利用脱细胞血管基质体外构建小口径组织工程血管

目的探讨利用犬的间充质干细胞诱导分化种子细胞,以异种脱细胞血管基质为基础体外构建小口径血管移植物。方法采用密度梯度离心和贴壁培养的方法从犬骨髓中分离出间充质干细胞并体外培养,诱导分化成内皮样细胞和平滑肌样细胞;采用非离子型去垢剂和胰蛋白酶去除猪颈动脉血管壁结构细胞,对脱细胞基质进行组织学、力学检测及孔隙率评估。在生物反应器内采用旋转种植的方法将犬骨髓间充质干细胞诱导的内皮样细胞种植到脱细胞基质上,体外构建小口径组织工程血管。结果犬的骨髓间充质干细胞体外能够定向诱导分化为平滑肌样细胞和内皮样细胞,可以作为血管组织工程的种子细胞。经过脱细胞处理后,光镜和电镜观察证实血管壁的细胞成分完全去除。具有良好的孔径和孔隙率。支架在生物力学、孔隙率等方面符合构建组织工程血管支架的要求。在生物反应器内剪切力条件下可以初步构建出组织工程血管。结论小口径血管移植物可以将间充质干细胞诱导种子细胞,以异种脱细胞血管支架作为基质,在搏动性生物反应器内培养的方法进行构建。     

动植物细胞或组织生物反应器悬浮培养过程中的通气方式

通气在动植物细胞或组织生物反应器培养过程中起着至关重要的作用,而同时通气过程所产生的机械损伤力亦可对细胞造成直接的伤害,因此,通气方式是动植物细胞或组织生物反应器培养过程设计与工程放大的关键技术之一。本文综述了动植物细胞或组织生物反应器悬浮培养过程中三种主要通气(异养培养时又称供氧)方式的结构特点,及其对气液传质、生物量、代谢产物量和细胞损伤的影响,以及改进的新型通气方式和几种通气方式的融合并用。     

同轴打印小直径组织工程血管*

随着人口老龄化问题的日益严重以及心血管疾病患病的增加,临床上对血管移植物的需求量也逐渐增大。利用涤纶和聚四氟乙烯制备大直径血管(>6mm)在临床上得到了广泛的应用,而小直径(< 6 mm)血管常因血栓和感染导致移植的失败,因此构建内皮细胞贴附的组织工程血管就显得至关重要。通过合成RGD修饰的海藻酸钠(RGD-alginate, RGD-ALG)以及甲基丙烯酸化的明胶(methacrylated gelatin,GelMA),利用氯化钙溶液溶解的普朗尼克F127作为牺牲材料,利用同轴打印制备出组织工程血管。通过选择不同直径的同轴打印喷嘴以及调节打印参数,可以制备出不同直径的组织工程血管。制备出的组织工程血管可以承受人生理状态下的血管压力,可以进行稳定的灌流培养,并且人脐静脉血管内皮细胞在通入组织工程血管中后可以稳定贴附在管壁上。     

组织工程皮肤生物反应器的研究与设计

目的设计一套生物反应器,能针对不同支架材料———细胞复合物进行构建组织工程皮肤。方法根据皮肤的自身生长特点和不同支架材料-细胞复合物的特性,模拟皮肤的生长环境和力学环境,通过生物反应器解决组织工程皮肤构建中支架的装夹和气液界面问题。结果生物反应器由控制系统和生物反应器主体两部分构成,能提供对多种皮肤细胞复合物的动态培养。结论皮肤生物反应器能够满足不同组织工程皮肤产品的需要。能够形成气液界面和模拟生物力学的刺激。     

光生物反应器培养大型海藻细胞或组织

许多大型海藻含有具潜在重要药用价值的次生代谢物质,通常这些物质在藻体中含量极微,大型海藻体本身也不像微藻那样易在短期内大量获取,并且这些物质化学结构复杂,这使得直接提取或者人工合成极为困难。利用光生物反应器培养大型海藻细胞或组织,可以经济、无限量和资源循环再利用的方式,在植物体外合成生产重要的海洋植物次生代谢物质。光生物反应器所提供的可调控和工程优化的培养环境有望成为优化次生代谢物生物合成的有效手段。光生物反应器培养大型海藻细胞或组织也是大型海藻养殖业育苗技术发展的一个重要方向。综述了近10年来光生物反应器培养大型海藻细胞或组织在培养条件以及生长动力学模型方面国内外的研究进展,并对该领域未来可能的研究方向作一展望。     

内皮祖细胞在血管组织工程研究中的应用现状

组织工程血管是修复紫绀型先心病右心室流出道的潜在替代材料,实验研究表明外周血内皮祖细胞(endothelial progenitor cell,EPC)可作为构建组织工程血管的良好种子细胞。现阶段国内外对组织工程血管的研究方兴未艾,EPC在血管组织工程研究中的应用还处在体外培养或动物实验阶段,尚无临床应用报道。近来某些基础研究的成果对于新生血管的形成和EPC的自动募集机制有了合理的解释,其中缺氧被认为是重要的始动因素,这些研究成果也为EPC作为种子细胞应用于血管组织工程提供了理论基础。所以,阐明EPC的低氧诱导机制及其在血管组织工程的应用必将有助于复杂紫绀型先心病的外科治疗。本文主要介绍了目前该领域研究现状及相关研究基础的进展,并总结提出了在EPC研究和未来临床应用中需解决的问题。     

细胞联合培养在血管构建中的作用

组织工程三大要素为种子细胞、支架材料和信号分子,干细胞因其多分化潜能成为热门的种子细胞。血管化问题是制约工程化组织应用于临床的问题之一。利用干细胞构建组织工程血管的手段之一是在分离培养得到足够的种子细胞后,通过生长因子、细胞外基质、外力作用、其他细胞等的调控实现内皮向分化。只有实现了成功的血管构建,工程化组织才能正常的发挥作用。近年来不少国内外专家学者通过细胞联合培养的方法,观察细胞间的相互作用对血管构建的影响,结果表明,细胞联合培养在血管的形成、存活、稳定方面起到了重要的作用,为组织工程血管化提供了有效的途径,本文就部分细胞联合培养在血管构建中的作用作一综述。     

《生物工程下游技术》(第二版)

“生物工程下游技术”目前还没有准确的定义 ,因此 ,考虑到我国发展生物技术的需要 ,本书界定了一个较宽的范围 ,几乎包括了整个生物技术的中、下游技术和最终产品的质量监测等诸多方面。全书分为三篇。在第一篇“生物反应器及大规模细胞培养”中 ,包括了生物反应器基本理论与参数、细胞生长及代谢动力学、生物反应器的基本类型、反应器的参数控制与优化等部分。鉴于基因工程药物的糖基化问题日益引起生物医药界的关注 ,哺乳动物细胞载体的重要性亦更加突出。故书中对动物细胞的大规模培养、细胞培养用微载体等 ,花了较大的篇幅 ,进行了较为…     

使用旋转式生物反应器体外扩增人表皮细胞

目的:使用Cytodex-3微载体和高截面纵横比的旋转式生物反应器容器作为培养系统大规模扩增人表皮细胞(hECs)。方法:使用中性蛋白酶和胰蛋白酶-EDTA两步骤法从人皮肤中分离出人表皮细胞,使用DIL标记细胞后结合微载体后在旋转式生物反应器(RCCS)中培养,细胞贴附微载体的生长状态使用倒置显微镜,扫描电镜观测。并且分析细胞群体倍增时间来比较微重力培养与平面培养的体外增殖能力差异。结果:在旋转式生物反应器的微重力培养体系中,人表皮细胞能快速贴附到微载体表面,在培养过程中达到很大的细胞密度,并且表现出很强的增殖能力和细胞活性。结论:使用旋转式生物反应器和微载体悬浮培养人表皮细胞,是大量制备皮肤组织工程种子细胞的一种有效方法。     

使用旋转式生物反应器体外扩增人表皮细胞

目的：使用Cytodex-3微载体和高截面纵横比的旋转式生物反应器容器作为培养系统大规模扩增人表皮细胞（hECs）。方法：使用中性蛋白酶和胰蛋白酶．EDTA两步骤法从人皮肤中分离出人表皮细胞,使用DIL标记细胞后结合微载体后在旋转式生物反应器（RCCS）中培养,细胞贴附微载体的生长状态使用倒置显微镜,扫描电镜观测。并且分析细胞群体倍增时间来比较微重力培养与平面培养的体外增殖能力差异。结果：在旋转式生物反应器的微重力培养体系中,人表皮细胞能快速贴附到微载体表面,在培养过程中达到很大的细胞密度,并且表现出很强的增殖能力和细胞活性。结论：使用旋转式生物反应器和微载体悬浮培养人表皮细胞,是大量制备皮肤组织工程种子细胞的一种有效方法。     

Simulated weightlessness in the design and exploitation of a NMR-compatible bioreactor

Mammalian cells cultured in simulated weightlessness take advantage of a favorable environment, experiencing low shear stress and reduced turbulence. NMR spectroscopy allows the on-line noninvasive monitoring of cell growth and metabolism. With this in mind, we developed a novel bioreactor that fits into a NMR instrument and in which the simulated weightlessness conditions are obtained by a suitable medium and a flow-lift suspension. In detail, the gravitational vector acting on cells is counterbalanced by the hydrodynamic thrusts created by a bottom-up spiral flow of a fluid having increased density. We validate its efficiency (a) by calculating the main physical parameters as relative velocity, shear stress, and oxygen transport, and (b) by comparing the experimental results of growing a cell culture in the proposed bioreactor with those obtained using an established simulated weightlessness system (rotating wall vessel, NASA). As a test study we focused on the proliferation of human umbilical vein endothelial cells (HUVEC) in terms of cell viability and organization of their cytoskeleton.     

Real-time dissolved carbon dioxide monitoring II: Surface aeration intensification for efficient CO2 removal in shake flasks and mini-bioreactors leads to superior growth and recombinant protein yields

Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O₂ (DO) and dissolved CO₂ (dCO₂) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small-scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real-time monitoring of DO and dCO₂ in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO₂ at low level, which significantly improved not only biomass growth but also protein yield. We expect that implementing such controlled smart shake flasks can minimize the process development gap which currently exists in shake flask level and bioreactor level results.     

Design and characterization of a rotating bed system bioreactor for tissue engineering applications

The main challenge in the development of bioreactors for tissue engineering is the delivery of a sufficient nutrient and oxygen supply for cell growth in a 3D environment. Thus, a new rotating bed system bioreactor for tissue engineering applications was developed. The system consists of a culture vessel as well as an integrated rotating bed of special porous ceramic discs and a process control unit connected with the reactor to ensure optimal culturing conditions. The aim of the project was the design and construction of a fully equipped rotating bed reactor, and in particular, the characterization and optimization of the system with regard to technical parameters such as mixing time and pH-control to guarantee optimal conditions for cell growth and differentiation. Furthermore, the applicability of the developed system was demonstrated by cultivation of osteoblast precursor cells. The porous structure of the ceramic discs and the external medium circulation loop provide an optimal environment for tissue generation in long-term cultivations. Mass transfer limitations were minimized by the slow rotation, which also provides the cells with sufficient nutrients and oxygen through alternate contact to air and medium. An osteoblast precursor cell line was successfully cultivated in this bioreactor for 28 days.     

Enhanced production of L-(+)-lactic acid in chemostat by Lactobacillus casei DSM 20011 using ion-exchange resins and cross-flow filtration in a fully automated pilot plant controlled via NIR

Due to the lack of suitable in-process sensors, on-line monitoring of fermentation processes is restricted almost exclusively to the measurement of physical parameters only indirectly related to key process variables, i.e., substrate, product, and biomass concentration. This obstacle can be overcome by near infrared (NIR) spectroscopy, which allows not only real-time process monitoring, but also automated process control, provided that NIR-generated information is fed to a suitable computerized bioreactor control system. Once the relevant calibrations have been obtained, substrate, biomass and product concentration can be evaluated on-line and used by the bioreactor control system to manage the fermentation. In this work, an NIR-based control system allowed the full automation of a small-scale pilot plant for lactic acid production and provided an excellent tool for process optimization. The growth-inhibiting effect of lactic acid present in the culture broth is enhanced when the growth-limiting substrate, glucose, is also present at relatively high concentrations. Both combined factors can result in a severe reduction of the performance of the lactate production process. A dedicated software enabling on-line NIR data acquisition and reduction, and automated process management through feed addition, culture removal and/or product recovery by microfiltration was developed in order to allow the implementation of continuous fermentation processes with recycling of culture medium and cell recycling. Both operation modes were tested at different dilution rates and the respective cultivation parameters observed were compared with those obtained in a conventional continuous fermentation. Steady states were obtained in both modes with high performance on lactate production. The highest lactate volumetric productivity, 138 g L(-1) h(-1), was obtained in continuous fermentation with cell recycling.     

Monitoring cellular state transitions in a production-scale CHO-cell process using an electronic nose

An electronic nose is used to monitor the bioreactor off-gas composition in perfused cultivations of a CHO-cell line producing recombinant human blood coagulation factor VIII. The applicability of the electronic nose for monitoring cellular state transitions and process control is explained. It is shown that the instrument can reveal characteristic process states related to product and lactate formation, and detect microbial infections in a very early stage of the infection. The visualization of ideal process conditions is realized by using principal component analysis (PCA) and the on-line applicability of this method is outlined. The results illustrate the potential of the electronic nose as on-line sensor for ensuring product and process quality in production-scale bioprocesses.     

A new method to model change in cutaneous blood flow due to mechanical skin irritation part II: parameter identification procedure

Mechanical skin irritation, for example a light scratch with a needle, induces histamine and neuropeptide release on the line of stroke and in the surrounding tissue. Both histamine and neuropeptides are vasodilators. They cause vasodilation by changing the contraction state of the vascular smooth muscles and hence vessel compliance. Smooth muscle contraction state is very difficult to measure in vivo. For that reason we propose in this article an identification procedure to establish an irritation law. The law gives change in vessel compliance as a function of space, time and the intensity of the stroke. We have showed that vessel compliance increases immediately after the stroke not only on the line of stroke, but also in the surrounding tissue. Then, after a short delay, vessel compliance starts decreasing in the surrounding tissue, whereas vessel compliance on the line of stroke keeps increasing. Hence, blood is transported from the surrounding tissue to the line of stroke. In this way, higher blood volume on the line of stroke can be obtained than by only changing vessel compliance locally.     

The use of multidimensional image-based analysis to accurately monitor cell growth in 3D bioreactor culture

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells.     

Bioreactors for 3-dimensional high-density culture of human cells

A bioreactor was developed as an instrument to culture human or animal cells that require attachment in a large quantity or at a high density. The purpose for developing such a bioreactor is two-fold: to produce a large quantity of animal or human cells that have been modified by gene recombination technology to accommodate manufacture of physiologically-active substances or human proteins on an industrial scale; and for research to culture animal cells to form a high-density 3-dimensional structure as a morphological or functional tissue or organ entity. In the current report, the circulatory flow bioreactor and radial flow bioreactor (RFB) are introduced, in which the former can be scaled up. As a small bioreactor produced for the latter purpose, a rotary cell culture system and novel multicoaxial hollow-fiber bioreactor are introduced. Finally, a small RFB culture system that was scaled down by the present author and his collaborators for the study of a 3-dimensional high density culture system is described. The RFB can be readily scaled up for manufacturing or scaled down for research purposes. This is a cell culturing system that can induce the functions of human tissues by preparing a high density 3-dimensional organization of cells of human origin.     

Doppler optical coherence tomography for measuring flow in engineered tissue

The engineering of human tissue represents a major paradigm shift in clinical medicine. Early embodiments of tissue engineering are currently being taken forward to the clinic by production methods that are essentially extensions of laboratory manual procedures. However, to achieve the status of routine large-scale clinical practice, automation and scale-out processes are required. This in turn will require the development of reliable on-line monitoring and control systems. This paper examines one demand of crucial importance, namely the real time in vitro monitoring of the flow characteristics through growing tissue since this has a complex interrelationship. Doppler optical coherence tomography (DOCT) is a recently developed imaging technique for studying the rheological properties of tissues in vivo. Capable of non-invasive imaging in real time with high resolution, it is potentially ideal for the continuous monitoring of engineered tissues in vitro. As a base line, the current status of DOCT in vivo is therefore reviewed. This paper also reports the first preliminary use of DOCT in tissue engineering. The application described involves the imaging of a fully developed laminar flow through a combined tissue fabrication/bioreactor with a tissue-engineered construct (substitute blood vessel) in situ.