浓度与压力梯度可调的三维细胞培养微流控芯片的研制

目的制作化学浓度梯度与压力梯度可调的细胞三维培养微流控芯片,构建可模拟在体细胞生长所处动态微环境的体外模型。方法利用光刻成型技术、模塑法以及等离子键合工艺,制作3通道结构的微流控细胞培养芯片。通过微注射泵控制微通道内溶液流动生成浓度梯度,利用液面高度差生成压力梯度,并通过骨架染色比较二维培养与三维培养下的细胞形态。结果获得了化学浓度梯度与压力梯度可调的微流控细胞培养芯片。在2μL.min-1的流速下,中间通道的浓度梯度3 h后可达到相对稳定。100 Pa的压力差在中间通道生成的视在压力梯度为0.11 Pa/μm,从而驱动三维支架内间隙渗流的生成。并在微流控芯片内实现脐静脉内皮细胞稳定的三维培养。结论该芯片结构简单,制作方便,能灵活调控细胞生长所处的微环境,可进一步用于研究不同的微环境参数对细胞行为的影响。     

仿生微流控肝芯片研究进展

仿生微流控器官芯片是生物医药研究领域的一个前沿热点,它在微型芯片上建立起模拟人体器官的微组织,并带有高通量检测功能,在药物研发与毒性评价中具有重要的应用前景。肝脏是人体的代谢中枢,易于受到化合物毒害,其肝毒性是药物毒性评价中的重要指标。近年来,有关仿生微流控肝芯片的研究有大量的研究成果。概括和总结这种芯片的基本设计理念、微通道灌流方式、细胞体系,并讨论其主要应用前景和未来的发展趋势。     

微流控芯片技术在免疫分析中的应用

微流控芯片已广泛用于生物医学、高通量药物合成筛选、环境监测和生物战剂侦检等领域,本文就微流控芯片在免疫分析中的应用做一综述。1微流控芯片技术分析概述微流控芯片技术是通过微细加工技术在芯片上构建由储液池、微反应室、微管道等微功能元件构成的微流路系统,加载生物样品和反应液后,在压力泵或者电场作用下形成微流路,于芯片上进行一种或连续多种的反应,达到对样品高通量快速分析的目的。微流控芯片技术由于具有高度集成性,可在一张芯片上完成采样、稀释、加试剂、反应、分离和检测等多种功能,又被称为微型全分析系统(micro total a…     

微血管模型的重建和生物力学设计

血管生成在许多生理和病理过程中发挥着重要作用,但其机理仍不清楚.血管细胞在体内同时受到多种生物化学和生物力学刺激,处于复杂的微环境中,因此,在体外构建血管模型并重现其在体微环境,对探究血管生成机制十分必要.近年来,随着微加工和微流控技术的进步,各种体外微血管模型应运而生,对剪切力、渗流、血管生成因子浓度梯度等变量进行准...     

体外诱导小鼠胚胎干细胞血管形成

目的探讨体外定向诱导胚胎干细胞向内皮细胞分化的条件,模拟体内血管生成和血管新生过程,为研究新血管的形成提供一种新思路。方法体外培养小鼠胚胎干细胞诱导形成胚胎小体,将11 d的胚胎小体种植到胶原中诱发出芽性血管新生;通过免疫组织化学染色方法检测胚胎小体及其出芽向内皮细胞和功能血管的分化。结果胚胎干细胞在体外能自发形成胚胎小体,其内部含有血管样结构,并伴有平滑肌的分化;种植到胶原中的胚胎小体能再现出芽性血管新生。结论胚胎干细胞存体外不但能定向分化成内皮细胞,还能再现体内血管生成、血管新生及动脉生成过程。     

仿生微流控肝芯片的应用

正1生理机制研究仿生微流控肝芯片可用于研究肝脏的生理机制。例如,Prodanov等研制了一种由多孔膜分隔的两个微流体室组成的微流控肝芯片,由其模拟了肝窦的微结构与机能。Mi等使用天然可降解的鼠尾胶原和生长因子,培养HepG2和人脐静脉内皮细胞(HUVEC),在芯片上成功构建了肝窦样结构,其HepG2肝板和可控且均匀分布的HUVEC单层结构很好地模拟了体内肝脏的形态结构     

动态三维组织培养系统驱动方法的研究现状

文题释义：动态三维培养：是一种高度仿生的体外模型培养方式,通过三维培养可以重建体内复杂微环境的模型来克服传统体外模型的局限性;动态培养下的培养基流动可以为细胞和组织提供各种生物力学刺激,这种生物力学刺激是细胞和组织分化和各种生理过程中的重要因素。 微流控技术：是一门在微流道(100 nm-100 μm)中操纵微量(10^-9-10^-18 L)流体的微系统技术,可以用于生成和精确调节流体流动和时空梯度,以及通过可控制的方式向细胞提供营养和其他化学信号。其中,微流控驱动方法可以用来控制和调节微流道内流体的流动方式,常见的微流控驱动方法有表面张力泵、渗透泵、重力泵、注射泵和蠕动泵。  摘要背景：相比于动物模型,体外模型因其实验周期短、成本低、种属差异小等优点在毒理学、病理学和药学研究中被广泛使用。目前,动态三维组织培养模式是体外模型的重要发展趋势,而借助于微流控技术中驱动液体方式可实现体外模型的动态三维培养。 目的：简述微流控领域中的微流体驱动方法和各自的优缺点,以及不同驱动方法在不同组织培养需求中的应用。 方法：应用计算机检索CNKI、Web of Science数据库中与动态三维组织培养及微流控驱动方法实现细胞或组织动态培养的相关文献,检索中、英文关键词为“Microfluidic;Micropump;Organ-on-Chip;Three-dimensional tissue culture;微流控技术;微泵;器官芯片;三维动态组织培养”。结果与结论：微流控驱动方法主要分被动驱动与主动驱动,被动驱动包括表面张力泵、渗透泵、重力泵,主动驱动包括注射泵、蠕动泵,每种驱动方法均有其优缺点。对于动态三维组织培养系统中需要实现培养基流速的精准控制,可以优先考虑注射泵和阀门式蠕动泵;对于动态三维组织培养系统中需要实现培养基的闭环流动,可优先考虑重力泵和蠕动泵;对于动态三维组织培养系统中需要实现实验过程中的无菌环境,可优先考虑表面张力泵、重力泵和气动式蠕动泵;对于动态三维组织培养系统中需要实现高通量培养,可优先考虑表面张力泵、重力泵和气动式蠕动泵。 ORCID: 0000-0002-0807-4288(杨勇) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

血管生成的体外三维培养

肿瘤的血管生成对肿瘤的发生发展与侵袭转移等一系列病理过程具有重要作用。血管生成的体外三维培养方法为研究血管生成构建了类似于体内的三维空间。体外血管三维培养方法已由定性分析方法发展到半定量、定量分析方法,为今后开拓肿瘤治疗提供了一个新的思路。     

基于微流控芯片模拟体内受精及早期胚胎发育环境的输卵管模型构建

文题释义： 微流控芯片：又被称作芯片实验室,是将传统的化学技术和生物技术结合,并将所有基本操作单元微缩集成在一块芯片上以自动完成全过程的一项新技术,它在生物、化学、医学等领域都有巨大潜力,目前广泛运用于各行各业。 输卵管：女性生殖系统的重要组成部分,体内受精及早期胚胎培养的场所,胚胎在输卵管壶腹部和峡部交界处完成受精过程后,在流动的输卵管液、摆动的输卵管上皮细胞纤毛、收缩的输卵管肌肉等的共同作用下移动至宫腔进行着床,另外输卵管上皮细胞会分泌各种细胞因子辅助胚胎的发育,对胚胎的发育和着床过程非常重要。 背景：胚胎受精和早期胚胎培养是辅助生殖技术中重要的一部分,然而近几十年来胚胎培养技术却基本没有更新,因此胚胎受精和早期胚胎培养的条件成为了限制辅助生殖技术发展的一个瓶颈。 目的：构建基于微流控芯片来模拟体内受精及早期胚胎发育环境的仿真输卵管模型。 方法：采用软光刻法制作微流控芯片,使芯片微通道在形状上符合输卵管的解剖结构;组织消化贴壁法进行小鼠输卵管原代上皮细胞的培养和提纯;用角蛋白免疫荧光法对提纯后的小鼠输卵管原代上皮细胞进行鉴定,并将鉴定后的上皮细胞种植在微流控芯片通道内壁上以模拟输卵管生化环境;将芯片接入自动换液装置以模拟输卵管液流环境。 结果与结论：①这款输卵管模型呈圆柱状,长度为2 cm,直径为1 cm,在形状上与体内输卵管峡部的解剖学特征比较相符合;②角蛋白免疫荧光结果为阳性,提示组织消化贴壁法可分离培养出小鼠输卵管原代上皮细胞;③提纯后的小鼠输卵管原代上皮细胞种植在模型内壁,为胚胎的受精和早期培养提供了与体内微环境类似的生化环境。微流控芯片接入自动换液装置后,通道内的代谢废物能被及时带走,新的营养物质得以补充,实现了对输卵管真实流体环境的模拟;④研究将微流控芯片应用于辅助生殖技术,通过模拟体内受精及早期胚胎发育环境,实现了输卵管解剖学和生化环境的的重建,构建了以输卵管为模型的器官芯片,为进一步改善辅助生殖技术和提高受精率和优胚率奠定基础。 ORCID: 0000-0002-8168-8999(汪萌) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

微流控浓度梯度芯片的开发与生物化学应用研究

在生物化学分析中系统研究样本与不同浓度组分间的相互作用是至关重要的。微流控芯片技术能够在微米级的通道内完成精确的液体控制，近年来被普遍应用于生物化学分析领域。微流控浓度梯度芯片是一种能够快速构建稳定生物化学浓度梯度的工具，能够与大多数细胞培养、化学分析等技术相结合，为传统的生化分析提供新平台。本文综述微流控浓度梯度芯片的形成机制及其在生物化学等领域的应用，为拓宽浓度梯度相关应用研究提供新思路。     

多通道微流控芯片的设计、仿真及细胞迁移应用研究

细胞迁移是指细胞朝着特定的化学浓度梯度发生定向迁移运动,其在胚胎发育、伤口愈合、肿瘤转移中发挥着至关重要的作用。当前研究手段大多通量低,难以综合考虑不同浓度梯度条件对细胞迁移行为的影响。针对上述问题,本文首先设计了一款四通道微流控芯片,其特征如下:借助层流和扩散机制在细胞迁移主通道中建立和维持浓度梯度;可在单一显微镜视野下同时观测四组细胞迁移现象;集成了宽度为20μm的细胞隔离带,可校准细胞初始位置,保证实验结果的准确性。随后,借助Comsol Multiphysics有限元分析软件完成了微流控芯片的仿真分析,证明了芯片上设计S型微通道和水平压力平衡通道有助于在细胞迁移主通道中形成稳定的浓度梯度。最后,采用不同浓度(0、0.2、0.5、1.0μmol·L^-1)与糖尿病及其并发症密切相关的晚期糖基化终末产物(AGEs)孵育中性粒细胞,研究了其在100 nmol·L^-1趋化因子fMLP浓度梯度环境中的迁移行为。结果表明,AGEs抑制了中性粒细胞的迁移能力,证明了四通道微流控芯片的可靠性和实用性。     

Characterization of in vitro endothelial linings grown within microfluidic channels

In vivo, endothelial cells grow on the inner surface of blood vessels and are shaped to conform to the vessel's geometry. In the smallest vessels this shape entails substantial bending within each cell. Microfabricated channels can replicate these small-scale geometries, but endothelial cells grown within them have not been fully characterized. In particular, the presence of focal adhesions and adherens junctions in endothelial cells grown in microchannels with corners has not been confirmed. We have fabricated square microfluidic channels (50 μm wide, 50 μm deep) and semicircular microfluidic channels (60 μm wide, 45 μm deep) in polydimethylsiloxane and cultured human umbilical vein endothelial cells (HUVEC) within them. Immunofluorescent staining and three-dimensional reconstruction of image stacks taken with confocal microscopy confirmed that HUVEC are capable of forming adherens junctions on all channel walls in both channel geometries, including the sidewalls of square profile channels. The presence of shear stress is critical for the cells to form focal adhesions within both channel geometries. Shear stress is also responsible for the conforming of HUVEC to the channel walls and produces a square cross-sectional geometry of in vitro endothelial linings within square profile channels. Thus, geometry and applied shear stress are important design criteria for the development of in vitro endothelial linings of microvessels.     

Wnt5a-mediating neurogenesis of human adipose tissue-derived stem cells in a 3D microfluidic cell culture system

In stem cell biology, cell plasticity refers to the ability of stem cells to differentiate into a variety of cell lineages. Recently, cell plasticity has been used to refer to the ability of a given cell type to reversibly de-differentiate, re-differentiate, or transdifferentiate in response to specific stimuli. These processes are regulated by multiple intracellular and extracellular growth and differentiation factors, including low oxygen. Our recent study showed that 3D microfluidic cell culture induces activation of the Wnt5A/β-catenin signaling pathway in hATSCs (human Adipose Tissue-derived Stem Cells). This resulted in self renewal and transdifferentiation of hATSCs into neurons. To improve neurogenic potency of hATSCs in response to low oxygen and other unknown physical factors, we developed a gel-free 3D microfluidic cell culture system (3D-μFCCS). The functional structure was developed for the immobilization of 3D multi-cellular aggregates in a microfluidic channel without the use of a matrix on the chip. Growth of hATSCs neurosphere grown on a chip was higher than the growth of control cells grown in a culture dish. Induction of differentiation in the Chip system resulted in a significant increase in the induction of neuronal-like cell structures and the presentation of TuJ or NF160 positive long neuritis compared to control cells after active migration from the center of the microfluidic channel layer to the outside of the microfluidic channel layer. We also observed that the chip neurogenesis system induced a significantly higher level of GABA secreting neurons and, in addition, almost 60% of cells were GABA + cells. Finally, we observed that 1 month of after the transplantation of each cell type in a mouse SCI lesion, chip cultured and neuronal differentiated hATSCs exhibited the ability to effectively transdifferentiate into NF160 + motor neurons at a high ratio. Interestingly, our CHIP/PCR analysis revealed that HIF1α-induced hATSCs neurogenesis on the chip. This induction was a result of the direct binding of HIF1α to the regulatory regions of the Oct4 and β-catenin genes in nucleus. In the Chip culture of hATSCs that we developed, a low oxygen microenvironment was induced. The low oxygen level induced HIF1α expression, which resulted in increased expression of Wnt5A/β-catenin and Oct4 via the direct binding of HIF1α to the regulatory regions of β-catenin and Oct4.     

A novel TNF receptor family member binds TWEAK and is implicated in angiogenesis.

TWEAK is a member of the TNF ligand family that induces angiogenesis in vivo. We report cloning of a receptor for TWEAK (TweakR) from a human umbilical vein endothelial cell (HUVEC) library. The mature form of TweakR has only one hundred and two amino acids and six cysteine residues in its extracellular region. Five different assays demonstrate TWEAK-TweakR binding, and the interaction affinity constant (Kd) is within a physiologically relevant range of 2.3 +/- 0.1 nM. The TweakR cytoplasmic domain binds TRAFs 1, 2, and 3. Cross-linking of TweakR induces HUVEC growth, and mRNA levels are upregulated in vitro by a variety of agents and in vivo following arterial injury. Soluble TweakR inhibits endothelial cell migration in vitro and corneal angiogenesis in vivo.     

Microfluidic assay of endothelial cell migration in 3D interpenetrating polymer semi-network HA-Collagen hydrogel

Cell migration through the extracellular matrix (ECM) is one of the key features for physiological and pathological processes such as angiogenesis, cancer metastasis, and wound healing. In particular, the quantitative assay of endothelial cell migration under the well-defined three dimensional (3D) microenvironment is important to analyze the angiogenesis mechanism. In this study, we report a microfluidic assay of endothelial cell sprouting and migration into an interpenetrating polymer semi-network HA-Collagen (SIPNs CH) hydrogel as ECM providing an enhanced in vivo mimicking 3D microenvironment to cells. The microfluidic chip could provide a well-controlled gradient of growth factor to cells, whereas the hydrogel could mimic a well-defined 3D microenvironment in vivo. (In addition/Furthermore, the microfluidic chip gives a well-controlled gradient of growth factor to cells) For this reason, three types of hydrogel, composed of semi-interpenetrating networks of collagen and hyaluronic acid were prepared, and firstly we proved the role of the hydrogel in endothelial cell migration. The diffusion property and swelling ratio of the hydrogel were characterized. It modulated the migration of endothelial cells in quantified manner, also being influenced by additional synthesis of Matrix metalloproteinase(MMP)-sensitive remodeling peptides and Arginine–glycine–lycinee (RGD) cell adhesion peptides. We successfully established a novel cell migration platform by changing major determinants such as ECM material under biochemical synthesis and under growth factor gradients in a microfluidic manner.     

HB-EGF stimulates eNOS expression and nitric oxide production and promotes eNOS dependent angiogenesis

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family of ligands that is expressed by many cell types including endothelial cells. We have previously shown that HB-EGF stimulates angiogenesis in vitro in human umbilical vein endothelial cells (HUVEC). Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is an important regulator of angiogenesis. However, the role of HB-EGF in regulation of eNOS has not yet been investigated. Whether HB-EGF-induced endothelial cell migration and vascular network formation are mediated via production of NO from eNOS is also unknown. To address these questions, we stimulated HUVEC with HB-EGF and evaluated the expression of eNOS at the mRNA and protein levels. HB-EGF significantly upregulated expression of eNOS mRNA, stimulated eNOS protein production, and increased NO release from HUVEC. HB-EGF phosphorylated eNOS in a phosphatidylinositol 3-kinase (PI3K) dependent fashion, and stimulated in vitro angiogenesis. eNOS siRNA inhibited HB-EGF-stimulated HUVEC migration in a scratch assay. NG-nitro-L-arginine-methyl-ester (L-NAME) and L-N5-(1-lminoethyl)ornithine,dihydochloride (L-NIO) (specific inhibitors of eNOS) also abolished HB-EGF-induced HUVEC migration and angiogenesis. More importantly, we found that HB-EGF also promotes angiogenesis in vivo in the Marigel plug assay. Lastly, inhibition of the p38 MAPK pathway enhanced HB-EGF-induced EC migration and angiogenesis. We conclude that HB-EGF, through its interaction with EGF receptors (EGFR), stimulates eNOS activation and NO production via a PI3K-dependent pathway. Thus, activation of eNOS appears to be one of the key signaling pathways necessary for HB-EGF mediated angiogenesis. These novel findings highlight an important role for HB-EGF as a regulator of endothelial cell function.     

基于细胞3D打印技术的肿瘤药物筛选细胞芯片研究

现有的药物筛选评价技术中,动物筛选模型存在种属差异和周期长等缺点,高通量筛选和细胞筛选模型则与体内环境差异大,药物筛选准确率低。细胞3D打印技术为在体外构建仿真的组织器官模型提供了可能,当其与细胞芯片技术结合则为体外构建高效准确的药物筛选模型提供了新的技术空间。本研究构建了含有多个叉指电极（IDEs）阵列的细胞芯片,用细胞3D打印技术在芯片上组装了卵巢癌细胞HO-8910和人肝间充质干细胞HMSC-H组织模型,并通过对组织模型内细胞阻抗变化的检测,反映细胞生长、贴附、增殖、凋亡的过程及药物对细胞活性的影响等,最终基于该模型检测了抗癌药物顺铂和环磷酰胺对肿瘤细胞的杀伤和肝毒性。结果显示：支架微丝直径和孔径约为200～300 μm,肿瘤细胞和肝细胞在三维结构里生长良好;DMEM作为电解液,芯片在10.4 Hz可准确检测到三维结构中细胞增殖引起的阻抗变化,20 h后阻抗升高69.6%;基于该筛选模型,能同步检测到药物的抗肿瘤作用和肝毒性,并筛选出需要肝的二次代谢产物才能产生抗肿瘤性的药物环磷酰胺。     

“Deep-media culture condition” promoted lumen formation of endothelial cells within engineered three-dimensional tissues in vitro

In the field of tissue engineering, the induction of microvessels into tissues is an important task because of the need to overcome diffusion limitations of oxygen and nutrients within tissues. Powerful methods to create vessels in engineered tissues are needed for creating real living tissues. In this study, we utilized three-dimensional (3D) highly cell dense tissues fabricated by cell sheet technology. The 3D tissue constructs are close to living-cell dense tissue in vivo. Additionally, creating an endothelial cell (EC) network within tissues promoted neovascularization promptly within the tissue after transplantation in vivo. Compared to the conditions in vivo, however, common in vitro cell culture conditions provide a poor environment for creating lumens within 3D tissue constructs. Therefore, for determining adequate conditions for vascularizing engineered tissue in vitro, our 3D tissue constructs were cultured under a “deep-media culture conditions.” Compared to the control conditions, the morphology of ECs showed a visibly strained cytoskeleton, and the density of lumen formation within tissues increased under hydrostatic pressure conditions. Moreover, the increasing expression of vascular endothelial cadherin in the lumens suggested that the vessels were stabilized in the stimulated tissues compared with the control. These findings suggested that deep-media culture conditions improved lumen formation in engineered tissues in vitro.     

Bioactive silicate materials stimulate angiogenesis in fibroblast and endothelial cell co-culture system through paracrine effect

Angiogenesis is critical in tissue engineering, and bioceramic-induced angiogenesis has been reported. However, the role of other types of cells such as fibroblasts in this bioceramic-induced angiogenesis process has not been reported, and is closer to the in vivo situation of tissue regeneration. In this study, the paracrine effect of silicate bioceramic-induced angiogenesis in the presence of fibroblasts was confirmed by investigating the effects of calcium silicate (CS), one of the simplest silicate bioactive ceramics, on angiogenesis in co-cultures of human dermal fibroblasts (HDF) and human umbilical vein endothelial cells (HUVEC). Results showed that CS extracts stimulated the expression of vascular endothelial growth factor (VEGF) from co-cultured HDF and subsequently enhanced the expression of VEGF receptor 2 on co-cultured HUVEC (co-HUVEC). The endothelial nitric oxide synthase and nitric oxide production in co-HUVEC was then increased to finally initiate the proangiogenesis. During this process, the expression of vascular endothelial cadherin from co-HUVEC was up-regulated, and cadherin proteins were concentrated at the cell junctions to facilitate tube formation. Silicon ions are confirmed to play an important role during silicate bioceramic-inducing angiogenesis, and effective silicon ion concentrations (0.7–1.8 μg ml^?1) are proposed.