微载体培养技术的研究与进展

背景:微载体培养技术是一种新兴的大规模细胞培养技术,广泛地应用于组织工程中种子细胞的扩增。微载体具有比表面积大等优点,在微载体培养技术中具有决定性作用。目的:文章就近年来制备微载体的生物材料和方法的研究进展做一综述,为微载体培养技术和组织工程的研究提供理论基础。方法:由第一作者于2009-10应用计算机检索PubMed数据库、万方数据库和维普数据库相关文献。英文资料的检索时间为1967/2009;中文资料的检索时间为1990/2009。英文检索词为"microcarrier,biomaterials cell culture,tissue engineering";中文检索词为"微载体,生物材料,细胞培养,组织工程"。纳入标准:①微载体材料、制备工艺及性能的研究。②微载体细胞培养的研究。③动物实验及临床应用。阅读标题和摘要进行筛选,共纳入34篇用于综述。结果与结论:虽然国内外研究人员在微载体的研究与制备方面做了很多工作,但迄今仍与临床应用有一定的距离。目前的主要工作是将不同种类的材料通过新型的工艺制备得到复合材料,通过表面改性来调节微载体的力学和生物降解性能。     

修复运动性肌腱损伤组织工程化肌腱及种子细胞和支架材料

背景：获得大规模、具有再生活力的种子细胞以及具有与正常人体肌腱组织相接近的力学性能的理想支架材料是当前组织工程化肌腱研究面临的最为关键的限制性因素。 目的：总结和分析组织工程肌腱研究中的种子细胞和支架材料的研究进展。 方法：查阅近年来肌腱组织工程研究的相关文献,综合国内外最新研究成果,就肌腱组织工程中合适的种子细胞来源、研究更为理想的支架材料及组织相容性等方面的进展进行概述。 结果与结论：肌腱组织工程中常用的种子细胞有间充质干细胞、肌腱干细胞及胚胎干细胞等,可以向骨、软骨和脂肪分化,修复肌腱损伤的理想细胞。肌腱组织工程支架材料有天然材料及人工合成材料等,肌腱组织工程支架材料应有良好的生物相容性和适度的机械性能,复合材料将是肌腱组织工程支架材料研究的重点。 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      

聚合物微载体及其在细胞培养方面的应用

微载体培养系统是当前贴壁依赖型细胞大规模培养的主要方法。介绍了微载体系统培养细胞的优越性，评述了近年来常用的几种制备微载体的天然聚合物材料，较为详尽地总结了微载体特别是大孔微载体的制备方法和微载体的改性方法。目前关于用微载体培养细胞来生产生物医药制品的研究较多，重点评述了天然聚合物材料制备的微载体在组织工程领域的应用前景。     

球形多孔微载体支持下高密度培养人肝细胞L-02的定时形态变化

背景：微载体培养技术作为一项体外高浓度细胞培养技术,近年来已在肝细胞的体外培养中得到应用。 目的：对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的形态学观察。 方法：以自制的壳聚糖球形多孔微载体样本为支架来培养人肝细胞L-02设为实验组;无壳聚糖球形多孔微载体支持下人肝细胞的培养设为对照组。对两组细胞进行定时的细胞计数,对实验组进行形态学观察,包括倒置相差生物显微镜观察和扫描电子显微镜观察。 结果与结论：两组培养的细胞数量均呈现前3 d增长,在培养第3天细胞数量达到最高值;实验组3个样本培养的细胞数明显高于对照组无微载体培养的细胞数量(P < 0.05),实验组各样本之间细胞数差异无显著性意义(P > 0.05)。倒置相差生物显微镜下动态观察,可见前3 d微载体表面黏附生长的肝细胞则逐渐增多,培养第3天可见大部分微载体表面有许多肝细胞黏附成团,总的存活率均在90%以上,且肝细胞保持着良好的形态学结构;扫描电子显微镜观察,微载体表面、切面和内部均可看到有许多球状肝细胞紧密黏附。结果说明,以自制的壳聚糖球形多孔微载体作为一种支架,在体外三维环境下可以进行高浓度细胞培养。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

软骨组织工程的研究现状及展望

目前,软骨组织工程的研究内容主要集中在以下几个方面:种子细胞;三维生物材料载体;细胞培养体系;人工软骨性能、实验动物模型和临床试验评价。我们综述了软骨组织工程的研究现状及其进展。     

微载体培养技术在骨与软骨组织工程研究中的应用☆

背景：骨与软骨组织工程学中增殖种子细胞和保持细胞特定表型是其难点,微载体生物反应器培养系统提供了很好的条件来解决这个问题。 目的：分析近年来国内外骨、软骨细胞微载体培养的研究进展,为骨与软骨细胞微载体培养技术和组织工程研究提供理论基础。 方法：由第一作者在2010-11进行检索。检索数据库：PubMed数据库(网址http://www.ncbi.nlm.gov/PubMed);万方数据库(网址http://www.wanfangdata.com.cn),资料的检索时间范围为1967/2011。英文检索词为“microcarrier,cartilage,tissue engineering”,中文检索词为“微载体,软骨,组织工程学”。排除与本文无关及陈旧、重复的文章,共保存32篇文献做进一步分析。 结果与结论：在微载体培养系统中,可较好的调控骨与软骨细胞培养条件,能在短时间内大量的增殖,并能保持其细胞的表型,甚至出现表型增强现象,在骨、软骨组织工程学研究和临床应用中有着巨大潜力。     

基因强化骨组织工程中的病毒载体

背景：不同的基因输送策略也被应用到骨组织工程中以修复破坏的骨组织,作为最有效率的基因转运载体,病毒载体在骨组织工程中的应用方兴未艾。 目的：系统回顾和讨论目前基因强化骨组织工程中常用的病毒载体相关应用。 方法：利用PubMed数据库对2002年1月至2015年1月的相关文献进行了检索,检索的文章主要聚焦在病毒载体基因转导方法和其在骨组织工程中的应用。对腺病毒、反转录病毒、腺相关病毒和嵌合病毒在骨组织工程的相关应用及不足进行了讨论。总共24篇相关文献被纳入此篇综述。 结果与结论：总结了近年来病毒载体联合基因治疗促进骨组织再生的研究工作。讨论了包括装载目的基因的病毒载体联合种子细胞例如间充质干细胞植入支架材料修复骨缺损。研究表明,基因强化的骨组织工程比传统组织工程具有更多的优点;病毒载体介导的基因转染效率比普通载体更高;病毒载体介导的基因强化骨组织工程用于人体的安全性仍需要漫长的临床观察研究。病毒载体系统仍然是最有效的将外源基因转入种子细胞的手段之一。 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;组织工程全文链接：     

平滑肌细胞在旋转生物反应器内的微载体培养与快速扩增

为获取足量数量活性良好的膀胱平滑肌种子细胞，本实验探索在旋转生物反应器内应用微载体技术快速扩增膀胱平滑肌细胞的方法。将培养的第二代新西兰兔膀胱不滑肌细胞应用Cytodex-3微载体在旋转生物反应器（RCCS）内进行动态培养，观测平滑肌细胞的生长情况和细胞代谢率，进行a-肌动蛋白免疫组化染色，并与常规方法培养平滑肌细胞进行比较。结果显示膀胱平滑肌细胞在Cytodex-3微载体上生长迅速、细胞代谢率高、生长倍增时间缩短。在培养第九天，细胞数量可达最初接种的23倍。免疫组化显示平滑肌细胞活性良好。结果表明利用微载体细胞培养技术可简便快速地在体外扩增平滑肌细胞，可为构建组织工程化人造膀胱提供大量活性良好的平滑肌细胞。     

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。     

Vero细胞微载体放大培养技术研究

<正>目前,国内大部分疫苗企业仍采用传统的转瓶细胞培养工艺来生产病毒性疫苗,该方法存在细胞密度低、病毒滴度低、劳动强度大等缺点。20世纪70年代,建立了微载体/生物反应器培养动物细胞的工艺~([1]),把悬浮培养和贴壁培养两种培养工艺融合在一起,兼有两者的优点,使得动物细胞工业化的大规模、高密度培养,以满足生物技术发展的需要,而细胞微载体放大培养就是其中的关键技术之一~([2])。本文通过胰酶消化微载体上Vero细胞,实现了5 L     

A microcarrier cell culture system for large scale production of hepatitis A virus

Hepatitis A virus (HAV) was isolated from human faeces using a fetal rhesus monkey kidney cell line (Frhk-4). Infectious medium from passage 12 was used to inoculate a large (5000 cm2) microcarrier cell culture maintained in suspension. The microcarriers used were swollen, collagen-coated dextran beads on which it was easy to propagate Frhk-4 cells. Intra- and extra-cellular virus levels were assayed and compared with conventional cultures in 25 cm2 plastic flasks. The results show that virus production per cell was similar in both systems. The number of cells per area unit in confluent cultures was initially lower in the microcarrier culture but subsequently increased. Two to three weeks post inoculation the virus yield per area unit in the microcarrier system was half of that of the conventional culture. The lower cell density per area unit in the microcarrier system was compensated by the large growth area that could be maintained in a single vessel and the total production of virus was substantial. Weekly harvests of medium with HAV antigen titres around 10(-2) contained antigenic material sufficient for several thousands of anti-HAV IgM tests. Propagation of HAV in microcarrier cell cultures thus seems a safe and simple way to produce large amounts of HAV.     

Expansion of chondroprogenitor cells on macroporous microcarriers as an alternative to conventional monolayer systems

Routine tissue culture methodologies can hardly cope with the scale of cell production required for the manufacture of engineered cartilage tissue products. In vitro cell expansion has become an essential step in the process of tissue engineering of articular cartilage and the optimization of expansion protocols is a fundamental issue that needs to be addressed. The expansion suitability of chondroprogenitor cells isolated from the superficial zone of articular cartilages was evaluated in both conventional monolayer and macroporous microcarrier in spinner flask cultures. Although monolayer systems promoted rapid in vitro expansion of undifferentiated cells, they present limited scalability. Alternatively, the use of CultiSpher-G microcarriers resulted in cell densities of 5.5 x 10(5) cell/ml, representing a 17-fold expansion in batch cultures. In addition, chondroprogenitor cells were capable of undergoing bead-to-bead migration, which allowed subcultivation to be performed without a harvesting step, thus improving the scalability of the expansion process. By employing macroporous microcarrier cultures it will be possible to obtain large number of chondroprogenitor cells for tissue engineering applications. Not only its satisfactory expansion potential, but more importantly the cost and operational advantages over traditional monolayer culture make this system a feasible alternative method for the extensive expansion of chondroprogenitor cells.     

A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR

The development of a rapid and sensitive assay for detection of replication-competent adenoviruses (RCAs) is described. This RCA assay consists of an incubation step of 4 days of adenoviral vectors on A549 cells in a microcarrier cell culture system followed by detection of amplified RCAs by E1-specific quantitative PCR. The detection limit of this assay is 3 RCAs in 1 x 10(10) vector particles per 70 ml of microcarrier cell culture. The main advantage of the combination of cell culture and PCR detection is that replicated virus can be detected long before cytopathic effects become visible and therefore, it is much faster than conventional cell culture-based assays. This assay was validated by spiking replication-incompetent adenoviral vectors with wild-type adenovirus serotype 5 (wt Ad5) as a positive control for RCA. It was found that the replication of wt Ad5 is hampered above a vector particle per cell ratio of 50. However, if microcarrier beads are used, many cells can be grown in a small suspension culture and consequently a large number of vector particles can be tested for contamination with RCA.     

Can microcarrier-expanded chondrocytes synthesize cartilaginous tissue in vitro?

Tissue engineering is a promising approach for articular cartilage repair; however, it is challenging to produce adequate amounts of tissue in vitro from the limited number of cells that can be extracted from an individual. Relatively few cell expansion methods exist without the problems of de-differentiation and/or loss of potency. Recently, however, several studies have noted the benefits of three-dimensional (3D) over monolayer expansion, but the ability of 3D expanded chondrocytes to synthesize cartilaginous tissue constructs has not been demonstrated. Thus, the purpose of this study was to compare the properties of engineered cartilage constructs from expanded cells (monolayer and 3D microcarriers) to those developed from primary chondrocytes. Isolated bovine chondrocytes were grown for 3 weeks in either monolayer (T-Flasks) or 3D microcarrier (Cytodex 3) expansion culture. Expanded and isolated primary cells were then seeded in high density culture on Millicell? filters for 4 weeks to evaluate the ability to synthesize cartilaginous tissue. While microcarrier expansion was twice as effective as monolayer expansion (microcarrier: 110-fold increase, monolayer: 52-fold increase), the expanded cells (monolayer and 3D microcarrier) were not effectively able to synthesize cartilaginous tissue in vitro. Tissues developed from primary cells were substantially thicker and accumulated significantly more extracellular matrix (proteoglycan content: 156%-292% increase; collagen content: 70%-191% increase). These results were attributed to phenotypic changes experienced during the expansion phase. Monolayer expanded chondrocytes lost their native morphology within 1 week, whereas microcarrier-expanded cells were spreading by 3 weeks of expansion. While the use of 3D microcarriers can lead to large cellular yields, preservation of chondrogenic phenotype during expansion is required in order to synthesize cartilaginous tissue.     

Biocompatible inkjet printing technique for designed seeding of individual living cells

Inkjet printers are capable of printing at high resolution by ejecting extremely small ink drops. Established printing technology will be able to seed living cells, at micrometer resolution, in arrangements similar to biological tissues. We describe the use of a biocompatible inkjet head and our investigation of the feasibility of microseeding with living cells. Living cells are easily damaged by heat; therefore, we used an electrostatically driven inkjet system that was able to eject ink without generating significant heat. Bovine vascular endothelial cells were prepared and suspended in culture medium, and the cell suspension was used as "ink" and ejected onto culture disks. Microscopic observation showed that the endothelial cells were situated in the ejected dots in the medium, and that the number of cells in each dot was dependent on the concentration of the cell suspension and ejection frequency chosen. After the ejected cells were incubated for a few hours, they adhered to the culture disks. Using our non-heat-generating, electrostatically driven inkjet system, living cells were safely ejected onto culture disks. This microseeding technique with living cells has the potential to advance the field of tissue engineering.     

Suspension culture of Marek’s disease virus and evaluation of its immunological effects

Marek’s disease virus (MDV) is a cell-associated α-herpesvirus of chickens. It is difficult to grow MDV in suspension culture. Therefore, MDV vaccines are currently produced using adherent primary chicken embryo fibroblasts, and on a large scale this is labour-intensive and costly. In this study, the CVI988 strain of MDV was inoculated into chicken fibroblast cell line UMNSAH/DF-1 (DF-1) cultured by microcarrier suspension for the proliferation experiment. Moreover, the effects of culture conditions, such as inoculation method, multiplicity of infection (MOI), microcarrier concentration, and pH value, on the proliferation of MDV were investigated. The results demonstrated that the maximum viral load of 64.76?±?2.64?×?10⁶ PFU/flask in a working volume of 100?ml could be obtained using synchronous cell seeding and inoculation method at an MOI of 0.02 and a microcarrier concentration of 5?g/l at pH 7.2. At the same time, the CVI988/DF-1 vaccines prepared by the microcarrier culture process and the traditional adherent cell culture process (CVI988/Rispens) were compared through bird experiments. We found a protective rate of 94.4% using the CVI988/DF-1 vaccine with specific pathogen-free chickens that was equivalent to that of the commercial vaccine CVI988/Rispens (protection rate of 94.1%). In this study, the MDV CVI988/DF-1 vaccine prepared by the microcarrier suspension culture of DF-1 cells could provide effective immune protection for specific pathogen-free chickens, providing a reference for the prevention and control of MD and further development of a large-scale bioreactor for producing the MD vaccine.     

Large-scale Vero cell culture on microcarriers in a twenty-liter stirred tank fermentor

In biotechnology, animal cell culture is an important process for the production of many biologicals such as vaccines, monoclonal antibodies, or other recombinant products. Among many established continuous cell lines, Vero cells can be maintained in many passages in cultures without inducing tumorigenicity and have been recommended by World Health Organization for the production of human biologicals. Owing to its anchorage-dependent growth characteristics, Vero cells can be grown on microcarrier in a suspension vessel where microcarrier provides the culture system with a high culture surface to volume ratio. In this paper we compared the growth kinetics of Vero cells on Cytodex 1 microcarrier in a 20-liter fermentor vs. 100 ml spinner flask culture. The kinetics of Vero cell growth in the 20-liter fermentor was similar to the results obtained from small spinner flask culture, as determined by cell specific growth rate or corresponding doubling time. The approximately 150-fold increase in culture vessel volume did not compromise the growth kinetics of Vero cells, suggesting the system is applicable for large stirred-tank fermentor cultures.     

Porous poly(lactic-co-glycolic acid) microsphere as cell culture substrate and cell transplantation vehicle for adipose tissue engineering

Tissue engineering often requires ex vivo cell expansion to obtain a large number of transplantable cells. However, the trypsinization process used to harvest ex vivo expanded cells for transplantation interrupts interactions between cultured cells and their extracellular matrices, facilitating apoptosis and consequently limiting the therapeutic efficacy of the transplanted cells. In the present study, open macroporous poly(lactic-co-glycolic acid) (PLGA) microspheres were used as a cell culture substrate to expand human adipose-derived stromal cells (ASCs) ex vivo and as a cell transplantation vehicle for adipose tissue engineering, thus avoiding the trypsinization necessary for transplantation of ex vivo expanded cells. Human ASCs cultured on macroporous PLGA microspheres in stirred suspension bioreactors expanded 3.8-fold over 7 days and differentiated into an adipogenic lineage. The apoptotic activity of ASCs cultured on microspheres was significantly lower than that of trypsinized ASCs. ASCs cultured on microspheres survived much better than trypsinized ASCs upon transplantation. The implantation of ASCs cultured on microspheres resulted in much more extensive adipose tissue formation than the implantation of ASCs cultured on plates, trypsinized, and subsequently mixed with microspheres. Ex vivo cell expansion and transplantation using this system would improve the therapeutic efficacy of cells over the current methods used for tissue engineering.     

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。     

Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation

Articular cartilage has a limited capacity for self-repair. To overcome this problem, it is expected that functional cartilage replacements can be created from expanded chondrocytes seeded in biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems often results in dedifferentiation. This investigation focuses on the post-expansion phenotype of human nasal chondrocytes expanded on macroporous gelatin CultiSpher G microcarriers. Redifferentiation was evaluated in vitro via pellet cultures in three different culture media. Furthermore, the chondrogenic potential of expanded cells seeded in polyethylene glycol terephthalate/ polybuthylene terephthalate (PEGT/PBT) scaffolds, cultured for 14 days in vitro, and subsequently implanted subcutaneously in nude mice, was assessed.

Chondrocytes remained viable during microcarrier culture and yielded doubling times (1.07±0.14 days) comparable to T-flask expansion (1.20±0.36 days). Safranin-O staining from pellet culture in different media demonstrated that production of GAG per cell was enhanced by microcarrier expansion. Chondrocyte–polymer constructs with cells expanded on microcarriers contained significantly more proteoglycans after subcutaneous implantation (288.5±29.2 μg) than those with T-flask-expanded cells (164.0±28.7 μg). Total collagen content was similar between the two groups.

This study suggests that macroporous gelatin microcarriers are effective matrices for nasal chondrocyte expansion, while maintaining the ability of chondrocyte differentiation. Although the exact mechanism by which chondrocyte redifferentiation is induced through microcarrier expansion has not yet been elucidated, this technique shows promise for cartilage tissue engineering approaches.