. In addition, in the prolonged incubation time, the percent of chondrocytes in their nature round morphology increased with an increase in the incubation period and they could synthesize the type II collagen and cartilage-specific proteoglycans. All of these results showed that the PHBV scaffolds had the potential to be used as chondrocytes carrier for cartilage engineering.     

Bioactive Phenylboronic Acid-Functionalized Hyaluronic Acid Hydrogels Induce Chondro-Aggregates and Promote Chondrocyte Phenotype

Hydrogels are extensively investigated as biomimetic extracellular matrix (ECM) scaffolds in tissue engineering. The physiological properties of ECM affect cellular behaviors, which is an inspiration for cell-based therapies. Photocurable hyaluronic acid (HA) hydrogel (AHAMA-PBA) modified with 3-aminophenylboronic acid, sodium periodate, and methacrylic anhydride simultaneously is constructed in this study. Chondrocytes are then cultured on the surface of the hydrogels to evaluate the effect of the physicochemical properties of the hydrogels on modulating cellular behaviors. Cell viability assays demonstrate that the hydrogel is non-toxic to chondrocytes. The existence of phenylboronic acid (PBA) moieties enhances the interaction of chondrocytes and hydrogel, promoting cell adhesion and aggregation through filopodia. RT-PCR indicates that the gene expression levels of type II collagen, Aggrecan, and Sox9 are significantly up-regulated in chondrocytes cultured on hydrogels. Moreover, the mechanical properties of the hydrogels have a significant effect on the cell phenotype, with soft gels (≈2 kPa) promoting chondrocytes to exhibit a hyaline phenotype. Overall, PBA-functionalized HA hydrogel with low stiffness exhibits the best effect on promoting the chondrocyte phenotype, which is a promising biomaterial for cartilage regeneration.     

Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.     

软骨细胞体外增殖去分化的拉曼光谱分析

基于显微拉曼光谱技术,对组织工程的软骨种子细胞在传代增殖过程中的去分化进行单细胞分析。首先,对体外单层培养的第1-4代(P1-P4)大鼠软骨细胞样本进行了单细胞拉曼光谱检测,由此识别出软骨细胞中各种碱基、糖基、氨基酸等主要物质分子结构的特征峰集合。随后,分析拉曼光谱中若干重点特征峰强度随细胞传代次数的变化,发现软骨细胞体外增殖过程中核酸(789、1094、1576 cm~(-1))含量降低、Ⅱ型胶原(特异组分为羟脯氨酸,1207 cm~(-1))和蛋白聚糖(特异组分为糖胺聚糖,1042、1063、1126、1160 cm~(-1))合成下降、脂质(1304 cm~(-1))及磷酸盐(957 cm~(-1))含量增加等分子水平变化,从而在活体单细胞层次初步揭示了去分化引起软骨细胞增殖变缓、分泌减弱、形态纤维化等现象的分子机制。     

Effects of adenovirus-mediated bFGF, IL-1Ra and IGF-1 gene transfer on human osteoarthritic chondrocytes and osteoarthritis in rabbits

The study investigated the effects of adenovirus-mediated gene transfection of basic fibroblast growth factor (bFGF), bFGF combined with interleukin-1 receptor antagonist protein (IL-Ra) and/or insulin-like growth factor-1 (IGF-1) both in human osteoarthritis (OA) chondrocytes and rabbits OA model. Human OA chondrocytes were delivered by adenovirus-mediated bFGF, IL-Ra and IGF-1 vectors, respectively. Chondrocyte proliferation, glycosaminoglycan (GAG) content, expression of type II collagen, ADAMTS-5, MMP-13, MMP-3 and TIMP-1 were determined. Rabbit OA model was induced by anterior cruciate ligament transaction (ACLT) in knees. Adenoviral vectors encoding human bFGF, IL-Ra and IGF-1 were injected intraarticularly into the knee joints after ACLT. The effects of adenovirus- mediated gene transfection on rabbit OA were evaluated. In vitro, the transfected genes were expressed in cell supernatant of human OA chondrocytes. AdbFGF group significantly promoted chondrocyte proliferation, and increased GAG and type II collagen synthesis than in the OA group. As two or three genes were transfected in different combinations, there was significant enhancement on the GAG content, type II collagen synthesis, and TIMP-1 levels, while ADAMTS-5, MMP-13, and MMP-3 levels were reduced. In vivo, the transfected genes were expressed in synovial fluid of rabbits. Intraarticular delivery of bFGF enhanced the expression of type II collagen in cartilage and decreased cartilage Mankin score compared with the OA control group (P = 0.047; P < 0.01, respectively). Multiple-gene transfection in different combinations showed better results than bFGF transfection alone. This study suggests that bFGF gene transfection is effective in treating experimental OA. Multiple gene transfection has better biologic effects on OA.     

In vitro growth and activity of chondrocytes on three dimensional polycaprolactone/chitosan scaffolds

Porous polycaprolactone/chitosan blend scaffolds with various compositional proportions were prepared using a particulate‐leaching method. The pore parameters of resultant scaffolds were found to be mainly modulated by porogen. The compressive mechanical properties and hydrophilicity of scaffolds were examined by measuring their compressive modulus and stress strength as well as swelling index. Selected chondrocytes isolated from articular cartilage of knee joints of rabbits were seeded on these scaffolds, and further in vitro cultured for various periods. The growth and activity of seeded cells were estimated by counting numbers of cells proliferated on scaffolds and measuring the amounts of proteoglycans and type II collagen synthesized by the seeded cells. It was found that some scaffolds composed of proper component ratios and having appropriate pore parameters exhibited promising characteristics for the adhesion and proliferation of seeded cells while maintaining the phenotype and activity of the cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Microscale control over collagen gradient on poly(L-lactide) membrane surface for manipulating chondrocyte distribution

A collagen gradient was constructed to interrogate cell adhesion on a poly(L-lactide) (PLLA) membrane surface. Utilizing a microinfusion pump, gradients of amino groups were generated on the PLLA surface by an aminolysis method. Immobilization of collagen onto the gradient surfaces was performed by glutaraldehyde (GA) coupling to form the collagen gradients. The -NH(2) and immobilized collagen density profiles on the PLLA membranes were quantitatively determined by ninhydrin and hydroproline (Hyp) analysis, respectively. By using fluorescein isothiocyanate (FITC) labeled collagen (FITC-Col), the profile of the as-prepared collagen gradient was directly monitored by a fluorescence microscope. The scanning force microscopy and water contact angle studies revealed that the morphology and wettability of the modified membranes changed progressively as a function of position along the gradient surface. Rabbit auricular chondrocytes were cultured on the collagen gradient membranes to test their cellular response. The attachment and spreading behaviors of the chondrocytes were dependent on the surface collagen density. These results indicate a surface on which the variation of collagen gradient strongly modifies the biological response of chondrocytes.