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。     

微流控免疫分析芯片的研究进展

在最近十几年中,微流控芯片技术得到了迅速发展,它可以提高分析速度、增加分析效率、减少样本和试剂的消耗。我们对微流控免疫分析芯片的设计、制作以及应用进行了综述。     

用于循环肿瘤细胞捕获的鱼骨型微流控芯片的模拟仿真与优化

目的通过对鱼骨型微流控芯片进行模拟仿真与优化,实现癌症患者外周血中循环肿瘤细胞的高效捕获。方法用Fluent 15.0软件对细胞在微流控芯片中的流动进行建模仿真,通过MATLAB编程统计芯片在不同结构参数(鱼骨宽度、鱼骨间隙、鱼骨高度、通道高度)、液体流动方向与流速等条件(共计250个条件)下所有细胞可被捕获的位置数目,预测细胞捕获效率,并进行实验验证。结果在鱼骨宽度75μm、间隙125μm、深度70μm、通道深度30μm、流体正向且流速1 m L/h的条件下,鱼骨芯片可以达到最高的细胞捕获效率。结论通过计算流体动力学方法对在不同芯片中的细胞捕获进行模拟,利用MATLAB建立捕获效率的统计模型并进行优化,快速筛选出可获得细胞高效捕获的参数组合,并通过实验,对优化的芯片参数进行验证,实现了循环肿瘤细胞的高效捕获。     

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     

人三维体外血管模型的构建

目的 利用HUVEC建立人体外三维血管模型。方法原代培养HUVEC，利用Ⅰ型胶原蛋白建立三维系统，在上面培养HUVEC，随着胶原蛋白的降解，人体外三维血管模型逐渐形成，HE染色三维血管样结构。结果HUVEC成功培养，人体外三维血管模型顺利构建。结论利用Ⅰ型胶原蛋白建立的三维系统，能成功构建人体外三维血管模型。     

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。     

微流控肺器官芯片的研究进展

近年来随着微制造技术的快速发展,推动了微流控器官芯片模型的建立。微流控器官芯片作为一种能够模拟器官中的组织和细胞结构及生理环境的微流控设备,得到了广泛的研究和发展。其中微流控肺器官芯片是最早研究的微流控器官芯片之一,与常规体外细胞和动物模型相比,微流控肺器官芯片模型模拟肺生理和病理条件更为精准,对呼吸系统疾病、药物开发及个体化治疗具有重要意义。文章介绍微流控肺器官芯片的原理、制造、模拟呼吸系统疾病和药物的研发及筛选,并总结微流控肺器官芯片发展所面临的挑战。微流控肺器官芯片旨在阐明复杂的病理生理学的肺疾病,并加快药物开发和药物筛选。随着肺部疾病和药物的社会和经济负担的增加,微流控肺器官芯片有望成为一个蓬勃发展的平台,也能减少制药公司和研究人员对传统体外细胞培养和动物模型的依赖。     

3D打印技术制备器官芯片的研究现状

器官芯片是一种新兴的体外生物模型,在生物医学领域有重要的应用前景。但是,相关研究的开展通常受限于器官芯片繁琐和昂贵的制备过程。近年来,科研工作者借助3D打印技术,实现器官芯片制备的简易化、低成本化,以及芯片结构复杂化和成型一体化。这一技术的突破,有力推动器官芯片相关研究的发展,为其在生物医学领域的广泛应用提供有力支持。综述3D打印制备器官芯片的研究现状,主要包括器官芯片的发展背景、传统制造方法的局限性、 3D打印器官芯片的技术分类及其生物医学应用。列举5种基于不同成型原理的3D打印方法,归纳比较各方法的工艺特点以及制备器官芯片时的适用范围,探讨3D打印制备肝、肾、血管、心脏等器官芯片的具体实例和效果。最后分析该技术的不足之处, 并展望这一领域的发展趋势。     

微流控法去除低温保护剂对卵母细胞发育的影响

为了减小低温保护剂去除过程对卵母细胞造成的渗透损伤和毒性损伤,本文利用微流控芯片对猪二次减数分裂中期(MⅡ-stage)卵母细胞低温保护剂的去除方案进行了优化研究。首先分析了微流控去除方法中去除时间、去除液成分及浓度对卵母细胞存活率及体外发育情况的影响,然后将微流控去除方法与传统的一步法、两步法进行了比较。研究结果表明,微流控法中去除总时间为8 min时,卵母细胞存活率(95.99%±4.64%)及桑椹胚率(74.17%±1.18%)与新鲜细胞(98.53%±2.94%;78.22%±1.34%)相比,差异无统计学意义;1 mol/L蔗糖去除液最有利于卵母细胞低温保护剂去除后的存活及体外发育;微流控法去除低温保护剂后,卵母细胞的存活率、体外发育情况等,均好于传统去除方法。本文研究结果提示,以微流控法去除低温保护剂可减小对卵母细胞的损伤,从而可能进一步提高卵母细胞的低温保存效果。     

Integration of optical fiber light guide,fluorescence detection system,and multichannel disposable microfluidic chip

A combination of fluorescence detection and microfluidic technology provides promising applications in life sciences. A prototype of an integrated fluorescence detection system and optical fiber light guide on a laminate-based multichannel microfluidic chip has been developed and tested. A blue LED, plastic optical fiber, photodiode, Mylar and PMMA, and fluorescein and BSA-FITC were used as an excitation source, light coupler and guide, detector, microfluidic substrate and sample, respectively. The results show that the system is capable of detecting weak fluorescence emission from a fluorescein solution at concentration down to 0.01 ng/ml, and gives linear response. The results were also reproducible, and no cross-talk between adjacent channels was observed. The test using BSA as a model analyte demonstrates its feasibility for on-chip immunosensor applications. The performance and applications can be developed further. This prototype can be used as a platform to develop a simple and compact bio-fluorescence detection system integrated with an inexpensive and disposable multichannel microfluidic chip for biomedical devices.     

VEGFR-3 in adult angiogenesis.

Vascular endothelial growth factor receptor 3 (VEGFR-3, Flt-4), the receptor for vascular endothelial growth factors (VEGFs) C and D, is expressed on lymphatic endothelium and may play a role in lymphangiogenesis. In embryonic life, VEGFR-3 is essential for blood vessel development. The purpose of this study was to investigate whether VEGFR-3 is also involved in blood vessel angiogenesis in the adult. This was studied in human tissues showing angiogenesis and in a model of VEGF-A-induced iris neovascularization in the monkey eye, by the use of immunohistochemistry at the light and electron microscopic level. VEGFR-3 was expressed on endothelium of proliferating blood vessels in tumours. In granulation tissue, staining was observed in the proliferative superficial zone in plump blood vessel sprouts, in the intermediate zone in blood vessels and long lymphatic sprouts, and in the deeper fibrous zone in large lymphatics, in a pattern demonstrating that lymphangiogenesis follows behind blood vessel angiogenesis in granulation tissue formation. At the ultrastructural level, VEGFR-3 was localized in the cytoplasm and on the cell membrane of endothelial cells of sprouting blood vessels and sprouting lymphatics. In monkey eyes injected with VEGF-A, blood vessel sprouts on the anterior iris surface and pre-existing blood vessels in the iris expressed VEGFR-3. In conclusion, these results support a role for VEGFR-3 and its ligands VEGF-C and/or VEGF-D in cell-to-cell signalling in adult blood vessel angiogenesis. The expression of VEGFR-3 in VEGF-A-induced iris neovascularization and in pre-existing blood vessels exposed to VEGF-A suggests that this receptor and possibly its ligands are recruited in VEGF-A-driven angiogenesis.     

Rapid remodeling of airway vascular architecture at birth

Recent advances have documented the development of lung vasculature before and after birth, but less is known of the growth and maturation of airway vasculature. We sought to determine whether airway vasculature changes during the perinatal period and when the typical adult pattern develops. On embryonic day 16.5 mouse tracheas had a primitive vascular plexus unlike the adult airway vasculature, but instead resembling the yolk sac vasculature. Soon after birth (P0), the primitive vascular plexus underwent abrupt and extensive remodeling. Blood vessels overlying tracheal cartilage rings regressed from P1 to P3 but regrew from P4 to P7 to form the hierarchical, segmented, ladder‐like adult pattern. Hypoxia and HIF‐1α were present in tracheal epithelium over vessels that survived but not where they regressed. These findings reveal the plasticity of airway vasculature after birth and show that these vessels can be used to elucidate factors that promote postnatal vascular remodeling and maturation. Developmental Dynamics 239:2354–2366, 2010. © 2010 Wiley‐Liss, Inc.     

A method of characterising the complex anatomy of vascular occlusions and 3D printing biomimetic analogues

Chronic total occlusions (CTOs) occur in approximately 40% of individuals with symptomatic peripheral arterial disease and are indicative of critical limb ischaemia. Currently, few medical devices can effectively treat CTOs long-term, with amputation often required. This is due to a lack of knowledge of CTO anatomy, making device design and testing difficult. This study is a proof-of-concept study, which aimed to develop a workflow for further characterising the complex multi-material anatomy of CTOs and creating 3D models of CTO components, which may be useful in producing a vascular CTO biomimetic for device testing. Here, we establish such a workflow using samples of atheromatous plaques. We focus on a high-resolution, non-destructive microcomputed tomography (μCT) technique which enables visualisation of occlusion anatomy at a greater resolution than computed tomography angiography (CTA), which is the typical modality used for CTO clinical visualisation. Four arteries (n = 2 superficial femoral; n = 2 popliteal) with evidence of atheromatous plaques were cut into 8 cm segments, which were then stained with iodine and scanned at low resolution, with calcified regions rescanned at high resolution. Resulting files were manually segmented to generate 3D models, which were then 3D printed in resin using a stereolithography printer to produce parts suitable for creating a biomimetic. In total, μCT files from three arterial segments (n = 2 high resolution, n = 1 low resolution) were deemed suitably calcified for segmentation, and thus were segmented to produce 3D models. 3D models of the arterial wall, intima and atheromatous calcium deposits from a high-resolution popliteal artery scan were successfully 3D printed at several scales. While this research is at an early stage, it holds great promise. The workflow for segmentation and 3D printing various components of an atheromatous plaque established here is replicable and uses software and equipment which are accessible to research laboratories in both academia and industry. The ability to print detailed models on a desktop 3D printer is unprecedented and can be improved further, which is promising for future development of biomimetics with multi-material detail of both soft tissue and calcified components of a vascular occlusion. Indeed, this workflow provides a solid foundation for future studies of CTO anatomy and the creation of true, multi-material CTO biomimetics. Such biomimetics may enable the development of improved interventional devices, as they would mimic the general in vivo CTO environment. As this method cannot be applied in vivo, we cannot yet produce patient-specific biomimetics, however, these analogues would still be important in device development, which would improve patient outcomes in critical limb ischaemia.     

Morphology of angiogenesis in human cancer: a conceptual overview, histoprognostic perspective and significance of neoangiogenesis

This paper reviews the histomorphological aspects of angiogenesis and neoangiogenesis, quantitative and qualitative, and their applications in prognostic evaluation of neoplastic diseases. The merits and weak points of intratumoral microvessel density (MVD), a widely regarded bona fide predictor of tumour growth, metastases and patient survival, are discussed. Total microvascular area (TVA) has been found useful in recent prognostic studies utilizing newer immunohistochemical vascular markers. Of particular significance is the fact that MVD and TVA are most predictive of patient outcome in those tumours that induce significant neoangiogenesis, namely carcinomas of breast and prostate, and haematological malignancies. In contrast, carcinomas of lung and urinary bladder do not show significant associations of MVD and TVA with poor prognosis, reflecting differences in angiogenic mechanisms. In gliomas, MVD appears to correlate with outcome in high-grade, but not low-grade tumours, and does not correlate with tumour cellularity in the infiltrating portions of the tumour, reflecting a paucity of neoangiogenesis and directional vascular growth. Recent studies have found CD105, Tie-2/Tek and vascular endothelial growth factor receptors to be the best markers of neoangiogenesis. The vascular parameters so measured correlate better with overall and disease-free survival in breast, colon and lung carcinoma than panendothelial markers such as CD31. A correlation of vascular patterns with prognosis has been established in ocular melanomas, glioblastomas and squamous carcinomas of head and neck region. Vascular networks with closed loops are closely associated with mortality due to metastases in uveal melanomas. Fewer bizarre glomeruloid vessels and prominent classical capillary pattern was an independent predictor of longer survival in glioblastoma. Therefore a judicious combination of quantitative and qualitative microscopic angiogenic parameters, with emphasis on neoangiogenesis and vascular patterns wherever applicable, should be an integral component of a more consistent tumour staging system for accurate prognostic evaluation of tumours, selection of optimal anti-angiogenic therapy and pertinent research.     

Polyurethane elastomer: a new material for the visualization of cadaveric blood vessels

A multitude of various materials are available for the visualization of cadaveric vessels, ranging from natural materials like gelatin and latex to synthetic materials like silicone rubber or acrylates. To achieve a detailed overview of the vascular architecture in microvascular studies in experimental flap surgery, the injected material should have low viscosity to assure perfusion of even the smallest vessels. In addition, the material ideally should have either no or only minimal shrinkage, and should harden within a reasonable time, but retain sufficient elasticity and resistance to withstand tearing off the delicate vessels during subsequent dissection or casting. Because none of the available injection materials adequately combines these attributes, we evaluated the polyurethane elastomer "PU4ii" in latissimus dorsi muscles as a new material for the visualization of cadaveric vessels in comparison with the frequently used silicone rubber. The dissection of vessels injected with PU4ii proved easy largely because of its exceptional hardness. Even if not visible before dissection, the completely perfused vessels were easily palpated in the surrounding fat or muscle tissue of the microsurgical latissimus dorsi model. Despite the significantly higher hardness of PU4ii over silicone rubber (98 Sh-A vs. 12 Sh-A), PU4ii proved enough elasticity (20-25 N/mm(2) E modulus) and a high tear resistance (64-68 N/mm vs. 15 N/mm) preventing breakage during dissection even within the smallest vessels. In contrast to silicone rubber (and latex or gelatin), the high corrosion resistance and form stability of PU4ii also allowed building of casts for qualitative examination by scanning electron microscopy and quantitative analysis of the vessel density using micro-computed tomography with accurate 3D representation. In this study we show that PU4ii has physical characteristics that make it a multi-purpose material that allows at the same breath an excellent gross visualization of the architecture of cadaveric blood vessels as well as a detailed evaluation of casts by modern microscopic and or radiologic tools. Thus, the new polyurethane elastomer PU4ii is in many respects superior to the widely used silicone rubber and can be strongly recommended as a visualization material for a comprehensive evaluation of cadaveric blood vessels in microsurgery.     

Interferon-α is a Potent Inhibitor of Basic Fibroblast Growth Factor-Stimulated Cell Proliferation in Human Uterine Cells

PROBLEM: Abnormal uterine bleeding is a significant health problem for many women and is the number-one reason for performing hysterectomy in the United States. Leiomyomas (uterine fibroids) are benign neoplams that are a frequent cause of abnormal uterine bleeding. The goal of this study was to assess the effects of the anti-angiogenic cytokine, interferon (INF)-α, on the proliferation of both leiomyoma and normal uterine cells. METHOD OF STUDY: Primary cultures of leiomyoma, myometrial, and endometrial stromal cells were established for in vitro study. The effects of INF-α (10, 100, and 1000 U/ml) were tested on serum-stimulated and basic fibroblast growth factor-stimulated cell proliferation using the [³H]thymidine incorporation assay. RESULTS: INF-α was a potent inhibitor of cell proliferation for all three cell types, with endometrial stromal cells showing the greatest sensitivity. The antiproliferative effect did not appear to result from toxic effects on the cells. CONCLUSION: INFs may prove to be useful therapeutic agents for the treatment of leiomyoma-related abnormal uterine bleeding.     

3D-reconstruction of craniofacial structures of a human anencephalic fetus. Case report

It was the purpose of this study to investigate the craniofacial bones, cartilages and major blood vessels of a human anencephalic fetus and to describe the malformations of these structures accompanying this developmental disruption. The head of a 16 week old human fetus with anencephaly was serially sectioned and the craniofacial bones, cartilages and major blood vessels were traced and reconstructed by 3D-computer technology. The sphenoidal, temporal and occipital bones showed severe malformations, whereas the bones of the facial cranium were normally developed. Both internal carotid arteries ended within the cerebro-vascular area with no further branches. The vertebral arteries also ended within the cerebro-vascular area. No arterial circle of Willis was developed. The notochord terminated normally in the sphenoid body. The observations indicate that the possible reason for this malformation was non-closure of the anterior neuropore of the neural tube. The normal termination of the notochord suggests that it had no influence on the pathogenesis of this malformation. The malformations of the temporal and occipital bones are contradictory to the hypothesis of Marin-Padilla (1991) that anencephaly is caused by a maldevelopment of the sphenoidal bone.