Cartmell Sarah H, Porter Blaise D, García Andrés J, Guldberg Robert E
School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA.
Tissue Eng. 2003 Dec;9(6):1197-203. doi: 10.1089/10763270360728107.
Cellular activity at the center of tissue-engineered constructs in static culture is typically decreased relative to the construct periphery because of transport limitations. We have designed a tissue culture system that perfuses culture medium through three-dimensional (3D) porous cellular constructs to improve nutrient delivery and waste removal within the constructs. This study examined the effects of medium perfusion rate on cell viability, proliferation, and gene expression within cell-seeded 3D bone scaffolds. Human trabecular bone scaffolds were seeded with MC3T3-E1 osteoblast-like cells and perfused for 1 week at flow rates of 0.01, 0.1, 0.2, and 1.0 mL/min. Confocal microscopy after 1 week of culture indicated that a flow rate of 1.0 mL/min resulted in substantial cell death throughout the constructs whereas lowering the flow rate led to an increasing proportion of viable cells, particularly at the center of the constructs. DNA analysis showed increases in cell proliferation at a flow rate of 0.01 mL/min relative to 0.2 mL/min and static controls. Conversely, mRNA expressions of Runx2, osteocalcin, and alkaline phosphatase were upregulated at 0.2 mL/min compared with lower flow rates as quantified by real-time RT-PCR. These data suggest that medium perfusion may benefit the development of 3-D tissues in vitro by enhancing transport of nutrients and waste within the constructs and providing flow-mediated mechanical stimuli.
由于传输限制，在静态培养中组织工程构建体中心的细胞活性相对于构建体周边通常会降低。我们设计了一种组织培养系统，该系统通过三维（3D）多孔细胞构建体灌注培养基，以改善构建体内的营养物质输送和废物清除。本研究考察了培养基灌注速率对接种细胞的3D骨支架内细胞活力、增殖和基因表达的影响。将人小梁骨支架接种MC3T3-E1成骨样细胞，并以0.01、0.1、0.2和1.0 mL/min的流速灌注1周。培养1周后的共聚焦显微镜检查表明，1.0 mL/min的流速导致整个构建体中大量细胞死亡，而降低流速会使活细胞比例增加，尤其是在构建体的中心。DNA分析显示，相对于0.2 mL/min的流速和静态对照，0.01 mL/min的流速下细胞增殖增加。相反，通过实时RT-PCR定量分析，与较低流速相比，0.2 mL/min时Runx2、骨钙素和碱性磷酸酶的mRNA表达上调。这些数据表明，培养基灌注可能通过增强构建体内营养物质和废物的传输以及提供流动介导的机械刺激，有利于体外三维组织的发育。
