组织工程生物反应器的力学环境特点

组织工程学是一门以细胞生物学和工程学为基础,应用工程学和生命科学的原理,开发器官缺损患者所需代替物,并构建和保持或增强其组织功能性的一门交叉学科。生物反应器作为组织工程中非常重要的工具,目前主要是从生物力学问题、三维空间培养问题、传质问题、培养的环境条件问题(pH、氧张力等)和物理因素(电场、磁场、应力场)等方面开展其研究。本文作者主要从生物力学角度介绍组织工程生物反应器研究的最新进展,重点探讨组织工程生物反应器的力学环境。     

组织工程化旋转生物反应器研究进展

概述了组织工程化水平旋转生物反应器的工作原理、培养环境、应用现状和发展趋势。水平旋转生物反应器为体外培养动物细胞保持其正常形态、结构、功能和遗传特性提供了一种新手段，得天独厚的微重力、高效物质传递和低剪应力环境、多孔立体网状支架材料、在线监测和控制细胞三维生长等优势，为离体细胞重建组织、实现人工构建组织和器官有望成为现实。     

组织工程化旋转生物反应器研究进展

概述了组织工程化水平旋转生物反应器的工作原理、培养环境、应用现状和发展趋势。水平旋转生物反应器为体外培养动物细胞保持其正常形态、结构、功能和遗传特性提供了一种新手段,得天独厚的微重力、高效物质传递和低剪应力环境、多孔立体网状支架材料、在线监测和控制细胞三维生长等优势,为离体细胞重建组织、实现人工构建组织和器官有望成为现实。     

生物反应器的设计与组织工程肌腱的构建

目的设计一套生物反应器,能构建具有较好形态和机械力学性能的肌腱组织。方法根据肌腱细胞体内的生物和力学环境,建立了细胞和可降解材料复合物的力学模型,设计了一套能够模拟体内力学环境的生物反应器,采用鸡肌腱为种子细胞培养扩增后接种于聚羟基乙酸(PGA)材料上,形成细胞-材料复合物并置于反应器中培养。设静态培养对照组,通过实验取材进行大体观察、组织学和生物力学检测。结果生物反应器能够构建出具有一定组织结构和机械强度的肌腱。检测结果显示恒定交变应力作用下培养的肌腱优于静态培养的肌腱组织。结论细胞和可降解材料复合物的力学模型具有一定的正确性,通过分析也存在某些局限性;应力作用下可以促进肌腱细胞基质的分泌,蠕变特性成为促进胶原定向排列的重要因素。     

旋转生物反应器的力学环境及其对细胞生长的影响

研究了旋转生物反应器的力学环境及其对细胞生长的影响。理论建模和微分方程求解反应器内重力和细胞所受剪应力的大小,扫描电镜评价细胞生长密度、生长速度和形貌。结果表明,自制的旋转生物反应器在理论上能提供微重力(K<8.38×10-2)和低剪应力(τ<1.62 dyn/cm2)环境,扫描电镜结果显示在反应器内培养的细胞生长速度、数量和形态明显优于24孔板静态培养。旋转生物反应器的力学环境有利于细胞保持良好形态和快速扩增,是一种明显优于静态培养的新方法和新技术。     

生物反应器在血管组织工程中的应用及进展

论述生物反应器的种类与发展,以及其在血管组织工程种子细胞培养和组织工程血管构建方面的主要研究进展。根据生物反应器领域的发展,分析了生物反应器对种子细胞培养、扩增的影响,尤其是对干细胞培养、定向分化方面的影响;阐述生物反应器内种植细胞的方法,以及机械力学对细胞生长、黏附的影响;探讨生物力学与血管构建的关系。最后提出生物反应器未来的发展趋势。     

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.     

动态力学因素在组织工程化血管构建中的作用及其研究进展

动态培养环境是成功构建具有生理功能和力学性能的工程化组织的重要因素之一.越来越多的研究证实,接近生物体内生理环境的动态力学条件对细胞在体外支架上的黏附、增殖、分化以及细胞外基质的重建等一系列生长行为均有重要的作用,通过模拟和优化动态培养条件,可以促进细胞的生长和细胞外基质的重建,从而提高组织工程化血管的性能.本文对近年来在组织工程化血管动态培养环境方面的主要研究进展做了综合评述,重点讨论了不同力学环境对血管细胞生长和细胞外基质重建的影响,以及生物反应器在组织工程化血管构建中的作用,并通过组织工程化血管的生物学性能分析,讨论了动态力学培养环境对血管组织的形成及其力学性能的作用.     

微重力反应器在细胞培养中的应用

模拟微重力培养是一种全新的组织培养技术,其核心技术是建立动物细胞的三维培养体系,在近10年取得了快速发展。微重力反应器源自美国航空航天局,是一种水平旋转的、无泡的旋转培养仪,可以提供模拟微重力环境,具有充分的氧和营养物质的交换、三维立体结构、低剪切力和独特的流体力学特征等优点。这种悬浮培养技术为多种细胞和组织块的生长和代谢提供良好的培养环境,可以进行高密度的组织培养,并保持所培养细胞的组织分化特异性。     

微重力反应器在细胞培养中的应用

模拟微重力培养是一种全新的组织培养技术，其核心技术是建立动物细胞的三维培养体系，在近10年取得了快速发展。微重力反应器源自美国航空航天局，是一种水平旋转的、无泡的旋转培养仪，可以提供模拟微重力环境，具有充分的氧和营养物质的交换、三维立体结构、低剪切力和独特的流体力学特征等优点。这种悬浮培养技术为多种细胞和组织块的生长和代谢提供良好的培养环境，可以进行高密度的组织培养，并保持所培养细胞的组织分化特异性。     

HEK 293 cell suspension culture using fibronectin-adsorbed polymer nanospheres in serum-free medium

Previously, we reported on suspension culture of anchorage-dependent animal cells using plain polymer nanospheres in serum-containing medium. For commercial cell culture, it is more advantageous to use serum-free medium than serum-containing medium. To culture anchorage-dependent animal cells using polymer nanospheres in serum-free medium, the nanospheres need to be coated with cell adhesion proteins. In this study, we utilized fibronectin-adsorbed polymer nanospheres for suspension culture of anchorage-dependent animal cells in serum-free medium. Fibronectin was adsorbed onto poly(lactic-co-glycolic acid) nanospheres (433 nm in average diameter) by immersing the nanospheres in fetal bovine serum. The nanospheres were used to culture human embryonic kidney (HEK) 293 cells in serum-free medium in stirred suspension bioreactors. Nanospheres attached between HEK 293 cells and promoted cell aggregate formation compared with culture without nanospheres. Most cells in the aggregates were viable over a 10-day culture period. Importantly, the use of poly(lactic-co-glycolic acid) nanospheres promoted the cell growth significantly, compared with culture without nanospheres (3.8- vs 1.8-fold growth). The nanosphere culture method developed in this study removes the time-consuming and costly process of adaptation of anchorage-dependent animal cells to suspension culture in serum-free medium. This culture method may be useful for the large-scale suspension culture of various types of anchorage-dependent animal cells in serum-free medium.     

Characterization of the distribution of matter in hybrid liver support devices where cells are cultured in a 3-D membrane network or on flat substrata

Bioreactors for liver assist tested on small animal models are generally scaled-up to treat humans by increasing their size to host a given liver cell mass. In this process, liver cell function in different culture devices is often established based on the metabolite concentration difference between the bioreactor inlet and outlet irrespective of how matter distributes in the bioreactor. In this paper, we report our investigation aimed at establishing whether bioreactor design and operating conditions influence the distribution of matter in two bioreactors proposed for liver assist. We investigated a clinical-scale bioreactor where liver cells are cultured around a three-dimensional network of hollow fiber membranes and a laboratory-scale bioreactor with cells adherent on collagen-coated flat substrata. The distribution of matter was characterized under different operating modes and conditions in terms of the bioreactor residence time distribution evaluated by means of tracer experiments and modeled as a cascade of N stirred tanks with the same volume. Under conditions recommended by the manufacturers, matter distributed uniformly in the clinical-scale bioreactor as a result of the intense backmixing (N=1) whereas axial mixing was negligible in the laboratory-scale bioreactor (N=8). Switching from recycle to single-pass operation definitely reduced axial mixing in the clinical-scale bioreactor (N=2). Increasing feed flow rate significantly enhanced axial mixing in the laboratory-scale bioreactor (N=4). The effects of design, operating mode and conditions on matter distribution in bioreactors for liver cell culture suggest that characterization of the distribution of matter is a necessary step in the scale-up of bioreactors for liver assist and when function of liver cells cultured in different bioreactors is evaluated and compared.     

Time course of primary liver cell reorganization in three-dimensional high-density bioreactors for extracorporeal liver support: an immunohistochemical and ultrastructural study

To enable extracorporeal liver support based on the use of primary liver cells, culture models supporting the maintenance of cell integrity and function in vitro are required. In this study the cell organization and ultrastructure of primary porcine hepatocytes cocultured with nonparenchymal cells in three-dimensional high-density bioreactors were analyzed after 10, 20, and 30 days of culture by immunohistochemistry and transmission electron microscopy. Biochemical data showed that metabolic activity of the cells in the system was relatively stable over at least 20 days. Immunohistochemical studies were performed in comparison with donor organ biopsies. They showed that hepatocytes and nonparenchymal cells reaggregated in bioreactors, forming structures partly resembling natural liver parenchyma. Bile duct-like structures characterized by cytokeratin 7 (CK-7) immunoreactivity (IR) were regularly detected. Nonparenchymal cells (vimentin IR) formed sinusoidal-like structures within parenchymal cell aggregates. Proliferative activity (Ki-67 IR) increased over time. The detection of collagen I and laminin indicated the production of extracellular matrix components within bioreactors. The results showed that primary liver cell reorganization and long-term maintenance of their differentiated state were achieved within the bioreactors The findings on cell proliferation indicated that the culture model is also of interest for further in vitro studies on cell regeneration and tissue formation.     

Design of a flow perfusion bioreactor system for bone tissue-engineering applications

Several different bioreactors have been investigated for tissue-engineering applications. Among these bioreactors are the spinner flask and the rotating wall vessel reactor. In addition, a new type of culture system has been developed and investigated, the flow perfusion culture bioreactor. Flow perfusion culture offers several advantages, notably the ability to mitigate both external and internal diffusional limitations as well as to apply mechanical stress to the cultured cells. For such investigation, a flow perfusion culture system was designed and built. This design is the outgrowth of important design requirements and incorporates features crucial to successful experimentation with such a system.     

Poly(acrylamide) Spheroids with Tunable Elasticity for Scalable Cell Culture Applications

Many biological tissues resemble hydrogels and display Young's moduli below 50 kPa, corresponding to most cell types for the natural environmental conditions to grow. Contrastingly, conventional cell culture usually involves rigid substrates resulting in stiff priming effects, which are of increasing concern when it comes to scalable culturing of adhesive cells for regenerative purposes. As a solution to this problem, the employment of synthetic poly(acryl amide) (PAAm)-based hydrogel beads with tissue-matched mechanical properties are proposed as soft matrix for culture modes suitable for tidal bioreactor culture. Herein, technology is described to generate spherical, mm-scaled PAAm hydrogel beads with adjustable, soft elastic properties that are produced by a continuous microfluidic approach. A simple and robust method is demonstrated to functionalize the spheroids with protein ligands, and the suitability of the matrix for cell cultivation is successfully demonstrated with three different cell types (murine mesenchymal stem cells, renal carcinoma cells, and human induced pluripotent stem cells) in model experiments and in a tidal bioreactor system. This versatile approach will pave the way toward novel cell culture systems based on bioreactors that allow scalable, soft carrier-based expansion of cells on matrices with tissue-matched elasticity.     

The multiparametric effects of hydrodynamic environments on stem cell culture

Stem cells possess the unique capacity to differentiate into many clinically relevant somatic cell types, making them a promising cell source for tissue engineering applications and regenerative medicine therapies. However, in order for the therapeutic promise of stem cells to be fully realized, scalable approaches to efficiently direct differentiation must be developed. Traditionally, suspension culture systems are employed for the scale-up manufacturing of biologics via bioprocessing systems that heavily rely upon various types of bioreactors. However, in contrast to conventional bench-scale static cultures, large-scale suspension cultures impart complex hydrodynamic forces on cells and aggregates due to fluid mixing conditions. Stem cells are exquisitely sensitive to environmental perturbations, thus motivating the need for a more systematic understanding of the effects of hydrodynamic environments on stem cell expansion and differentiation. This article discusses the interdependent relationships between stem cell aggregation, metabolism, and phenotype in the context of hydrodynamic culture environments. Ultimately, an improved understanding of the multifactorial response of stem cells to mixed culture conditions will enable the design of bioreactors and bioprocessing systems for scalable directed differentiation approaches.     

Bioreactors for tissue engineering: focus on mechanical constraints. A comparative review

Considering the current techniques in cell culture, the stimulation of cellular proliferation and the formation of bidimensional tissues such as skin are widely performed in academic and industrial research laboratories. However, the formation of cohesive, organized, and functional tissues by three-dimensional (3D) cell culture is complex. A suitable environment is required, which is achieved and maintained in a specific bioreactor, a device that reproduces the physiological environment (including biochemical and mechanical functions) specific to the tissue that is to be regenerated. Bioreactors can also be used to apply mechanical constraints during maturation of the regenerating tissue for studying and understanding the mechanical factors influencing tissue regeneration. In this work, the main types of bioreactors used for tissue engineering and regeneration, as well as their most common applications, were reviewed and compared. The importance of the mechanical properties applied to the scaffolds and the regenerating constructs has been often neglected. This review focused on the influence of mechanical stresses and strains during the culture period that leads to the final mechanical properties of the construct.     

Production of recombinant adeno-associated vectors using two bioreactor configurations at different scales

The conventional methods for producing recombinant adeno-associated virus (rAAV) rely on transient transfection of adherent mammalian cells. To gain acceptance and achieve current good manufacturing process (cGMP) compliance, clinical grade rAAV production process should have the following qualities: simplicity, consistency, cost effectiveness, and scalability. Currently, the only viable method for producing rAAV in large-scale, e.g. > or =10(16) particles per production run, utilizes baculovirus expression vectors (BEVs) and insect cells suspension cultures. The previously described rAAV production in 40 L culture using a stirred tank bioreactor requires special conditions for implementation and operation not available in all laboratories. Alternatives to producing rAAV in stirred tank bioreactors are single-use, disposable bioreactors, e.g. Wave. The disposable bags are purchased pre-sterilized thereby eliminating the need for end-user sterilization and also avoiding cleaning steps between production runs thus facilitating the production process. In this study, rAAV production in stirred tank and Wave bioreactors was compared. The working volumes were 10 L and 40 L for the stirred tank bioreactors and 5 L and 20 L for the Wave bioreactors. Comparable yields of rAAV, approximately 2E+13 particles per liter of cell culture were obtained in all volumes and configurations. These results demonstrate that producing rAAV in large scale using BEVs is reproducible, scalable, and independent of the bioreactor configuration.     

Chondrogenesis in perfusion bioreactors using porous silk scaffolds and hESC-derived MSCs

Tissue engineered cartilage can be grown in vitro with the use of cell-scaffold constructs and bioreactors. The present study was designed to investigate the effects of perfusion bioreactors on the chondrogenic potential of engineered constructs prepared from porous silk fibroin scaffolds seeded with human embryonic stem cell (hESC)-derived mesencyhmal stem cells (MSCs). After four weeks of incubation, constructs cultured in perfusion bioreactors showed significantly higher amounts of glycosaminoglycans (GAGs) (p < 0.001), DNA (p < 0.001), total collagen (p < 0.01), and collagen II (p < 0.01) in comparison to static culture. Mechanical stiffness of constructs increased 3.7-fold under dynamic culture conditions and RT-PCR results concluded that cells cultured in perfusion bioreactors highly expressed (p < 0.001) cartilage-related genes when compared with static culture. Distinct differences were noted in tissue morphology, including polygonal extracellular matrix structure of engineered constructs in thin superficial zones and an inner zone under static and dynamic conditions, respectively. The results suggest that the utility of perfusion bioreactors to modulate the growth of tissue-engineered cartilage and enhance tissue growth in vitro.     

Optimization of dynamic culture conditions: effects on biosynthetic activities of chondrocytes grown in collagen sponges

Application of mechanical stimulation, using dynamic bioreactors, is considered an effective strategy to enhance cellular behavior in load-bearing tissues. In this study, two types of perfusion mode (direct and free flow) are investigated in terms of the biosynthetic activities of chondrocytes grown in collagen sponges by assessment of cell proliferation rate, matrix production, and tissue morphology. Effects of the duration of preculture and dynamic conditioning are further determined. Our results have demonstrated that both bovine and human-derived chondrocytes demonstrate a dose-dependent response to flow rate (0-1 mL/min) in terms of cell number and glycosaminoglycan (GAG) content. This may reflect the weak adhesion of cells to the sponge scaffolds and the immature state of the constructs even after 3 weeks of proliferative culture. Our studies define an optimal flow rate between 0.1 and 0.3 mL/min for direct perfusion and free flow bioreactors. Using fresh bovine chondrocytes and a lower flow rate of 0.1 mL/min, a comparison was made between free flow system and direct perfusion system. In the free flow bioreactor, no cell loss was observed and higher GAG production was measured compared with static cultured controls. However, as with direct perfusion, the enhancement effect of free flow perfusion was strongly dependent on the maturation and organization of the constructs before the stimulation. To address the maturation of the matrix, preculture periods were varied before mechanical conditioning. An increase in culture duration of 18 days before mechanical conditioning resulted in enhanced GAG production compared with controls. Interestingly, additional enhancement was found in specimens that were further subjected to a prolonged duration of perfusion (63% increase after an additional 4 days of perfusion) after prematuration. The free flow system has an advantage over the direct perfusion system, especially when using sponge scaffolds, which have lower mechanical properties; however, mass transfer of nutrients is still more optimal throughout the scaffolds in a direct perfusion system as demonstrated by histological analysis.