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。     

成骨细胞在旋转壁式生物反应器内的大规模扩增

目的 进行SD大鼠成骨细胞在旋转壁式生物反应器内大规模扩增培养的研究。方法利用微载体悬浮培养法在旋转壁式生物反应器内大规模扩增成骨细胞，检测其组织形态和生物功能后。作为接种到支架材料上并于反应器内三维环境中培养组织工程骨的种子细胞。结果成骨细胞在生物反应器中每代可以扩增十倍以上，同时经过倒置相差显微镜、SEM(扫描电镜)以及ALP(碱性磷酸酶)和MTT等生物学性能检测后，发现在旋转壁式生物反应器中三维培养的成骨细胞各种生物指标性能良好。结论新型旋转壁式生物反应器可以提供低剪切力的培养环境，而且细胞之间有三维联系的机会，成骨细胞表现出良好的体外扩增能力，适于建立一种理想的成骨细胞体外扩增的三维培养体系。     

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。     

组织工程生物反应器嵌入式实时检测控制系统的设计

背景:组织工程生物反应器通过模拟体内环境,可为细胞或组织提供适宜的生长条件,并能培养出与体内结构和功能相似的三维细胞或组织。目的:对水平旋转生物反应器培养细胞或组织的环境需求进行分析,针对其特殊的环境需求提出了基于Linux和ARM9嵌入式处理器的生物反应器检测控制系统设计。方法:以系统对实验室自制的生物反应器进行控制监测。实验主要完成支架材料制备,实验用主要仪器(自动台式灭菌器,气液膜,液液膜电子分析天平等)准备,实验材料-骨髓间充质干细胞的原代、传代培养及制备,以及系统对反应器的转速和蠕动泵控制,温度,pO2,pH值的检测。结果与结论:与单片机控制检测系统相比,改进系统驱动控制方式,提高了控制精度,增强检测灵敏度。同时新设计增加了养分压以及pH值检测,进一步完善了反应器的功能。     

血管生物反应器流场的数值分析

目的 研究一种新型小口径血管生物反应器在不同工况下的流场分布。方法 采用数值方法模拟反应器外筒单独旋转、内筒单独旋转和内外筒同向等速旋转情况下其内部的流场分布,对流速和切应力等参数进行比较分析。结果 血管生物反应器工作时,培养液随内外筒的旋转而旋转流动,速度分布均匀;可为培养液内的细胞提供非破坏性的低切应力环境;并且反应器内部的切应力大小与转速呈二次函数关系。结论 该反应器能为血管培养提供良好的培养环境,本研究可为其实验研究提供指导意见和理论基础。     

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     

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.     

旋转生物反应器内球形体培养对人胎盘间充质干细胞炎症因子分泌的影响

背景：生物反应器内球形体培养是既能维持间充质干细胞干性又能大规模扩增的体外培养方法，明确其对干细胞免疫调节作用的影响有利于指导临床应用。目的：探讨旋转生物反应器内球形体培养对人胎盘间充质干细胞炎症因子分泌的影响。方法：从人胎盘组织中分离培养人胎盘间充质干细胞，分别进行二维培养和旋转生物反应器培养，利用倒置相差显微镜、免疫组化染色和CCK-8实验观察细胞形态与增殖能力，RT-qPCR和流式荧光法检测多种炎症因子基因表达与蛋白分泌量。结果与结论：(1)旋转生物反应器内培养的人胎盘间充质干细胞聚集成多细胞球形体，数量与体积逐渐增加；(2)苏木精-伊红染色可见旋转生物反应器内培养4 d的人胎盘间充质干细胞在球形体内分布均匀，形态正常；(3)免疫组化染色显示球形体内有大量人胎盘间充质干细胞的细胞核Ki-67阳性；(4)CCK-8实验结果显示，生物反应器内球形体培养的人胎盘间充质干细胞活力显著高于二维培养的细胞；(5)RT-q PCR和流式免疫荧光法检测结果显示，旋转生物反应器内球形体培养的人胎盘间充质干细胞中白细胞介素1β、白细胞介素4、白细胞介素6、白细胞介素8、白细胞介素10、白细胞介素17...     

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。     

旋转生物反应器培养对组织工程气管软骨力学强度的影响

目的研究旋转生物反应器培养对组织工程气管软骨力学强度的影响，探索适宜的组织工程气管软骨培养方法。 方法分离2周龄Lewis大鼠剑突软骨细胞传代培养，收集第3代软骨细胞种植到DegraPol管状支架上，静态培养7d，然后将软骨细胞-支架复合物分别置于旋转生物反应器内培养（生物反应器组）或继续静态培养培养3周（静态培养组）。取出软骨细胞-支架复合物，以噻唑蓝（MTT）法测定软骨细胞增殖活性，结果以吸光度（A）值表示（每组n=6）；以Zwick1445型材料试验机测定软骨细胞-支架复合物的最大应变值和应力值（每组n=4）；并制备扫描电镜标本，观察软骨细胞在DegraPol支架中培养后的超微结构变化。 结果不同条件下培养3周，生物反应器组和静态培养组A值分别0.17±0.05、0.12±0.01，最大应力值分别为（0.33±0.04）和（0.26±0.01）MPa，最大应变值分别为（3.53±0.91）和（1.71±0.13）mm/mm，2组间3项指标的差异均有统计学意义（均P〈0.05）。扫描电镜观察显示生物反应器组获得更好的软骨样结构和更多的细胞外基质。 结论旋转生物反应器能够提供更好的体外培养条件，有利于组织工程气管软骨的形成。     

Optimizing of Culture Conditionin Horizontal Rotating Bioreactor

1 IntroductionBioreactor is the most important equipment in tissue engineering. It can mimic the micro-environment of cell growth in vitro. At present, horizontal rotating bioreactor is the most advanced equipment for cell culture in the world. 2 Rotating bioreactors2.1 Working principleThere are two kinds of horizontal rotating bioreactor: HARV(high aspect ratio vessel) and RCCS (rotary cell culture system). It is drived by step motor with horizontal rotation, the culture medium and cell is filled between ...     

Engineering of rat articular cartilage on porous sponges: effects of tgf-beta 1 and microgravity bioreactor culture

The objective of this study was to develop an engineered rat hyaline cartilage by culturing articular chondrocytes on three-dimensional (3D) macroporous poly(DL-lactic-co-glycolic acid) (PLGA) sponges under chondrogenic induction and microgravity bioreactor conditions. Experimental groups consisted of 3D static and dynamic cultures, while a single cell monolayer (2D) served as the control. The effect of seeding conditions (static vs. dynamic) on cellularization of the scaffolds was investigated. MTT assay was used to evaluate the number of viable cells in each group at different time points. Formation of a hyaline-like cartilage was evaluated for up to 4 weeks in vitro. While 2D culture resulted in cell sheets with very poor matrix production, 3D culture was in the favor of tissue formation. A higher yield of cell attachment and spatially uniform cell distribution was achieved when dynamic seeding technique was used. Dynamic culture promoted cell growth and infiltration throughout the sponge structure and showed the formation of cartilage tissue, while chondrogenesis appeared attenuated more towards the outer region of the constructs in the static culture group. Medium supplemented with TGF-beta 1 (5 ng/ml) had a positive impact on proteoglycan production as confirmed by histochemical analyses with Alcian blue and Safranin-O stainings. Formation of hyaline-like tissue was demonstrated by immunohistochemistry performed with antibodies against type II collagen and aggrecan. SEM confirmed higher level of cellularization and cartilage tissue formation in bioreactor cultures induced by TGF-beta 1. The data suggest that PLGA sponge inside rotating bioreactor with chondrogenic medium provides an environment that mediates isolated rat chondrocytes to redifferentiate and form hyaline-like rat cartilage, in vitro.     

Bone tissue engineering in a rotating bioreactor using a microcarrier matrix system

A novel approach was utilized to grow in vitro mineralized bone tissue using lighter-than-water, polymeric scaffolds in a high aspect ratio rotating bioreactor. We have adapted polymer microencapsulation methods for the formation of hollow, lighter-than-water microcarriers of degradable poly(lactic-co-glycolic acid). Scaffolds were fabricated by sintering together lighter-than-water microcarriers from 500 to 860 microm in diameter to create a fully interconnected, three-dimensional network with an average pore size of 187 microm and aggregate density of 0.65 g/mL. Motion in the rotating bioreactor was characterized by numerical simulation and by direct measurement using an in situ particle tracking system. Scaffold constructs established a near circular trajectory in the fluid medium with a terminal velocity of 98 mm/s while avoiding collision with the bioreactor wall. Preliminary cell culture studies on these scaffolds show that osteoblast-like cells readily attached to microcarrier scaffolds using controlled seeding conditions with an average cell density of 6.5 x 10(4) cells/cm(2). The maximum shear stress imparted to attached cells was estimated to be 3.9 dynes/cm(2). In addition, cells cultured in vitro on these lighter-than-water scaffolds retained their osteoblastic phenotype and showed significant increases in alkaline phosphatase expression and alizarin red staining by day 7 as compared with statically cultured controls.     

Chondrogenesis of aged human articular cartilage in a scaffold-free bioreactor

Chondrogenesis of aged human articular chondrocytes was evaluated under controlled in vitro conditions, using a rotating bioreactor vessel. Articular chondrocytes isolated from 10 aged patients (median age, 84 years) were increased in monolayer culture. A single-cell suspension of dedifferentiated chondrocytes was inoculated in a rotating wall vessel, without the use of any scaffold or supporting gel material. After 90 days of cultivation, a three-dimensional cartilage-like tissue was formed, encapsulated by fibrous tissue resembling a perichondrial membrane. Morphological examination revealed differentiated chondrocytes ordered in clusters within a continuous dense cartilaginous matrix demonstrating a strong positive staining with monoclonal antibodies against collagen type II and articular proteoglycan. The surrounding fibrous membrane consisted of fibroblast-like cells, and showed a clear distinction from the cartilaginous areas when stained against collagen type I. Transmission electron microscopy revealed differentiated and highly metabolically active chondrocytes, producing an extracellular matrix consisting of a fine network of randomly distributed cross-banded collagen fibrils. Chondrogenesis of aged human articular chondrocytes can be induced in vitro in a rotating bioreactor vessel using low shear and efficient mass transfer. Moreover, the tissue-engineered constructs may be used for further in vitro studies of differentiation, aging, and regeneration of human articular cartilage.     

Use of a rotating bioreactor toward tissue engineering the temporomandibular joint disc

This objective of this study was to determine the effects of a rotating bioreactor in temporomandibular joint (TMJ) disc tissue engineering. Porcine TMJ disc cells were seeded at a density of 20 million cells/mL onto nonwoven poly(glycolic acid) (PGA) scaffolds in spinner flasks for 1 week and then cultured either under static conditions or in a rotating bioreactor for a period of 6 weeks. A series of analyses was performed, including mechanical testing, measurement of cellularity, quantification of matrix biosynthesis with a hydroxyproline assay and enzyme-linked immunosorbent assays, and observation of matrix distribution with immunohistochemistry. Between the bioreactor and static cultures, there were marked differences in gross appearance, histological structure, and distribution of collagen types I and II. Engineered constructs from the bioreactor contracted earlier and to a greater extent, resulting in a denser matrix and cell composition. In addition, immunostaining intensity was generally uniform in static constructs, in contrast to higher intensity around the periphery of bioreactor constructs. Moreover, bioreactor constructs had higher amounts of collagen II than did static constructs. However, differences in total matrix content and compressive stiffness were generally not significant. On the basis of the results of this study there is no clear benefit from use of the rotating bioreactor, although a sequence of static culture followed by rotating bioreactor culture may prove in the future to be more beneficial than either alone.     

A bioreactor model of mouse tumor progression

The present study represents an investigation of a novel stirred bioreactor for culture of a transformed cell line under defined hydrodynamic conditions in vitro. Cell colonies of the EL-4 mouse lymphoma cell line grown for the first time in a rotating disc bioreactor (RDB), were observed to undergo changes in phenotype in comparison to standard, static flask cultures. RDB cultures, with or without agitation, promoted the formation of adherent EL-4 cell plaques that merged to form contiguous tumor-like masses in longer-term cultures, unlike the unattached spheroid aggregates of flask cultures. Plaques grown under agitated conditions were further altered in morphology and distribution in direct response to fluid mechanical stimuli. Plaque colonies growth in RDBs with or without agitation also exhibited significant increases in production of interleukin-4 (IL-4) and lactate, suggesting an inducible "Warburg effect." Increases in cell biomass in RDB cultures were no different to flask cultures, though a trend toward a marginal increase was observed at specific rotational speeds. The RDB may therefore be a suitable alternative method to study mechanisms of tumor progression and invasiveness in vitro, under more complex physicochemical conditions that may approximate natural tissue environments.