旋转式三维细胞培养装置的研制

背景：内皮细胞体外培养形成的单层内皮细胞能否抵抗血流的冲击,如何实现细胞均匀分布生长是实验中的常见问题。 目的：针对背景问题,研制一种细胞培养装置,可提供细胞生长的三维环境,实现细胞贴硅胶弹性腔管壁生长,改善细胞的分布不均。 方法：从控制单元、机械单元和细胞培养单元介绍了该装置的结构与功能,并利用该装置进行一例脐静脉内皮细胞培养实验。 结果与结论：旋转式三维细胞培养装置为细胞代谢提供了一定条件,该装置培养的细胞能贴壁均匀,生长良好。     

一种新型机械应变细胞加载装置的研制

目的研究开发一套具有自主知识产权的数控机械应变细胞加载装置,为细胞力学研究提供必要的研究手段。方法加载装置基于圆形基底形变技术,采用数字式测控系统和基于PC机平台的专用软件,实现对体外培养细胞加载牵张应变。采用MTT比色实验检测人牙周膜细胞在弹性硅橡胶细胞培养膜上的附着生长能力。采用该装置对体外培养人牙周膜细胞加载1%、10%、20%拉伸应变0.5、1和24 h,倒置相差显微镜观察细胞形态、排列的变化。结果数控机械应变细胞加载装置可对体外培养细胞加载不同强度、频率和时间的拉伸应变,具有输出应变范围大、精度高、操作方便、显示直观等优点。硅橡胶膜和对照细胞培养板接种细胞1、2、4、7、8 d后,MTT比色实验光吸收值之间无统计学差异(P>0.05),显示弹性硅橡胶膜具有良好的细胞附着生长能力。人牙周膜细胞加载10%、20%拉伸应变24 h后,细胞形态、排列发生改变,胞体呈长梭形,并成栅栏状平行排列,细胞长轴垂直于拉伸应变方向。结论数控机械应变细胞加载装置可有效地对体外培养细胞加载动态机械拉伸应变,为体外细胞力学研究提供了必要的研究手段。     

新型动态载荷与循环灌流生物反应器系统的设计

目的设计一套动态应变加载系统和三维灌流培养系统,并测试其性能。方法明确动态载荷与循环灌流生物反应器的设计原则;设计并构建动态应变加载系统和三维灌流培养系统,研制供三维灌流与加载专用的培养舱;测定培养系统的无菌效果以及其应变加载的精确度和稳定性,进行组织工程骨的初步培养观察。结果该生物反应器可为组织培养物提供不同大小和频率的压应变以及不同流量的灌流条件,可控精度高、操作简单可行、性能稳定;连续运转5 d后,培养液中无细菌生长。初步培养结果表明,体外构建的组织工程骨在生物反应器中培养10 d,其成骨细胞的增殖和成骨活性明显高于静态培养和单纯灌流培养。结论该生物反应器可以成为三维条件下进行骨细胞生物力学研究的一种较理想的动态培养与应变加载装置。     

一种应用于应变场三维细胞培养的组织工程支架

我们从组织工程化组织构建的角度，提出了一种可用于应变场细胞三维培养的组织工程支架。采用表面化学和材料力学方法，探讨了支架的组成、结构、表面特性、力学性质及细胞相容性。利用生物医用聚乙烯醇（PVA）耐水泡沫，表面裱衬生物可降解聚乳酸羟基乙酸共聚物（PLGA）后，具有良好的表面特性和适宜的孔隙率，既能产生一定的弹性回缩，又具有良好的细胞相容性。结果表明，PVA耐水泡沫表面裱衬PLGA为组织工程研究应变对细胞三维培养的影响提供了一种良好的三维支架。     

一种用于应变场三维细胞培养的组织工程支架

我们从组织工程化组织构建的角度 ,提出了一种可用于应变场细胞三维培养的组织工程支架。采用表面化学和材料力学方法 ,探讨了支架的组成、结构、表面特性、力学性质及细胞相容性。利用生物医用聚乙烯醇(PVA)耐水泡沫 ,表面裱衬生物可降解聚乳酸羟基乙酸共聚物 (PL GA)后 ,具有良好的表面特性和适宜的孔隙率 ,既能产生一定的弹性回缩 ,又具有良好的细胞相容性。结果表明 ,PVA耐水泡沫表面裱衬 PL GA为组织工程研究应变对细胞三维培养的影响提供了一种良好的三维支架。     

一种体外贴壁细胞应变加载装置的研制

目的开发一套新型的应变加载装置,用于贴壁细胞力学生物学研究。方法该装置基于基底形变加载技术,采用可控制编程器驱动步进器,引起硅橡胶小室变形,实现多单元大应变的细胞加载;研制该装置,检测机械性能;建立硅橡胶小室的三维模型,利用有限元技术对硅橡胶小室进行仿真,分析该小室的应变场均匀性问题;采用该装置对骨髓间充质干细胞(bone marrow stromal cells,BMSCs)加载5%机械应变,频率0.5 Hz,2 h/d,持续5 d,并在倒置显微镜下观察细胞形态的变化。结果所研制的适用于体外细胞加载装置可对3组细胞加载基底实现最大至50%机械单向应变;在10%应变范围内,硅橡胶小室底部的均匀应变场面积占比保持在50%以上,保证了细胞受力均匀; BMSCs形态发生明显变化,排列方向趋于垂直主应变加载方向。结论该装置运行可靠,应变范围宽,频率可调,操作方便,可同时对多组细胞培养基底进行应变加载,为细胞力学生物学研究提供了便利条件。     

生物反应器在组织工程中的应用

组织工程学是利用生命科学与工程科学原理,体外制备用于修复受损组织的移植物,以满足器官移植需求.目前制备的工程化组织存在生物活性差和经济成本高的缺陷,限制了其临床应用.生物反应器是制备工程化组织的重要设备,它可以促进营养物质交换和增加机械应力刺激等.通过对培养微环境的精细调控,生物反应器可以促进工程化组织的熟化,降低成本,从而推动工程化组织的实际应用.     

小口径组织工程血管生物反应器的制造

生物反应器在构建小口径组织工程血管中起着重要的作用.本文设计并造了能够模拟人体小口径动脉脉动流的生物反应器.该反应器的波形发生器输出成年人左心室容积变化信号驱动直线电机作为动力源,通过调节后负荷,从而产生近生理的脉动流.特殊设计的旋转培养室可以对三维的管壁支架进行二次的细胞接种.并且使得旋转接种和脉动培养能够连续进行.与现有的组织工程血管生物反应器相比,在理论和实践上有较大的创新性.     

动态力学应变对人骨膜细胞的生物学作用

探讨动态力学应变对体外分离培养的人骨膜细胞的生物学效应。使用Flexercell应力系统，将频率为1Hz、振幅为5％变形、正弦波状力学应变作用于体外培养的人骨膜细胞，在不同时间段检测细胞总蛋白含量、DNA合成量、ALP活性及骨钙素分泌量，并与空白对照组相比较，观察动态力学应变对人骨膜细胞生长、分化指标的影响。结果表明：所施加的动态力学应变对骨膜细胞的生长增殖无明显影响，但显著促进骨膜细胞向成骨细胞的分化。动态力学应变能够促进人骨膜细胞向成骨细胞分化，这可能是其对骨的生物学作用的机制之一。     

一种组合式组织工程动态培养仪

随着组织工程技术应用的多样化,为不同的目标组织提供不同的培养条件,改进工程组织的化学和生物力学性能,或通过不同的培养条件诱导干细胞向不同方向分化,这些得到了学者们越来越多的关注。结合中国现有的实验室条件,中国医学科学院,北京协和医学院整形外科医院自主研发了一种组合式组织工程动态培养仪,以为不同的目标工程组织提供不同的动态培养环境。     

采用生物反应器体外构建大面积组织的接种技术

目的　采用培养袋生物反应器进行大面积支架材料细胞接种技术的研究。方法　以人体成纤维细胞和PET无纺布支架材料为模型,考察水平摇床的转速和起始细胞悬液密度对细胞接种动力学、细胞接种率、支架中细胞密度以及细胞分布的影响。结果　在实验条件范围内,成纤维细胞接种大面积PET支架材料的过程基本符合一级反应动力学;低细胞悬液密度接种时,接种率和支架中细胞密度随转速的增加而下降,高细胞悬液密度接种时则反之;细胞分布均匀度都随着转速的增加而下降,起始细胞悬液密度对细胞分布影响较小。结论　采用培养袋生物反应器进行大面积支架材料的细胞接种是可行的,研究结果为进一步优化适合大面积支架材料接种的方法奠定基础。     

动物细胞培养用生物反应器及其力学环境

动物细胞体外培养时 ,生物反应器是整个培养过程的关键设备 ,为细胞提供一个适宜的生长环境 ,使之快速增殖并形成所需的生物组织制品。由于动物细胞在其形态结构、培养方法以及所需的力学环境等方面均不同于微生物细胞 ,因而传统的微生物反应器显然已不适用于动物细胞大规模培养 ,特别是组织工程的需要 ,促使新型生物反应器的研究与开发。本文针对动物细胞培养的基本特点 ,综述了动物细胞培养用生物反应器 ,并探讨了生物反应器中的力学问题     

Mechanical Characterization of an In Vitro Device Designed to Quantitatively Injure Living Brain Tissue

Due to the nonlinear, viscoelastic material properties of brain, its mechanical response is dependent upon its total strain history. Therefore, a low strain rate, large strain will likely produce a tissue injury unique from that due to a high strain rate, moderate strain. Due to a lack of current understanding of specific in vivo physiological injury mechanisms, a priori assumptions cannot be made that a low strain rate injury induced by currently employed in vitro injury devices is representative of clinical, nonimpact, inertial head injuries. In the present study, an in vitro system capable of mechanically injuring cultured tissue at high strain rates was designed and characterized. The design of the device was based upon existing systems in which a clamped membrane, on which cells have been cultured, is deformed. However, the present system incorporates three substantial improvements: (1) noncontact measurement of the membrane deflection during injury; (2) precise and independent control over several characteristics of the deflection; and (3) generation of mechanical insults over a wide range of strains (up to 0.65) and strain rates (up to 15s^–1). Such a system will be valuable in the elucidation of the mechanisms of mechanical trauma and determination of injury tolerance criteria on a cellular level utilizing appropriate mechanical injury parameters.     

力学应变对体外培养动物细胞的影响

介绍了生物力学领域中细胞生长、增殖与分泌方面的实验研究进展.着重介绍了基底加载实验中不同环境对细胞分裂与增殖的作用;力学载荷对细胞黏附及生长的影响;力学应变对体外培养成骨细胞增殖与分泌的影响;流体环境对细胞生长增殖的作用.     

A Strain Device Imposing Dynamic and Uniform Equi-Biaxial Strain to Cultured Cells

The objective of this study is to design a new apparatus to allow the control of the magnitude and frequency of dynamic stretch applied uniformly to cells cultured on a silicon elastic membrane. The apparatus is designed to produce equi-biaxial dynamic stretches with area changes ranging from 0% to 55% and frequencies ranging from 0 to 2 Hz. Homogeneous finite strain analysis using triangles of markers was performed to compute the symmetric two-dimensional Lagrangian strain tensor on the membrane. Measurements of strain in both static and dynamic conditions showed that the shear component of the strain tensor (E_rc) was near zero, and that there was no significant difference between radial (E_rr) and circumferential (E_cc) components, indicating the attainment of equi-biaxial strain. Bovine aortic endothelial cells were transiently transfected with a chimeric construct in which the luciferase reporter is driven by TPA-responsive elements (TRE). The transfected cells cultured on the membrane were stretched. The luciferase activity increased significantly only when the cells were stretched by 15% or more in area. Cells in different locations of the membrane showed similar induction of luciferase activities, confirming that strain is uniform and equi-biaxial across the membrane. © 1998 Biomedical Engineering Society. PAC98: 8780+s, 8745-k, 8722-q     

旋转灌注式生物反应器系统构建及在骨组织工程中的应用

构建一种旋转灌注式生物反应器系统，并设计制造具有相互连通微管道结构的大段支架。生物反应器在旋转的同时实现对大段支架的灌注，系统内外气体的实时交换通过安装在反应器两端的半透膜和可透气的蠕动泵软管实现。容器内的氧气和营养物质在得到充分混合之后，连续不断地输送到支架微管道内。使黏附在微管道内的细胞获得充足营养的同时受到一定流体剪应力的刺激，调节细胞功能的发挥。该系统克服了静态培养中存在的各种缺点，改善了培养环境，增加了培养过程的可控性，有助于促进黏附在微管道内部的细胞增殖、分化和产生大量基质。将成骨细胞／支架结构体在该生物反应器系统中培养14d后，用扫描电镜观察细胞在大段支架微管道内的生长情况，结果表明，支架微管道内有大量细胞长入。     

The Role of Matrix Metalloproteinase-2 in the Remodeling of Cell-Seeded Vascular Constructs Subjected to Cyclic Strain

Tissue engineering offers the opportunity to develop vascular substitutes that mimic the responsive nature of native arteries. A good blood vessel substitute should be able to remodel its matrix in response to mechanical stimulation, as imposed by the hemodynamic environment. We have developed a novel method of studying the influence of mechanical strain on the remodeling of cell-seeded collagen gel blood vessel analogs. We assessed the remodeling capacity by examining the effect of mechanical conditioning upon the expression of enzymes which remodel the extracellular matrix, called matrix metalloproteinases (MMPs), and upon the mechanical properties of the constructs. We found that subjecting collagen constructs to a 10% cyclic radial distention, over a course of 4 days, resulted in an overall increase in the production of MMP-2. Cyclic mechanical strain also stimulated enzymatic activation of latent MMP-2. We found that cyclic strain also significantly increased the mechanical strength and material modulus, as indicated by an increase in circumferential tensile properties of the constructs. These observations suggested that MMP-2-dependent remodeling affects the material properties of vascular tissue analogs. To further investigate this possible connection we examined the effects of dynamic conditioning in the presence of two nonspecific inhibitors of MMP activity. Interestingly, we found that nonspecific inhibition of MMP ablated the benefits of mechanical conditioning upon mechanical properties. Our observations suggest that a better understanding of the complex relation between mechanical stimulation and construct remodeling is key for the proper design of tissue-engineered blood vessel substitutes. © 2001 Biomedical Engineering Society. PAC01: 8719Rr, 8714Ee, 8717-d     

Rotating-wall vessels, promising bioreactors for osteoblastic cell culture: comparison with other 3D conditions

Osteoblastic cells cultured on microcarriers in bioreactors are a potentially useful tool to reproduce the in vivo three-dimensional (3D) bone network. The aim is to compare different types of 3D and two-dimensional (2D) osteoblastic culture. ROS17/2.8 cells are cultured in a bioreactor (rotating-wall vessel) or in two kinds of control (3D petri dish, 3D Percoll) and on two types of microcarrier (Cytodex 3 and Biosilon). Growth and morphology are determined by cell count and SEM, and differentiation is determined by dosage of alkaline phosphatase (ALP) activity and northern blots (ALP and osteocalcin (OC)). SEM shows that Biosilon microcarriers are the best substrate. Proliferation in the RWV and 3D petri dish is still in the exponential phase, whereas growth in the 2D culture reaches a plateau after eight days of culture. ALP activity and the ALP and OC mRNA levels are similar at day 8 for both the RWV and 3D petri dish. However, at day 10, cells are more differentiated in the RWV. The study shows that osteoblasts are both proliferate and differentiate in 3D structures. A BrDU immunocytochemical approach shows that only the cells in the periphery of the aggregates proliferate. Therefore the bioreactor may be a suitable tissue culture model for investigation of growth and differentiation processes in tissue engineering.